Quaternized polypyridine ruthenium complex as well as preparation method and antibacterial application thereof

Abstract

The invention discloses a quaternized polypyridine ruthenium complex as well as a preparation method and application thereof, and relates to the technical field of antibacterial medicines. The quaternized polypyridine ruthenium complex has a structure as shown in a formula I-1 or a formula I-2. The invention also provides an application of the complex in preparation of bacteriostatic or antibacterial drugs. The complex contains metal ions and has charges, compared with traditional small organic molecules, the ability of penetrating through bacterial cell membranes and the retention effect of the complex are enhanced, and the metal complex can be modified by different ligands due to the multi-coordination configuration of the metal complex, so that the biological activity of the metal complex is higher. Tests prove that the quaternized polypyridine ruthenium complex with the broad-spectrum antibacterial activity can effectively reduce the hemolysis effect of toxin released by staphylococcus aureus on red blood cells, and the ruthenium complex does not have the tendency of triggering drug resistance of bacteria, has the broad-spectrum antibacterial activity, and can be used for preparing the antibacterial medicine. The sensitivity of various bacteria to the medicine can be obviously enhanced.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial pharmaceutical technology, specifically relating to a quaternized polypyridine ruthenium complex, its preparation method, and its antibacterial applications. Background Technology

[0002] Staphylococcus aureus ( S. aureus Acinetobacter baumannii is one of the most common pathogens causing infectious diseases in clinical practice. It can cause infections in multiple sites, including the skin and soft tissues, the blood system, and the lower respiratory tract, leading to a range of diseases such as pneumonia and sepsis. In recent years, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains, which exhibit severe resistance to many antibiotics, has made the treatment of these infections increasingly difficult. Acinetobacter baumannii is a Gram-negative pathogen that often exhibits multidrug resistance due to its highly impermeable outer membrane and its remarkable ability to acquire and retain extracellular DNA, which usually contains the determinants of antibiotic resistance. Acinetobacter baumannii is the most prevalent opportunistic bacterial pathogen, causing invasive infections in hospitalized and critically ill patients, primarily manifesting as nosocomial pneumonia and bloodstream infections.

[0003] The development of novel antimicrobial drugs is currently facing a severe and urgent challenge. Traditional antibiotics primarily work by inhibiting the function of specific bacterial targets (such as key macromolecules), but bacteria readily develop resistance through target mutations, rendering these drugs ineffective. Even more problematic is that drug-resistant bacteria (such as Staphylococcus aureus) often form biofilms to enhance their resistance. These biofilms not only significantly hinder antibiotic penetration but also protect the bacteria from the host's immune system, making them a key factor in chronic, recurrent infections and significantly increasing the difficulty of treatment. Even if antibiotics successfully eliminate some bacteria, the extracellular polymers and other residual substances they induce still provide a breeding ground for subsequent bacterial attachment and biofilm regeneration, leading to infection recurrence, prolonged disease duration, and increased healthcare burden. Therefore, breaking through the limitations of existing mechanisms of action and developing novel targets such as the intact cellular structures essential for bacterial survival (such as the cell membrane) has become a core strategic direction for addressing the drug resistance crisis and eradicating intractable infections. Thus, developing new antimicrobial drugs that target the bacterial cell membrane has become a crucial technology. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a quaternized polypyridine ruthenium complex, its preparation method and antibacterial application.

[0005] The polypyridine ruthenium complex of this invention contains metal ions carrying a charge. The positively charged organic molecular chains combine with anions on the surface of bacterial cell membranes, thereby disrupting the cell membrane composition, causing intracellular leakage, and preventing the bacteria from producing toxins. Furthermore, the multi-coordination configuration of the metal complex allows for modification with different ligands, resulting in improved biological activity. Therefore, by modifying the polypyridine ruthenium complex, bacterial disruption can be achieved, thereby inhibiting the production of toxins by drug-resistant strains.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a quaternized polypyridine ruthenium complex having the structure shown in Formula I-1 or Formula I-2: Formula I-1; Formula I-2: in, Choose from any of the following structures: ; Linker can be any chemically feasible linker structure.

[0007] Optionally, the Linker is or , where m is 1~5 and n is 1~10.

[0008] Optionally, the quaternized polypyridine ruthenium complex has the structure shown in Ru-1 to Ru-9: .

[0009] Secondly, the present invention provides a method for preparing the aforementioned quaternized polypyridine ruthenium complex. The preparation method of the quaternized polypyridine ruthenium complex shown in Formula I-1 includes the following steps: reacting p-fluorobenzaldehyde with 4-dimethylaminopyridine in a solvent under heat to obtain an intermediate; reacting the intermediate with 1,10-phenanthroline-5,6-dione in a solvent under heat to obtain a ligand; and reacting the ligand with the ruthenium complex in a solvent to obtain the quaternized polypyridine ruthenium complex shown in Formula I-1. The preparation method of the quaternized polypyridine ruthenium complex shown in Formula I-2 includes the following steps: under protective conditions, p-hydroxybenzaldehyde and a brominated compound are reacted by heating in a solvent to obtain a first intermediate; the intermediate is reacted by heating with 4-dimethylaminopyridine in a solvent to obtain a second intermediate; the second intermediate is reacted by heating with 1,10-phenanthroline-5,6-dione in a solvent to obtain a ligand; the ligand is reacted with the ruthenium complex in a solvent to obtain the quaternized polypyridine ruthenium complex shown in Formula I-2.

[0010] Optionally, the preparation method of the quaternized polypyridine ruthenium complex of formula Ru-7 includes the following steps: S1. The p-fluorobenzaldehyde of formula Ib and 4-dimethylaminopyridine were heated under reflux in acetonitrile. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of formula II-b. S2. The compound of formula II-b was reacted with 1,10-phenanthroline-5,6-dione in ethanol under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was then purified by recrystallization from ethanol to obtain the ligand of formula III-b. S3. The ligand of the III-b structure and the ruthenium complex were heated and reacted in ethylene glycol. After the reaction was completed, the mixture was purified to obtain the quaternized polypyridine ruthenium complex shown in formula Ru-7.

[0011] Optionally, the preparation method of the quaternized polypyridine ruthenium complexes represented by formulas Ru-1~Ru-6 and Ru-8~Ru-9 includes the following steps: (1) Under protective conditions, p-fluorobenzaldehyde of formula Ia and a brominated compound were heated and refluxed in acetonitrile. After cooling to room temperature, the mixture was filtered and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediates of formula Ic~If were obtained. (2) The intermediates of the Ic ~ If structure were reacted with 4-dimethylaminopyridine in acetonitrile under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of the II-c ~ II-f structure. (3) The compound with the structure of formula II-c ~ II-f was reacted with 1,10-phenanthroline-5,6-dione in ethanol under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand with the structure of formula III-c ~ III-f. (4) The ligands of the III-c ~ III-f structures were heated and reacted with ruthenium complexes in ethylene glycol. After the reaction was completed, the mixtures were purified to obtain the quaternized polypyridine ruthenium complexes shown in the formulas Ru-1 ~ Ru-6 and Ru-8 ~ Ru-9. The structural formulas of each raw material, intermediate, and quaternized polypyridine ruthenium complex are as follows: .

[0012] Optionally, in step (1), the temperature of the reflux reaction is 85 ~ 95℃ and the time is 7 ~ 9 h; the purification of the compound with the Ic~If structure is carried out by elution on a silica gel chromatography column with an eluent of petroleum ether / ethyl acetate volume ratio of 20:1.

[0013] Optionally, in steps S1 and (2), the temperature of the reflux reaction is 85 ~ 95℃ and the time is 44 ~ 52h, and the purification is carried out by extraction with dichloromethane and water.

[0014] Optionally, in steps S2 and (3), the temperature of the reflux reaction is 125 ~ 135℃ and the time is 2 ~ 4h; In steps S3 and (4), the temperature of the reflux reaction is 145 ~ 155℃ and the time is 9 ~ 11 h. The purification is carried out by elution with acetonitrile and xylene on a silica gel chromatography column.

[0015] Thirdly, the present invention provides the application of the aforementioned quaternized polypyridine ruthenium complex in the preparation of antibacterial or antimicrobial drugs.

[0016] Optionally, the antibacterial or antimicrobial drug includes drugs that inhibit or kill Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii.

[0017] This invention has at least one of the following beneficial effects: (1) This invention introduces a quaternary ammonium structure into the ruthenium polypyridine ligand. The positive charge of the quaternary ammonium structure binds to the negative ions on the bacterial cell membrane, thereby disrupting the bacterial cell membrane. By introducing alkyl chains of different lengths, the lipid solubility of the drug is adjusted, significantly enhancing its ability to disrupt the bacterial cell membrane, thus improving its antibacterial activity. Therefore, the ruthenium complex of this invention can disrupt the bacterial cell membrane structure, causing leakage of cell contents, thereby effectively inhibiting bacterial growth and preventing bacteria from forming a protective biofilm on the host surface.

[0018] (2) Experimental results show that the formula Ru-1 provided by the present invention can rapidly kill Staphylococcus aureus in dynamic growth within 90 minutes at a concentration of 6.25 μg / mL, rapidly kill methicillin-resistant Staphylococcus aureus in dynamic growth within 30 minutes at a concentration of 1.56 μg / mL, and rapidly kill Acinetobacter baumannii in dynamic growth within 60 minutes at a concentration of 50 μg / mL. Ru-1 can effectively inhibit and remove bacterial mature biofilms in vitro, thereby reducing the formation rate of drug-resistant bacteria. Importantly, the toxicity of Ru-1 in the body of the giant wax moth is comparable to that of vancomycin, and it does not induce bacteria to develop drug resistance. Compared with the prior art, the polypyridine ruthenium complex with quaternization modification can effectively inhibit bacterial growth and avoid the emergence of bacterial drug resistance. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This invention provides a synthetic route for quaternized polypyridine ruthenium complexes; Figure 2 This is a graph showing the time-dependent killing effect of the polypyridine ruthenium complex Ru-1 on Staphylococcus aureus provided by this invention. Figure 3 This is a graph showing the time-dependent killing effect of the polypyridine ruthenium complex Ru-1 provided by this invention on methicillin-resistant Staphylococcus aureus; Figure 4 This is a graph showing the time-dependent killing effect of the polypyridine ruthenium complex Ru-1 on Acinetobacter baumannii provided by this invention. Figure 5 This is a graph showing the determination of Staphylococcus aureus resistance and commonly used antibiotic resistance induced by the polypyridine ruthenium complex Ru-1 provided by this invention; Figure 6 This is a graph showing the inhibition and eradication of mature Staphylococcus aureus biofilm by the polypyridine ruthenium complex Ru-1 provided by the present invention; Figure 7 This is a graph showing the toxicity and therapeutic effects of the polypyridine ruthenium complex Ru-1 in *Hemiberlesia lataniae* provided by this invention. Figure 8 This is the mass spectrum of the polypyridine ruthenium complex Ru-1 provided by the present invention; Figure 9 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-1 provided by this invention; Figure 10 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-1 provided by the present invention; Figure 11 This is the mass spectrum of the polypyridine ruthenium complex Ru-2 provided by the present invention; Figure 12 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-2 provided by this invention; Figure 13 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-2 provided by the present invention; Figure 14 This is the mass spectrum of the polypyridine ruthenium complex Ru-3 provided by the present invention; Figure 15 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-3 provided by this invention; Figure 16This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-3 provided by the present invention; Figure 17 This is the mass spectrum of the polypyridine ruthenium complex Ru-4 provided by the present invention; Figure 18 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-4 provided by this invention; Figure 19 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-4 provided by the present invention; Figure 20 This is the mass spectrum of the polypyridine ruthenium complex Ru-5 provided by the present invention; Figure 21 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-5 provided by this invention; Figure 22 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-5 provided by the present invention; Figure 23 This is the mass spectrum of the polypyridine ruthenium complex Ru-6 provided by the present invention; Figure 24 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-6 provided by this invention; Figure 25 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-6 provided by the present invention; Figure 26 This is the mass spectrum of the polypyridine ruthenium complex Ru-7 provided by the present invention; Figure 27 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-7 provided by this invention; Figure 28 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-7 provided by the present invention; Figure 29 This is the mass spectrum of the polypyridine ruthenium complex Ru-8 provided by the present invention; Figure 30 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-8 provided by this invention; Figure 31 This is the HPLC chromatogram of the polypyridine ruthenium complex Ru-8 provided by the present invention; Figure 32 This is the mass spectrum of the polypyridine ruthenium complex Ru-9 provided by the present invention; Figure 33 This is the proton NMR spectrum of the polypyridine ruthenium complex Ru-9 provided by this invention; Figure 34 This is an HPLC chromatogram of the polypyridine ruthenium complex Ru-9 provided by the present invention. Detailed Implementation

[0021] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] This invention provides a method for preparing quaternized polypyridine ruthenium complexes, the synthetic route being as follows: Figure 1 As shown, it includes the following steps: (1) Under argon conditions, p-hydroxybenzaldehyde of formula Ia and the brominated compound were heated and refluxed in acetonitrile, cooled to room temperature, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediate of formula Ic~If structure was obtained. (2) The intermediates of the Ic ~ If structure and the p-fluorobenzaldehyde of the Ib structure were respectively reacted with the 4-dimethylaminopyridine (DMAP) shown in the Ig structure in acetonitrile solvent and heated under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of the II-b ~ II-f structure. (3) The compounds of formula II-b ~ II-f and 1,10-phenanthroline-5,6-dione of formula Ih were heated and refluxed in ethanol solvent. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligands of formula III-b ~ III-f. (4) The ligands of the III-b ~ III-f structures were reacted with the ruthenium complex in ethylene glycol. After the reaction was completed, the mixture was purified to obtain the quaternized polypyridine ruthenium complexes shown in Ru-1 ~ Ru-9.

[0023] Among them, the compounds of formula Ia, Ib, Ic, Id, Ie, If, Ig, Ih, II-b, II-c, II-d, II-e, II-f, III-b, III-c, III-d, III-e, III-f, and the quaternized polypyridine ruthenium complexes shown in Ru-1 to Ru-9 have the following structural formulas: Figure 1 As shown.

[0024] In one specific embodiment of the present invention, the structural formulas of Ru-1, Ru-2, Ru-3, Ru-4, Ru-5, Ru-6, Ru-7, Ru-8, and Ru-9 are as follows: The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.

[0025] Example 1 A method for preparing quaternized polypyridine ruthenium complexes (Ru-1, Ru-2, Ru-3) includes the following steps: (1) Under argon conditions, (1.0 g, 8.2 mmol) of the compound of formula Ia and (8.9 g, 41 mmol) of the brominated compound (1,4-dibromobutane) were heated to reflux at 90 °C for 8 h in acetonitrile. After cooling to room temperature, the mixture was filtered and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediate of formula Id was obtained. (2) The intermediate compound of formula Id (515 mg, 2 mmol) was reacted with DMAP in 30 mL acetonitrile solvent under reflux at 90 °C for 48 h. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of formula II-d. (3) The compound of formula II-d (300 mg, 1 mmol) and the compound of formula Ih (210 mg, 1 mmol) were heated and refluxed at 80 °C for 9 h in 30 mL of ethanol solvent. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand of formula III-d. (4) The ligand of the (245 mg, 0.5 mmol) formula III-d structure was reacted with (354 mg, 0.5 mmol) dtb, (270 mg, 0.5 mmol) dmb, and (242 mg, 0.5 mmol) dpy ruthenium complexes in 20 mL ethylene glycol at 150 °C for 10 h. After the reaction was completed, the mixture was purified to obtain quaternized polypyridine ruthenium complexes Ru-1, Ru-2, and Ru-3.

[0026] The 1H NMR, mass, and HPLC spectra of the quaternized polypyridine ruthenium complexes Ru-1, Ru-2, and Ru-3 are shown below. Figures 8-16 Thus, the structural formulas of Ru-1, Ru-2, and Ru-3 can be determined.

[0027] Example 2 A method for preparing quaternized polypyridine ruthenium complexes (Ru-4, Ru-5, Ru-6) includes the following steps: (1) Under argon conditions, (1.0 g, 8.2 mmol) of the compound of formula Ia and (7.7 g, 41 mmol) of the brominated compound (1,2-dibromoethane) were heated to reflux at 90 °C for 8 h in acetonitrile. After cooling to room temperature, the mixture was filtered and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediate of formula Ic was obtained. (2) The intermediate of formula Ic (454 mg, 2 mmol) was reacted with DMAP (244 mg, 2 mmol) in 30 mL of acetonitrile solvent under reflux at 90 °C for 8 h. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of formula II-c. (3) The compound of formula II-c (271 mg, 1 mmol) and the compound of formula Ih (210 mg, 1 mmol) were heated and refluxed at 80 °C for 9 h in 30 mL of ethanol solvent. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand of formula III-c. (4) The ligands of the (230 mg, 0.5 mmol) formula III-c structure, namely (354 mg, 0.5 mmol) dtb, (270 mg, 0.5 mmol) dmb, and (242 mg, 0.5 mmol) dpy ruthenium complexes, were reacted in 20 mL of ethylene glycol at 150 °C for 10 h. After the reaction was completed, the mixture was purified to obtain quaternized polypyridine ruthenium complexes Ru-4, Ru-5, and Ru-6.

[0028] The 1H NMR, mass, and HPLC spectra of the quaternized polypyridine ruthenium complexes Ru-4, Ru-5, and Ru-6 are shown below. Figures 17-25 Thus, the structural formulas of Ru-4, Ru-5, and Ru-6 can be determined.

[0029] Example 3 A method for preparing a quaternized polypyridine ruthenium complex (Ru-7) includes the following steps: (1) The compound of formula Ib (1.2 g, 10 mmol) was reacted with DMAP in 30 mL of acetonitrile solvent under reflux at 90 °C for 48 h. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of formula II-b. (2) The compound of formula II-b (227 mg, 1 mmol) and the compound of formula Ih (210 mg, 1 mmol) were heated to reflux at 80 °C for 9 h in 30 mL of ethanol solvent. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand of formula III-b. (3) The ligand of the (209 mg, 0.5 mmol) formula III-b structure was reacted with the (354 mg, 0.5 mmol) dtb ruthenium complex in 20 mL ethylene glycol at 150 °C for 10 h. After the reaction was completed, the mixture was purified to obtain the quaternized polypyridine ruthenium complex Ru-7.

[0030] The 1H NMR, mass, and HPLC spectra of the quaternized polypyridine ruthenium complex Ru-7 are shown below. Figures 26-28 Therefore, the structural formula of Ru-7 can be determined.

[0031] Example 4 A method for preparing a quaternized polypyridine ruthenium complex (Ru-8) includes the following steps: (1) Under argon conditions, (1.0 g, 8.2 mmol) of the compound of formula Ia and (10 g, 41 mmol) of the brominated compound (1,6-dibromohexane) were heated to reflux at 90 °C for 8 h in acetonitrile. After cooling to room temperature, the mixture was filtered and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediate of formula Ie was obtained. (2) The compound with the structure of formula Ie (568 mg, 2 mmol) was reacted with DMAP (244 mg, 2 mmol) in 30 mL of acetonitrile solvent under reflux at 90 °C for 48 h. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound with the structure of formula II-e. (3) The compound of formula II-e (327 mg, 1 mmol) and the compound of formula Ih (210 mg, 1 mmol) were heated to reflux at 80 °C for 9 h in 30 mL of ethanol solvent. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand of formula III-e. (4) The ligand of the (259 mg, 0.5 mmol) formula III-e structure was reacted with the (354 mg, 0.5 mmol) dtb ruthenium complex in 20 mL ethylene glycol at 150 °C for 10 h. After the reaction was completed, the mixture was purified to obtain the quaternized polypyridine ruthenium complex Ru-8.

[0032] The 1H NMR and HPLC chromatograms of the quaternized polypyridine ruthenium complex Ru-8 are shown below. Figures 29-31 Therefore, the structural formula of Ru-8 can be determined.

[0033] Example 5 A method for preparing a quaternized polypyridine ruthenium complex (Ru-9) includes the following steps: (1) Under argon conditions, (1.0 g, 8.2 mmol) of the compound of formula Ia and (9.5 g, 41 mmol) of the brominated compound (bis(2-bromoethyl) ether) were heated to reflux in acetonitrile, cooled to room temperature, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediate of formula If was obtained. (2) The compound with the formula If structure (546 mg, 2 mmol) was reacted with DMAP in 30 mL of acetonitrile solvent under reflux at 90 °C for 48 h. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound with the formula II-f structure. (3) The compound of formula II-f (315 mg, 1 mmol) and the compound of formula Ih (210 mg, 1 mmol) were heated to reflux at 80 °C for 9 h in 30 mL of ethanol solvent. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand of formula III-f. (4) The ligand of the (209 mg, 0.5 mmol) formula III-f structure was reacted with the (354 mg, 0.5 mmol) dtb ruthenium complex in 20 mL ethylene glycol at 150 °C for 10 h. After the reaction was completed, the mixture was purified to obtain the quaternized polypyridine ruthenium complex Ru-9.

[0034] The 1H NMR, mass, and HPLC spectra of the quaternized polypyridine ruthenium complex Ru-9 are shown below. Figures 32-34 Therefore, the structural formula of Ru-9 can be determined.

[0035] Example 6 The minimum concentrations of the polypyridine ruthenium complexes Ru-1 to Ru-9 that inhibited the in vitro growth of Staphylococcus aureus in the logarithmic growth phase were determined, and the specific process is as follows: 5 mg of the complexes Ru-1 to Ru-9 (analyte compounds) were dissolved in DMSO (10 mg / mL), and the drug was diluted with sterile water to 1 mg / mL for later use. Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii cultured to the logarithmic growth phase were diluted 1000-fold in MH medium to prepare bacterial suspensions; vancomycin (Van) or polymyxin (Poly B) were used as positive controls. 200 μL of the bacterial suspension was mixed with 50 μL of drug solutions of different concentration gradients and added to 96-well plates. The 96-well plates were incubated at 37°C for 20 hours, and the minimum inhibitory concentration (MIC) of the drug was then determined. The experimental results are shown in Tables 1 and 2. The MIC of Ru-1 against Acinetobacter baumannii was 6.25 μg / mL.

[0036] Table 1. Antibacterial activity and physicochemical properties of nine ruthenium complexes. Table 2 Antibacterial activity of nine ruthenium complexes against MRSA a Methicillin-resistant S. aureus ATCC BAA-1747. b Methicillin-resistant S. aureus ATCC BAA-41. c Methicillin-resistant S. aureus ATCC 43300. d Methicillin-resistant S. aureus ATCC 33591. e Methicillin-resistant S. aureus ATCC BAA-1720. Table 1 shows that the nine ruthenium complexes exhibited antibacterial activities against Staphylococcus aureus ranging from 0.78 to 25 μg / mL, essentially no activity against Pseudomonas aeruginosa, and inhibitory concentrations against Gram-negative Escherichia coli ranging from 12.5 to 200 μg / mL. Overall, Ru-1 showed the most significant antibacterial activity among the nine compounds. Further analysis revealed that Ru-1 had an inhibitory concentration of 6.25 μg / mL against Acinetobacter baumannii.

[0037] Table 2 shows that the nine ruthenium complexes all have good antibacterial activity against various methicillin-resistant Staphylococcus aureus (MRSA). Among them, the best compound Ru-1 showed particularly significant performance with a minimum inhibitory concentration of 0.78 to 1.56 μg / mL against five MRSAs.

[0038] Example 7 The hemolytic activity of the prepared polypyridine ruthenium complexes Ru-1 to Ru-9 was determined by the following process: The hemolytic activity of ruthenium complexes was measured using rabbit erythrocytes. Fresh rabbit erythrocytes (RBCs) were centrifuged (2000 r / min, 2 min) and washed three times with PBS (phosphate-buffered saline). Different concentrations of ruthenium complexes and 5% of blood cells (RBCs) were incubated at 37°C for 30 min. 1% Triton X-100 solution served as a positive control, and PBS solution served as a negative control. The suspension was centrifuged (2000 r / min, 2 min) to obtain the supernatant. The absorbance of the supernatant was measured at 543 nm. The hemolysis rate was measured as follows: Hemolysis rate = [(OD sample - OD negative) / (OD positive – OD negative)]. All experiments were performed in triplicate.

[0039] As shown in Table 1, HC 50 As shown by the SI (membrane selectivity) results, the hemolytic concentrations of all nine ruthenium complexes were above 250 μg / mL, with Ru-1 exhibiting a membrane selectivity exceeding 320 μg / mL. These results indicate that all nine ruthenium complexes demonstrated good biocompatibility and membrane selectivity.

[0040] Example 8 The study investigated the time-dependent bactericidal activity of the polypyridine ruthenium complex Ru-1 against bacteria during the logarithmic growth phase. The specific procedure is as follows: The overnight bacteria were diluted at a ratio of 1:1000 and cultured to the logarithmic growth phase. The bacterial suspension was then diluted 1000 times, and the bacterial suspension was mixed with complex Ru-1 to prepare suspensions with final concentrations of 1 ×MIC, 2 ×MIC, 4 ×MIC, and 8 ×MIC. Every 30 minutes, 50 μL of the bacterial suspension was evenly spread on an agar plate and incubated at 37°C for 20 h. The colony count was then recorded.

[0041] The result is as follows Figure 2 , Figure 3 and Figure 4 As shown, in Figure 2 In (A, B), when the concentration of Ru-1 reaches 8 × MIC, it has a significant bactericidal effect on Staphylococcus aureus. Figure 3 In (A, B), the scavenging effect of compound Ru-1 was significantly enhanced at a concentration of 2 × MIC. Figure 4 In (A, B), compound Ru-1 completely killed Acinetobacter baumannii within one hour at a concentration of 8×MIC. Therefore, it can be concluded that Ru-1 kills bacteria efficiently and rapidly in a concentration-dependent manner and has a broad-spectrum bactericidal effect.

[0042] Example 9 The investigation into whether the polypyridine ruthenium complex Ru-1 induces drug resistance in Staphylococcus aureus was conducted as follows: Staphylococcus aureus cultured to the logarithmic growth phase was diluted 1000-fold in culture medium to prepare a bacterial suspension. 200 μL of the bacterial suspension was mixed with 50 μL of drug solutions at different concentration gradients and added to 96-well plates. The 96-well plates were incubated at 37°C for 20 hours, and the minimum inhibitory concentration (MIC) of the drug was determined. The bacterial suspension at 0.5 × MIC wells was inoculated into TSB medium and incubated for 5–6 hours. The MIC of the ruthenium complex was then determined using the method described above. The experiment was repeated 20 times, with ampicillin sodium as a control.

[0043] The result is as follows Figure 5 As shown in the results, after 20 days of continuous induction of Staphylococcus aureus with Ru-1, the MIC value of Ru-1 did not change significantly, while that of the control, ampicillin sodium, increased to 128 times the original MIC value. Therefore, it can be seen that Staphylococcus aureus is not likely to develop resistance to Ru-1 complex, and Ru-1 has a good prospect of becoming a marketed antibiotic.

[0044] Furthermore, the 20th generation strains cultured with traditional antibiotics (gentamicin, vancomycin, ofloxacin, ciprofloxacin, polymyxin B, tetracycline, kanamycin, and ampicillin) were used as test strains. The MIC values ​​of Ru-1 against these drug-resistant strains were determined, revealing that Ru-1 exhibited good antibacterial activity against the 20th generation strains of traditional antibiotics. This indicates that Ru-1 possesses low resistance characteristics.

[0045] Example 10 The inhibition and eradication rates of the polypyridine ruthenium complex Ru-1 against mature Staphylococcus aureus and methicillin-resistant Staphylococcus aureus biofilms were determined. The specific procedure is as follows: After culturing bacteria in TSB medium for 18–20 hours, they were diluted 1:1000 and ready for use. The bacterial suspension was then transferred to 24-well plates and incubated with different concentrations of Ru-1 for 48 h. The plates were then dried for approximately 6 h and stained with 500 μL of 1% crystal violet solution for 30 min. Excess crystal violet staining was removed from the wells with water. The crystal violet staining adhering to the wells was then dissolved in 50% acetic acid, and the absorbance was measured at 595 nm using a microplate reader. Each set of data was performed in triplicate.

[0046] Overnight cultured bacteria were diluted 1000-fold in culture medium to prepare a bacterial suspension. 200 μL of the suspension was added to each well of a 96-well plate and incubated at 37 °C for 24 h. Ru-1 was serially diluted to the desired concentration (1×MIC ~ 64×MIC) for later use; vancomycin (Van) served as a positive control. After washing away floating bacteria with sterile PBS, 200 μL of different concentrations of the compound or 200 μL of fresh TBS medium was added to each well, and incubation continued at 37 °C for 24 h. Then, the culture medium was removed, and the plates were washed twice with PBS. 200 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazole bromide (MTT) (1%) was added to each well, and incubation was carried out at 37 °C for 4 h. After removing the MTT solution, 200 μL of DMSO was added to each well, and the absorbance at 540 nm was measured using a microplate reader. The corresponding eradication rate was calculated.

[0047] The result is as follows Figure 6 As shown in the results, the higher the concentration of Ru-1, the stronger its ability to inhibit bacterial biofilm. Furthermore, at a concentration of 25 μg / mL, the bacterial biofilm could be almost completely removed. These results confirm that Ru-1 not only effectively inhibits the formation of Staphylococcus aureus biofilm, but also has a significant effect on mature, stubborn biofilms.

[0048] Example 11 The toxicity of Ru-1 in Galleria mellonella was determined to assess its in vivo safety.

[0049] Different concentrations (16 mg / kg, 32 mg / kg, 64 mg / kg, 128 mg / kg) of Ru-1 solution and vancomycin solution were prepared for acute toxicity testing. 5 μL of each concentration of sample was injected into the second pair of abdominal legs at the tail of each larva. The control group was treated with dimethyl sulfoxide (DMSO). Subsequently, the larvae were randomly divided into 12 groups of 10 larvae each, and the survival rate of the larvae was recorded over 7 days. The experimental model protocol is described below. Figure 7 (A, D) in the list.

[0050] The result is as follows Figure 7 As shown, by Figure 7 As shown in (B, C), different concentrations of Ru-1 and vancomycin were injected into the larvae of the large wax moth, and their survival rates were recorded after 7 consecutive days of observation. At a Ru-1 concentration of 64 mg / kg, the survival rate of the large wax moth larvae was 92%, and even when the concentration was increased to 128 mg / kg, the survival rate remained at 83%. Furthermore, as... Figure 7(E) shows that the survival rate of large wax moth larvae treated with Ru-1 at 32 mg / kg and 64 mg / kg was higher than that treated with the same concentration of vancomycin.

[0051] In summary, the polypyridine ruthenium complex provided by this invention enhances the antibacterial effect by inhibiting and eliminating the formation of bacterial biofilms. The introduction of the quaternary ammonium structure improves the affinity of the complex for bacterial biofilms and effectively enhances its ability to disrupt biofilms, thereby improving its antibacterial activity.

[0052] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A quaternized polypyridine ruthenium complex, characterized in that, The quaternized polypyridine ruthenium complex has the structure shown in Formula I-1 or Formula I-2: Equation I-1; Formula I-2: in, Choose from any of the following structures: ; Linker can be any chemically feasible linker structure.

2. The quaternized polypyridine ruthenium complex according to claim 1, characterized in that, Linker is or , where m is 1~5 and n is 1~10.

3. The quaternized polypyridine ruthenium complex according to claim 1, characterized in that, The quaternized polypyridine ruthenium complex has the structures shown in Ru-1 to Ru-9: 。 4. The method for preparing the quaternized polypyridine ruthenium complex according to any one of claims 1 to 3, characterized in that, The preparation method of the quaternized polypyridine ruthenium complex shown in Formula I-1 includes the following steps: reacting p-fluorobenzaldehyde with 4-dimethylaminopyridine in a solvent under heat to obtain an intermediate; reacting the intermediate with 1,10-phenanthroline-5,6-dione in a solvent under heat to obtain a ligand; and reacting the ligand with the ruthenium complex in a solvent to obtain the quaternized polypyridine ruthenium complex shown in Formula I-1. The preparation method of the quaternized polypyridine ruthenium complex shown in Formula I-2 includes the following steps: under protective conditions, p-hydroxybenzaldehyde and a brominated compound are reacted by heating in a solvent to obtain a first intermediate; the intermediate is reacted by heating with 4-dimethylaminopyridine in a solvent to obtain a second intermediate; the second intermediate is reacted by heating with 1,10-phenanthroline-5,6-dione in a solvent to obtain a ligand; the ligand is reacted with the ruthenium complex in a solvent to obtain the quaternized polypyridine ruthenium complex shown in Formula I-2.

5. The preparation method according to claim 4, characterized in that, The preparation method of the quaternized polypyridine ruthenium complex represented by formula Ru-7 includes the following steps: S1. The p-fluorobenzaldehyde of formula Ib and 4-dimethylaminopyridine were heated under reflux in acetonitrile. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of formula II-b. S2. The compound of formula II-b was reacted with 1,10-phenanthroline-5,6-dione in ethanol under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was then purified by recrystallization from ethanol to obtain the ligand of formula III-b. S3. The ligand of the III-b structure and the ruthenium complex were heated and reacted in ethylene glycol. After the reaction was completed, the mixture was purified to obtain the quaternized polypyridine ruthenium complex shown in the formula Ru-7. The preparation method of the quaternized polypyridine ruthenium complexes shown in formulas Ru-1~Ru-6 and Ru-8~Ru-9 includes the following steps: (1) Under protective conditions, p-fluorobenzaldehyde of formula Ia and a brominated compound were heated and refluxed in acetonitrile. After cooling to room temperature, the mixture was filtered and the solvent was evaporated under reduced pressure to obtain the crude product. After purification, the intermediates of formula Ic~If were obtained. (2) The intermediates of the Ic~If structure were reacted with 4-dimethylaminopyridine in acetonitrile under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was extracted and separated, and the organic phases were combined to obtain the compound of the II-c~II-f structure. (3) The compound with the structure of formula II-c~II-f was heated and refluxed in ethanol with 1,10-phenanthroline-5,6-dione. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from ethanol to obtain the ligand with the structure of formula III-c~III-f. (4) The ligands of the III-c~III-f structure and the ruthenium complex were heated in ethylene glycol and purified after the reaction to obtain the quaternized polypyridine ruthenium complexes shown in the formulas Ru-1~Ru-6 and Ru-8~Ru-9; The structural formulas of each raw material, intermediate, and quaternized polypyridine ruthenium complex are as follows: 。 6. The preparation method according to claim 5, characterized in that, In step (1), the temperature of the reflux reaction is 85~95℃ and the time is 7~9h; the purification of the compound with the Ic~If structure is carried out by elution and purification on a silica gel chromatography column with a petroleum ether / ethyl acetate volume ratio of 20:

1.

7. The preparation method according to claim 5, characterized in that, In steps S1 and (2), the temperature of the reflux reaction is 85~95℃ and the time is 44~52h. The purification is carried out by extraction with dichloromethane and water.

8. The preparation method according to claim 5, characterized in that, In steps S2 and (3), the temperature of the reflux reaction is 125~135℃ and the time is 2~4h; In steps S3 and (4), the reflux reaction is carried out at a temperature of 145-155°C for 9-11 hours. The purification is carried out by elution with acetonitrile and xylene on an alumina chromatography column.

9. The use of the quaternized polypyridine ruthenium complex according to any one of claims 1 to 3 in the preparation of antibacterial or antimicrobial drugs.

10. The application according to claim 9, characterized in that, The antibacterial or antimicrobial drugs include those that inhibit or kill Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii.

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