A pyrrole-modified iridium polypyridine complex for antibacterial purposes, its preparation method and application.

Abstract

This invention belongs to the field of antibacterial pharmaceutical technology, and discloses a pyrrole-modified iridium polypyridine complex for antibacterial purposes, its preparation method, and its applications. In the compound structure of the pyrrole-modified iridium polypyridine complex, pyrrole is a penta-nitrogen heterocycle with significant research value. Combined with the metallic element iridium, compared to traditional small organic molecules, it enhances its ability to penetrate bacterial cell membranes, improves transmembrane activity, and effectively penetrates into the bacterial cell membrane, generating ROS-induced oxidative stress, thereby leading to bacterial death. Experiments have demonstrated that the complex of this invention can produce ROS-induced oxidative stress and inhibit biofilm formation.

Description

Technical Field

[0001] This invention relates to the field of antibacterial pharmaceutical technology, specifically to a pyrrole-modified iridium polypyridine complex for antibacterial purposes, its preparation method, and its application. Background Technology

[0002] Biofilms are commonly found on the surfaces of hospital instruments and human tissues, in industrial and food processing plants, and in the natural environment. Almost all bacteria can form biofilms. A biofilm is a sessile bacterial community surrounded by an extracellular polymeric substance (EPS) matrix released by the microorganism itself. Biofilms have a rigid structure and high resistance, preventing the entry of antimicrobial drugs. The EPS of Staphylococcus aureus plays a crucial role in biofilm formation, regulating various functions through intracellular signaling molecules, such as the production of virulence factors, physiological functions, and adaptation to antibiotic resistance mechanisms. Furthermore, EPS serves as a channel for water and nutrients to enter the biofilm. The highly metabolically active Staphylococcus aureus cells on the outer layer and the non-growing, dormant cells in the center are difficult to eradicate. The vast majority of bacteria in nature exist in the form of biofilms. Therefore, inhibiting biofilm formation is key to antibacterial activity.

[0003] To date, many researchers and teams have discovered the antibacterial activity of metallic iridium. E. Sauvageot et al. reported that cyclic metallized iridium(II) dipyridylamine complexes have antibacterial activity against Pseudomonas aeruginosa, and that the antibacterial activity of the iridium(III) dipyridylamine complexes increases after binding to biotin (see Sauvageot E, Elie M, Gaillard S, et al. Antipseudomonal activity enhancement of luminescentiridium (iii) Dipyridylamine complexes under visible blue light[J]. Metallomics . 2017; 9: 1820–1827.).

[0004] The team led by Guojian Liao reported that the polypyridine-iridium dimer complex [Ir(PPy)2CI]2 (ppy-phenylpyridine) exhibits activity against exponentially growing and non-replicating Mycobacterium smegmatis, with a minimum inhibitory concentration (MIC) of 2 μg / mL. It demonstrates rapid bactericidal kinetics, killing the pathogen within 30-60 minutes. Simultaneously, [Ir(PPy)2CI]2 can generate a large amount of reactive oxygen species (ROS) within the bacteria (see Liao, G., Peng, X., Li, I. Ye, Z., Xiang, X., Fu, C. The Discovery of an Iridium(III) Dimer Complex as a Potent Antibacterial Agent against Non-Replicating Mycobacterium smegmatis [J]. Polymers , 2018, 10:297.).

[0005] Staphylococcus aureus is a biofilm-forming bacterium. As one of the most common pathogens, it can cause various infections, including fatal pneumonia and sepsis. In recent years, overcoming existing antibacterial technologies has become an important topic in antimicrobial research. The destruction of bacterial intracellular components by ROS-induced oxidative stress has become a hot research topic. Therefore, this study will explore how to generate ROS-induced oxidative stress effects to inhibit biofilm formation, which is key to the development of antimicrobial drugs. Summary of the Invention

[0006] To address the technical problems of the difficulty in inhibiting Staphylococcus aureus biofilm and the limitations of existing bactericidal technologies, this invention provides a pyrrole-modified iridium polypyridine complex for antibacterial purposes, its preparation method, and its application. Specifically, it provides a pyrrole-modified iridium polypyridine complex with ROS-induced oxidative stress effect and biofilm inhibition, along with its preparation method and application. Pyrrole contains a nitrogen-containing heterocycle and is a highly valuable antibacterial group. The iridium-containing complex modified with the pyrrole group enhances its transmembrane activity against bacteria, effectively penetrating the bacterial cell membrane to generate ROS-induced oxidative stress, thereby disrupting the integrity of the bacterial structure and leading to bacterial death. This invention combines iridium polypyridine with a pyrrole group to form a novel antibacterial agent with excellent anti-biofilm activity, providing a new strategy for the development of antibacterial drugs.

[0007] To achieve the above objectives, the present invention provides a pyrrole-modified iridium polypyridine complex, wherein the complex is a cyclic metallic iridium complex having the structure shown in Formula I. I.

[0008] Furthermore, when the complex is irradiated with visible light at 400-700 nm, it is targeted and enriched within the bacterial cell and catalyzes the production of ROS, thereby mediating bacterial oxidative stress, inhibiting bacterial toxin secretion, and inhibiting the formation and maturation of bacterial biofilms; the mediating of bacterial oxidative stress includes: disrupting the integrity of the bacterial cell membrane.

[0009] A second aspect of the present invention provides a method for preparing the pyrrole-modified iridium polypyridine complex of the present invention, the method comprising the following steps: S1. The compound with the structure shown in Formula Ia and the compound with the structure shown in Formula Ib are placed in a mixed system of ammonium acetate and glacial acetic acid, and heated under a protective atmosphere and refluxed. After the reaction solution is cooled to room temperature, it is filtered and dried to obtain the compound with the structure shown in Formula Ic. S2, IrCl3 and C 11 H7F2N was added to a mixture of 2-ethoxyethanol and water, and the mixture was heated under a protective atmosphere and refluxed. After the reaction solution was cooled to room temperature, it was filtered and dried to obtain a compound with the structure shown in formula Id. S3. The compound with the structure shown in Formula Ic and the compound with the structure shown in Formula Id are added to an organic solvent and heated under a protective atmosphere and refluxed. After cooling to room temperature, a salt containing the target counter anion is added for ion exchange. After filtration and drying, the mixture is purified by column chromatography to obtain the pyrrole group-modified iridium polypyridine complex shown in Formula I.

[0010] Further, in step S1, the molar ratio of the compound with the structure shown in formula Ib to the compound with the structure shown in formula Ia is 1:1.2~1.3.

[0011] Furthermore, in step S1, the volume ratio of ammonium acetate to glacial acetic acid is 25~35:1.

[0012] Furthermore, in step S1, the concentrations of the compound with the structure shown in formula Ib and the compound with the structure shown in formula Ia in the mixed system of ammonium acetate and glacial acetic acid are 0.025~0.04 mol / L and 0.03~0.05 mol / L, respectively.

[0013] Furthermore, in step S1, the temperature of the heating reflux is 120℃~130℃, and the time is 3h~4h.

[0014] Furthermore, in step S2, IrCl3 and C11 The molar ratio of the H7F2N compound is 1:2.1~2.2.

[0015] Furthermore, in step S2, the volume ratio of 2-ethoxyethanol to water is 2~4:1.

[0016] Furthermore, in step S2, IrCl3 and C 11 The concentrations of the H7F2N compound in a mixture of 2-ethoxyethanol and water were 0.03–0.06 mol / L and 0.06–0.12 mol / L, respectively.

[0017] Furthermore, in step S2, the temperature of the heating reflux is 120℃~130℃, and the time is 24h~26h.

[0018] Further, in step S3, the molar ratio of the compound with the structure shown in formula Ic to the compound with the structure shown in formula Id is 2~2.1:1.

[0019] Further, in step S3, the organic solvent is methanol and dichloromethane; the volume ratio of methanol to dichloromethane is 1:1~3.

[0020] Furthermore, in step S3, the concentrations of the compound with the structure shown in formula Ic and the compound with the structure shown in formula Id in the organic solvent are 0.005~0.02 mol / L and 0.005~0.02 mol / L, respectively.

[0021] Furthermore, in step S3, the temperature of the heating reflux reaction is 60℃~65℃, and the time is 8h~10h.

[0022] Furthermore, in step S3, the salt containing the target counter anion is a saturated KPF6 aqueous solution.

[0023] Further, in step S3, the mixture of acetonitrile and dichloromethane is used as the eluent for separation and purification by neutral alumina column chromatography.

[0024] A third aspect of the present invention provides an antibacterial composition comprising the pyrrole-modified iridium polypyridine complex of the present invention, and a pharmaceutically or chemically acceptable carrier, excipient or excipient.

[0025] Furthermore, the dosage form of the antibacterial composition is selected from any one of injections, topical creams, gels, sprays, lotions, medical dressings, oral care preparations, and disinfectants.

[0026] The fourth aspect of the present invention provides the application of the pyrrole group-modified iridium polypyridine complex of the present invention in the preparation of products for inducing bacterial ROS production, mediating bacterial oxidative stress effects, and inhibiting bacterial biofilm formation.

[0027] Furthermore, the product is any one of the following: antibacterial drugs, antibacterial biofilm preparations, medical disinfection products, food preservatives and antibacterial products, and daily chemical antibacterial products.

[0028] Furthermore, the bacteria include Staphylococcus aureus.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Compared with the prior art, the present invention provides a pyrrole group modified iridium polypyridine complex, which enhances transmembrane interaction compared with traditional antibiotics due to the pyrrole group in its molecule.

[0030] (2) Experimental results show that the novel antibacterial agent with thiophene modification of the present invention can effectively inhibit the growth of Staphylococcus aureus at a content of 8µg / mL.

[0031] (3) The pyrrole group-modified iridium polypyridine complex of the present invention has the effect of inhibiting Staphylococcus aureus biofilm.

[0032] (4) In addition, the pyrrole structure in the pyrrole group-modified iridium polypyridine complex of the present invention has a strong ROS-induced oxidative stress effect, which leads to bacterial death. Attached Figure Description

[0033] Figure 1 shows a simulation diagram of the MIC effect of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention on inhibiting bacterial growth. Figure 2 shows a physical image of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention inhibiting the biofilm formation of Staphylococcus aureus. Figure 3 shows the effect of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention on inhibiting the biofilm formation of Staphylococcus aureus. Figure 4 shows a physical image of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention, which induces ROS-induced oxidative stress in Staphylococcus aureus. Figure 5 shows the effect of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention on the ROS-induced oxidative stress effect in Staphylococcus aureus. Figure 6 shows the mass spectrum of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention; Figure 7 shows the 1H NMR spectrum of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention. Figure 8 shows the structural formula of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this invention. Detailed Implementation

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0036] In this paper, IrF5 represents the final product. The protective atmosphere used in this paper is not limited to argon or helium.

[0037] The embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the invention. For those skilled in the art, other equivalent embodiments based on the content of the present invention can be obtained without creative effort, and all such embodiments should be considered to fall within the protection scope of the present invention.

[0038] Unless otherwise specified, the raw materials used in the following examples are all publicly available in the prior art, such as those that can be directly purchased or prepared according to publicly available methods. The reagents used in the examples are shown in Table 1 below, and the instruments are shown in Table 2 below.

[0039] Table 1 Table 2 Example 1: (1) Intermediate for preparing the Ic structure: The reaction mixture of the compound of formula Ib (0.5 g, 1.00 mmol) and the compound of formula Ia (0.3 g, 1.21 mmol) was heated under reflux for 3 h in a mixture of 30 mL ammonium acetate and 1 mL glacial acetic acid under argon protection. After cooling to room temperature, the pH was adjusted to 7 with ammonia. A large amount of green solid precipitated out. The solid was filtered off, wrapped in laboratory paper, and dried in an 85 °C oven for 4 h. It was then stored in a sealed container to obtain the intermediate of formula Ic, which was a green, flaky solid. Yield: 90%.

[0040] (2) Intermediates for preparing the Id structure: IrCl3 (0.5 g, 1.67 mmol) and C 11 H7F2N (0.65 g, 3.40 mmol), a mixture of 30 mL 2-ethoxyethanol and 10 mL water was heated under argon protection and refluxed for 24 h. After cooling to room temperature, the solid was filtered off, wrapped in laboratory paper, and dried in an 85 °C oven for 4 h. It was then stored in a sealed container to obtain the intermediate of formula Id, which was a yellow powder. Yield: 90%.

[0041] (3) Preparation of pyrrole-modified iridium polypyridine complex IrF5: Compounds of formula Ic (0.15 g, 0.35 mmol) and formula Id (0.23 g, 0.32 mmol) were dissolved in 10 mL MeOH and 20 mL DCM, and reacted in a dry 50 mL reaction flask equipped with a stir bar. The mixture was heated under argon protection at 60 °C for 8 h (adding more solution if necessary). After the reaction was complete, the MeOH solvent was evaporated, and 15 mL of saturated KPF6 aqueous solution was added. The mixture was filtered and dried to obtain a yellow-green precipitate. The crude product was purified by neutral alumina column chromatography using acetonitrile / dichloromethane (1 / 10, v / v) as eluent to give the final product IrF5, a yellow-brown solid. Yield: 60%.

[0042] The structural formula of the intermediate of the Ic structure is as follows: ; like Figure 6 , Figure 7 The image shows the mass spectrum and the hydrogen nuclear magnetic resonance spectrum of the pyrrole-modified iridium polypyridine complex IrF5 prepared in this embodiment. Figure 8 The figure shows the structural formula of IrF5, an iridium polypyridine complex modified with a pyrrole group.

[0043] The synthetic route for the pyrrole-modified iridium polypyridine complex IrF5 is as follows: Example 2: The minimum inhibitory concentration (MIC) of the complex IrF5 prepared in Example 1 was evaluated using a serial dilution method, as follows: (1) Dilute the bacteria cultured overnight with TSB medium 1000 times (0.5-1×10⁻⁶). 7IrF5 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg / mL.

[0044] (2) Add 100 μL of IrF5 to the first well of the 96-well plate, and add 50 μL of sterile water to the second to twelfth wells. Take out 50 μL from the first well and serially dilute it to make the drug volume in each well 50 μL. The last well contains 50 μL of sterile water as a blank control.

[0045] (3) Add 200 μL of diluted bacterial solution to drugs of different concentrations, for a total volume of 250 μL.

[0046] (4) The mixture of drug and bacterial culture was incubated in a constant temperature incubator at 37 ℃ for 18-20 h. The results were observed, and the minimum inhibitory concentration (MIC) of IrF5 was found to be 8 μg / mL. The experimental results were as follows: Figure 1 As shown.

[0047] Example 3: The effect of the IrF5 complex prepared in Example 1 on inhibiting Staphylococcus aureus biofilm was determined by the following method: (1) Staphylococcus aureus Newman strain, which has grown to the logarithmic phase and has been diluted 1000 times, was cultured in TSB medium in a 24-well plate and incubated with IrF5 and TSB mixture at concentrations of 0.25 MIC, 0.5 MIC, 0.75 MIC, and 0 MIC (control) for 48 h, 2 mL per well.

[0048] (2) Remove the liquid, wash away airborne bacteria with PBS, dry, add thiazolyl bromide blue tetrazolium staining agent, after complete staining, remove the staining agent, dry, add 200 μL DMSO to each well to dissolve the biofilm, and measure the OD of each group. 595 Absorbance.

[0049] (3) In Figure 2 In this study, the biofilms grown in each well were stained with STYO 9 dye (green). Observation using a laser confocal microscope clearly showed that a concentration of 0.75 MIC of compound IrF5 inhibited the formation of Staphylococcus aureus biofilms in a concentration-dependent manner, thus determining the anti-biofilm activity of compound IrF5. The experimental results indicate that compound IrF5 has good biofilm inhibitory activity.

[0050] (4) In Figure 3In this study, the inhibitory effect of compound IrF5 at concentrations of 0.25 MIC, 0.5 MIC, 0.75 MIC, and 0 MIC on biofilm formation by Staphylococcus aureus was determined to assess the anti-biofilm activity of compound IrF5. The experimental results indicate that compound IrF5 possesses good biofilm inhibitory activity.

[0051] Example 4: The effect of the ROS-induced death of Staphylococcus aureus by the IrF5 complex prepared in Example 1 was determined by the following method. (1) IrF5 complex was prepared in 96-well plates using a serial dilution method. N-acetyl-L-cysteine ​​(NAC, 2 mg / mL) and bacterial suspension (150 μL) were added sequentially, and the cells were incubated at 37°C for 18 hours before determining the minimum inhibitory concentration (MIC). In short, logarithmic-phase Staphylococcus aureus cells were incubated with an equal volume of 10 μM DCFH-DA in PBS at 37°C for 30 minutes. Then, the cells were washed three times with PBS to remove excess DCFH-DA. The treated Staphylococcus aureus cells were resuspended and added to black 96-well plates. Subsequently, different concentrations of IrF5 were added to each well. After incubation for 1 hour, fluorescence intensity was measured for 60 min at excitation / emission wavelengths of 488 / 530 nm using a microplate reader. Vancomycin was used as a positive control.

[0052] (2) In Figure 4 and Figure 5 In the study, it can be clearly seen that IrF5 exhibits green fluorescence as the concentration increases, proving that the complex has a good ability to generate ROS, causing oxidative stress in bacteria, oxidizing and destroying bacterial components, and thus leading to bacterial death.

[0053] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A pyrrole-modified iridium polypyridine complex, characterized in that, The complex is a cyclic metallic iridium complex, having the structure shown in Formula I: I 。 2. The pyrrole-modified iridium polypyridine complex according to claim 1, characterized in that, The complex, when irradiated with visible light at 400-700 nm, is targeted and enriched within bacterial cells and catalyzes the production of ROS, thereby mediating bacterial oxidative stress, inhibiting bacterial toxin secretion, and inhibiting the formation and maturation of bacterial biofilms; the mediating of bacterial oxidative stress includes: disrupting the integrity of bacterial cell membranes.

3. A method for preparing the pyrrole-modified iridium polypyridine complex according to claim 1 or 2, characterized in that, The method includes the following steps: S1. The compound with the structure shown in Formula Ia and the compound with the structure shown in Formula Ib are placed in a mixed system of ammonium acetate and glacial acetic acid, and heated under a protective atmosphere and refluxed. After the reaction solution is cooled to room temperature, it is filtered and dried to obtain the compound with the structure shown in Formula Ic. S2, IrCl3 and C 11 H7F2N was added to a mixture of 2-ethoxyethanol and water, and the mixture was heated under a protective atmosphere and refluxed. After the reaction solution was cooled to room temperature, it was filtered and dried to obtain a compound with the structure shown in formula Id. S3. The compound with the structure shown in Formula Ic and the compound with the structure shown in Formula Id are added to an organic solvent and heated under a protective atmosphere and refluxed. After cooling to room temperature, a salt containing the target counter anion is added for ion exchange. After filtration and drying, the mixture is purified by column chromatography to obtain the pyrrole group-modified iridium polypyridine complex shown in Formula I.

4. The method according to claim 3, characterized in that, In step S1, The molar ratio of the compound with the structure shown in Formula Ib to the compound with the structure shown in Formula Ia is 1:1.2~1.3; and / or The volume ratio of ammonium acetate to glacial acetic acid is 25-35:1; and / or The heating reflux temperature is 120℃~130℃, and the time is 3h~4h.

5. The method according to claim 3, characterized in that, In step S2, IrCl3 and C 11 The molar ratio of the H7F2N compound is 1:2.1~2.2; and / or The volume ratio of 2-ethoxyethanol to water is 2-4:1; and / or The heating reflux temperature is 120℃~130℃, and the time is 24h~26h.

6. The method according to claim 3, characterized in that, In step S3, The molar ratio of the compound with the structure shown in Formula Ic to the compound with the structure shown in Formula Id is 2~2.1:1; and / or The organic solvent is methanol and dichloromethane; the volume ratio of methanol to dichloromethane is 1:1~3; and / or The heating reflux reaction is carried out at a temperature of 60°C to 65°C for a duration of 8 to 10 hours; and / or The salt containing the target counter anion is a saturated aqueous solution of KPF6; and / or The mixture of acetonitrile and dichloromethane was used as the eluent for separation and purification by neutral alumina column chromatography.

7. An antibacterial composition, characterized in that, It comprises the pyrrole-modified iridium polypyridine complex as described in claim 1 or 2, and a pharmaceutically or chemically acceptable carrier, excipient, or excipient.

8. The antibacterial composition according to claim 7, characterized in that, The dosage form of the antibacterial composition is selected from any one of the following: injection, topical cream, gel, spray, lotion, medical dressing, oral care preparation, and disinfectant preparation.

9. The use of the pyrrole-modified iridium polypyridine complex of claim 1 or 2 in the preparation of products for inducing bacterial ROS production, mediating bacterial oxidative stress effects, inhibiting bacterial toxin secretion, or inhibiting bacterial biofilm formation.

10. The application according to claim 9, characterized in that, The product is any one of the following: antibacterial drugs, antibacterial biofilm preparations, medical disinfection products, food preservatives and antibacterial products, and daily chemical antibacterial products; the bacteria include Staphylococcus aureus.

Images