Application of cyclometalated palladium complex in preparation of antibacterial drugs

Abstract

The invention discloses application of a cyclometalated palladium complex to preparation of antibacterial drugs. The complex has a structure as shown in a formula I in the specification. According to the cyclometalated palladium complex, the metal palladium complex with different ring tension is obtained from biphenyl amino and metal palladium through different second phosphine ligands, and the structure type of the metal palladium complex is greatly different from that of other metal palladium complexes; the application of the cyclometalated palladium complex is enriched; meanwhile, the diversity of a metal complex structure with antibacterial activity is enriched; an activity experiment result shows that the cyclometalated palladium complex provided by the invention can realize efficient inhibition on various bacteria under the condition of lower concentration (1.25-10 mu M), has lower toxicity on various cells, is higher in safety, has a selection index SI of more than 18, and can be used for preparing a medicine for treating various bacteria. Wherein one of the cyclometalated palladium complexes also has a certain inhibition capability on a mycobacterium tuberculosis H37Rv attenuated strain Ra, so that the clinical practical application effect of the cyclometalated palladium complexes is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, relates to the application of a cyclometallated palladium complex in the preparation of antibacterial drugs, and deeply explores the potential influence of its structural characteristics on different antibacterial activities. Background Art

[0002] Respiratory tract infection is an acute disease caused by a variety of pathogens, including the new coronavirus, influenza virus, and Mycobacterium tuberculosis. Among them, Mycobacterium tuberculosis poses a serious threat to human health. According to data from the World Health Organization in 2009, about 3 million people die from tuberculosis each year worldwide, and about one-third of the population carries latent Mycobacterium tuberculosis. At present, the treatment of tuberculosis faces many challenges, such as drug resistance, long treatment cycles, and drug side effects. Especially multidrug-resistant tuberculosis (MDR-TB), due to the increasing resistance of Mycobacterium tuberculosis to second-line anti-tuberculosis drugs, its treatment has become a major problem in the field of global public health. Therefore, it is particularly urgent to develop new drug targets and diagnostic tools to effectively combat tuberculosis.

[0003] Due to the diversity of their constituent elements and the controllable structure, metal complexes have led to a wealth of research on coordination compounds in terms of synthesis, properties, and structure. In recent years, the potential application value of complexes in biological activity has increasingly attracted the attention of researchers. Studies have found that various metal complexes have certain antibacterial effects. For example, patent CN115677734A discloses that various metal binuclear complexes have antibacterial activity, but the antibacterial process requires light. Patent CN111732610 discloses a series of cyclometallated iridium complexes with antibacterial activity. These complexes also have inhibitory activity against a variety of cells. Therefore, we believe that it is of great significance to discover more metal complexes with different structures so that they have antibacterial activity while causing less damage to other cells, that is, metal complexes with a higher selectivity index. Summary of the Invention

[0004] The present invention provides a cyclometallated palladium complex for use in the preparation of an antibacterial agent. The complex comprises a counteranion (chloride ion), an amino ligand, a phosphine ligand, and a carbon coordination site on an aromatic ring. The complex exhibits antibacterial activity, particularly against Mycobacterium tuberculosis, and possesses a high selectivity index.

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

[0006] The cyclometalated palladium complex has a structure shown in the following formula I:

[0007]

[0008] in, Select from any of the following structures:

[0009]

[0010] The present invention synthesizes cyclometalated palladium complexes Pd-1 to Pd-4 by referring to the synthesis method in ACS Macro Lett. 2013, 2, 10-13, and then performs preliminary screening of the antibacterial activity of the cyclometalated palladium complexes against different bacteria at a concentration of 10 μM.

[0011] The present invention provides an application of a cyclometalated palladium complex for preparing an antibacterial drug. The bacterial species are Staphylococcus aureus (Sa), Mycobacterium smegmatis (Ms), and Mycobacterium tuberculosis H37Rv attenuated strain Ra.

[0012] The present invention tested the anti-tumor and antibacterial activities of the cyclometallated iridium complexes by the following steps: the cyclometallated palladium complexes were diluted in a gradient and added to the target bacteria to determine the optimal screening product. Specifically, the metal complexes were diluted in a gradient and added to the bacterial culture and cultured at 37°C for 3 days. The OD values were read. 600 The value is then compared with the control group to determine whether bacterial growth is inhibited. A control group without compound treatment and a control group treated with streptomycin are set up, and the antibacterial activity of the metal complex is determined by measuring the minimum inhibitory concentration (MIC), which is the lowest concentration of the antibacterial drug that can inhibit bacterial growth in the culture medium.

[0013] Furthermore, in order to explore the biosafety of metal complexes with good antibacterial effect, the present invention conducted a cytotoxicity experiment on the metal complexes. The metal complexes were diluted in gradients and added to the cell culture, cultured at 37°C for 2 days, and the OD was read by CCK8 colorimetry. 450 The value was compared with that of the control group to determine whether cell growth was inhibited. The control group was not treated with the compound.

[0014] Furthermore, in order to explore the antibacterial activity and clinical application possibility of the metal complex, the present invention used the attenuated strain Ra of Mycobacterium tuberculosis H37Rv to conduct experiments. The compound was diluted in series and added to the bacterial culture of the attenuated strain Ra of H37Rv, and cultured at 37°C for 7 days. The OD was read by color development with resazurin solution. 600 The value is compared with the control to determine whether bacterial growth is inhibited. A control without compound treatment and a control treated with streptomycin are set up.

[0015] Furthermore, the present invention evaluates the efficacy and safety of the compound through MIC value and selection index (SI).

[0016] Furthermore, the present invention conducts structural analysis on the screened metal complexes with antibacterial activity and summarizes the preliminary structure-activity relationship between the structure of the metal complexes with Pd(P^P)-type structure and their antibacterial activity.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] (1) The cyclometallic palladium complex provided by the present invention has different ring strain stability obtained by reacting benzylamino and metal palladium through different second phosphine ligands, thereby enriching the application of cyclometallic palladium complexes and enriching the diversity of metal complex structures with antibacterial activity;

[0019] (2) The cyclometal palladium complexes screened by the present invention can achieve high efficiency inhibition of various bacteria at relatively low concentrations (1.25-10 μM), and have low toxicity to various cells, high safety, and a selectivity index SI greater than 18. Among them, one cyclometal palladium complex also has a certain inhibitory ability against the attenuated strain Ra of Mycobacterium tuberculosis H37Rv, which greatly increases the effect of the practical clinical application of the cyclometal palladium complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the cyclometallic palladium complex of the present invention;

[0021] Figure 2 The results of antibacterial screening of cyclometallated palladium complexes using a concentration of 10 μM in two bacteria showed that three cyclometallated palladium complexes showed significant inhibitory effects on Mycobacterium smegmatis Ms at a concentration of 10 μM, while only Pd-2 had no inhibitory effect on Staphylococcus aureus Sa.

[0022] Figure 3 The minimum inhibitory concentration (MIC) test of cyclometallated palladium complexes at different concentrations was conducted in Mycobacterium smegmatis. It was found that all four cyclometallated palladium complexes had good antibacterial activity against Ms, among which the complex Pd-2 had the lowest MIC value.

[0023] Figure 4 Cytotoxicity evaluation of cyclometallated palladium complexes was conducted in human cervical cancer cells (HeLa) and canine kidney cells (MDCK) at different concentrations. It was found that complexes Pd-1, Pd-3, and Pd-4 all had certain cytotoxicity against both cell types, while complex Pd-2 had the lowest cytotoxicity and the best effect compared to the other three complexes.

[0024] Figure 5 The results of antibacterial screening of the cyclometalated palladium complex Pd-2 in the attenuated strain Ra of Mycobacterium tuberculosis H37Rv using different concentrations were presented. It was found that the complex Pd-2 had an inhibitory effect on the attenuated strain Ra at a concentration of 20 μM.

[0025] Figure 6 Data analysis for cyclometallated palladium complexes in bacteria and cells;

[0026] Figure 7 The antibacterial results of the cyclometallated palladium complexes in Comparative Example 1 were tested on two bacteria at a concentration of 10 μM. It was found that except for the complex Pd-6, the other complexes had no inhibitory effect on Ms. 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

[0027] 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.

[0028] All cyclometalated palladium complexes and comparative (cyclo)metalated palladium complexes were synthesized and purified by the synthesis methods in the following two references: Chem. Commun., 2013, 49, 7010 and ACS Macro Lett. 2013, 2, 10-13. The reagents and experimental methods used in the following examples are as follows:

[0029] ① Components and preparation of liquid culture medium

[0030] 0.94g 7H9 powder (Middlebrook 7H9 broth base, BD Company, USA) was added to 200mL sterile water and sterilized, followed by addition of 0.8mL 50% glycerol solution, 1mL 40% glucose solution, 1mL 10% Tween 80 solution and 1mL 3M NaCl solution. The 7H9 liquid culture medium prepared is a liquid culture medium for Mycobacterium smegmatis. 6g TSB powder (211825 Tryptone Soy Broth Base, BD Company, USA) was added to 200mL sterile water and sterilized, and the TSB liquid culture medium prepared is a liquid culture medium for Staphylococcus aureus. 0.94g 7H9 powder was added to 200mL sterile water and sterilized, followed by addition of 0.8mL 50% glycerol solution, 1mL 10% Tween 80 solution and 20mL OADC growth medium to obtain a liquid culture medium for attenuated strain Ra.

[0031] ② Preparation of cyclometallated palladium complexes (Pd-1 to Pd-4)

[0032] The present invention obtains four different cyclometalated palladium complexes by purchasing from Bid Pharmaceuticals, wherein Pd-1, cas: 1375325-77-1, item number: BD449921; Pd-2, cas: 1375325-71-5, item number: BD280382; Pd-3, cas: 1375325-68-0, item number: BD266347, and Pd-4, cas: 1375325-64-6, item number: BD294922; the purchased cyclometalated palladium complexes Pd-1 to Pd-4 are prepared into solutions or suspensions for experiments. Dimethyl sulfoxide (DMSO) is used to dissolve the cyclometalated palladium complexes (powder) for use in subsequent experiments. Specific method: First, prepare a cyclometalated palladium complex mother solution with an original concentration of 4mmol / L (4mM), then use DMSO to dilute it to a cyclometalated palladium complex dilution with a working concentration of 1mM. When determining the minimum inhibitory concentration (MIC) of the cyclometalated palladium complex, use the cyclometalated palladium complex dilution with the working concentration to perform a concentration gradient experiment. The concentration gradients of the cyclometalated palladium complex dilutions are: 40μM, 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM. Figure 1 The four cyclometallated palladium complexes shown are set up as four groups of drugs.

[0033] ③Inoculation and culture methods

[0034] The above three bacteria were inoculated into the corresponding liquid culture medium and cultured at 37°C until OD 600The value was about 0.6-0.8, the bacterial solution was diluted and inoculated into a 96-well plate according to the specific settings in the embodiment. The entire experimental operation was carried out in a common biosafety cabinet.

[0035] Example 1: In vitro antibacterial activity experiment of cyclometallated palladium complexes

[0036] First, two bacteria (Mycobacterium smegmatis Ms and Staphylococcus aureus Sa) were cultured separately and diluted with culture medium to a CFU of 10 5 -10 6 / mL, the drug concentration is 10μM, then add 1uL of a drug (1mM) to each well. First add 1uL of a single drug to a sterile 96-well plate, then add 49uL of culture medium, and finally add 50uL of diluted bacterial solution. Make 3 replica wells for each drug group, add sterile water to the blank well to keep it moist, and culture it in a 37℃ incubator. Ms needs to be cultured for 72h, and Sa needs 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.

[0037] Results: As Figure 2 As shown, the four cyclometallated palladium complexes, Pd-1 to Pd-4, exhibited varying inhibitory effects against the two bacterial species. Specifically, with the exception of Pd-3, all exhibited significant inhibitory effects against Mycobacterium smegmatis, with inhibition rates exceeding 85% for Pd-1, Pd-2, and Pd-4. Furthermore, with the exception of Pd-2, all exhibited significant inhibitory effects against Staphylococcus aureus, with inhibition rates exceeding 90% for Pd-1, Pd-3, and Pd-4. This indicates that the four cyclometallated palladium complexes exhibited varying degrees of inhibitory activity against both bacteria.

[0038] Example 2: Minimum inhibitory concentration of cyclometallated palladium complexes against Mycobacterium smegmatis

[0039] Methods: First, Mycobacterium smegmatis Ms was cultured and diluted with culture medium to a CFU of 10 5 -10 6 / mL, first add 8uL of 1mM cyclometal palladium complexes Pd-1 to Pd-4 to the first column of a sterile 96-well plate, then add 92uL of culture medium, first add 50uL of culture medium to the remaining wells, mix the liquid in the first column, then draw 50uL to the second column and mix, repeat the operation to the last column, draw 50uL and discard, then add 50uL 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, culture in a 37°C incubator for 72h, and measure the OD 600By observing the well plate, it can be seen that the concentration of the cyclometallated palladium 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.

[0040] Results: As Figure 3 As shown, we can see that the minimum inhibitory concentration (MIC) of complexes Pd-3 and Pd-4 is 10 μM, the MIC value of complex Pd-1 is lower than 10 μM, and the MIC value of complex Pd-2 is 1.25 μM, showing the best inhibitory effect. This result shows that the four cyclometallated palladium complexes Pd-1 to Pd-4 have good antibacterial activity against Ms.

[0041] Example 3: Determination of the cytotoxicity of cyclometallated palladium complexes in cells

[0042] Method: First, in a 96-well plate, 5 Cells / well, 3 replica wells for each concentration were plated, and cells (HeLa cells and MDCK cells) were cultured in a 37°C incubator overnight until the cells (HeLa cells and MDCK cells) were completely attached. 20uL of each drug group at a concentration of 4mM was first added to the first column of the 48-well plate, and then 780uL of DMEM culture medium containing 10% serum and 1% double antibody P / S was added. 400uL of DMEM culture medium containing 10% serum and 1% double antibody P / S was first added to the remaining wells. After mixing the liquid in the first column, 400uL was pipetted into the second column and mixed. Repeat the operation to the last column, 400uL was pipetted and discarded. 100uL of the liquid in the 48-well plate was added to each well of the 96-well plate. 3 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 48h. The liquid in the well plate was then aspirated and the prepared CCK8 (prepared with DMEM culture medium at a ratio of 1:9) was added. After culture in a 37°C incubator for 1h, the OD was measured using a microplate reader. 450 The software was used to draw the relevant bar graph.

[0043] Results: In this experiment, we conducted cytotoxicity tests on four cyclometallated palladium complexes. As shown in the figure, the results show that complexes Pd-1, Pd-3, and Pd-4 exhibit some cytotoxicity against HeLa and MDCK cells at lower concentrations, while complex Pd-2 exhibits cytotoxicity against both cell types only at higher concentrations. This suggests that Pd-2 has the lowest cytotoxicity among the cyclometallated palladium complexes and is more effective.

[0044] Example 4: Determination of the antibacterial effect of cyclometallated palladium complexes on the attenuated strain Ra of Mycobacterium tuberculosis H37Rv

[0045] Method: First, the attenuated strain Ra was cultured in the liquid culture medium described above for the attenuated strain Ra and diluted to a CFU of 10 5 -106 / mL, first add 16uL of each drug group at a concentration of 1mM to a sterile 96-well plate, then add 184uL of the same culture medium, add 100uL of the same culture medium to the remaining wells, mix the liquid in the first column, then pipette 100uL into the second column and mix it, repeat the operation to the last column, pipette 100uL and discard it, then add 100uL of the diluted bacterial solution to all wells, make three replicate wells for each drug group, and add sterile water to the blank wells to keep them moist. Ms used streptomycin sm (concentration of both is 20ug / mL) as the positive control group, and cultured in a 37℃ incubator for 7 days. Then add 30uL of 1% resazurin solution to each well and culture in a 37℃ incubator until the solution changes color. After the incubation period, remove the 96-well culture plate, observe and photograph the color changes 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.

[0046] Results: Based on the previous results, we found that the cyclometalated palladium complex Pd-2 was more effective than the other three. Therefore, this experiment focused on the effect of this cyclometalated palladium complex on Ra. As shown in the figure, the results show that the cyclometalated palladium complex Pd-2 appears blue in the wells with a concentration of 20 μM, indicating that Ra growth is inhibited at this concentration. In other words, the minimum inhibitory concentration of the cyclometalated palladium complex Pd-2 is 20 μM. Although the minimum inhibitory concentration is higher than 10 μM, it still has some inhibitory effect on Ra.

[0047] Example 5: Determination of the Antibacterial Activity and Safety of Cyclometallated Palladium Complexes

[0048] 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.

[0049] Results: As Figure 6 As shown, we obtained the MIC value, IC50, CC through the software 50 and selection index SI (CC50 / IC 50 ). After comparison, we can see that the selectivity indexes of the cyclometallated palladium complexes Pd-3 and Pd-4 are low and do not meet our selection criteria; the Hela cell selectivity index of the cyclometallated palladium complex Pd-1 meets the requirements, but the MDCK cell selectivity index does not meet the requirements; and the selectivity index of the cyclometallated palladium complex Pd-2 is much higher than 18, and the MIC value is also at a low level. In summary, among the four cyclometallated palladium complexes, Pd-2 shows the best effect.

[0050] Example 6: Possible structure-activity relationship between the structure of cyclometallated palladium complexes and their antibacterial activity

[0051] In the antibacterial activity test on different bacteria, the study found that the antibacterial activity of cyclometal palladium complexes Pd-1 to Pd-4 is regulated by multiple factors. Among them, the hydrophobicity of the ligand (such as aromatic rings, long-chain alkyl substituents) can enhance the ability of the complex to penetrate the bacterial cell membrane, thereby improving the antibacterial activity. Therefore, the activity of the cyclometal palladium complex Pd-3 is relatively poor. Secondly, cyclometal palladium complexes are often planar tetragonal structures, similar to cisplatin, and may interfere with bacterial replication through similar mechanisms (such as DNA cross-linking), which is conducive to inserting DNA base pairs or binding to thiol groups. Similarly, cyclometal palladium complexes have a certain ring strain stability and good catalytic activity. Therefore, they may also achieve antibacterial properties based on the catalytic generation of ROS, resulting in radical oxidative stress. At the same time, cyclometal palladium complexes are very different from traditional antibiotics in structure, and can achieve better inhibitory activity against a variety of drug-resistant bacteria.

[0052] It can be seen from the examples and test results that the cyclometallated palladium complex provided by the present invention has good inhibitory activity against a variety of bacteria when used as an antibacterial agent in research, among which the inhibitory activity against Mycobacterium tuberculosis is relatively high, and it also has good inhibitory activity against streptomycin-resistant Mycobacterium tuberculosis.

[0053] 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 cyclometallated palladium complexes are different, the corresponding complexes have different ring strain stability and different inhibitory activities; complexes with the same structure also have different inhibitory activities against different types of bacteria.

[0054] Comparative Example 1: In order to verify the obvious technical advantages of the antibacterial ability of Pd-1 to Pd-4 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.

[0055]

[0056] Comparison of palladium complexes Inhibitory activity against Ms Inhibitory activity against Sa Pd-6 =10uM >10uM Pd-7 >10uM >10uM Pd-8 >10uM >10uM Pd-9 >10uM >10uM Pd-10 >10uM >10uM Pd-11 >10uM >10uM Pd-12 >10uM >10uM

[0057] From the above comparative experimental results, it can be seen that the cyclic metal palladium complexes (Pd-1 to Pd-4) in this application have very obvious technical advantages over other (cyclic) metal palladium complexes in terms of antibacterial activity against Ms, Sa, etc.

[0058] 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. A use of a cyclometalated palladium complex for preparing an antibacterial drug, characterized in that: The cyclometalated palladium complex has the structure shown in Formula I: in, Select from any of the following structures: The pathogenic bacteria used in the application include Staphylococcus aureus Sa, Mycobacterium smegmatis Ms and Mycobacterium tuberculosis H37Rv attenuated strain Ra.

2. Use of a cyclometallated palladium complex according to claim 1 or 2 for preparing an antibacterial drug, characterized in that: The minimum inhibitory concentration (MIC) values of the cyclometallated palladium complexes Pd-3 and Pd-4 are both 10 μM.

3. Use of a cyclometallated palladium complex according to claim 1 or 2 for preparing an antibacterial drug, characterized in that: The minimum inhibitory concentration (MIC) value of the cyclometallic palladium complex Pd-1 is lower than 10 μM, and the minimum inhibitory concentration (MIC) value of the cyclometallic palladium complex Pd-2 is 1.25 μM.

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