Bisimidazole-modified polypyridine ruthenium complexes, their preparation methods and applications

Abstract

This application belongs to the field of antibacterial pharmaceutical technology, specifically relating to a ruthenium-pyridine complex modified with bisimidazole, its preparation method, and its application. The structure of the bisimidazole-modified ruthenium-pyridine complex is shown in Formula I. This invention modifies the electron cloud distribution and targeting by introducing a bisimidazole group into the ruthenium polypyridine ligand. Utilizing the nucleophilic binding properties of bisimidazole and the spatial synergistic effect of the polypyridine ligand, it significantly enhances the interaction between bisimidazole and bacterial cell membrane phospholipids and virulence factors, thereby improving antibacterial activity and anti-biofilm properties, providing a new approach for developing novel and highly effective drugs against drug-resistant bacteria.

Description

Technical Field

[0001] This application belongs to the field of antibacterial pharmaceutical technology, specifically relating to a polypyridine ruthenium complex modified with diimidazole, its preparation method and application. Background Technology

[0002] The global spread of antibiotic resistance has become a major challenge threatening public health. Methicillin-resistant Staphylococcus aureus (MRSA), a common multidrug-resistant bacterium in clinical practice, can cause various serious illnesses such as skin infections, pneumonia, and sepsis. Its resistance rate to traditional antibiotics continues to rise, leading to a growing shortage of clinical treatment options. Vancomycin, considered the "last line of defense" for treating MRSA infections, has also seen the emergence of resistant strains, and the formation of bacterial biofilms can further enhance resistance by 10 to 1000 times, placing enormous pressure on clinical treatment. Statistics show that more than one million people die globally each year from antibiotic resistance-related diseases, and this number is expected to continue to rise in the coming decades.

[0003] Metal complexes, with their rich structural diversity and unique mechanisms of action, have become an important research direction for overcoming antibiotic resistance. Ruthenium-based complexes, as an important branch of transition metal complexes, possess strong antibacterial activity, good biocompatibility, and are less prone to cross-resistance, and have been proven to have significant inhibitory effects on Staphylococcus aureus. However, existing ruthenium-based antibacterial complexes still suffer from insufficient targeting, limited anti-biofilm activity, and poor in vivo metabolic stability, which restricts their clinical translation and application. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polypyridine ruthenium complex modified with diimidazole, its preparation method, and its application. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a polypyridine ruthenium complex modified with diimidazole, the structural formula of which is shown in Formula I: Formula I; in, Choose from any of the following structures: .

[0005] This invention provides a ruthenium(II) complex modified with diimidazole, which exhibits excellent antibacterial activity and anti-biofilm properties against Staphylococcus aureus. It has a unique mechanism of action, is not prone to inducing drug resistance, and has high in vivo safety, making it suitable for treating drug-resistant bacterial infections.

[0006] The core of the structural design of the above-mentioned complex lies in: by introducing a biimidazole group into the polypyridine ligand, the electronic effect and targeted binding characteristics of biimidazole are utilized to adjust the lipid-water partition coefficient and electron cloud distribution of the complex; at the same time, through the spatial synergistic effect of biimidazole and different substituent bipyridines, the interaction between the complex and bacterial cell membrane phospholipids, virulence factors and nucleic acids is enhanced, thereby improving antibacterial activity, targeting and anti-biofilm properties.

[0007] As a further preferred embodiment, the structure of the polypyridine ruthenium complex is shown as Ru-1, Ru-2, Ru-3, or Ru-4: ; ; ; .

[0008] Secondly, the present invention provides a method for preparing the above-mentioned polypyridine ruthenium complex modified with diimidazole, characterized by comprising the following steps: S1. Dissolve the compound of formula Ia in a solvent, and under the protection of an inert gas, then carry out a nucleophilic substitution reaction with the compound of formula Ib to obtain the intermediate of formula Ic. S2. Under the protection of an inert gas, the intermediate of the Ic structure and the compound of the Id structure are subjected to a condensation reaction under the conditions of ammonium acetate and acetic acid to obtain the main ligand of the Ie structure. S3. The main ligand of the Ie structure is mixed with a compound of the If structure, the Ig structure, the Ih structure, or the Ii structure and an organic solvent, and then a coordination reaction is carried out under the protection of an inert gas. After purification, the polypyridine ruthenium complex with diimidazole modification is obtained. The structural formulas of the compounds of formula Ia, Ib, Ic, Id, Ie, If, Ig, Ih, and Ii are shown below: .

[0009] This invention employs imidazole to modify ruthenium-based complexes. Imidazole, as a heterocyclic group with unique electronic structure and biological activity, possesses excellent nucleophilic binding ability and targeted recognition potential. In drug molecule design, it can enhance the interaction between drugs and targets by adjusting the electron cloud distribution, lipid-water partition coefficient, and biocompatibility of the compound. Introducing imidazole into the structural design of ruthenium polypyridine complexes can synergistically improve the antibacterial activity, targeting, and anti-biofilm properties of the complexes, providing new ideas for developing novel and highly effective drugs against drug-resistant bacteria.

[0010] As a further preferred embodiment, the solvent in S1 is anhydrous ethanol; The nucleophilic substitution reaction is carried out at a temperature of 70℃-80℃ for 3-5 days.

[0011] As a further preferred embodiment, the molar ratio of the intermediate of formula Ic structure to the compound of formula Id structure in S2 is 1:1.

[0012] As a further preferred embodiment, the condensation reaction is carried out at a temperature of 120°C-150°C for 5-10 hours.

[0013] As a further preferred embodiment, the inert gas in S1, S2, and S3 is at least one of helium, argon, and nitrogen.

[0014] As a further preferred embodiment, the organic solvent in S3 is ethylene glycol; The coordination reaction was carried out at a temperature of 140℃-160℃ for 8h-12h.

[0015] Thirdly, the present invention provides the application of the above-mentioned pyridine ruthenium complex modified with diimidazole in the preparation of drugs for inhibiting drug-resistant bacteria.

[0016] As a further preferred embodiment, the drug-resistant bacteria is Staphylococcus aureus.

[0017] The beneficial effects of this invention are as follows: (1) Strong antibacterial activity: The MIC of the ruthenium-pyridine complex modified with diimidazole provided by the present invention against Staphylococcus aureus Newman is 3.125 μg / mL and the MBC is 12.5 μg / mL, which is significantly better than the clinical drug vancomycin.

[0018] (2) Fast sterilization speed: At concentrations of 2×MIC, 4×MIC and 8×MIC, the polypyridine ruthenium complex with diimidazole modification provided by the present invention can completely kill Staphylococcus aureus within 2 hours, which can effectively reduce the risk of bacterial resistance induction.

[0019] (3) Not easy to induce drug resistance: After 20 days of continuous passage experiments, the MIC value of the pyridine ruthenium complex with diimidazole modification provided by the present invention against Staphylococcus aureus remained stable (3.125 μg / mL-12.5 μg / mL), while the MIC value of the positive control ampicillin sodium increased by 128 times under the same conditions.

[0020] (4) Outstanding anti-pathogenicity: The pyridine ruthenium complex modified with diimidazole provided by the present invention can effectively inhibit the formation of bacterial biofilm at sub-inhibitory concentrations and can remove mature biofilms in a concentration-dependent manner, significantly reducing bacterial pathogenicity. Attached Figure Description

[0021] 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.

[0022] Figure 1 The diagram shows the synthetic route for the polypyridine ruthenium complex modified with diimidazole provided by this invention; Figure 2 The figure shown is a graph illustrating the inhibition of Staphylococcus aureus biofilm formation by the polypyridine ruthenium complex Ru-3 provided by this invention. Figure 3 The figure shown is a time-dependent killing effect of the polypyridine ruthenium complex Ru-3 provided by the present invention on Staphylococcus aureus. Figure 4 The figure shown is a graph illustrating the determination of Staphylococcus aureus resistance induced by the polypyridine ruthenium complex Ru-3 provided by the present invention. Figure 5 The image shown is the proton NMR spectrum of the polypyridine ruthenium complex Ru-1 provided by this invention; Figure 6 The image shown is the proton NMR spectrum of the polypyridine ruthenium complex Ru-2 provided by this invention; Figure 7 The image shown is the proton NMR spectrum of the polypyridine ruthenium complex Ru-3 provided by this invention; Figure 8 The image shown is the proton NMR spectrum of the polypyridine ruthenium complex Ru-4 provided by this invention. Detailed Implementation

[0023] 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.

[0024] The structural formulas of formulas Ia, Ib, Ic, Id, Ie, If, Ig, Ih, and Ii in the preparation process of the following embodiments of the present invention are shown below: Example 1 A polypyridine ruthenium complex modified with diimidazole (its synthetic route is as follows) Figure 1 As shown), its preparation method specifically includes the following steps: (1) Preparation of the host ligand of the Ie structure: Step 1: Preparation of intermediate Ic: Starting with 4-bis(2-chloroethyl)aminobenzaldehyde (Formula Ia), 15 mL of anhydrous ethanol was added as a solvent, and under argon protection, it was refluxed with N-methylimidazolium (Formula Ib) at 80 °C for 3 to 5 days to obtain the intermediate 3,3'-[(4-formylphenyl)azadiylbis(ethane-2,1-diyl)]bis(1-methyl-1H-imidazol-3-onium) (Formula Ic).

[0025] Step 2: Preparation of the host ligand Ie: Intermediate Ic was mixed with 1,10-phenanthroline-5,6-dione (Formula Id) at a molar ratio of 1:1, and 15 mL of acetic acid was added as a reaction solvent. Ten equivalents of ammonium acetate were also added. Under argon protection, the mixture was refluxed at 130 °C for 6 hours, resulting in a condensation reaction to generate the main ligand 3,3'-[(4-(1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)phenyl)azonidibis(ethane-2,1-diyl)]bis(1-methyl-1H-imidazoline-3-onium) (Formula Ie).

[0026] (2) Preparation of polypyridine ruthenium complexes modified with bisimidazole (Ru-1): The main ligand, Ie (0.1 mmol), [Ru(bpy)₂Cl₂]H₂O (If) (0.1 mmol), was mixed with ethylene glycol and reacted at 150 °C for 10 hours under an argon atmosphere. After the reaction was complete, 10 equivalents of potassium hexafluorophosphate were added to obtain a reddish-brown solid, which was then centrifuged or filtered to obtain the crude product. The crude product was then separated and purified by neutral alumina column chromatography to obtain compound Ru-1. Its characterization results are as follows: Figure 5 As shown.

[0027] The structure of Ru-1 is shown below: .

[0028] Example 2 A polypyridine ruthenium complex modified with diimidazole (its synthetic route is as follows) Figure 1 As shown), its preparation method specifically includes the following steps: (1) Preparation of the main ligand of the Ie structure: The preparation process is exactly the same as in Example 1.

[0029] (2) Preparation of polypyridine ruthenium complexes modified with bisimidazole (Ru-2): The main ligand Ie (0.1 mmol), [Ru(dmb)2Cl2]H2O (Ig) (0.1 mmol), and ethylene glycol were mixed and reacted at 150 °C for 10 hours under an argon atmosphere. After the reaction was complete, 10 equivalents of potassium hexafluorophosphate were added to obtain a reddish-brown solid, which was then centrifuged or filtered to obtain the crude product. The crude product was then separated and purified by neutral alumina column chromatography to obtain compound Ru-2. The characterization results are as follows: Figure 6 As shown.

[0030] The structure of Ru-2 is shown below: .

[0031] Example 3 A polypyridine ruthenium complex modified with diimidazole (its synthetic route is as follows) Figure 1 As shown), its preparation method specifically includes the following steps: (1) Preparation of the main ligand of the Ie structure: The preparation process is exactly the same as in Example 1.

[0032] (2) Preparation of polypyridine ruthenium complexes modified with imidazolium (Ru-3): The main ligand Ie (0.1 mmol), [Ru(dtb)₂Cl₂]H₂O (Ii) (0.1 mmol) were mixed with ethylene glycol and reacted at 150 °C for 10 hours under an argon atmosphere. After the reaction was complete, 10 equivalents of potassium hexafluorophosphate were added to obtain a reddish-brown solid, which was then centrifuged or filtered to obtain the crude product. The crude product was then separated and purified by neutral alumina column chromatography to obtain compound Ru-3. The characterization results are as follows: Figure 7 As shown.

[0033] The structure of Ru-3 is shown below: .

[0034] Example 4 A polypyridine ruthenium complex modified with diimidazole (its synthetic route is as follows) Figure 1 As shown), its preparation method specifically includes the following steps: (1) Preparation of the main ligand of the Ie structure: The preparation process is exactly the same as in Example 1.

[0035] (2) Preparation of polypyridine ruthenium complexes modified with diimidazole (Ru4): The main ligand Ie (0.1 mmol), [Ru(dmob)2Cl2]H2O (Ih) (0.1 mmol) were mixed with ethylene glycol and reacted at 150 °C for 10 hours under an argon atmosphere. After the reaction was complete, 10 equivalents of potassium hexafluorophosphate were added to obtain a reddish-brown solid, which was then centrifuged or filtered to obtain the crude product. The crude product was then separated and purified by neutral alumina column chromatography to obtain compound Ru-4. The characterization results are as follows: Figure 8 As shown.

[0036] The structure of Ru-4 is shown below: .

[0037] Example 5 The minimum concentrations of the polypyridine ruthenium complexes Ru-1, Ru-2, Ru-3, and Ru-4 prepared in Examples 1-4 above that inhibit the in vitro growth of Staphylococcus aureus in the logarithmic phase are as follows: The complex shown in formula Ru-3 (the test compound) was dissolved in DMSO. Staphylococcus aureus cultured to the logarithmic growth phase was diluted 1000-fold in TSB medium to prepare a bacterial suspension. Vancomycin (Van) was used as a positive control. 200 μL of the bacterial suspension was mixed with 50 μL of drug solutions at different concentration gradients and added to 96-well plates. The 96-well plates were incubated at 37°C for 20 hours, and the minimum inhibitory concentration (MIC) of the drug was determined. The MIC of the complex shown in formula Ru-3 was found to be 3.125 μg / mL.

[0038] The complex Ru-3 was replaced with Ru-1, Ru-2, and Ru-4 respectively, and the remaining steps were the same. The MIC values ​​of Ru-1, Ru-2, and Ru-4 were measured to be 200 μg / mL, 100 μg / mL, and 100 μg / mL, respectively.

[0039] Example 6 The inhibition rate of Staphylococcus aureus biofilm formation by the polypyridine ruthenium complex Ru-3 prepared in Example 3 was determined, and the specific procedure is as follows: The inhibitory effect of compounds on bacterial biofilm formation was evaluated using crystal violet staining. Subinhibitory concentrations (0.25 MIC, 0.5 MIC, 0.75 MIC) of Ru-3 and diluted bacterial suspensions were added to 24-well plates. Ctrl(PBS) was used as a blank control, and 0.75 MIC vancomycin was used as a positive control. The plates were incubated at 37°C for 48 h. After incubation, the suspended bacteria were removed and the plates were washed with PBS. Staining was performed with 0.1% crystal violet solution for 30 min. Finally, acetic acid was added to dissolve the crystal violet adhering to the biofilm. The OD was then measured using a microplate reader. 600 value.

[0040] The result is as follows Figure 2 As shown in the results, Ru-3 at a concentration of 0.75 MIC has a significantly better inhibitory effect on biofilm than Vancomycim at a concentration of 0.75 MIC.

[0041] Example 7 The cytotoxic effect of the polypyridine ruthenium complex Ru-3 on Staphylococcus aureus during the logarithmic growth phase was investigated. The specific procedure 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 2×MIC, 4×MIC, and 8×MIC using complex Ru-3. Vancomycin (Van) was used as a positive control, and (Ctrl:PBS) was used as a blank control. Every 30 minutes, 50 μL of bacterial suspension was evenly spread on agar plates and incubated at 37°C for 20 hours. The colony count was then recorded.

[0042] The result is as follows Figure 3 As shown, in Figure 3 In (A) and (B), different concentrations of Ru-3 completely killed Staphylococcus aureus within 2 hours. These data indicate that Ru-3 has rapid and thorough bactericidal properties, which will effectively reduce the risk of inducing bacterial resistance.

[0043] Example 8 The investigation into whether the polypyridine ruthenium complex Ru-3 induces drug resistance in Staphylococcus aureus was conducted as follows: The complex shown in formula Ru-3 (the test compound) 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 and added to 96-well plates. The 96-well plates were incubated at 37°C for 20 hours, and the minimum inhibitory concentration (MIC) of the drug was determined. This procedure was repeated for 20 consecutive days of induction, using ampicillin sodium as a control.

[0044] The result is as follows Figure 4 As shown in the results, after Staphylococcus aureus was continuously induced by the complex Ru-3 for 20 days, the MIC value of Ru-3 increased by 4 times, while that of the control, ampicillin sodium, increased to 128 times the original MIC value. Therefore, it can be seen that Staphylococcus aureus is not likely to develop resistance to the Ru-3 complex, and Ru-3 has a good prospect of becoming a marketed antibiotic.

[0045] Therefore, in summary, this invention successfully prepared a ruthenium(II) complex with a diamide structure, Ru-3, through molecular structure design. This complex possesses the following significant advantages: 1) strong antibacterial activity, with an inhibitory effect on Staphylococcus aureus superior to the clinical drug vancomycin; 2) rapid bactericidal speed, and low likelihood of inducing bacterial resistance; 3) eradication of mature bacterial biofilms, enhancing the antibacterial effect. This ruthenium(II) complex provides a new technical solution to the problem of antibiotic resistance and can be used as a novel metal-based antibacterial drug for treating diseases related to drug-resistant bacterial infections, possessing significant clinical application value and market prospects.

[0046] 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 polypyridyl ruthenium complex having a bis-imidazole modification, characterized in that, The structural formula of the polypyridine ruthenium complex is shown in Formula I: Formula I; wherein is selected from any one of the following structures: 。 2. The polypyridyl ruthenium complex according to claim 1, wherein The structures of the polypyridine ruthenium complexes are shown as Ru-1, Ru-2, Ru-3, or Ru-4: ； ； ； 。 3. Process for the preparation of a polypyridine ruthenium complex with bis- imidazole modification according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Dissolve the compound of formula Ia in a solvent, and under the protection of an inert gas, then carry out a nucleophilic substitution reaction with the compound of formula Ib to obtain the intermediate of formula Ic. S2. Under the protection of an inert gas, the intermediate of the Ic structure and the compound of the Id structure are subjected to a condensation reaction under the conditions of ammonium acetate and acetic acid to obtain the main ligand of the Ie structure. S3. The main ligand of the Ie structure is mixed with a compound of the If structure, the Ig structure, the Ih structure, or the Ii structure and an organic solvent, and then a coordination reaction is carried out under the protection of an inert gas. After purification, the polypyridine ruthenium complex with diimidazole modification is obtained. The structural formulas of the compounds of formula Ia, Ib, Ic, Id, Ie, If, Ig, Ih, and Ii are shown below: 。 4. The preparation method according to claim 3, characterized in that, The solvent mentioned in S1 is anhydrous ethanol; The nucleophilic substitution reaction is carried out at a temperature of 70℃-80℃ for 3-5 days.

5. The preparation method according to claim 3, characterized in that, The molar ratio of the intermediate of the Ic structure to the compound of the Id structure in S2 is 1:

1.

6. The preparation method according to claim 5, characterized in that, The condensation reaction is carried out at a temperature of 120℃-150℃ for 5-10 hours.

7. The preparation method according to claim 3, characterized in that, The inert gas mentioned in S1, S2, and S3 is at least one of helium, argon, and nitrogen.

8. The preparation method according to claim 7, characterized in that, The organic solvent mentioned in S3 is ethylene glycol; The coordination reaction was carried out at a temperature of 140℃-160℃ for 8h-12h.

9. The use of the bisimidazole-modified polypyridine ruthenium complex according to any one of claims 1-2 in the preparation of a drug for inhibiting drug-resistant bacteria.

10. The application according to claim 9, characterized in that, The drug-resistant bacteria is Staphylococcus aureus.

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