Pyrazole functional group modified metal iridium complex as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibacterial medicines, and particularly relates to a pyrazole functional group modified metal iridium complex as well as a preparation method and application thereof. The structural formula of the pyrazole functional group modified metal iridium complex is as shown in formula I in the specification. The pyrazole group is introduced into the ligand of the cyclometalated iridium complex, so that the capability of penetrating and destroying bacterial cell membranes is remarkably enhanced, and the antibacterial activity is improved. Tests prove that the metal iridium complex plays a bactericidal role by destroying membrane integrity, inducing cellular content leakage and depolarization of a membrane, can remarkably inhibit formation of golden yellow pigment and a biological membrane of staphylococcus aureus, is not easy to induce bacteria to generate drug resistance, and has a good application prospect in clinical anti-infection treatment. Formula I.

Description

Technical Field

[0001] This application belongs to the field of antibacterial pharmaceutical technology, specifically relating to a metal iridium complex modified with pyrazole functional groups, its preparation method, and its application. Background Technology

[0002] Staphylococcus aureus is one of the most common pathogens in clinical practice, causing everything from minor skin infections to serious systemic diseases such as pneumonia, sepsis, and endocarditis. With the long-term and irregular use of traditional antibiotics, bacterial resistance is becoming an increasingly serious problem.

[0003] Studies have found that the pathogenicity of Staphylococcus aureus is closely related to the various virulence factors it secretes. Among them, α-hemolysin can penetrate the host cell membrane to form pores, leading to cell lysis; while the aurea pigment, as an important antioxidant, can help bacteria resist oxidative damage from the host's immune system. In addition, this bacterium has an extremely strong ability to form biofilms. The presence of biofilms increases the bacteria's resistance to environmental stress and antibiotics by hundreds of times, leading to recurrent infections and making it difficult to eradicate.

[0004] Traditional antibacterial drug development often focuses on single bactericidal effects and easily induces bacterial resistance through gene mutations. In recent years, metal complexes have shown great potential in the antibacterial field due to their unique stereostructure and multi-target mechanism of action. In particular, cyclometalated iridium complexes possess excellent photophysical properties and structural tunability. However, existing iridium complexes often suffer from poor hydrophilicity-hydrophobicity balance and weak ability to penetrate bacterial cell membranes.

[0005] Therefore, developing a novel "multifunctional" antibacterial agent that can kill bacteria while inhibiting toxin secretion and biofilm formation has become an urgent problem to be solved in the field of antibacterial drug research. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a metal iridium complex modified with pyrazole functional groups, its preparation method, and its application. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a metal iridium complex modified with pyrazole functional groups, the structural formula of which is shown in Formula I: Formula I.

[0007] This invention provides an iridium(III) complex modified with a pyrazole structure. This complex exhibits excellent antibacterial activity against Staphylococcus aureus, with a unique mechanism of action, low susceptibility to inducing drug resistance, and high in vivo safety, making it suitable for treating bacterial infections. The core of this complex's structural design lies in the pyrazole group, a nitrogen-containing five-membered heterocycle with unique hydrogen bond donor and acceptor sites, enabling rich interactions with biomolecules. By precisely introducing the pyrazole functional group into the iridium complex ligand, not only can the electron distribution of the metal center be modulated, but more importantly, the pyrazole ring can specifically bind to the phospholipid components on the bacterial cell membrane surface. This structural modification significantly reduces the energy barrier for the complex to penetrate the membrane barrier, inducing membrane depolarization and causing physical damage. This results in rapid bactericidal activity while inhibiting Staphylococcus aureus secretion and biofilm formation. Furthermore, due to its physical membrane-damaging mechanism, bacteria are extremely difficult to develop drug resistance.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned metal iridium complex modified with pyrazole functional groups, comprising the following steps: Under inert protective gas conditions, the compound represented by structure Ia and the compound represented by structure Ib are subjected to a condensation reaction in a reaction solvent to obtain the intermediate compound represented by structure Ic. The compound represented by intermediate formula Ic was mixed with the compound represented by formula Id, and the reaction was carried out under an inert protective gas. After the reaction was completed, potassium hexafluorophosphate was added, and the mixture was centrifuged or filtered to purify it, thereby obtaining the metal iridium complex modified with pyrazole functional groups. The structural formulas of the compounds represented by formula Ia, formula Ib, intermediate formula Ic, and formula Id are shown below: .

[0009] As a further preferred embodiment, the molar ratio of the compound represented by Formula Ia, the compound represented by Formula Ib, and ammonium acetate is 1:1:30.

[0010] As a further preferred embodiment, the reaction solvent is an acetic acid solution.

[0011] As a further preferred embodiment, the inert protective gas is at least one of helium, argon, and nitrogen.

[0012] As a further preferred embodiment, the ratio of the compound represented by intermediate formula Ic to the compound represented by formula Id is 0.1 mmol: 0.2 mmol.

[0013] As a further preferred embodiment, the condensation reaction is carried out at a temperature of 120°C-150°C for 2 h-5 h.

[0014] Thirdly, the present invention provides the use of the above-mentioned metal iridium complex modified with pyrazole functional groups in the preparation of drugs for inhibiting Staphylococcus aureus.

[0015] Fourthly, the present invention provides the use of the above-mentioned metal iridium complex modified with pyrazole functional groups in the preparation of a drug for inhibiting the production of golden pigment by Staphylococcus aureus.

[0016] Fifthly, the present invention provides the use of the above-mentioned metal iridium complex modified with pyrazole functional groups in the preparation of drugs for inhibiting or eradicating Staphylococcus aureus biofilms.

[0017] The beneficial effects of this invention are as follows: This invention significantly enhances the ability of cyclometalated iridium complexes to penetrate and disrupt bacterial cell membranes by introducing a pyrazole group into the ligand, thereby improving their antibacterial activity. Experiments have demonstrated that the iridium complexes of this invention exert their bactericidal effect by disrupting membrane integrity, inducing leakage of cell contents, and causing membrane depolarization. Simultaneously, they significantly inhibit the formation of the aureotropic pigment and biofilm in Staphylococcus aureus, and are less likely to induce bacterial resistance, showing promising application prospects in clinical anti-infective therapy. Furthermore, data from embodiments of this invention show that the iridium complexes provided by this invention possess the following characteristics: (1) Strong antibacterial activity: The minimum inhibitory concentration against Staphylococcus aureus is 5 μg / mL; (2) Fast sterilization speed: At concentrations of 1×MIC, 2×MIC and 4×MIC, Staphylococcus aureus can be completely killed within 1 hour, which can effectively reduce the risk of bacterial resistance induction.

[0018] (3) Not easy to induce drug resistance: After 20 days of continuous passage experiments, the MIC value of Staphylococcus aureus remained stable, while the MIC value of the positive control ampicillin sodium increased by 64 times. (4) Effectively combats bacterial toxins: It can effectively inhibit the formation of golden pigment at sub-inhibitory concentrations; (5) Outstanding anti-pathogenicity: It can effectively inhibit the formation of bacterial biofilm at sub-inhibitory concentrations and can effectively remove mature biofilms at minimum bactericidal concentrations, significantly reducing bacterial pathogenicity. 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 The diagram shows the synthetic route for phenylpyridine iridium complexes modified with pyrazole groups provided by this invention; Figure 2 The figure shown is a graph illustrating the inhibition of Staphylococcus aureus biofilm by the phenylpyridine-iridium complex provided by this invention. Figure 3 The figure shown is a time-dependent killing effect diagram of the phenylpyridine-iridium complex provided by the present invention on Staphylococcus aureus. Figure 4 The figure shown is a graph illustrating the determination of Staphylococcus aureus drug resistance induced by the phenylpyridine-iridium complex provided by the present invention. Figure 5 The figure shown is a graph illustrating the inhibition of Staphylococcus aureus pigment by the phenylpyridine-iridium complex provided by the present invention. Figure 6 The image shows the mass spectrum of the phenylpyridine-iridium complex provided by this invention; Figure 7 The image shown is the proton NMR spectrum of the phenylpyridine-iridium complex provided by this invention; Figure 8 The image shown is a carbon spectrum of the phenylpyridine-iridium complex provided by this invention; Figure 9 The image shown is the fluorine spectrum of the phenylpyridine-iridium complex provided by this invention. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0022] Example 1 A metal iridium complex modified with pyrazole functional groups is prepared by the following steps (the synthetic route is as follows). Figure 1 (as shown) (1) Preparation of the host ligand of the Ic structure: Formula Ia (1,10-phenanthroline-5,6-dione), Formula Ib, and ammonium acetate were mixed in a molar ratio of 1:1:30. 10 mL of acetic acid was added as the reaction system. The mixture was refluxed at 130 °C for 3 hours under an argon atmosphere, resulting in a condensation reaction that produces the main ligand 2-(5-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (Formula Ic).

[0023] (2) Preparation of phenylpyridine-iridium complexes modified with pyrazole functional groups: Intermediate Ic (0.1 mmol) and Id (0.2 mmol) were mixed and reacted at 65 °C for 8 hours under an argon atmosphere. After the reaction was complete, 10 equivalents of potassium hexafluorophosphate of compound Ic were added to obtain an orange-yellow solid. The crude product was then obtained by centrifugation or filtration. The crude product was then separated and purified by neutral alumina column chromatography to obtain a phenylpyridine-iridium complex modified with a pyrazole functional group. The characterization results are as follows: Figure 6-9 As shown.

[0024] The structural formulas of formulas Ia, Ib, Ic, and Id in the above preparation process are shown below: The structure of the metal iridium complex modified with pyrazole functional groups is shown below: .

[0025] Example 2 The minimum concentration of the phenylpyridine-iridium complex prepared in Example 1 that inhibited the in vitro growth of Staphylococcus aureus in the logarithmic growth phase was determined, and the specific procedure is as follows: The iridium complex was dissolved in DMSO, and Staphylococcus aureus cultured to the logarithmic growth phase was diluted 1000-fold in TSB medium to prepare a bacterial suspension. 200 μL of the bacterial suspension was mixed with 50 μL of drug solutions at different concentration gradients (1.6 mg / mL, 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.56 μg / mL), and then added to 96-well plates. The 96-well plates were incubated at 37°C for 18-20 hours, and the minimum inhibitory concentration (MIC) of the drug was determined. The MIC of the iridium complex was found to be 5 μg / mL.

[0026] Example 3 The eradication rate of the large phenylpyridine-iridium complex prepared in Example 1 against mature Staphylococcus aureus biofilm was determined, and the specific procedure is as follows: The phenylpyridine-iridium complex was dissolved in DMSO, and overnight cultured Staphylococcus aureus was diluted 100-fold in TSB medium to prepare a bacterial suspension. 2 mL of bacterial suspension and different concentrations of the complex solution were added to each well of a 24-well plate to achieve final complex concentrations of 1 μg / mL, 2 μg / mL, and 3 μg / mL. After incubation at 37 °C for 48 h, airborne bacteria were removed, and the plate was washed three times with PBS. Adhering bacteria were air-dried overnight at room temperature and then stained with 0.1% crystal violet solution for 15 min. The crystal violet solution was removed, and the plate was washed three times with PBS. Subsequently, the crystal violet adhering to the biofilm was dissolved using 50% acetic acid, and the absorbance at 595 nm was measured (PBS was used as a control group).

[0027] The result is as follows Figure 2 As shown in the results, the phenylpyridine-iridium complex prepared in this invention has a strong inhibitory effect on biofilms.

[0028] Example 4 The efficacy of the phenylpyridine-iridium complex prepared in Example 1 against Staphylococcus aureus during the logarithmic growth phase was investigated. The specific process is as follows: Dilute overnight Staphylococcus aureus at a ratio of 1:1000, culture to the logarithmic growth phase, and then measure the OD... 600 Bacterial suspensions with a concentration of 0.1 were prepared into suspensions with final concentrations of 1×MIC, 2×MIC, and 4×MIC by mixing with complexes, with pure bacterial suspensions as controls. Every 30 minutes, 50 μL of bacterial suspension was evenly spread on agar plates and incubated at 37°C for 20–24 hours, after which the colony count was recorded.

[0029] The result is as follows Figure 3 As shown, different concentrations of iridium complexes completely killed Staphylococcus aureus within 1 hour. These data indicate that iridium complexes possess rapid and thorough bactericidal properties, which will effectively reduce the risk of inducing bacterial resistance.

[0030] Example 5 The investigation into whether the pyridine-iridium complex prepared in Example 1 of the benzene-based study induced drug resistance in Staphylococcus aureus was conducted as follows: Iridium pyridine (Ir) complex was dissolved in DMSO. Staphylococcus aureus cultured to the logarithmic growth phase was diluted 1000-fold in TSB medium to prepare a bacterial suspension. 200 μL of the bacterial suspension was mixed with 50 μL of drug solutions at different concentration gradients (1.6 mg / mL, 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.56 μg / mL), and then added to 96-well plates. The plates were incubated at 37°C for 20 hours, and the minimum inhibitory concentration (MIC) of the drug was determined. This process was repeated for 20 consecutive days of induction, using ampicillin sodium as a control.

[0031] The result is as follows Figure 4 As shown in the results, after Staphylococcus aureus was continuously induced by the iridium pyridine complex for 20 days, the minimum inhibitory concentration (MIC) did not increase, while that of the control, ampicillin sodium, increased to 64 times the original MIC value. Therefore, it can be seen that Staphylococcus aureus is not likely to develop resistance to the complex, and the complex has a good prospect of becoming a marketable antibiotic.

[0032] Example 6 The inhibitory effect of phenylpyridine-iridium complex on the auricularia pigment of Staphylococcus aureus was investigated, and the specific process is as follows: Overnight Staphylococcus aureus was diluted 1:1000 and co-incubated for 48 h with different concentrations (1 μg / mL, 2 μg / mL, and 3 μg / mL) of drug dissolved in DMSO, using pure bacterial culture as a control. 3 mL of bacterial culture was centrifuged, the supernatant was discarded, and the sample was washed twice with PBS buffer. The pigment was extracted three times with 1 mL of methanol, and the extracts were combined and the absorbance of the pigment at 450 nm was measured using a microplate reader. Relative level of pigment synthesis = A 450 mm (Sample) / A 450mm (Negative control) × 100% Test results Figure 5 This indicates that 3 μg / mL can significantly inhibit the production of Staphylococcus aureus pigment, and its effect is better than that of the positive control naftifine hydrochloride (2 μg / mL).

[0033] Therefore, in summary, this invention successfully prepared a phenylpyridine-iridium complex through molecular structure design. This complex has the following significant advantages: 1) strong antibacterial activity and rapid bactericidal speed, with low likelihood of inducing bacterial resistance; 2) effective inhibition of bacterial biofilms, enhancing the antibacterial effect; 3) effective inhibition of Staphylococcus aureus pigment, counteracting bacterial toxins. The iridium(III) complex of this invention provides a new technical solution to the problem of antibiotic resistance and can be used as a novel metal-based antibacterial drug to treat diseases related to drug-resistant bacterial infections, possessing significant clinical application value and market prospects.

[0034] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A metal iridium complex modified with a pyrazole functional group, characterized in that, The structural formula of the metal iridium complex modified with pyrazole functional groups is shown in Formula I: Equation I.

2. The method for preparing the pyrazole-functionalized metal iridium complex according to claim 1, characterized in that, Includes the following steps: Under inert protective gas conditions, the compound represented by structure Ia and the compound represented by structure Ib are mixed with ammonium acetate, and then a condensation reaction is carried out in the reaction solvent to obtain the intermediate compound represented by structure Ic. The compound represented by intermediate formula Ic was mixed with the compound represented by formula Id, and the reaction was carried out under an inert protective gas. After the reaction was completed, potassium hexafluorophosphate was added, and the mixture was centrifuged or filtered to purify it, thereby obtaining the metal iridium complex modified with pyrazole functional groups. The structural formulas of the compounds represented by formula Ia, formula Ib, intermediate formula Ic, and formula Id are shown below: 。 3. The preparation method according to claim 2, characterized in that, The molar ratio of the compound represented by Formula Ia and the compound represented by Formula Ib to ammonium acetate is 1:1:

30.

4. The preparation method according to claim 3, characterized in that, The reaction solvent is acetic acid solution.

5. The preparation method according to claim 2, characterized in that, The inert protective gas is at least one of helium, argon, and nitrogen.

6. The preparation method according to claim 2, characterized in that, The ratio of the amount of the intermediate compound of formula Ic to the amount of the compound of formula Id is 0.1 mmol: 0.2 mmol.

7. The preparation method according to claim 2, characterized in that, The condensation reaction is carried out at a temperature of 120℃-150℃ for 2 h-5 h.

8. The use of the pyrazole functional group-modified iridium metal complex of claim 1 in the preparation of a drug for inhibiting Staphylococcus aureus.

9. The use of the pyrazole-functionalized iridium complex of claim 1 in the preparation of a drug for inhibiting the production of auric pigment by Staphylococcus aureus.

10. The use of the pyrazole-functionalized iridium complex of claim 1 in the preparation of a medicament for inhibiting or eradicating Staphylococcus aureus biofilm.

Images