Drug-resistant bacterium-resistant metal ruthenium complex with dual-core structure as well as preparation method and application of drug-resistant bacterium-resistant metal ruthenium complex

Abstract

The invention discloses an anti-drug-resistant bacterium metal ruthenium complex with a dual-core structure as well as a preparation method and application thereof, and relates to the technical field of antibacterial medicines. The structure of the complex is as follows: # imgabs0 #, and the complex can be used for preparing drugs for resisting drug-resistant bacteria, relieving red blood cell hemolysis, inhibiting MRSA and / or inhibiting MRSA biofilm formation. The complex contains metal ions and has four positive 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 due to the multi-coordination configuration of the metal complex, the metal complex can be modified by different ligands, so that the biological activity of the metal complex is higher. Experiments prove that the diruthenium polypyridine complex disclosed by the invention can effectively reduce the hemolysis effect of toxin released by MRSA on red blood cells, and can inhibit the formation of an MRSA biological membrane.

Description

Technical Field

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

[0002] Staphylococcus aureus (S. aureus) is one of the most common pathogenic bacteria in clinical infectious diseases, which can cause infections in multiple parts such as skin and soft tissues, blood system, and lower respiratory tract, resulting in a series of diseases, such as pericarditis, pseudomembranous enteritis, pneumonia, and sepsis. In recent years, due to the emergence of drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), which has serious drug resistance to many antibiotics, the treatment of such infections has become increasingly difficult.

[0003] Hemolysis refers to the phenomenon that the cell membrane of red blood cells is damaged and ruptured due to physical factors, chemical factors, biological factors, etc., and the internal protoplasm leaks out of the cell, causing the death of red blood cells. The hemolysis reaction is the reaction in which the red blood cell membrane is damaged, resulting in the leakage of hemoglobin from the red blood cells. Most hemolysins are produced by Gram-positive bacteria. The toxin inserts into the phospholipid bilayer structure of the membrane to form a channel, leading to the release of potassium ions, and then the leakage of linear hemoglobin. For example, the α-hemolysin of Staphylococcus aureus can damage human blood cells and nucleated cells. Bacterial biofilm (or bacterial biofilm, BF) refers to a large amount of bacterial aggregation membrane-like substance formed by bacteria adhering to the contact surface and secreting polysaccharide matrix, fibrin, lipoproteins, etc., and surrounding themselves with it. The formation of biofilm provides a relatively stable environment for bacteria, making it easy for them to develop drug resistance. Therefore, how to reduce hemolysis and inhibit the formation of bacterial biofilm has become one of the key points in the research and development of antibiotics. 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 an anti-drug-resistant bacteria metal ruthenium complex with a binuclear structure and a preparation method thereof.

[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides an anti-drug-resistant bacteria metal ruthenium complex with a binuclear structure, and the complex has the structure shown in Formula I: Formula I; Wherein, the is selected from any one of the following structures: , , .

[0006] The antibacterial metal ruthenium complex with a dual-core structure of the present invention contains metal ions with four positive charges, and the positively charged organic molecular chain combines with the anions on the surface of the bacterial cell membrane, thereby destroying the composition of the microbial cell membrane, causing the leakage of intracellular substances, and resulting in the inability of the bacterial cells to produce toxins. Moreover, the multi-coordination configuration of the metal complex enables it to be modified with different ligands, thereby achieving a better biological activity effect. Therefore, by modifying the diruthenium polypyridine complex, the destruction of bacteria can be achieved, thereby inhibiting the production of toxins by drug-resistant strains.

[0007] 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: Ru-1; Ru-2; Ru-3.

[0008] The second aspect of the present invention provides a preparation method of an antibacterial metal ruthenium complex with a dual-core structure, including the following steps: Reacting the main ligand of the formula I-c structure with the compound of the formula I-d structure or the compound of the formula I-e structure or the compound of the formula I-f structure, and purifying to obtain the complex; .

[0009] In a preferred embodiment of the present invention, the preparation method of the main ligand includes the following steps: Under argon conditions, heating and refluxing the compound of the formula I-a structure and the compound of the formula I-b structure in a solvent. After the reaction is complete, diluting with water and neutralizing with a pH regulator, centrifuging and drying, and purifying to obtain the main ligand of the formula I-c structure; .

[0010] In a preferred embodiment of the present invention, the molar ratio of the compound of the formula I-a structure to the compound of the formula I-b structure is 1:1.5 - 2.5.

[0011] In a preferred embodiment of the present invention, in the preparation method of the main ligand, ammonium acetate is also added to react with the compound of the formula I-a structure and the compound of the formula I-b structure by heating and refluxing in a solvent; the molar ratio of ammonium acetate to the compound of the formula I-a structure is 25 - 35:1; The solvent is acetic acid solvent, and the pH regulator is concentrated ammonia water; The purification method of the main ligand is recrystallization purification using ethanol.

[0012] In a preferred embodiment of the present invention, the preparation method of the complex specifically comprises the following steps: Add the main ligand of formula I-c and the compound of formula I-d or the compound of formula I-e or the compound of formula I-f into DMF solvent, heat at 140 °C to 160 °C for 8 h to 12 h, after cooling, add solid KPF6 to obtain a precipitate, evaporate the solvent under reduced pressure to obtain a crude product; purify the crude product by column chromatography on alumina, use a mixture of xylene and acetonitrile as the eluent to obtain the complex shown in formula I; Among them, 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 to 2.5 In the mixture of xylene and acetonitrile, the volume ratio of xylene to acetonitrile is 2 to 4:1.

[0013] The third aspect of the present invention provides an application of an anti-drug-resistant bacteria metal ruthenium complex with a binuclear structure in the preparation of anti-drug-resistant bacteria drugs.

[0014] The fourth aspect of the present invention provides an application of an anti-drug-resistant bacteria metal ruthenium complex with a binuclear structure in the preparation of drugs for reducing red blood cell hemolysis.

[0015] The fifth aspect of the present invention provides an application of an anti-drug-resistant bacteria metal ruthenium complex with a binuclear structure in the preparation of drugs for inhibiting MRSA and / or inhibiting the formation of MRSA biofilms.

[0016] The present invention has at least one of the following beneficial effects: Compared with the prior art, the present invention provides an anti-drug-resistant bacteria metal ruthenium complex with a binuclear structure. Since it contains metal ions and has four positive charges, compared with traditional organic small molecules, it enhances the transmembrane effect and retention effect, and the multi-coordination configuration of the metal complex enables it to be modified with different ligands, thereby achieving a better biological activity effect. Experimental results show that the ruthenium metal complex with a binuclear structure of the present invention can effectively reduce the hemolytic effect of toxins released by MRSA on red blood cells at concentrations of 0.25 MIC, 0.5 MIC, and 0.75 MIC, and has an inhibitory effect on the formation of its biofilm. Thus, it can be seen that the bis-ruthenium polypyridine complex provided by the present invention has certain potential in the development of antibacterial agents. Description of the Drawings

[0017] Figure 1 The high-resolution mass spectrum of the main ligand of the present invention is shown.

[0018] Figure 2 The high-resolution mass spectrum of the complex Ru-1 of the present invention is shown.

[0019] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the complex Ru-1 of the present invention is shown as follows.

[0020] Figure 4 The high-resolution mass spectrum of the complex Ru-2 of the present invention is shown as follows.

[0021] Figure 5 The hydrogen nuclear magnetic resonance spectrum of the complex Ru-2 of the present invention is shown as follows.

[0022] Figure 6 The high-resolution mass spectrum of the complex Ru-3 of the present invention is shown as follows.

[0023] Figure 7 The hydrogen nuclear magnetic resonance spectrum of the complex Ru-3 of the present invention is shown as follows.

[0024] Figure 8 The hemolytic effect diagrams of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on MRSA are shown as follows.

[0025] Figure 9 The determination diagrams of the hemolytic effect of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on MRSA are shown as follows.

[0026] Figure 10 The inhibition effect diagrams of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on the MRSA biofilm are shown as follows.

[0027] Figure 11 The determination diagrams of the inhibition effect of the complexes Ru-1, Ru-2, and Ru-3 of the present invention on the MRSA biofilm are shown as follows. Detailed implementation manners

[0028] In order to make the technical problems, technical solutions, and beneficial effects solved by the present invention clearer and more understandable, the present 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 only used to explain the present invention and are not used to limit the present invention.

[0029] Example 1: (1) Preparation of the main ligand: 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) solvent for 3 hours. The cooled solution was diluted with water and neutralized with concentrated ammonia water. It was filtered by suction and then dried to obtain a crude product, which was recrystallized with ethanol to obtain the compound main ligand, which was a pale yellow solid powder. Yield: 40%.

[0030] (2) Preparation of Complex Ru-1: Under argon, a mixture of cis-[Ru(bpy)2Cl2] (Formula I-d, 96.8 mg, 0.2 mmol) and the main ligand (38.8 mg, 0.1 mmol) was heated in DMF at 150 °C for 10 h. After cooling, a red-brown precipitate was obtained by adding solid KPF6. 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-acetonitrile (2:1, v / v) as the eluent. A red product was obtained. Yield: 23.9%.

[0031] (3) Preparation of Complex Ru-2: Under argon, a mixture of cis-[Ru(dmp)2Cl2] (Formula I-e, 108 mg, 0.2 mmol) and the main ligand (38.8 mg, 0.1 mmol) was heated in DMF at 150 °C for 10 h. After cooling, a red-brown precipitate was obtained by adding solid KPF6. 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-acetonitrile (3:1, v / v) as the eluent. A red-brown product was obtained. Yield: 24%.

[0032] (4) Preparation of Complex Ru-3: Under argon, a mixture of cis-[Ru(dtb)2Cl2] (Formula I-f, 141.6 mg, 0.2 mmol) and the main ligand (38.8 mg, 0.1 mmol) was heated in DMF at 150 °C for 10 h. After cooling, a red-brown precipitate was obtained by adding solid KPF6. 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-acetonitrile (4:1, v / v) as the eluent. A red-brown product was obtained. Yield: 10.9%.

[0033] The high-resolution mass spectra and 1H NMR spectra of the main ligand, Ru-1, Ru-2, and Ru-3 complexes prepared in this example are as Figures 1 to 7 shown, and thus the structural formulas of the main ligand, Ru-1, Ru-2, and Ru-3 complexes can be determined as follows: Main ligand; Ru-1; Ru-2; Ru-3; The reaction routes of the above reactions are as shown below: Determine the MIC values (minimum inhibitory concentration) of Ru-1, Ru-2, and Ru-3. The specific method is as follows: Cultivate the Staphylococcus aureus strain in TSB medium until the logarithmic growth phase; dilute it 1000 times with fresh medium to obtain a bacterial suspension. Add 50 µL of different concentrations of Ru-1 to the 96-well plate in sequence, and then add 200 µL of the bacterial suspension to each well, so that the final concentration of Ru-1 in each well is 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, 0.195 µg / mL from left to right in turn. Add 50 µL of sterile water to the last well as a blank control, and measure 3 parallel groups for each drug. After incubating at 37 °C for 20 h, monitor the growth of bacteria by observing the turbidity of the culture. The lowest drug concentration corresponding to the clear drug-added well is the MIC (minimum inhibitory concentration).

[0034] Determine the MIC values of Ru-2 and Ru-3 according to the same method as above.

[0035] After determination, the MIC values of Ru-1, Ru-2, and Ru-3 are 100 µg / mL, 6.25 µg / mL, and 3.125 µg / mL, respectively.

[0036] Example 2: Perform hemolysis experiments on the complexes Ru-1, Ru-2, and Ru-3 prepared in Example 1. The method is as follows: (1) Dissolve the complexes Ru-1, Ru-2, and Ru-3 in DMSO and co-incubate them with MRSA cultured in TSB medium until OD 600 reaches 1. Aspirate the bacterial solution into an EP tube and centrifuge it at 2500 r / min for 1 minute. Fresh red blood cells are separated from rabbit blood by centrifugation and washed 3 times with PBS. Subsequently, add the centrifuged rabbit blood, PBS, and the supernatant of the bacterial solution to the EP tube together. Incubate in a water bath at 37 °C for 30 min, and then observe the results. The Ctrl group uses PBS instead of the drug as a blank control group.

[0037] Figure 8Among them, the hemolysis inhibition effects of complexes Ru-1, Ru-2, and Ru-3 on MRSA were judged by observing the depth of the color of the supernatant. The lighter the color of the blood in the supernatant, the stronger the inhibitory effect on hemolysis. The experimental results showed that complex Ru-1 could inhibit the production of MRSA toxins at a concentration of 0.75 MIC, and complexes Ru-2 and Ru-3 could significantly inhibit the production of MRSA toxins at a concentration of 0.75 MIC.

[0038] Figure 9 Among them, by pipetting Figure 8 the supernatant in the EP tube shown and measuring the OD 540 absorbance at to judge the hemolysis volume after Ru-1, Ru-2, and Ru-3 acted on bacteria. The experimental results showed that complexes Ru-1, Ru-2, and Ru-3 had strong hemolysis inhibition effects on MRSA. At a concentration of 0.5 MIC, compared with the blank control group, the absorbance value decreased by one order of magnitude, and more obvious hemolysis inhibition was shown at 0.75 MIC, indicating that complexes Ru-1, Ru-2, and Ru-3 could inhibit the production of MRSA toxins to a certain extent.

[0039] Example 3: The complexes Ru-1, Ru-2, and Ru-3 prepared in Example 1 were subjected to a biofilm experiment, and the method was as follows: (1) Inhibiting the formation of bacterial biofilms was mainly to verify that complexes Ru-1, Ru-2, and Ru-3 could effectively inhibit the formation of bacterial biofilms at a certain concentration, thereby reducing the drug resistance of bacteria. The experiment was mainly carried out in a 24-well plate. Add 0.5 mL of sterile water or complexes Ru-1, Ru-2, and Ru-3 at different concentrations (0.25 MIC, 0.5 MIC, 0.75 MIC) to the 24-well plate. Add 1.5 mL of MRSA bacterial solution diluted 1000 times to each well. Set 3 parallels in each group, and use sterile water as the blank control group (Ctrl group). Place the 24-well plate in a constant temperature incubator at 37 °C for about 48 h, take it out, wash away the planktonic bacteria, and dry it.

[0040] (2) Stain the dried biofilm with 5% crystal violet for 30 min - 60 min, wash away the excess crystal violet solution, and dry it slightly. Add 2 mL of 50% glacial acetic acid solution to dissolve the crystal violet fixed on the biofilm, and measure the OD 595 , draw the experimental result graph according to the experimental data, and analyze the inhibitory ability of complexes Ru-1, Ru-2, and Ru-3 at different concentrations on the MRSA biofilm.

[0041] Figure 10Among them, the inhibitory ability of complexes Ru-1, Ru-2 and Ru-3 against MRSA biofilm was judged by observing the depth of crystal violet on the biofilm. The lighter the color, the stronger the inhibitory ability against MRSA biofilm. The experimental results showed that complexes Ru-1, Ru-2 and Ru-3 could significantly inhibit the formation of MRSA biofilm at a concentration of 0.75 MIC.

[0042] Figure 11 Among them, for Figure 10 the microtiter plate in [previous context] was measured. The microtiter plate was placed on an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance at 595 nm 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, a more obvious effect of inhibiting the formation of bacterial biofilm was shown, indicating that complexes Ru-1, Ru-2 and Ru-3 could inhibit the formation of MRSA biofilm.

[0043] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A metal ruthenium complex with a dual-core structure for combating drug-resistant bacteria, characterized in that, The complex has the structure shown in Formula I: Formula I; Among them, the is selected from any one of the following structures: , , .

2. The complex according to claim 1, characterized in that, The complex has the structure shown in any one of Ru-1, Ru-2, and Ru-3: Ru-1; Ru-2; Ru-3.

3. A method for preparing the complex according to any one of claims 1 to 2, characterized in that, It includes the following steps: React the main ligand of Formula I-c with the compound of Formula I-d or the compound of Formula I-e or the compound of Formula I-f, and purify to obtain the complex; 。 4. The preparation method according to claim 3, characterized in that, The preparation method of the main ligand includes the following steps: Under argon atmosphere, heat and reflux the compound of Formula I-a and the compound of Formula I-b in a solvent. After the reaction is complete, dilute with water and neutralize with a pH regulator, then centrifuge, dry, and purify to obtain the main 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, In the preparation method of the main ligand, ammonium acetate is also added to react with the compound of Formula I-a and the compound of Formula I-b by heating and refluxing in a solvent; the molar ratio of ammonium acetate to the compound of Formula I-a is 25 - 35:1; The solvent is acetic acid solvent, and the pH regulator is concentrated ammonia water; The purification method of the main ligand is recrystallization purification using ethanol.

7. The preparation method according to claim 3, characterized in that, The preparation method of the complex specifically includes the following steps: Add the main ligand of Formula I-c and the compound of Formula I-d or the compound of Formula I-e or the compound of Formula I-f into DMF solvent, heat at 140°C - 160°C for 8 h - 12 h, after cooling, add solid KPF6 to obtain a precipitate, evaporate the solvent under reduced pressure to obtain a crude product; purify the crude product by column chromatography on alumina, using a mixture of xylene - acetonitrile as the eluent to obtain the complex shown in Formula I; Among them, 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; In the mixture of xylene - acetonitrile, the volume ratio of xylene to acetonitrile is 2 - 4:

1.

8. Use of the complex according to any one of claims 1 - 2 in the preparation of anti-drug-resistant bacteria drugs.

9. Use of the complex according to any one of claims 1 - 2 in the preparation of drugs for reducing red blood cell hemolysis.

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

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