A biguanide-ruthenium complex and its preparation method and application

Abstract

The present invention relates to the technical field of organic synthesis, and in particular to a biguanide-ruthenium complex and a preparation method and application thereof. The structural formula of the biguanide-ruthenium complex is shown in Formula I or Formula II; the biguanide-ruthenium complex of the present invention can be effectively absorbed by bacteria, thereby having significant antibacterial activity. In addition, a series of complexes with biguanide as the main ligand have the physicochemical properties of photoactivation, and the ligand will be cracked after light irradiation, and the bacteria are incubated with the complex and then illuminated, and the antibacterial activity of the complex before illumination is significantly improved. The relevant experimental results show that the complex has PDT and PACT activities, can produce ROS, and cause an imbalance in the redox metabolism of bacteria, thereby achieving antibacterial and bactericidal effects.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis, and in particular to a biguanide-ruthenium complex and a preparation method and application thereof. Background Art

[0002] The advent of the antibiotic penicillin ushered in a new era in the treatment of infectious diseases. However, this has two sides. On the one hand, the emergence of antibiotics is a major step forward in human history. Diseases caused by bacterial infections have been controlled, greatly reducing mortality. On the other hand, the abuse and misuse of antibiotics have led to increased bacterial resistance and the emergence of "super bacteria." Despite this, traditional organic medicinal chemistry has failed to fill the gap in antibacterial drugs: a 2022 analysis showed that as of June 2021, there were only 45 "traditional" antibiotics in clinical development. Therefore, there is an urgent need to develop a new generation of antibiotics. Summary of the invention

[0003] Based on the above content, the present invention provides a biguanide-ruthenium complex and its preparation method and application. The biguanide-ruthenium complex of the present invention has photodynamic antibacterial and photoactivated antibacterial effects and can effectively kill bacteria.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a biguanide-ruthenium complex, the structural formula of which is shown in Formula I or Formula II;

[0006]

[0007] The second technical solution of the present invention is a method for preparing the above-mentioned biguanide-ruthenium complex, which is method one or method two; when the biguanide-ruthenium complex is a structure shown in formula I, method one is adopted, comprising the following steps;

[0008] The ruthenium salt and the lithium salt are added to an organic solvent and heated under reflux, and then 2,2-biquinoline is added under reflux to obtain substance 2;

[0009] The substance 2 is heated to react with a biguanide derivative to obtain a biguanide-ruthenium complex having a structure shown in formula I;

[0010] When the biguanide-ruthenium complex is a structure shown in Formula II, method 2 is adopted, comprising the following steps:

[0011] The ruthenium salt is dissolved in an organic solvent, and then isopropanol is added and refluxed to obtain product 4;

[0012] The product 4 and the biguanide derivative are dissolved in a mixed solvent and refluxed to obtain the product 5;

[0013] The product 5 is reacted with an auxiliary ligand by reflux to obtain the biguanide-ruthenium complex.

[0014] In a preferred embodiment of the present invention, in the method 1, the molar ratio of the ruthenium salt to the lithium salt is 1:15; the molar ratio of the ruthenium salt to the 2,2-biquinoline is 1:2; the temperature of the heating reflux is 90°C, the time is 2h; the temperature of the reflux reaction is 140°C, the time is 8h; the ruthenium salt is ruthenium trichloride; the lithium salt is lithium chloride; the organic solvent is N,N'-dimethylformamide. After the reflux reaction is completed, the steps of acetone precipitation, suction filtration, cold water, cold acetone washing, and suction filtration are also included.

[0015] In a preferred embodiment of the present invention, in the method 1, the molar ratio of the substance 2 to the biguanide derivative is 1:1; the biguanide derivative is 1-o-tolylbiguanide; the temperature of the heating reaction is 90°C and the time is 8 hours. Anhydrous ethanol is also added during the heating reaction, that is, the substance 2 and the biguanide derivative are dissolved in anhydrous ethanol and heated for reaction; after the heating reaction, the steps of diluting with water, precipitating with a saturated potassium hexafluorophosphate KPF6 solution, filtering, and purifying by neutral alumina column chromatography are also included.

[0016] In a preferred embodiment of the present invention, in the method 2, the ruthenium salt is ruthenium trichloride; the organic solvent is dimethyl sulfoxide; the biguanide derivative is 1-o-tolylbiguanide; the auxiliary ligand is 2,9-dimethylbipyridine; and the mixed solvent is a mixture of ethanol and dimethyl sulfoxide in a volume ratio of 9:1.

[0017] In a preferred embodiment of the present invention, in the method 2, when preparing the product 4, the reflux temperature is 85°C and the time is 30 hours; when preparing the product 4, after the reflux is completed, the step of cooling to room temperature, filtering and collecting the solid, washing the solid with isopropanol, and vacuum drying is included; when preparing the product 5, the reflux temperature is 90°C and the time is 3 hours; when preparing the product 5, after the reflux is completed, the step of cooling to room temperature and removing the solvent by vortexing is included; the temperature of the reflux reaction is 130°C and the time is 12 hours; after the reflux reaction is completed, the step of cooling to room temperature, adding an appropriate amount of water, adding a saturated solution of potassium hexafluorophosphate dropwise, letting it stand in a refrigerator, filtering and drying, and purifying the crude product with a neutral alumina column is included.

[0018] The third technical solution of the present invention is the use of the above-mentioned biguanide-ruthenium complex in the preparation of antibacterial drugs.

[0019] A fourth technical solution of the present invention is an antibacterial drug, the raw materials of which include the above-mentioned biguanide-ruthenium complex.

[0020] In a preferred embodiment of the present invention, the antibacterial drug is a drug against methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae and / or Klebsiella pneumoniae.

[0021] The present invention discloses the following technical effects:

[0022] The invention provides a biguanide-ruthenium complex, which has photodynamic antibacterial and photoactivated antibacterial effects and can effectively kill bacteria. The antibacterial effect of the complex can be significantly improved after illumination.

[0023] The invention uses a derivative of a biguanide compound as a starting material to synthesize a ruthenium metal complex of 1-o-tolylbiguanide. The invention further uses different ligands and ruthenium complexes to synthesize Ru2. The method of the invention is simple and easy to operate and prepare. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The mass spectrum of Ru1 prepared in Example 1 of the present invention;

[0026] Figure 2 The mass spectrum of Ru2 prepared in Example 2 of the present invention;

[0027] Figure 3 This is the mass spectrum of Ru2 prepared in Example 2 of the present invention after illumination;

[0028] Figure 4 The UV-visible absorption spectrum of Ru1 in PBS buffer in Example 1 of the present invention is verified;

[0029] Figure 5 The UV-visible absorption spectrum of Ru2 in PBS buffer in Example 1 of the present invention is verified;

[0030] Figure 6 The UV absorption curves of Ru2 in PBS buffer after being irradiated with light for different time periods in Example 1 of the present invention;

[0031] Figure 7 The flat plate coating of Ru1 and Ru2 in Example 2 for verifying the effect of the present invention;

[0032] Figure 8 The inhibition of biofilm formation and its quantitative analysis of Ru2 in Example 2 of the present invention are verified;

[0033] Fig. 9 Live-dead staining of MRSA after being treated with Ru2 in Example 2 of the present invention;

[0034] Fig.10 This is a scanning electron microscope image of MRSA treated with Ru2 in Example 2 of the present invention;

[0035] Fig.11 The ROS detection of MRSA after being treated with Ru2 in Example 2 of the present invention is verified;

[0036] Fig.12 In Example 2, DPBF was used to detect the singlet oxygen of Ru1 and Ru2. 1 Production of O2; (a) and (c) are [Ru(bpy)3] 2+ , The fluorescence intensity change of DPBF after irradiation with 520nm light source in the presence of Ru2; (b) and (d) are [Ru(bpy)3] 2+ , the fluorescence intensity change of DPBF after irradiation with 630nm light source in the presence of Ru1; (e) is the change of fluorescence intensity of DPBF at 479nm with different irradiation time of 520nm light source, (f) is the change of fluorescence intensity of DPBF at 479nm with different irradiation time of 630nm light source;

[0037] Fig.13 This is the hemolytic experiment of Ru1 and Ru2 in Example 2 for verifying the effect of the present invention; wherein, (a) and (b) are the hemolytic conditions of Ru1 and Ru2 at different concentrations, respectively; and (c) is the quantitative statistics of the hemolytic rates of Ru1 and Ru2. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] One of the technical solutions of the present invention is a biguanide-ruthenium complex, the structural formula of which is shown in Formula I or Formula II;

[0044]

[0045] The present invention detected that the formation of bacterial biofilm after treatment with the biguanide-ruthenium complex (Ru1, Ru2) shown in the above formula I and formula II was inhibited, while the biguanide derivatives had almost no antibacterial and biofilm inhibition effects. Ultimately, Ru1 and Ru2 can induce bacterial cell death in multiple ways, such as destroying bacterial cell membranes and interfering with bacterial intracellular redox imbalance. In addition, the complex can inhibit the formation of bacterial biofilm, and can effectively reduce the production of bacterial resistance caused by the large amount of antibiotics used due to the bacterial biofilm barrier.

[0046] The second technical solution of the present invention is a method for preparing the above-mentioned biguanide-ruthenium complex. When the biguanide-ruthenium complex has a structure shown in Formula I, the preparation method comprises the following steps:

[0047] (1) Add ruthenium trichloride and anhydrous lithium chloride to N,N'-dimethylformamide (DMF), heat and reflux at 90°C for 2h, add 2,9-dimethylbipyridine, program the temperature to 140°C, reflux for 8h, after the reaction is completed, precipitate with acetone, filter, wash with cold water and cold acetone, and filter to obtain substance 2;

[0048] (2) dissolving substance 2 and 1-o-tolylbiguanide in anhydrous ethanol, heating the reaction for 8 h, diluting with water, precipitating with saturated potassium hexafluorophosphate solution, filtering with suction, and purifying with neutral alumina column chromatography to obtain product 3, i.e., a biguanide-ruthenium complex (Ru1) having a structure shown in formula I;

[0049] When the biguanide-ruthenium complex has a structure shown in Formula II, the preparation method comprises the following steps:

[0050] (1) Dissolve ruthenium trichloride in dimethyl sulfoxide (DMSO), then add isopropanol, heat and reflux for 30 hours, and after the reaction is completed, filter and wash with isopropanol to obtain substance 4;

[0051] (2) dissolving substance 4 and 1-o-tolylbiguanide in anhydrous ethanol, heating for reaction, and drying by rotation under reduced pressure to obtain product 5;

[0052] (3) Product 5 and 2,2'-biquinoline are added to ethylene glycol, heated and stirred overnight; diluted with water, saturated potassium hexafluorophosphate solution is added dropwise, allowed to stand for precipitation, filtered and dried, and purified by neutral alumina column chromatography to obtain product 6, i.e., a biguanide-ruthenium complex (Ru2) with a structure shown in Formula II.

[0053] The third technical solution of the present invention is the use of the above-mentioned biguanide-ruthenium complex in the preparation of antibacterial drugs.

[0054] The biguanide-ruthenium complex of the present invention can be effectively absorbed by bacteria, thereby having significant antibacterial activity. In addition, the series of complexes with biguanide as the main ligand of the present invention have the physicochemical property of photoactivation, and the ligand will be cracked after being irradiated with light, and the antibacterial activity of the complex before irradiation is significantly improved after the bacteria are incubated with the complex and then illuminated. The relevant experimental results show that the biguanide-ruthenium complex of the present invention has PDT and PACT activities, can generate ROS, and cause the redox metabolism of bacteria to be unbalanced, thereby achieving antibacterial and bactericidal effects.

[0055] A fourth technical solution of the present invention is an antibacterial drug, the raw materials of which include the above-mentioned biguanide-ruthenium complex; the antibacterial drug is a drug against methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae and / or Klebsiella pneumoniae.

[0056] The present invention is further described below by way of examples.

[0057] Example 1

[0058] (1) Ruthenium trichloride RuCl3·nH2O (MW=260.84, 1 mmol) and anhydrous lithium chloride LiCl (MW=42.39, 15 mmol) were added to 5 mL of N,N'-dimethylformamide DMF, and the mixture was heated to reflux at 90°C for 2 h. 2,2-Biquinoline biq (MW=256.31, 2 mmol) was added, and the temperature was raised to 140°C and refluxed for 8 h. After the reaction was completed, acetone was used for precipitation, which was filtered and washed with cold water and cold acetone. Substance 2 was obtained by filtration with a yield of 60.8%.

[0059] (2) Substance 2 (MW = 684.04, 0.1 mmol) and 1-o-tolylbiguanide (MW = 191.11, 0.1 mmol) were dissolved in 10 mL of anhydrous ethanol and heated at 90°C for 8 h. After the reaction, the mixture was diluted with water and precipitated with saturated potassium hexafluorophosphate (KPF6) solution. The mixture was filtered and purified by neutral alumina column chromatography to obtain product 3, i.e., biguanide-ruthenium complex (referred to as complex, denoted as Ru1), with a yield of 75.1%.

[0060] In this embodiment, the synthetic route of Ru1 is as follows:

[0061]

[0062] Example 2

[0063] First, take 2 ml of dimethyl sulfoxide (DMSO) solution and pass argon in a reaction tube. After removing the oxygen, add RuCl3·nH2O (1.0 mmol) and wait for it to dissolve. Then add about 5 ml of isopropanol and reflux at 85°C for 30 h in a reaction tube under argon protection. After the reaction is completed and cooled to room temperature, the solid is collected by suction filtration, washed with isopropanol, and dried in vacuo. Finally, the light yellow solid is product 4 with a yield of 62.2%. Product 4 (MW = 483, 0.1 mmol) and 1-o-tolylbiguanide TolBig (0.1 mmol) are dissolved in a mixed solvent of 4.5 ml of ethanol and 0.5 ml of DMSO, refluxed at 90°C for 3 h under argon, cooled to room temperature after the reaction is completed, and the solvent is removed by vortexing to obtain product 5 (MW = 423). Product 5 (0.1 mmol) and auxiliary ligand 2,9-dimethylbipyridine dmb (MW=184.1, 0.2 mmol) were dissolved in 5 ml of ethylene glycol, and the reaction was refluxed at 130°C for 12 h (reaction overnight). After the reaction was completed, the mixture was cooled to room temperature, an appropriate amount of water was added, and a saturated solution of potassium hexafluorophosphate was added dropwise. The mixture was allowed to stand in a refrigerator, filtered and dried, and the crude product was purified by a neutral alumina column to obtain product 6, i.e., biguanide-ruthenium complex (referred to as complex, denoted as Ru2), with a yield of 69.6%.

[0064] In this embodiment, the Ru2 synthesis route is as follows:

[0065]

[0066] Effect verification example 1

[0067] The complexes Ru1 and Ru2 obtained in Example 1 and Example 2 were subjected to relevant experimental characterizations, such as Figure 1 and Figure 2 As shown, the mass spectrometry characterization of Ru1 and Ru2 proves that their synthesis is successful: Ru1-ESI-MS(CH3CN):m / z calc.for([M-2(PF6)] 2+ )805.2215; found:([MH + ] + )804.2169.(1 / 2[M] 2+ )402.6070, Ru2-ESI-MS(CH3CN):m / zcalc.for([M-2(PF6)] 2+ )661.2215; found:([MH + ] + )660.2120. (1 / 2[M] 2+ )330.6081. Among them, for Ru2, if it is irradiated with 520nm light, the ligand will dissociate, as shown in Figure 3 As shown, in addition to the presence of the relevant molecular ion peaks of Ru2, the fragment peaks of the ligand dmb were also found: ([dmb+H + ] + ): 185.1093, and the remaining Ru2 fragment peak after dmb dissociation ([M-dmb-H + ] + )476.1166.

[0068] Furthermore, the UV-visible absorption spectra of the complexes Ru1 and Ru2 were detected. Figure 4 , 5 As shown in Figure 2, it has good stability in the dark, but after exposure to light, multiple isosbestic points appear in the ultraviolet absorption curve of Ru2, such as Figure 6 As shown, it means that Ru2 dissociates after being irradiated with light, producing new substances. Ligand dissociation often produces free ligands and ruthenium hydrates. The former will bind to DNA in a non-covalent manner such as insertion or embedding, while the latter will often bind to DNA in a covalent manner, thereby interfering with nucleic acid metabolism to achieve its antibacterial effect.

[0069] Effect Verification Example 2 Biological Activity Experiment

[0070] Bacterial culture

[0071] Four common pathogens: methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Staphylococcus aureus CMCC (B) 26003 (S. aureus), Escherichia coli ATCC 8739 (E. coli), Enterobacter cloacae ATCC 700323 (E. cloa), and Klebsiella pneumoniae ATCC 700603 (K. peneu) were all from Guangdong Provincial Culture Collection Center. The bacteria were placed in LB medium, cultured in a shaker at 37°C and 180 rpm, and maintained on LB agar.

[0072] Measurement of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0073] The MIC and MBC of the complexes Ru1 and Ru2 against various Gram-positive and Gram-negative bacteria were studied by the half-dilution method. The overnight cultured bacteria were diluted to 1×10 6 CFU / mL, add 100 μL of bacterial solution to each well, then add 100 μL of diluted complex solution of different concentrations to each well, the concentration of the complex is in the range of 0.25-256 μM, incubate the 96-well plate at 37°C for 2 hours, illuminate with the corresponding wavelength excitation light for 0 minutes and 30 minutes respectively, and continue to incubate for 24 hours. After incubation, use a multifunctional microplate reader to measure OD 600 The blank culture medium on the same plate is the negative control, and the wells containing only bacterial solution are the positive control. When the inhibition rate is greater than 90%, it is considered that an antibacterial effect has been produced. Compare the difference in antibacterial activity of the complex before and after illumination. Take out 50μL of bacterial solution from each well and spread it on the plate to determine the minimum bactericidal concentration (MBC).

[0074] Table 1 Antibacterial activity of complexes Ru1 and Ru2 against different bacterial species

[0075]

[0076] Dilution spread plate method

[0077] The complexes containing different concentrations of Ru1, Ru2 and 5×10 5 200 μL of LB medium with 500 CFU / mL of bacteria (n=3) was added to a 96-well plate. After culturing for 12 h, bacterial suspensions with different concentration gradients were taken, the suspensions were diluted 1000 times, and 50 μL of bacterial suspension was spread on LB agar plates. The plates were cultured at 37°C for 24 h, and the colonies were counted.

[0078] The antibacterial effects of the two ruthenium complexes are stronger than those of the biguanide derivative: 1-o-tolylbiguanide; the antibacterial activity of Ru1 is better than that of Ru2, and the antibacterial activities of the two complexes (Ru1, Ru2) are significantly improved after irradiation with light, which may be attributed to the ROS generated by their unique structure.

[0079] Biofilm formation inhibition test

[0080] The biomass of the biofilm was determined by crystal violet staining. 7 CFU / mL, 250μL) was added to a 12-well plate, and then equal amounts of different components (PBS, Ru1, Ru2) were added to the LB medium. After mixing, culture at 37°C, discard the supernatant and suspended bacteria every 24h, and re-dose. Discard the culture medium after 48h and 72h, respectively, and wash with PBS three times. After the 12-well plate is dried, stain the biofilm with crystal violet (0.1%, 500μL) for 30min. Remove excess crystal violet dye and wash with PBS several times until the supernatant is clear. Afterwards, add glacial acetic acid aqueous solution (33%, 500μL) to dissolve the crystal violet dye and measure OD600.

[0081] Figure 8 The results showed that compared with the dense and thick biofilm of the control group (phosphate buffer PBS), the biofilm treated with Ru1 was loose, and the inhibitory effect of biofilm was more obvious after increasing the light. These results show that Ru1 can inhibit the formation of biofilm, but its ability to destroy biofilm is poor. The possible reason is that when the bacteria are in a suspended state, Ru1 tightly binds to MRSA and catalyzes the production of ROS at a close distance, thereby eliminating MRSA and inhibiting the formation of biofilm.

[0082] Study on the staining of bacterial life and death by complex

[0083] The bacterial cell viability was assessed using a bacterial viability kit consisting of two stains (SYTO9 and PI) using a laser confocal fluorescence microscope. 6 CFU / mL) was incubated at 37°C for 2h with 100μL of complex at 2 times the MIC concentration, and the corresponding wavelength excitation light was illuminated for 0min and 30min respectively. After centrifugation and washing, 3μL of 1:1 (v / v) SYTO9 (0.5mM) and PI (1mM) mixture was added to 200μL of bacterial suspension, and incubated in the dark at 37°C for 20-30min, and then the bacterial sample was washed with PBS buffer. Then 10μL of the stained bacterial suspension was dropped into a clean slide and imaged with a fluorescence microscope using a 40x objective. S. aureus bacterial solution without any complex was used as a negative control.

[0084] like Fig. 9The results showed that compared with the control group, the red fluorescence of MRSA dead cells treated with Ru1 was significantly enhanced and concentration-dependent, indicating that Ru1 has good antibacterial activity and has antibacterial or bactericidal effects at low concentrations.

[0085] Study on the effects of complexes on the morphology of bacteria and biofilm cells.

[0086] Take the bacterial liquid MRSA in the logarithmic growth phase and the complex with 2×MIC concentration and incubate at 37℃ for 2h, and illuminate with the corresponding wavelength excitation light for 0min and 30min respectively. After the treated bacteria are centrifuged at 8000rpm for 3min, the bacteria are collected, washed with PBS, and then fixed with 2.5% glutaraldehyde at 4℃ for 4h. After removing the glutaraldehyde, the collected cells are graded and dehydrated in different gradients of ethanol (30%, 50%, 70%, 90% and 100%, each dehydration for 15min). Finally, the dehydrated cells are dripped on clean tin foil, freeze-dried, spray-plated with gold, and observed under a scanning electron microscope. At the same time, the untreated bacteria are observed for comparison.

[0087] Based on the excellent antibacterial activity in vitro, the antibacterial mechanism of the complex Ru2 was further evaluated. First, the bacterial morphology was observed using a scanning electron microscope (SEM). Fig.10 As shown, compared with the smooth and round bacterial morphology of the control group, Ru2 and Ru2 plus light groups were able to destroy the bacterial cell wall membrane, and compared with the small amount of wrinkling in the Ru2 group, the bacterial cell membrane in the Ru2 plus light group showed obvious wrinkles, collapse and damage.

[0088] Active oxygen level detection

[0089] Take bacterial liquid MRSA in the logarithmic growth phase, incubate it with the synthesized complex at 37°C for 2 hours, irradiate with corresponding wavelength excitation light for 0 minutes and 30 minutes respectively, centrifuge and wash, add DCFH-DA working solution, incubate in the dark in an incubator for 0.5 hours, wash with PBS 2-3 times, and observe and photograph with an inverted fluorescence microscope.

[0090] Depend on Fig.11 , 12 It can be seen that the complex Ru1 can produce ROS after incubation with MRSA, but after irradiation with light, its ROS fluorescence intensity is significantly increased: that is, the fluorescence intensity of low-concentration Ru1 after illumination is higher than that of high-concentration Ru1 without illumination; further, DPBF was used to detect the type of ROS: singlet oxygen. It is speculated that due to light, Ru1 may produce more ROS, causing redox imbalance in MRSA cells and leading to its death.

[0091] Biosafety

[0092] Fresh red blood cells were separated by centrifugation at 3500rpm for 5min, washed 3 times with PBS, and diluted to the final concentration (5%, v / v). Different concentrations of Ru1 and Ru2 were added to 450μL of red blood cells, incubated at 37°C for 4h with gentle shaking at 150rpm. At the end of the incubation, the solution was centrifuged at 3500rpm for 5min, and the hemolytic activity was measured at 540nm using an enzyme reader. Triton X-100 (0.1% in PBS), which can completely lyse red blood cells, was used as a positive control, while PBS was used as a negative control. The hemolysis rate of red blood cells was calculated by the following equation: Hemolysis rate (%) = (sample group - negative control) / (positive control - negative control) × 100%.

[0093] The results are as follows Fig.13 As shown in the figure, the hemolysis rates of Ru1 and Ru2 at 2MIC were both less than 5%, indicating that the complexes were relatively safe within the concentration range used.

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biguanide-ruthenium complex, characterized in that: The structural formula is shown in Formula Ⅰ or Formula Ⅱ; 2. A method for preparing the biguanide-ruthenium complex according to claim 1, characterized in that: It is method 1 or method 2; when the biguanide-ruthenium complex has a structure as shown in formula I, method 1 is adopted, comprising the following steps; The ruthenium salt and the lithium salt are added to an organic solvent and heated under reflux, and then 2,2-biquinoline is added under reflux to obtain substance 2; The substance 2 is heated to react with a biguanide derivative to obtain a biguanide-ruthenium complex having a structure shown in formula I; When the biguanide-ruthenium complex is a structure shown in Formula II, method 2 is adopted, comprising the following steps: The ruthenium salt is dissolved in an organic solvent, and then isopropanol is added and refluxed to obtain product 4; The product 4 and the biguanide derivative are dissolved in a mixed solvent and refluxed to obtain the product 5; The product 5 is reacted with an auxiliary ligand by reflux to obtain the biguanide-ruthenium complex.

3. The method for preparing the biguanide-ruthenium complex according to claim 2, characterized in that: In the method one, the molar ratio of the ruthenium salt to the lithium salt is 1:15; the molar ratio of the ruthenium salt to the 2,2-biquinoline is 1:2; the temperature of the heating reflux is 90°C and the time is 2 hours; the temperature of the reflux reaction is 140°C and the time is 8 hours; the ruthenium salt is ruthenium trichloride; the lithium salt is lithium chloride; and the organic solvent is N,N'-dimethylformamide.

4. The method for preparing the biguanide-ruthenium complex according to claim 2, characterized in that: In the method 1, the molar ratio of the substance 2 to the biguanide derivative is 1:1; the biguanide derivative is 1-o-tolylbiguanide; the temperature of the heating reaction is 90° C. and the time is 8 hours.

5. The method for preparing the biguanide-ruthenium complex according to claim 2, characterized in that: In the second method, the ruthenium salt is ruthenium trichloride; the organic solvent is dimethyl sulfoxide; the biguanide derivative is 1-o-tolylbiguanide; the auxiliary ligand is 2,9-dimethylbipyridine; and the mixed solvent is a mixture of ethanol and dimethyl sulfoxide in a volume ratio of 9:

1.

6. The method for preparing the biguanide-ruthenium complex according to claim 2, characterized in that: In the method 2, when preparing the product 4, the reflux temperature is 85°C and the time is 30 hours; when preparing the product 4, after the reflux is completed, the steps of cooling to room temperature, filtering to collect the solid, washing the solid with isopropanol, and vacuum drying are also included.

7. The method for preparing the biguanide-ruthenium complex according to claim 2, characterized in that: In the method 2, when preparing the product 5, the reflux temperature is 90°C and the time is 3 hours; when preparing the product 5, after the reflux is completed, the steps of cooling to room temperature and removing the solvent by vortexing are also included; the temperature of the reflux reaction is 130°C and the time is 12 hours; after the reflux reaction is completed, the steps of cooling to room temperature, adding an appropriate amount of water, dripping a saturated solution of potassium hexafluorophosphate, standing in a refrigerator, filtering and drying, and purifying the crude product with a neutral alumina column are also included.

8. Use of the biguanide-ruthenium complex according to claim 1 in the preparation of a drug for resisting methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae and / or Klebsiella pneumoniae.

9. An antibacterial drug, characterized in that: The raw materials include the biguanide-ruthenium complex described in claim 1; the antibacterial drug is a drug against methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae and / or Klebsiella pneumoniae.

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