Application of metal palladium diphosphine complex with antibacterial activity in preparation of antibacterial drugs

Abstract

The invention discloses an application of metal palladium diphosphine complexes Pd-1 to Pd-5 with antibacterial activity in preparation of antibacterial drugs. According to the metal palladium diphosphine complex, the diphosphine ligand and the metal palladium form different dihedral angles through different chain lengths, so that the structure type of the metal palladium diphosphine complex is greatly different from that of other metal palladium complexes; pd-1 to Pd-5 can achieve efficient inhibition on various bacteria under the condition of low concentration (5-10 mu M), toxicity to various cells is small, safety is good, the selection index is larger than 18, one metal palladium diphosphine complex has good inhibition capacity on a mycobacterium tuberculosis H37Rv attenuated strain Ra, and the metal palladium diphosphine complex can be used for preparing a medicine for treating mycobacterium tuberculosis H37Rv attenuated strain Ra. The clinical practical application effect of the metal palladium diphosphine complex is greatly improved; meanwhile, the diversity of metal complex structures with antibacterial activity is enriched.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of metal palladium bisphosphine complexes with antibacterial activity for the preparation of antibacterial drugs. Specifically, the present invention relates to the use of Pd(P^P) metal complexes in the antibacterial field, and further explores the potential impact of their structural characteristics on different antibacterial activities. Background Art

[0002] Respiratory infection is a general term for acute respiratory infections caused by a variety of pathogens. The new coronavirus, influenza virus, and Mycobacterium tuberculosis are important pathogens that cause respiratory infections, among which Mycobacterium tuberculosis is a pathogen that seriously endangers human health. According to statistics from the World Health Organization in 2009, approximately three million people die from tuberculosis each year worldwide, and about one-third of the population carries latent Mycobacterium tuberculosis. The current treatment of tuberculosis is complicated by problems such as drug resistance, long treatment cycles, and side effects. In particular, the treatment of multidrug-resistant tuberculosis (MDR-TB) has become a major challenge to global public health due to the increased resistance of Mycobacterium tuberculosis to second-line drugs (Gandhi et al, 2010). Therefore, there is an urgent need to develop new drug targets and diagnostic tools to combat tuberculosis.

[0003] Due to the diversity of their composition and the controllability of their structure, metal complexes have led to a wealth of research on the synthesis, properties, and structure of coordination compounds. In recent years, the potential application value of complexes in biological activity has attracted increasing attention from researchers. Studies have shown that palladium (Pd) complexes have a significant inhibitory effect on bacterial growth, and palladium combined with conventional antibiotics (including tetracycline, doxycycline, and chlortetracycline) has shown greater potential against sensitive and resistant strains (JInorg Biochem. 2005, 99, 2348-2354.). Based on this, we hope to explore other metal complexes with different structures containing Pd elements and study their antibacterial properties against a variety of bacteria and possible structure-activity relationships. Summary of the Invention

[0004] The present invention aims to provide a metal palladium bisphosphine complex with antibacterial activity for use in the preparation of antibacterial drugs; the metal palladium bisphosphine complex is a type of Pd(P^P) metal complex, which has antibacterial activity, especially anti-tuberculosis activity.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The metal palladium bisphosphine complex has the following structure:

[0007]

[0008] The present invention obtains five different bisphosphine ligand metal palladium complexes by purchasing from Bid Pharmaceutical, wherein Pd-1, cas: 205319-10-4, article number: BD290313; Pd-2, cas: 205319-06-8, article number: BD434439; Pd-3, cas: 149796-59-8, article number: BD293210, Pd-4, cas: 19978-61-1, article number: BD19194; Pd-5, cas: 59831-02-6, article number: BD165234; and the biological activities of the metal palladium bisphosphine complexes Pd-1 to Pd-5 in different bacterial species are studied, preferably the antibacterial activity in different bacteria is studied. The bacterial species are selected from Staphylococcus aureus (Sa), Klebsiella pneumoniae (kpw1), Pseudomonas aeruginosa (PAD1), Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis H37Rv attenuated strain Ra.

[0009] Furthermore, the present invention performs gradient dilution of metal palladium bisphosphine complexes Pd-1 to Pd-5 with antibacterial activity and adds them to Mycobacterium smegmatis to determine the optimal screening product. Specifically, metal palladium bisphosphine complexes Pd-1 to Pd-5 with antibacterial activity are gradient diluted and added to bacterial culture and cultured at 37°C for 3 days. The OD value is read. 600 The antibacterial activity of the palladium bisphosphine complexes Pd-1 to Pd-5 was determined by measuring the minimum inhibitory concentration (MIC), the lowest concentration at which the antibacterial drug can inhibit bacterial growth in the culture medium.

[0010] Furthermore, in order to explore the biosafety of metal palladium bisphosphine complexes with good antibacterial effect, the present invention conducted a cytotoxicity experiment on metal palladium bisphosphine complexes Pd-1 to Pd-5 with antibacterial activity. Metal palladium bisphosphine complexes Pd-1 to Pd-5 with antibacterial activity were diluted in a gradient and added to cell culture, cultured at 37°C for 2 days, and OD was read by CCK8 colorimetry. 450 The values ​​were compared with those of the control group to determine whether cell growth was inhibited.

[0011] Furthermore, to explore the antibacterial activity and potential clinical applications of palladium bisphosphine complexes, the present invention conducted experiments using the attenuated Mycobacterium tuberculosis H37Rv strain Ra. The compound was added to a culture of the attenuated H37Rv strain Ra in a gradient dilution. The culture was incubated at 37°C for 7 days. The color development with resazurin solution was compared with that of a control to determine whether bacterial growth was inhibited.

[0012] Furthermore, the present invention evaluates the efficacy and safety of the compound through MIC value and selection index (SI).

[0013] Furthermore, the present invention conducted structural analysis on the screened metal palladium bisphosphine complexes Pd-1 to Pd-5 with antibacterial activity, and summarized the preliminary structure-activity relationship between the structure of metal complexes with Pd(P^P)-like structural characteristics and their antibacterial activity.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The use of the palladium bisphosphine complexes Pd-1 to Pd-5 provided by the present invention for preparing antibacterial drugs. Since the bisphosphine ligand and the palladium metal form different dihedral angles due to different chain lengths, this is very different from the structural types of other palladium metal complexes, enriching the application of palladium bisphosphine complexes while enriching the diversity of metal complex structures with antibacterial activity;

[0016] (2) The metal palladium bisphosphine complexes Pd-1 to Pd-5 provided by the present invention are used in the preparation of antibacterial drugs. The above-mentioned metal palladium bisphosphine complexes can achieve high efficiency in inhibiting a variety of bacteria at a relatively low concentration (5-10 μM), and have low toxicity to a variety of cells, good safety, and a selectivity index greater than 18. Among them, one metal palladium bisphosphine complex also has good inhibitory ability against the attenuated strain Ra of Mycobacterium tuberculosis H37Rv, which greatly increases the effect of the metal palladium bisphosphine complex in practical clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic structural diagram of the metal palladium bisphosphine complex provided by the present invention;

[0018] Figure 2 The metal palladium bisphosphine complexes provided by the present invention were screened for antibacterial effects in four bacteria at a concentration of 10 μM. It was found that all five metal palladium bisphosphine complexes showed good inhibitory effects in Mycobacterium smegmatis Ms, only Pd-1 showed an inhibitory effect in Staphylococcus aureus Sa, and no effective compounds were found in Pseudomonas aeruginosa PAD1 and Klebsiella pneumoniae.

[0019] Figure 3The metal palladium bisphosphine complex provided by the present invention was used in a minimum inhibitory concentration test in Mycobacterium smegmatis at different concentrations. It was found that only the metal palladium bisphosphine complexes Pd-1 and Pd-4 had three MIC values ​​within 10 μM, indicating better and more stable effects.

[0020] Figure 4 The metal palladium bisphosphine complex provided by the present invention was used to perform cytotoxicity evaluation in human cervical cancer cells (HeLa) and canine kidney cells (MDCK) at different concentrations, and it was found that the cytotoxicity of the metal palladium bisphosphine complexes Pd-1 and Pd-4 was within the normal range;

[0021] Figure 5 The metal palladium bisphosphine complex provided by the present invention was used in the attenuated strain Ra of Mycobacterium tuberculosis H37Rv at different concentrations for antibacterial screening results. It was found that the metal palladium bisphosphine complex Pd-1 had an inhibitory effect on the attenuated strain Ra at a concentration of 10 μM, while Pd-4 showed no inhibitory effect;

[0022] Figure 6 Data analysis of the metal palladium bisphosphine complex provided by the present invention in bacteria and cells;

[0023] Figure 7 The results of the antibacterial tests on two types of bacteria using the metal palladium bisphosphine complexes in Comparative Example 1 at a concentration of 10 μM showed that the other complexes had no inhibitory effect on Ms except the complex Pd-6. The complex Pd-6 showed an inhibitory effect on Ms but it was not strong, while none of the five complexes showed an inhibitory effect on Sa. DETAILED DESCRIPTION

[0024] The technical solutions of the present invention are further described below with reference to specific examples. However, the examples are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product instructions shall be followed. Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used, unless otherwise specified, are conventional biochemical reagents. The experimental methods described, unless otherwise specified, are conventional methods.

[0025] The reagents and experimental methods used in the following examples are:

[0026] All bisphosphine ligand metal palladium complexes and comparative bis / non-phosphine ligand metal palladium complexes were synthesized and purified by the synthesis methods in the following two references: Chem. Commun., 2013, 49, 7010 and ACS MacroLett. 2013, 2, 10-13.

[0027] ① Components and preparation of liquid culture medium

[0028] In 200mL sterile water, add 0.94g 7H9 powder (Middlebrook7H9 broth culture medium basis, BD company of the United States) sterilization and add 0.8mL 50% glycerol solution, 1mL 40% glucose solution, 1mL 10% Tween 80 solution and 1mL3M NaCl solution, the 7H9 liquid culture medium prepared is a liquid culture medium for Mycobacterium smegmatis. In 200mL sterile water, add 6g TSB powder (211825 tryptone soy broth basis, BD company of the United States) sterilization, the liquid culture medium (TSB) prepared is a liquid culture medium for Staphylococcus aureus. In 200mL sterile water, add 2g NaCl powder, 2g peptone powder, 1g yeast extract powder sterilization, the liquid culture medium (LB) prepared is a liquid culture medium for Pseudomonas aeruginosa and Klebsiella pneumonia. 0.94 g of 7H9 powder was added to 200 mL of sterile water for sterilization, followed by addition of 0.8 mL of 50% glycerol solution, 1 mL of 10% Tween 80 solution and 20 mL of OADC growth medium to prepare a liquid culture medium for the attenuated strain Ra.

[0029] ② Preparation of metal palladium bisphosphine complexes (Pd-1 to Pd-5)

[0030] The metal palladium bisphosphine complexes Pd-1 to Pd-5 are used as solutions or suspensions. Dimethyl sulfoxide (DMSO) is used to dissolve the metal palladium bisphosphine complexes Pd-1 to Pd-5 powders for subsequent experimental use. Specific method: first prepare a metal palladium bisphosphine complex mother liquor with an original concentration of 4mmol / L (4mM), and then use DMSO to dilute it to a metal palladium bisphosphine complex dilution with a working concentration of 1mM. When determining the minimum inhibitory concentration (MIC) of the compound, a concentration gradient experiment is performed using the working concentration of the metal palladium bisphosphine complex dilution, and the concentration gradients are: 40μM, 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM. All of the above are used in the examples. Figure 1 The metal palladium bisphosphine complexes Pd-1 to Pd-5 shown are respectively set as 5 groups of drugs.

[0031] ③Inoculation and culture methods

[0032] The above four bacteria were inoculated into the corresponding liquid culture medium and cultured at 37°C until OD 600 The value was around 0.6-0.8. The bacterial solution was diluted and inoculated into a 96-well plate as required. The entire experimental operation was carried out in a conventional biosafety cabinet.

[0033] Example 1: In vitro antibacterial activity experiment of the metal palladium bisphosphine complex of the present invention.

[0034] Method: First, the bacteria were cultured and diluted with culture medium to a CFU of 10 5 -10 6 / mL, according to the calculation, we can calculate that if the drug concentration is 1mM, then add 1μL of drug to each well (so that the final drug concentration reaches 10μM). First add 1μL of drug to a sterile 96-well plate, then add 49μL of culture medium, and finally add 50μL of diluted bacterial solution. Make 3 replica wells for each group of drugs, add sterile water to the blank wells to keep them moist, and culture them in a 37℃ incubator. Except for Ms, which needs to be cultured for 72h, the other bacteria need to be cultured for 24h. After the culture is completed, observe the clarity of the liquid in the well and measure the OD with a microplate reader. 600 To indicate the bacterial growth status of each well and draw a related bar graph through the software.

[0035] Results: As Figure 2 As shown, the metal palladium bisphosphine complexes Pd-1 to Pd-5 exhibited different inhibitory effects on the four bacteria. Specifically, the metal palladium bisphosphine complexes Pd-1 to Pd-5 all exhibited good inhibitory effects on Mycobacterium smegmatis, with Pd-3 and Pd-4 achieving inhibition rates of 77%, while those of Pd-1, Pd-2, and Pd-5 were all above 80%. Only Pd-1 exhibited an inhibitory effect on Staphylococcus aureus, with an inhibition rate of 94%. None of the five complexes exhibited inhibitory effects on Pseudomonas aeruginosa PAD1 or Klebsiella pneumoniae. This result indicates that only Pd-1 exhibited inhibitory effects on both Mycobacterium smegmatis and S. aureus, demonstrating superior performance.

[0036] Example 2: Minimum inhibitory concentrations of metal palladium bisphosphine complexes Pd-1 to Pd-5 against Mycobacterium smegmatis.

[0037] Method: First, the bacteria were cultured and diluted with culture medium to a CFU of 10 5 -10 6 / mL, first add 8μL of 1mM metal palladium bisphosphine complex to the first column of a sterile 96-well plate, then add 92μL of culture medium, first add 50μL of culture medium to the remaining wells, mix the liquid in the first column, then draw 50μL to the second column and mix, repeat the operation to the last column, draw 50μL and discard, then add 50μL of diluted bacterial solution to all wells, make 3 replica wells for each group of drugs, add sterile water to the blank wells to keep them moist, use streptomycin sm (concentration of 20ug / mL) as the positive control group for Ms, culture in a 37℃ incubator for 72h, and measure the OD 600By observing the well plate, it can be seen that the concentration of metal palladium bisphosphine complex at which bacterial growth is significantly inhibited is the minimum inhibitory concentration. The experiment was repeated three times, and the relevant curve graph was drawn using software.

[0038] Results: As Figure 3 As shown, we can see that the results show that the minimum inhibitory concentration MIC of metal palladium bisphosphine complex Pd-3 and metal palladium bisphosphine complex Pd-5 are both greater than 10μM, which does not meet the criteria we selected; in the three repeated experiments, the MIC result of metal palladium bisphosphine complex Pd-2 was greater than 10μM once, and only the metal palladium bisphosphine complex Pd-1 and metal palladium bisphosphine complex Pd-4 had stable data in the three experiments, and the MIC values ​​were all within 10μM. This result shows that metal palladium bisphosphine complex Pd-1 and metal palladium bisphosphine complex Pd-4 have better and more stable effects on Ms bacteria.

[0039] Example 3: Determination of the cytotoxicity of metal palladium bisphosphine complexes Pd-1 to Pd-5 in cells.

[0040] Method: First, in a 96-well plate, 5 Cells / well, 3 replica wells for each concentration were plated, and cultured in a 37°C incubator overnight until the cells were completely attached. 20 μL of 4 mM compound was added to the first column of the 48-well plate, and then 780 μL of DMEM medium containing 10% serum and 1% double antibody P / S was added. 400 μL of DMEM medium containing 10% serum and 1% double antibody P / S was added to the remaining wells. After mixing the liquid in the first column, 400 μL was pipetted into the second column and mixed. Repeat the operation to the last column, and 400 μL was pipetted and discarded. 100 μL of the liquid in the 48-well plate was added to each well of the 96-well plate. Three replica wells were made for each group of drugs. PBS was added to the blank wells for moisturizing. The cells were cultured at 37°C incubator for 48 h. The liquid in the well plate was then aspirated, and the prepared CCK8 (prepared with DMEM at a 1:9 ratio) was added. After culture in a 37°C incubator for 1 h, the OD was measured using a microplate reader. 450 The software was used to draw the relevant bar graph.

[0041] Results: In this experiment, based on previous research results, we focused on the metal palladium bisphosphine complex Pd-1 and metal palladium bisphosphine complex Pd-4. Figure 4 As shown in the figure, the results show that the metal palladium bisphosphine complexes Pd-1 and Pd-4 have certain cytotoxicity to HeLa cells at high concentrations, while the cytotoxicity of the metal palladium bisphosphine complex Pd-1 in MDCK cells is slightly higher than that of the metal palladium bisphosphine complex Pd-4, but within the normal range. This shows that these two metal palladium bisphosphine complexes have certain biosafety at the cellular level.

[0042] Example 4: Determination of the antibacterial effect of metal palladium bisphosphine complexes Pd-1 to Pd-5 on the attenuated strain Ra of Mycobacterium tuberculosis H37Rv.

[0043] Method: First, culture Ra and dilute it with culture medium to a CFU of 10 5 -10 6 / mL, first add 16 μL of 1 mM compound to a sterile 96-well plate, then add 184 μL of culture medium, add 100 μL of culture medium to the remaining wells, mix the liquid in the first column, then pipette 100 μL into the second column and mix, repeat the operation to the last column, pipette 100 μL and discard, then add 100 μL of diluted bacterial solution to all wells, make three replicate wells for each drug group, add sterile water to keep the blank wells moist, Ms used streptomycin sm (concentration of 20 ug / mL) as the positive control, and culture in a 37°C incubator for 7 days. Then add 30 μL of 1% resazurin solution to each well, and culture in a 37°C incubator until the solution changes color. After the incubation period, remove the 96-well culture plate, observe and photograph the color change of each well in the 96-well plate (blue indicates no strain growth, red indicates strain growth). The MIC is defined as the lowest drug concentration that prevents the color from changing from blue to pink.

[0044] Results: According to the previous results, we can see that Pd-1 compound and Pd-4 compound are better than the other three compounds, so in this experiment we mainly study the effect of these two compounds on Ra. Figure 5 As shown in the figure, the results show that the Pd-1 compound shows blue in the 10 μM concentration well, indicating that the growth of Ra is inhibited at this concentration, that is, the minimum inhibitory concentration of Pd-1 is 10 μM, while Pd-4 does not show an inhibitory effect on Ra. Therefore, the antibacterial effect of Pd-1 is better than that of Pd-4 compound.

[0045] Example 5: Determination of the antibacterial activity and safety of metal palladium bisphosphine complexes Pd-1 to Pd-5.

[0046] Method: Analyze the data from the previous experiment to obtain the IC value of the effective compound. 50 and CC 50 , calculate the ratio to get the selection index.

[0047] Results: As shown in the figure, we obtained the MIC value, IC 50 、CC 50 and selection index SI(CC 50 / IC 50Comparison reveals that Pd-1 and Pd-4 compounds are more effective. Furthermore, Pd-1 exhibits significant inhibitory effects in both the Staphylococcus aureus and Ra bacteria inhibition tests, while Pd-4 does not. In summary, Pd-1 exhibits the best performance among Pd(P^P) complexes.

[0048] Example 6: Possible structure-activity relationship between the structures of metal palladium bisphosphine complexes Pd-1 to Pd-5 and their antibacterial activities.

[0049] In tests of antibacterial activity against various bacteria, the study found a correlation between the natural bite angle (βn) formed between the metal atom and the ligand of the palladium bisphosphine complexes Pd-1 to Pd-5 and their antibacterial activity. Furthermore, the electronic properties of the metal center, the steric hindrance and electrical properties of the ligands surrounding the metal, and the bond length between the metal and the coordinating anion all significantly influenced the antibacterial activity. Pd-1 has a wide dihedral angle, typically around 110°, and its rigid structure and large dihedral angle also contribute to its excellent antibacterial activity. Furthermore, we infer that throughout the antibacterial activity process, the ability of the palladium bisphosphine complex to generate reactive oxygen species is directly correlated with its antibacterial properties. The palladium bisphosphine complex can regulate the electronegativity of the metal center through different ligands, thereby affecting charge redistribution and catalytic activity, ultimately affecting antibacterial performance.

[0050] It can be seen from the examples and test results that the metal palladium complex screened by the present invention has good inhibitory activity against various bacteria when used as an antibacterial agent in application research, among which the inhibitory activity against Mycobacterium tuberculosis is relatively high, and it also has good inhibitory activity against streptomycin-resistant Mycobacterium tuberculosis.

[0051] As can be seen from the examples and test results, in the present invention, as the corresponding bisphosphine ligand structure, the angle between the bisphosphine ligand and the metal center, etc. of the metal palladium complex are different, the corresponding complex has different inhibitory activities; complexes with the same structure also have different inhibitory activities against different types of bacteria.

[0052] Comparative Example 1: In order to verify the obvious technical advantages of the antibacterial ability of Pd-1 to Pd-5 provided in this application over the antibacterial ability of other disclosed palladium complexes, we tested the antibacterial activity of other palladium complexes. The specific experimental results are as follows: We selected 7 palladium complexes in Ms and 5 palladium complexes in Sa for antibacterial experiments, and the concentration of the complexes was 10 μM. In Ms bacteria, we can see that except for Pd-6, the other 6 complexes did not show an inhibitory effect in Ms. The inhibition rate of Pd-6 was 49%, but it was also higher than Pd-1, Pd-3 and Pd-4 in this application; in Sa bacteria, we can see that all 5 complexes showed an inhibitory effect, but the effect was far inferior to Pd-1, Pd-2 and Pd-4 in this application. The above results show that the cyclic metal palladium complexes (Pd-1 to Pd-4) in this application and other (cyclic) metal palladium complexes have very obvious technical advantages in antibacterial activity against Ms, Sa, etc.

[0053]

[0054]

[0055]

[0056] From the above comparative experimental results, it can be seen that the metal palladium bisphosphine complexes (Pd-1 to Pd-5) in this application have very obvious technical advantages over other (bis / non)phosphine metal palladium complexes in terms of antibacterial activity against Ms, Sa, etc.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a metal palladium bisphosphine complex having antibacterial activity for preparing an antibacterial drug, characterized in that: The metal palladium bisphosphine complex has the following structure: The pathogenic bacteria used in the application include Staphylococcus aureus Sa, Klebsiella pneumoniae kpw1, Pseudomonas aeruginosa PAD1, Mycobacterium smegmatis Ms and Mycobacterium tuberculosis H37Rv attenuated strain Ra.

2. The use of the metal palladium bisphosphine complex with antibacterial activity according to claim 1 for preparing antibacterial drugs, characterized in that: The effective antibacterial concentration of the metal palladium bisphosphine complex is 5-10 μM.

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