Metal ruthenium complex with binuclear structure for resisting drug-resistant bacteria, preparation method and application thereof

Abstract

The present invention discloses a metal ruthenium complex with a binuclear structure for resisting drug-resistant bacteria, a preparation method and an application thereof, and relates to the field of antibacterial medicine technology. The structure of the complex is as follows:, which can be used to prepare drugs for resisting drug-resistant bacteria, reducing hemolysis of red blood cells, inhibiting MRSA and / or inhibiting the formation of MRSA biofilms. Since the complex contains metal ions and carries four positive charges, compared with traditional organic small molecules, it enhances its ability to penetrate bacterial cell membranes and retention effect, and the multi-coordinate configuration of the metal complex allows it to be modified with different ligands, thereby making it more biologically active. Experiments have shown that the bis-ruthenium polypyridine complex of the present invention can effectively reduce the hemolytic effect of toxins released by MRSA on red blood cells, and can inhibit the formation of MRSA biofilms.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial medicine, and specifically relates to a metal ruthenium complex with a binuclear structure for resisting drug-resistant bacteria, as well as a preparation method and application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is the most common pathogen causing clinical infectious diseases. It can cause infections in multiple areas, including the skin and soft tissues, the blood system, and the lower respiratory tract, leading to a range of diseases such as pericarditis, pseudomembranous colitis, pneumonia, and sepsis. In recent years, the emergence of drug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), which are severely resistant to many antibiotics, has made the treatment of such infections increasingly difficult.

[0003] Hemolysis occurs when the red blood cell membrane is damaged and ruptured due to physical, chemical, or biological factors, leading to the leakage of protoplasm from the cell and the cell's death. Hemolysis occurs when the red blood cell membrane is disrupted, causing hemoglobin to leak out of the cell. Hemolytic toxins are mostly produced by Gram-positive bacteria. These toxins insert into the membrane's phospholipid bilayer, forming channels that release potassium ions and subsequently release linear hemoglobin. For example, the α-hemolytic toxin of Staphylococcus aureus can damage human blood cells and nucleated cells. A bacterial biofilm (BF) is a membrane-like aggregate formed by bacteria adhering to a surface and surrounding themselves with a polysaccharide matrix, fibrin, and lipid proteins. Biofilm formation provides a relatively stable environment for bacteria, making them susceptible to developing drug resistance. Therefore, mitigating hemolysis and inhibiting biofilm formation are key areas of antibiotic research and development. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a metal ruthenium complex with a binuclear structure for resisting drug-resistant bacteria and a preparation method thereof.

[0005] The technical solutions of the present invention are as follows:

[0006] The first aspect of the present invention provides a metal ruthenium complex with a binuclear structure for use against drug-resistant bacteria, wherein the complex has a structure shown in Formula I:

[0007] Formula I;

[0008] Among them, the Select from any of the following structures: 、 、 .

[0009] The present invention's dual-core ruthenium complex for combating drug-resistant bacteria contains metal ions with four positive charges. The positively charged organic molecular chains bind to anions on the surface of bacterial cell membranes, disrupting the microbial cell membranes and leaking intracellular substances, preventing the bacteria from producing toxins. Furthermore, the metal complex's multi-coordinate configuration allows for modification with different ligands, resulting in enhanced biological activity. Therefore, by modifying the bis-ruthenium polypyridine complex, bacterial destruction can be achieved, thereby inhibiting toxin production by drug-resistant strains.

[0010] In a preferred embodiment of the present invention, the complex has a structure shown in any one of Ru-1, Ru-2 and Ru-3:

[0011] Ru-1;

[0012] Ru-2;

[0013] Ru-3.

[0014] The second aspect of the present invention provides a method for preparing a metal ruthenium complex having a binuclear structure for use against drug-resistant bacteria, comprising the following steps:

[0015] The main ligand of formula Ⅰ-c is reacted with the compound of formula Ⅰ-d, the compound of formula Ⅰ-e, or the compound of formula Ⅰ-f, and purified to obtain the complex;

[0016] .

[0017] In a preferred embodiment of the present invention, the method for preparing the primary ligand comprises the following steps:

[0018] Under argon conditions, the compound of formula I-a and the compound of formula I-b are heated under reflux in a solvent for reaction. After the reaction is complete, the mixture is diluted with water and neutralized with a pH adjuster, centrifuged and dried, and purified to obtain a primary ligand of formula I-c.

[0019] .

[0020] In a preferred embodiment of the present invention, the molar ratio of the compound of formula I-a to the compound of formula I-b is 1:1.5-2.5.

[0021] In a preferred embodiment of the present invention, the method for preparing the primary ligand further comprises adding ammonium acetate, the compound of formula I-a, and the compound of formula I-b to a solvent for heating under reflux reaction; the molar ratio of ammonium acetate to the compound of formula I-a is 25 to 35:1;

[0022] The solvent is acetic acid solvent, and the pH regulator is concentrated ammonia water;

[0023] The main ligand is purified by recrystallization using ethanol.

[0024] In a preferred embodiment of the present invention, the preparation method of the complex specifically comprises the following steps:

[0025] The primary ligand of formula I-c and the compound of formula I-d, formula I-e, or formula I-f are added to a DMF solvent, heated at 140°C to 160°C for 8 to 12 hours, cooled, and then solid KPF6 is added to obtain a precipitate. The solvent is evaporated under reduced pressure to obtain a crude product. The crude product is purified by column chromatography on alumina using a xylene-acetonitrile mixture as an eluent to obtain a complex represented by formula I.

[0026] The molar ratio of the main ligand of formula I-c to the compound of formula I-d or the compound of formula I-e or the compound of formula I-f is 1:1.5-2.5.

[0027] In the xylene-acetonitrile mixture, the volume ratio of xylene to acetonitrile is 2~4:1.

[0028] The third aspect of the present invention provides an application of a metal ruthenium complex having a binuclear structure for use in the preparation of drugs for use against drug-resistant bacteria.

[0029] A fourth aspect of the present invention provides an application of a metal ruthenium complex having a binuclear structure for use in preparing a drug for reducing hemolysis of red blood cells.

[0030] A fifth aspect of the present invention provides a use of a metal ruthenium complex having a binuclear structure for use in preparing a drug for inhibiting MRSA and / or inhibiting MRSA biofilm formation.

[0031] The present invention has at least one of the following beneficial effects:

[0032] Compared with the prior art, the present invention provides a metal ruthenium complex with a binuclear structure for resisting drug-resistant bacteria. Since it contains metal ions and carries four positive charges, it enhances the transmembrane effect and retention effect compared to traditional organic small molecules, and the multi-coordinate configuration of the metal complex allows it to be modified with different ligands, thereby achieving better biological activity. Experimental results show that the ruthenium metal complex with a binuclear structure of the present invention can effectively reduce the hemolytic effect of the toxins released by MRSA on red blood cells at concentrations of 0.25MIC, 0.5MIC and 0.75MIC, and has an inhibitory effect on the formation of its biofilm. It can be seen that the bis-ruthenium polypyridine complex provided by the present invention has certain potential in the development of the preparation of antibacterial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown is the high-resolution mass spectrum of the main ligand of the present invention.

[0034] Figure 2 Shown is the high-resolution mass spectrum of the complex Ru-1 of the present invention.

[0035] Figure 3 Shown is the hydrogen nuclear magnetic resonance spectrum of the complex Ru-1 of the present invention.

[0036] Figure 4 Shown is the high-resolution mass spectrum of the complex Ru-2 of the present invention.

[0037] Figure 5 Shown is the hydrogen nuclear magnetic resonance spectrum of the complex Ru-2 of the present invention.

[0038] Figure 6 Shown is the high-resolution mass spectrum of the complex Ru-3 of the present invention.

[0039] Figure 7 Shown is the hydrogen nuclear magnetic resonance spectrum of the complex Ru-3 of the present invention.

[0040] Figure 8 Shown are diagrams showing the hemolytic effects of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on MRSA.

[0041] Figure 9 Shown is a graph showing the hemolytic effect of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on MRSA.

[0042] Figure 10 Shown are diagrams showing the inhibitory effects of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on MRSA biofilm.

[0043] Figure 11 Shown is a graph showing the inhibitory effect of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on MRSA biofilm. DETAILED DESCRIPTION

[0044] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] Example 1:

[0046] (1) Preparation of primary ligand:

[0047] A mixture of 1,10-phenanthroline-5,6-dione (Formula I-b, 0.4 g, 2.00 mmol), 4'-methyl-[2,2'-bipyridine]-4-carbaldehyde (Formula I-a, 0.2 g, 1 mmol), and ammonium acetate (2.3 g, 30 mmol) was heated under reflux in acetic acid (15 mL) for 3 hours. The cooled solution was diluted with water and neutralized with concentrated ammonia. Filtration and drying afforded the crude product, which was then recrystallized from ethanol to obtain the primary ligand as a pale yellow solid powder. Yield: 40%.

[0048]

[0049] (2) Preparation of complex Ru-1:

[0050] Under argon, a mixture of cis-[Ru(bpy)2Cl2] (Formula I-d, 96.8 mg, 0.2 mmol) and the primary ligand (38.8 mg, 0.1 mmol) in DMF was heated at 150°C for 10 h. After cooling, solid KPF6 was added to yield a reddish-brown precipitate. The solvent was evaporated under reduced pressure to yield the crude product. The crude product was purified by column chromatography on alumina using a xylene-acetonitrile mixture (2:1, v / v) as the eluent. A red product was obtained in a yield of 23.9%.

[0051] (3) Preparation of complex Ru-2:

[0052] Under argon, a mixture of cis-[Ru(dmp)2Cl2] (Formula I-e, 108 mg, 0.2 mmol) and the primary ligand (38.8 mg, 0.1 mmol) in DMF was heated at 150°C for 10 h. After cooling, solid KPF6 was added to yield a reddish-brown precipitate. The solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography on alumina using a mixture of xylene and acetonitrile (3:1, v / v) as the eluent. A reddish-brown product was obtained in a 24% yield.

[0053] (4) Preparation of complex Ru-3:

[0054] Under argon, a mixture of cis-[Ru(dtb)2Cl2] (Formula I-f, 141.6 mg, 0.2 mmol) and the primary ligand (38.8 mg, 0.1 mmol) in DMF was heated at 150°C for 10 h. After cooling, solid KPF6 was added to yield a reddish-brown precipitate. The solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography on alumina using a xylene-acetonitrile mixture (4:1, v / v) as the eluent. A reddish-brown product was obtained in a yield of 10.9%.

[0055]

[0056] The high-resolution mass spectra and hydrogen nuclear magnetic resonance spectra of the main ligand, Ru-1, Ru-2, and Ru-3 complexes prepared in this example are as follows: Figures 1 to 7 As shown, the structural formulas of the main ligand, Ru-1, Ru-2, and Ru-3 complexes can be determined as follows:

[0057] main ligand;

[0058] Ru-1;

[0059] Ru-2;

[0060] Ru-3;

[0061] The reaction scheme of the above reaction is as follows:

[0062]

[0063] The MIC values ​​(minimum inhibitory concentrations) of Ru-1, Ru-2, and Ru-3 were determined as follows:

[0064] Staphylococcus aureus strains were cultured in TSB medium until the logarithmic growth phase and diluted 1000-fold with fresh medium to obtain a bacterial suspension. 50 µL of Ru-1 at varying concentrations was added sequentially to each well of a 96-well plate. Then, 200 µL of the bacterial suspension was added to each well, achieving the following final concentrations of Ru-1 (from left to right): 200 µg / mL, 100 µg / mL, 50 µg / mL, 25 µg / mL, 12.5 µg / mL, 6.25 µg / mL, 3.125 µg / mL, 1.56 µg / mL, 0.78 µg / mL, 0.39 µg / mL, and 0.195 µg / mL. 50 µL of sterile water was added to the last well as a blank control. Each drug was tested in triplicate. After incubation at 37°C for 20 hours, bacterial growth was monitored by observing the turbidity of the culture. The lowest drug concentration corresponding to the clear well was the minimum inhibitory concentration (MIC).

[0065] The MIC values ​​of Ru-2 and Ru-3 were determined using the same method as above.

[0066] The MIC values ​​of Ru-1, Ru-2 and Ru-3 were determined to be 100 μg / mL, 6.25 μg / mL and 3.125 μg / mL, respectively.

[0067] Example 2:

[0068] The complexes Ru-1, Ru-2 and Ru-3 prepared in Example 1 were subjected to a hemolysis test as follows:

[0069] (1) The complexes Ru-1, Ru-2 and Ru-3 were dissolved in DMSO and incubated with MRSA cultured in TSB medium until OD 600 Reach 1. The bacterial solution was pipetted into an EP tube and centrifuged at 2500 rpm for 1 minute. Fresh red blood cells were isolated from the rabbit blood by centrifugation and washed three times with PBS. Subsequently, the rabbit blood, PBS, and bacterial supernatant were added to the EP tube. Incubate in a 37°C water bath for 30 minutes, and then observe the results. The control group received PBS instead of drug, serving as a blank control.

[0070] Figure 8 The inhibitory effect of the Ru-1, Ru-2, and Ru-3 complexes on MRSA hemolysis was determined by observing the color of the supernatant. A lighter color indicates a stronger inhibitory effect. The results showed that Ru-1 inhibited MRSA toxin production at a concentration of 0.75 MIC, while both Ru-2 and Ru-3 significantly inhibited MRSA toxin production at a concentration of 0.75 MIC.

[0071] Figure 9 In, by absorbing Figure 8 The supernatant in the indicated EP tube was collected and the OD was determined. 540 The absorbance at 1 MIC was used to determine the amount of hemolysis after Ru-1, Ru-2, and Ru-3 acted on bacteria. The experimental results showed that the complexes Ru-1, Ru-2, and Ru-3 had a strong inhibitory effect on MRSA hemolysis. At a concentration of 0.5 MIC, the absorbance value decreased by one order of magnitude compared to the blank control group, and at a concentration of 0.75 MIC, the inhibitory effect was even more pronounced, indicating that the complexes Ru-1, Ru-2, and Ru-3 can inhibit the production of MRSA toxins to a certain extent.

[0072] Example 3:

[0073] The complexes Ru-1, Ru-2 and Ru-3 prepared in Example 1 were subjected to a biofilm experiment as follows:

[0074] (1) The purpose of inhibiting bacterial biofilm formation was to verify that the complexes Ru-1, Ru-2, and Ru-3 could effectively inhibit bacterial biofilm formation at a certain concentration, thereby reducing bacterial resistance. The experiment was mainly conducted in a 24-well plate. 0.5 mL of sterile water or complexes Ru-1, Ru-2, and Ru-3 at different concentrations (0.25MIC, 0.5MIC, 0.75MIC) were added to the 24-well plate. 1.5 mL of 1000-fold diluted MRSA bacterial solution was added to each well. Three parallel experiments were set up for each group, and sterile water was used as the blank control group (Ctrl group). The 24-well plate was placed in a constant temperature incubator at 37°C for about 48 hours, then removed, the floating bacteria were washed off, and the plate was dried.

[0075] (2) After drying, the biofilm was stained with 5% crystal violet for 30 min-60 min, the excess crystal violet solution was washed off, and the biofilm was dried slightly. 2 mL of 50% glacial acetic acid solution was added to dissolve the crystal violet fixed on the biofilm, and the OD was measured by microplate reader. 595 , based on the experimental data, the experimental results were plotted to analyze the inhibitory ability of different concentrations of complexes Ru-1, Ru-2 and Ru-3 on MRSA biofilm.

[0076] Figure 10 The inhibitory ability of the Ru-1, Ru-2, and Ru-3 complexes against MRSA biofilms was determined by observing the depth of the crystal violet on the biofilms. Lighter colors indicate stronger inhibition. The results showed that the Ru-1, Ru-2, and Ru-3 complexes significantly inhibited MRSA biofilm formation at a concentration of 0.75 MIC.

[0077] Figure 11 In, right Figure 10 The plate was placed on a microplate reader and the absorbance at 595 nm was read as the residual amount of bacterial biofilm. The data showed that each concentration group was lower than the blank control group, and at 0.75 MIC, it showed a more obvious inhibitory effect on bacterial biofilm formation, indicating that the complexes Ru-1, Ru-2 and Ru-3 can inhibit MRSA biofilm formation.

[0078] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A ruthenium complex with a binuclear structure for use against drug-resistant bacteria, characterized in that: The complex has a structure shown in Formula I: Formula I; Among them, the Select from any of the following structures: 、 .

2. The complex according to claim 1, characterized in that The complex has the structure shown in either Ru-2 or Ru-3: Ru-2; Ru-3。 3. A method for preparing the complex according to any one of claims 1 to 2, characterized in that: The following steps are involved: The main ligand of formula I-c is reacted with the compound of formula I-e or the compound of formula I-f, and purified to obtain the complex; 。 4. The preparation method according to claim 3, characterized in that The preparation method of the main ligand comprises the following steps: Under argon conditions, the compound of formula I-a and the compound of formula I-b are heated under reflux in a solvent for reaction. After the reaction is complete, the mixture is diluted with water and neutralized with a pH adjuster, centrifuged and dried, and purified to obtain a primary ligand of formula I-c. 。 5. The preparation method according to claim 4, characterized in that The molar ratio of the compound of formula I-a to the compound of formula I-b is 1:1.5-2.

5.

6. The preparation method according to claim 4, characterized in that The preparation method of the main ligand further includes adding ammonium acetate, a compound of formula I-a, and a compound of formula I-b to a solvent for heating and reflux reaction; the molar ratio of ammonium acetate to the compound of formula I-a is 25 to 35:1; The solvent is acetic acid solvent, and the pH regulator is concentrated ammonia water; The main ligand is purified by recrystallization using ethanol.

7. The preparation method according to claim 3, characterized in that The preparation method of the complex specifically comprises the following steps: The primary ligand of formula I-c and the compound of formula I-e or formula I-f are added to a DMF solvent, heated at 140°C to 160°C for 8 to 12 hours, cooled, and then KPF6 solid is added to obtain a precipitate. The solvent is evaporated under reduced pressure to obtain a crude product. The crude product is purified by column chromatography on alumina using a xylene-acetonitrile mixture as an eluent to obtain a complex represented by formula I. Wherein, the molar ratio of the main ligand of formula I-c to the compound of formula I-e or the compound of formula I-f is 1:1.5-2.5; In the xylene-acetonitrile mixture, the volume ratio of xylene to acetonitrile is 2~4:

1.

8. Use of the complex according to any one of claims 1 to 2 in the preparation of drugs against drug-resistant bacteria, characterized in that: The anti-drug-resistant bacteria is drug-resistant Staphylococcus aureus.

9. Use of the complex according to any one of claims 1 to 2 in the preparation of a drug for reducing hemolysis of red blood cells.

10. Use of the complex according to any one of claims 1 to 2 in the preparation of a medicament for inhibiting MRSA and / or inhibiting MRSA biofilm formation.

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