Ruthenium polypyridine complex containing naphthylpyridine skeleton structure as well as preparation method and application of ruthenium polypyridine complex
By developing ruthenium polypyridine complexes containing naphthyridine skeleton structure, the problem that existing antibiotics are difficult to penetrate bacterial biofilms is solved, effective antibacterial and antibiofilm activities against Staphylococcus aureus were achieved, and good antibacterial potential was shown.
Patent Information
- Application Number
- CN202510152765.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-12
- Publication Date
- 2025-05-23
AI Technical Summary
Existing antibiotics are difficult to effectively penetrate or remove bacterial biofilms, resulting in poor therapeutic effects on drug-resistant bacteria.
A ruthenium polypyridine complex containing a naphthyridine backbone structure was developed to synthesize a series of ruthenium polypyridine complexes with ligand L3 and [Ru(dtb)2Cl2], [Ru(dmb)2Cl2] or [Ru(bpy)2Cl2] to inhibit the growth, biofilm formation and hemolysin secretion of Staphylococcus aureus.
The prepared ruthenium polypyridine complex has excellent antibacterial activity and anti-biofilm activity against Staphylococcus aureus, which can effectively inhibit the secretion of hemolysin and show its potential in antibacterial.
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Figure CN120025382A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of antibacterial medicine, and specifically relates to a ruthenium polypyridine complex containing a naphthyridine skeleton structure, and a preparation method and application thereof. Background Art
[0002] Staphylococcus aureus is a gram-positive bacterium with a spherical shape (about 0.8 µm in diameter), most of which can produce hemolytic toxins. Staphylococcus aureus is a common colonizing bacterium that often appears in various parts of the body, such as the mouth, axilla, mucous membranes, anterior nares, intestines, etc. The characteristics of colonization make it a common bacterium in clinical infections. As a common pathogen, Staphylococcus aureus is the main cause of skin and soft tissue infections, endocarditis, osteomyelitis and bacteremia, and bacterial resistance exacerbates this problem.
[0003] The reason why Staphylococcus aureus can cause disease is that it can produce a large number of virulence factors, which can help bacteria obtain nutrition, adhere and escape host immunity. Staphylococcus aureus can produce a variety of virulence factors, including pore-forming toxins, exfoliating toxins, superantigens, biofilms, etc., and these virulence factors are related to the drug resistance of Staphylococcus aureus. The extracellular polymers (EPS) in bacterial biofilms form a dense and negatively charged natural barrier that can hinder the penetration of antimicrobial drugs, especially positively charged antibiotics, into the biofilm, thereby reducing the concentration of antimicrobial drugs inside the biofilm. The nutrient content and oxygen concentration in the biofilm gradually decrease from the outside to the inside. Nutritional deficiency slows down the metabolic rate of bacteria deep in the biofilm or even puts them into a dormant state, increasing their resistance to fungicides during the reproductive period, and hypoxia inside the biofilm also reduces the bactericidal ability of the antibiotics themselves. Typical antibiotic treatment is usually ineffective for biofilm-related infections because bacteria in the biofilm have formed various mechanisms to resist antibiotics. For example, in clinical treatment of drug-resistant bacteria, antibiotics such as polymyxin, ciprofloxacin, and vancomycin are often used, but these antibiotics cannot effectively penetrate or remove biofilms and have serious toxic side effects. Due to the complexity of bacterial biofilms and the diversity of drug resistance mechanisms, the development of alternative antibiotics that can effectively destroy bacterial biofilms faces huge challenges. Summary of the invention
[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a ruthenium polypyridine complex containing a naphthyridine skeleton structure and a preparation method and application thereof.
[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a ruthenium polypyridine complex containing a naphthylpyridine skeleton structure, wherein the ruthenium polypyridine complex has a structure shown in Formula I: Formula I; in, Select one of the following structures: .
[0006] As a preferred embodiment of the present invention, the ruthenium polypyridine complex has one of the structures shown in Formula II: Formula II.
[0007] In a second aspect, the present invention provides a method for preparing the ruthenium polypyridine complex, comprising the following steps: S1. Under inert gas conditions, heating the compound of formula Ia and selenium dioxide in dioxane and water to react to obtain a compound of formula Ib; S2. Under inert gas conditions, heating the compound of formula Ib, the compound of formula Ic and ammonium acetate to react to obtain an intermediate of formula Id; S3, under inert gas conditions, heating the intermediate of formula Id and the compound of formula Ie, the compound of formula If, or the compound of formula Ig to react, cooling after the reaction, adding KPF 6 solution, and obtain a ruthenium polypyridine complex containing a naphthylpyridine skeleton structure; .
[0008] As a preferred embodiment of the present invention, in step S1, The molar ratio of the compound of formula Ia to selenium dioxide is 2.5-2.7:5.0-5.2; The temperature of the heating reaction is 70°C to 90°C.
[0009] As a preferred embodiment of the present invention, in step S2, The molar ratio of the compound of formula Ib, the compound of formula Ic and ammonium acetate is 0.4-0.6:0.4-0.6:4-6; The temperature of the heating reaction is 120° C. to 140° C., and the heating time is 2 h to 4 h.
[0010] As a preferred embodiment of the present invention, in step S3, The intermediate of formula Id and the compound of formula Ie or the compound of formula If or the compound of formula Ig, KPF 6 The molar ratio is 0.9~1.1:0.9~1.1:18~22.
[0011] As a preferred embodiment of the present invention, in step S3, The intermediate of formula Id and the compound of formula Ie, the compound of formula If or the compound of formula Ig are subjected to heating reflux reaction in ethylene glycol; The addition ratio of the intermediate of formula Id structure to ethylene glycol is 0.04 mol~0.06 mol: 4 mL~6 mL, the heating reflux reaction temperature is 140° C.~160° C., and the heating reflux reaction is for 6 h~10 h.
[0012] As a preferred embodiment of the present invention, KPF is added in step S3. 6 The solution also includes: About joining KPF 6 The crude product obtained after the solution is purified, wherein the purification is performed on an alumina chromatography column using acetonitrile / xylene as an eluent.
[0013] In a third aspect, the present invention provides a use of the ruthenium polypyridine complex in the preparation of antibacterial drugs or antibiotics.
[0014] As a preferred embodiment of the present invention, the antibacterial drug or antibiotic drug is a drug that inhibits the growth, biofilm formation and hemolysin secretion of Staphylococcus aureus. The present invention has at least one of the following beneficial effects: The present invention uses 4-methyl-[1,8]-naphthyridine as a raw material to synthesize naphthyridine-4-carboxaldehyde, and then synthesizes ligand L3 through naphthyridine-4-carboxaldehyde and 1,10-phenanthroline-5,6-dione, and finally synthesizes ligand L3 by reacting ligand L3 with [Ru(dtb) 2 Cl 2 ] or [Ru (dmb) 2 Cl 2 ] or [[Ru(bpy) 2 Cl 2 ] A series of ruthenium polypyridine complexes containing a naphthyridine skeleton structure were synthesized, and the antibacterial activity was studied. The complex has a structure shown in Formula I. The antibacterial activity experimental results show that the ruthenium polypyridine complex containing a naphthyridine skeleton structure provided by the present invention has excellent antibacterial activity and anti-biofilm activity against Staphylococcus aureus, and can better inhibit the secretion of hemolysin. It can be seen that the ruthenium polypyridine complex containing a naphthyridine skeleton structure provided by the present invention has certain potential in antibacterial properties. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The reaction scheme of ruthenium polypyridine complexes containing naphthylpyridine skeleton structure; Figure 2 Shown is a graph showing the killing kinetics of ligands L3, Ru-1, Ru-2 and Ru-3 against Staphylococcus aureus; Figure 3 Shown is a graph showing the inhibitory effect of the complex Ru-1 on the α-hemolytic toxin of Staphylococcus aureus; Figure 4 Shown is the inhibition assay of biofilm formation of the complex Ru-1 against Staphylococcus aureus. DETAILED DESCRIPTION
[0016] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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.
[0017] Example 1 A ruthenium polypyridine complex containing a naphthyridine skeleton structure, the reaction route of which is as follows Figure 1 As shown, the preparation method comprises the following steps: (1) Preparation of ligand First, the compound 4-methyl-[1,8]-naphthyridine (370 mg, 2.57 mmol) of formula Ia and selenium dioxide (569 mg, 5.13 mmol) were heated to 80°C in dioxane (16 mL) and water (2 mL) for reaction. After cooling to room temperature, extraction was performed and the solvent was evaporated under reduced pressure to obtain a crude product. After purification, the compound naphthyridine-4-carboxaldehyde of formula Ib was obtained. The ligand (1,8-naphthyridine-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) of formula Id was synthesized from a mixture of the compound naphthyridine-4-carboxaldehyde (82.9 mg, 0.5 mmol) of formula Ib, the compound 1,10-phenanthroline-5,6-dione (118.0 mg, 0.5 mmol) of formula Ic, ammonium acetate (3854.15 mg, 5 mmol) and acetic acid (20 mL). After reflux at 130 °C for 3 h, the residue was cooled to room temperature and the pH was adjusted to about 7.0 with aqueous ammonia. The resulting solid was collected and dried at 60 °C to obtain the crude product L3 (ligand L3).
[0018] (2) Preparation of complex Ru-1 The compound of formula Ie [Ru(dtb) 2 Cl 2 A mixture of ] (35.5 mg, 0.05 mmol) and L3 (17.4 mg, 0.05 mmol) in ethylene glycol (5 mL) was heated at 150 °C under argon for 8 h to obtain a clear red solution. After cooling to room temperature, 1 mmol KPF 6A red precipitate was obtained after addition of aqueous solution (50 mL). The crude product was purified by column chromatography on neutral alumina using acetonitrile / xylene system as eluent to obtain red powder Ru-1, a ruthenium polypyridine complex containing a naphthyridine skeleton structure.
[0019] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.08 (d, J = 8.4 Hz, 2H), 8.92 (d, J = 10.8Hz, 5H), 8.30 – 8.07 (m, 5H), 7.65 (s, 8H), 7.39 (t, J = 6.4 Hz, 3H), 1.44 (s, 18H), 1.35 (s, 18H).
[0020] (3) Preparation of complex Ru-2 Using the compound of formula If [Ru (dmb) 2 Cl 2 A mixture of ] (27.0 mg, 0.05 mmol) and L3 (17.4 mg, 0.05 mmol) in ethylene glycol (5 mL) was heated at 150 °C under argon for 8 h to obtain a clear red solution. After cooling to room temperature, 1 mmol KPF 6 A red precipitate was obtained after addition of aqueous solution (50 mL). The crude product was purified by column chromatography on neutral alumina using acetonitrile / xylene system as eluent to obtain red powder Ru-2, i.e., a ruthenium polypyridine complex containing a naphthyridine skeleton structure.
[0021] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.05 (d, J = 8.0 Hz, 2H), 8.77 (d, J = 14.0Hz, 5H), 8.35 (s, 2H), 8.24 – 7.99 (m, 3H), 7.72 – 7.56 (m, 5H), 7.46 (d, J =5.6 Hz, 3H), 7.22 (d, J = 5.2 Hz, 3H), 2.58 (s, 6H), 2.48 (s, 6H).
[0022] (4) Preparation of complex Ru-3 The compound of formula I-g [Ru(bpy) 2 Cl 2 (24.2 mg, 0.05 mmol) and L3 (17.4 mg, 0.05 mmol) in ethylene glycol (5 mL) were heated at 150 °C under argon for 8 h to obtain a clear red solution. After cooling to room temperature, 1 mmol of aqueous KPF 6 solution (50 mL) was added to obtain a red precipitate. The crude product was purified by column chromatography on neutral alumina using an acetonitrile / xylene system as the eluent to obtain a red powder Ru-3, which is a ruthenium polypyridyl complex containing a naphthyridine skeleton structure.
[0023] 1 1H NMR (400 MHz, DMSO- d 6 ) δ 10.29 (d, J J = 8.0 Hz, 2H), 10.06 (d, J J = 8.5Hz, 1H), 9.38 (d, J J = 4.4 Hz, 1H), 9.25 (d, J J = 4.1 Hz, 2H), 9.18 (d, J J = 9.2 Hz,2H), 9.04 – 8.94 (m, 8H), 8.59 (d, J J = 4.3 Hz, 1H), 8.35 (d, J J = 4.4 Hz, 2H),8.08 (s, 1H), 7.96 (d, J J = 4.2 Hz, 2H), 7.82 (dd, J J = 8.1, 4.2 Hz, 5H).
[0024] Example 2 The ruthenium polypyridyl complexes Ru-1, Ru-2, and Ru-3 containing naphthyridine skeleton structures prepared in Example 1 were subjected to an in vitro antibacterial activity test as follows: (1) Determination of MIC by the microbroth dilution method: Staphylococcus aureus Newman strain was cultured in TSB medium until the logarithmic growth phase; it was diluted 1000 times with fresh medium to obtain a bacterial suspension. 50 µL of different concentrations of metal ruthenium complexes and ligands were added to the 96-well plate in turn, and then 200 µL of bacterial suspension was added to each well, so that the final concentrations of each well were 256 µg / mL, 128 µg / mL, 64 µg / mL, 32 µg / mL, 16 µg / mL, 8 µg / mL, 4 µg / mL, 2 µg / mL, 1 µg / mL, 0.5 µg / mL, and 0.25 µg / mL from left to right. 50 µL of sterile water was added to the last well as a blank control. Each drug was tested in 3 groups in parallel. After incubation at 37°C for 20 h, the bacterial growth was monitored by observing the turbidity of the culture. The lowest drug concentration corresponding to the clear dosing well was the MIC (minimum inhibitory concentration).
[0025] The results are shown in Table 1. Table 1 Minimum inhibitory concentration (MIC) of naphthyridine-modified ruthenium complexes Ru1 to Ru3 According to the test results, the three complexes showed different antibacterial abilities, among which Ru-1 showed the best antibacterial activity against Staphylococcus aureus (MIC = 1 µg / mL), Ru-2 showed a certain antibacterial effect against Staphylococcus aureus (MIC = 8 µg / mL), and Ru-3 did not show good antibacterial activity against Staphylococcus aureus.
[0026] (2) Time-killing dynamics: The Staphylococcus aureus grown to the logarithmic phase was diluted to 1×10 6 CFU / mL, different concentrations of compounds were added to 24-well plates, so that the final concentrations in the wells were 8µg / mL, 4µg / mL, 2µg / mL, and 1µg / mL. Incubated at 37°C in a shaker. 50µL of the mixture samples at different time periods (0, 15, 30, 60, 90, 120 min) were evenly spread on TSB agar plates and incubated at 37°C for 20 h. The number of colonies was calculated using the CFU counting method.
[0027] Figure 2 The figure shows the killing kinetics of Ru-1 against Staphylococcus aureus. According to the test, within 2 hours, the untreated bacteria ( Figure 2The control in the sample showed a steady growth. With the increase of Ru-1 concentration, the number of Staphylococcus aureus colonies gradually decreased; at a concentration of 4µg / mL, Staphylococcus aureus was completely killed in 2 hours, and at a concentration of 8µg / mL, Staphylococcus aureus was completely killed in 1.5 hours. This shows that compound Ru-1 has a significant rapid bactericidal effect and has clinical anti-infection potential.
[0028] (3) Determination of hemolytic toxicity: Red blood cells were obtained from fresh sterile rabbit blood, and the rabbit blood was washed three times with PBS. 950 μL of phosphate buffered saline (PBS) and 50 μL of red blood cells and different concentrations of Ru-1 compounds were added to a 1.5 mL sample tube and incubated at 37°C for 30 min. The negative control was a red blood cell suspension containing only PBS, and the positive control was PBS containing 0.1% (V / V) Triton X-100. After incubation, the mixture was centrifuged (2000 rpm, 2 min), and the supernatant (200 μL) was transferred to another 96-well plate. Finally, the hemolysis rate was calculated by measuring the absorbance at 540 nm.
[0029] Figure 3 The figure shows the inhibitory effect of the complex Ru-1 on the α-hemolytic toxin of Staphylococcus aureus, with ofloxacin ( Figure 3 Oflo in the sample was used as a control; OD 540 The inhibitory effect of Ru-1 on bacterial α-hemolytic toxin production after treatment was determined by the absorbance at 100 nm. The higher the absorbance value, the more rabbit red blood cells were ruptured, that is, the more α-hemolytic toxins produced by bacteria, resulting in hemolysis of the toxin. According to the test results, Ru-1 showed good inhibition of toxin production at sub-inhibitory concentrations, and as the concentration of Ru-1 increased, its inhibitory effect was significantly enhanced, showing a concentration-dependent trend. When the concentration of Ru-1 was 0.5 MIC, compared with the blank ( Figure 3 Compared with the positive control ofloxacin, Ru-1 has a stronger ability to inhibit hemolytic toxins. The above results show that Ru-1 has a good ability to inhibit hemolytic toxins.
[0030] (4) Inhibit the formation of biofilm: Biofilm assay was performed using 24-well plates. Staphylococcus aureus grown to logarithmic phase was diluted 1000-fold with fresh TSB medium, and then 2 mL of the diluted bacterial solution was mixed with 500 μL of different concentrations of Ru-1 in a 24-well plate. After incubation at 37 °C for 48 h, the bacterial suspension was removed and the plate was washed three times with PBS. The adhered bacteria were dried overnight at 37 °C and then stained with 0.1% crystal violet solution for 1 h, the crystal violet solution was removed, and the plate was washed again with PBS. Then, 1 mL of acetic acid and 1 mL of water were added, and the absorbance at 595 nm was monitored by UV to determine the formation of biofilm.
[0031] Figure 4 The complex Ru-1 and the blank group ( Figure 4 The results show that Ru-1 has a good ability to inhibit biofilm formation at sub-inhibitory concentrations, and as the concentration increases, the inhibition of biofilm formation becomes more obvious, showing a certain dose dependence.
[0032] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A ruthenium polypyridine complex containing a naphthylpyridine skeleton structure, characterized in that: The ruthenium polypyridine complex has a structure as shown in Formula I: Formula I; in, Select one of the following structures: 。 2. The ruthenium polypyridine complex according to claim 1, characterized in that The ruthenium polypyridine complex has one of the structures shown in Formula II: Formula II.
3. The method for preparing the ruthenium polypyridine complex according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Under inert gas conditions, heating the compound of formula Ia and selenium dioxide in dioxane and water to react to obtain a compound of formula Ib; S2. Under inert gas conditions, heating the compound of formula Ib, the compound of formula Ic and ammonium acetate to react to obtain an intermediate of formula Id; S3, under inert gas conditions, heating the intermediate of formula Id and the compound of formula Ie, the compound of formula If, or the compound of formula Ig to react, cooling after the reaction, adding KPF6 solution, to obtain a ruthenium polypyridine complex containing a naphthyridine skeleton structure; 。 4. The preparation method according to claim 3, characterized in that: In step S1, The molar ratio of the compound of formula Ia to selenium dioxide is 2.5-2.7:5.0-5.2; The temperature of the heating reaction is 70°C to 90°C.
5. The preparation method according to claim 3, characterized in that: In step S2, The molar ratio of the compound of formula Ib, the compound of formula Ic and ammonium acetate is 0.4-0.6:0.4-0.6:4-6; The temperature of the heating reaction is 120° C. to 140° C., and the heating time is 2 h to 4 h.
6. The preparation method according to claim 3, characterized in that: In step S3, The molar ratio of the intermediate of formula Id to the compound of formula Ie or the compound of formula If or the compound of formula Ig and KPF6 is 0.9-1.1:0.9-1.1:18-22.
7. The preparation method according to claim 3, characterized in that: In step S3, The intermediate of formula Id and the compound of formula Ie, the compound of formula If or the compound of formula Ig are subjected to heating reflux reaction in ethylene glycol; The addition ratio of the intermediate of formula Id structure to ethylene glycol is 0.04 mol~0.06 mol: 4 mL~6 mL, the heating reflux reaction temperature is 140° C.~160° C., and the heating reflux reaction is for 6 h~10 h.
8. The preparation method according to claim 3, characterized in that: After adding the KPF6 solution in step S3, the method further comprises: The crude product obtained after adding the KPF6 solution is purified, wherein the purification is performed on an alumina chromatography column using acetonitrile / xylene as an eluent.
9. Use of the ruthenium polypyridine complex according to any one of claims 1 to 2 in the preparation of antibacterial drugs or antibacterial drugs.
10. The use according to claim 9, characterized in that: The antibacterial drug or antibiotic drug is a drug that inhibits the growth, biofilm formation and hemolysin secretion of Staphylococcus aureus.