Cinnamaldehyde-modified polypyridine ruthenium complexes for inhibiting hemolysis and preparation method and application thereof

Abstract

The application belongs to the technical field of antibacterial medicine, and particularly relates to a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis, a preparation method and application thereof. The cinnamaldehyde-modified polypyridine ruthenium complex has a structure as shown in formula I. 1 , formula I 2 or formula I 3 The cinnamaldehyde-modified polypyridine ruthenium complex can effectively reduce the hemolysis of red blood cells caused by toxins released by Staphylococcus aureus, and the ruthenium complex does not induce the drug resistance of bacteria, and can significantly increase the sensitivity of Staphylococcus aureus to aminoglycoside antibiotics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial medicine, and particularly relates to a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis and a preparation method and application thereof. BACKGROUND

[0002] Hemoglobin escape is called hemolysis, which is simply referred to as hemolysis. Hemolysis can be caused by toxins, and most of the hemolytic toxins are produced by Staphylococcus aureus. This is because the alpha hemolysin of Staphylococcus aureus can affect the permeability of vascular endothelial cells and interact with various cells of the host, and is the main virulence factor causing skin necrosis and severe infection.

[0003] However, with the abuse of antibiotics, more and more methicillin-resistant Staphylococcus aureus is detected, which causes drug resistance to various antibiotics, thereby increasing the difficulty of clinical treatment of such infection. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis and a preparation method and application thereof.

[0005] The first object of the present application is to provide a cinnamic acid-modified polypyridine ruthenium complex for inhibiting hemolysis. The cinnamic acid-modified polypyridine ruthenium complex of the present application has a structure as shown in formula I:

[0006]

[0007] In formula I, has a structure as shown in formula I 1 , formula I 2 or formula I 3 .

[0008]

[0009] It should be noted that the cinnamic acid-modified polypyridine ruthenium complex of the present application contains a ruthenium ion, which is coordinated with the N atom in formula I 1 , formula I 2 or formula I 3 . At the same time, the ruthenium ion has a positive charge, while the bacterial cell membrane usually has a negative charge. The positively charged organic molecules can interact with the phospholipid bilayer of the bacterial cell membrane through electrostatic interaction, leading to the destruction of the cell membrane, the leakage of intracellular substances, and the inability of the bacterial cells to produce toxins, thereby inhibiting the growth of bacteria. At the same time, the ruthenium complex has a multi-coordination configuration, which can be modified with different ligands, thereby making it have higher biological activity and lower toxicity.

[0010] The second object of the present application is to provide a preparation method of the above-mentioned cinnamaldehyde-modified ruthenium polypyridyl complex, comprising the following steps:

[0011] The main ligand shown as formula a and the auxiliary ligand shown as formula b are dissolved in a solvent, and the N atom of the pyridyl group in the main ligand and the Cl atom in the auxiliary ligand are subjected to a coordination substitution reaction under a protective atmosphere, so that the main ligand is introduced into the auxiliary ligand to obtain the cinnamaldehyde-modified ruthenium polypyridyl complex shown as formula I; the synthetic route is shown as reaction formula 1:

[0012]

[0013] Reaction formula 1.

[0014] It should be noted that the present application uses a ruthenium complex as an auxiliary ligand, and ruthenium belongs to transition metals. The ruthenium complex has good biocompatibility, electrochemical and optical physical properties, and can form small molecule compounds and nanomaterials. In addition, the rigid octahedral geometry of the ruthenium complex is easy to modify the structure and has high plasticity. The ruthenium element has a variable valence and an extended p-electron structure, as well as excellent charge transfer characteristics, and can be used as a catalyst for chemical reactions in biological systems. This polypyridyl ruthenium complex enhances its ability to penetrate bacterial cell membranes and its retention effect. Therefore, by modifying the polypyridyl ruthenium complex, the present application can achieve the disruption of the bacterial membrane and thus inhibit the production of bacterial toxins.

[0015] Preferably, the molar ratio of the main ligand to the auxiliary ligand is 1:0.9-1.1.

[0016] Preferably, when is the structure shown as formula I 1 , the auxiliary ligand has the structure shown as formula b-1, and the chemical formula is Ru(bpy)2Cl2·2H2O; when is the structure shown as formula I 2 , the auxiliary ligand has the structure shown as formula b-2, and the chemical formula is Ru(dmb)2Cl2·2H2O; when is the structure shown as formula I 3 , the auxiliary ligand has the structure shown as formula b-3, and the chemical formula is Ru(dtb)2Cl2·2H2O; wherein bpy represents 2,2'-bipyridine, dmb represents 4,4'-dimethyl-2,2'-bipyridine, and dtb represents 4,4'-di-tert-butyl-2,2'-bipyridine:

[0017]

[0018] Preferably, the temperature of the coordination substitution reaction is 140-160℃, and the time is 6-10h.

[0019] Preferably, the solvent is ethylene glycol.

[0020] It should also be noted that the main ligand described in this invention is prepared through the following steps: 1,10-o-phenanthroline-5,6-dione, α-pentylcinnamaldehyde, and ammonium acetate are mixed, and under acid catalysis, the carbonyl group provided by 1,10-o-phenanthroline-5,6-dione undergoes a ketaldehyde condensation reaction with the aldehyde group provided by α-pentylcinnamaldehyde to form a main ligand containing cinnamaldehyde. During the reaction, ammonium acetate undergoes a certain degree of dissociation to generate ammonium ions, which then undergo a nucleophilic addition reaction with α-pentylcinnamaldehyde, followed by dehydration to form an imine product. Simultaneously, the nitrogen atom of the imine attacks the carbonyl carbon of 1,10-o-phenanthroline-5,6-dione, forming a new negatively charged oxygen intermediate. The negatively charged oxygen intermediate can react with another carbonyl group of 1,10-o-phenanthroline-5,6-dione through intramolecular electron and proton transfer processes to form a new carbon-oxygen bond. Simultaneously, a double bond forms between the nitrogen atom and the other carbonyl carbon, resulting in an intramolecular cyclization reaction that generates a product containing a heterocyclic structure, yielding a main ligand containing cinnamaldehyde. Preferably, the molar ratio of 1,10-o-phenanthroline-5,6-dione, α-pentylcinnamaldehyde, and ammonium acetate is 1:1:29–31.

[0021] Preferably, the synthesis temperature of the main ligand is 120℃~140℃, and the time is 2h~4h.

[0022] Preferably, the α-pentylcinnamaldehyde is prepared by the following steps: benzylidene (pentyl)-λ 3 -Oxyalkyl and 2-(triphenyl-λ) 5 -phosphite)acetaldehyde undergoes a ylide reaction in toluene, benzylidene(pentyl)-λ 3 -Oxyalkyl and 2-(triphenyl-λ) 5 (-phosphorylated)acetaldehyde forms a phosphorus ylide intermediate in toluene. The negatively charged carbon in this intermediate attacks the benzylidene (pentyl)-λ. 3 The alkyl group, similar to the carbonyl group, forms a four-membered ring intermediate, which then undergoes ring cleavage, eliminating triphenylphosphine oxide and generating a carbon-carbon double bond to give α-pentylcinnamaldehyde.

[0023] Preferably, the benzylidene (pentyl)-λ 3 -Oxyalkyl and 2-(triphenyl-λ) 5 The molar ratio of (-phosphite)acetaldehyde is 1:0.9 to 1.1.

[0024] A third objective of this invention is to provide the application of the above-mentioned cinnamaldehyde-modified ruthenium polypyridine complex in the preparation of antibacterial drugs.

[0025] Preferably, the antibacterial activity is the inhibition of Staphylococcus aureus.

[0026] A fourth object of the present application is to provide the use of the above-mentioned cinnamaldehyde-modified ruthenium polypyridyl complex in the preparation of a medicine for reducing hemolysis of red blood cells.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application provides a polypyridyl ruthenium complex with a cinnamaldehyde structure modification. Since ruthenium is a transition metal and has a positive charge, and the bacterial cell membrane usually has a negative charge, the positively charged organic molecules can interact with the phospholipid bilayer of the bacterial cell membrane through electrostatic interaction, leading to the destruction of the cell membrane and thus inhibiting bacterial growth. Meanwhile, compared with traditional organic small molecules, the polypyridyl ruthenium complex modified by the cinnamaldehyde structure enhances its ability to penetrate the bacterial cell membrane and the retention effect. Since the ruthenium complex has a rigid octahedral geometry, it is easier to modify the structure, so it can more easily optimize the binding affinity of the cell target than organic drugs. The polypyridyl structure is a typical nitrogen-containing heterocyclic compound, which allows it to have more targets and achieve the disruption of the bacterial cell membrane to inhibit the production of bacterial toxins.

[0029] The present application utilizes 1,10-phenanthroline-5,6-dione and alpha-pentyl cinnamaldehyde to perform a ketone-aldehyde condensation reaction to form a main ligand containing cinnamaldehyde; then the main ligand containing the cinnamaldehyde small molecule is introduced into an auxiliary ligand containing a ruthenium element to synthesize three different auxiliary ligand polypyridyl ruthenium complexes. The polypyridyl ruthenium complex of the present application can effectively reduce the hemolytic effect of the toxins released by Staphylococcus aureus on red blood cells, and the polypyridyl ruthenium complex does not induce the tendency of bacterial drug resistance, and can significantly increase the sensitivity of Staphylococcus aureus to aminoglycoside antibiotics. Therefore, the polypyridyl ruthenium complex with a cinnamaldehyde structure provided by the present application has certain potential in inhibiting hemolysis. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 3 is a graph of the hemolytic effect of the Ru-3 complex of the present application on Staphylococcus aureus under different concentrations; from left to right, they are 0.25xMIC, 0.5xMIC, 1xMIC, CTRL, PBS and Culture medium.

[0031] Figure 2 Figure 3 is a graph of the hemolytic effect of the Ru-3 complex of the present application on Staphylococcus aureus under different concentrations; from left to right, they are 0.25xMIC, 0.5xMIC, 1xMIC, CTRL, PBS and Culture medium. DETAILED DESCRIPTION

[0032] In order for those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific embodiments and drawings.

[0033] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0034] Example 1

[0035] This embodiment provides a method for preparing cinnamaldehyde-modified polypyridine ruthenium complexes for inhibiting hemolysis.

[0036] (1) Preparation of intermediates:

[0037] 177.27 mg of benzylidene (pentyl)-λ 3 -Oxyalkyl and 204.33 mg of 2-(triphenyl-λ) 5 (-phosphite)acetaldehyde was dissolved in toluene and reacted at 80°C for 1 h; the product obtained was extracted and separated by ethyl acetate and aqueous solution to give α-pentylcinnamaldehyde, the intermediate, with a yield of 78%; the preparation reaction route is shown in reaction formula 2:

[0038]

[0039] Reaction 2.

[0040] (2) Preparation of the host ligand:

[0041] 210.0 mg of 1,10-o-phenanthroline-5,6-dione, 202.3 mg of α-pentylcinnamaldehyde, and 2312.4 mg of ammonium acetate were weighed and placed in a 250 mL three-necked flask. 60 mL of acetic acid was added, and the mixture was refluxed at 130 °C for 3 h. After the reaction solution cooled, water was added for dilution, and the pH was adjusted to neutral with ammonia to precipitate a yellow precipitate. The precipitate was filtered under reduced pressure and purified by silica gel column chromatography using ethanol as the eluent. The purified product was then dried under vacuum to obtain (E)-2-(1-phenylhept-1-en-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, the main ligand, as shown in formula a, with a yield of 67%. The preparation reaction route is shown in reaction formula 3.

[0042]

[0043] Reaction 3.

[0044] (3) Preparation of cinnamaldehyde-modified polypyridine ruthenium complexes:

[0045] In a 50 mL three-necked flask, 117.75 mg of the main ligand shown in formula a and 158.7 mg of Ru(bpy)2Cl2·2H2O were dissolved in ethylene glycol; under the protection of argon, 8 h of stirring reflux at 150°C, after the reaction was completed, the reaction was cooled to room temperature, and a large amount of red-brown solid was precipitated after the saturated potassium hexafluorophosphate solution was added; the precipitate was filtered and collected, and the crude product was obtained by vacuum drying; the crude product was separated and purified by a neutral alumina column with a mixed solution of xylene and acetonitrile as the concentration gradient eluent; the volume ratio of toluene and acetonitrile was 10:1, and a cinnamaldehyde-modified polypyridine ruthenium complex, denoted as Ru-1, was obtained, and the yield was 72%; the preparation reaction route is shown in reaction formula 4:

[0046]

[0047] Reaction formula 4.

[0048] Example 2

[0049] The present embodiment provides a preparation method of a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis.

[0050] In a 50 mL three-necked flask, 117.75 mg of the main ligand shown in formula a and 162.1 mg of Ru(dmb)2Cl2·2H2O were dissolved in ethylene glycol; under the protection of argon, 8 h of stirring reflux at 150°C, after the reaction was completed, the reaction was cooled to room temperature, and a large amount of red-brown solid was precipitated after the saturated potassium hexafluorophosphate solution was added; the precipitate was filtered and collected, and the crude product was obtained by vacuum drying; the crude product was separated and purified by a neutral alumina column with a mixed solution of xylene and acetonitrile as the concentration gradient eluent; the volume ratio of toluene and acetonitrile was 10:1, and a cinnamaldehyde-modified polypyridine ruthenium complex, denoted as Ru-2, was obtained, and the yield was 53%; the preparation reaction route is shown in reaction formula 5:

[0051]

[0052] Reaction formula 5.

[0053] Example 3

[0054] The present embodiment provides a preparation method of a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis.

[0055] In a 50 mL three-necked flask, 117.75 mg of the main ligand shown in formula a and 212.6 mg of Ru(dtb)2Cl2·2H2O were dissolved in ethylene glycol; under the protection of argon, 8 h of stirring reflux at 150°C was performed, and after the reaction was completed, it was cooled to room temperature, and a large amount of red-brown solid was precipitated after the addition of saturated potassium hexafluorophosphate solution; the precipitate was filtered and collected, and vacuum dried to obtain a crude product; the crude product was separated and purified by a neutral alumina column with a mixed solution of xylene and acetonitrile as the eluent with a concentration gradient; the volume ratio of toluene and acetonitrile was 10:1, and a cinnamaldehyde-modified polypyridine ruthenium complex, denoted as Ru-3, was obtained, with a yield of 65%; the preparation reaction route is shown in reaction formula 6:

[0056]

[0057] Reaction formula 6.

[0058] Bacterial hemolysin secretion inhibition experiment:

[0059] The MIC value of Ru-3 was determined by experiment to be 1.56 μg / mL; 0.25×MIC, 0.5×MIC and 1×MIC concentrations of Ru-3 were taken and mixed with staphylococcus aureus culture solution, and the suspension was placed in a 37°C shaking bed at a speed of 220 r / min for 10 h; then 1 mL of the suspension was first taken with a pipette, and after high-speed centrifugation at a speed of 5000 rpm for 2 min, the bacterial supernatant was obtained. 2 mL of defibering rabbit blood was taken in a centrifuge tube, and after high-speed centrifugation at a speed of 2000 rpm for 2 min, the supernatant was discarded, and the rabbit red blood cells were stored, 150 μL of the bacterial supernatant and 25 μL of the rabbit red blood cells were added to 1 mL of PBS buffer, and incubated at 37°C for 30 min to obtain three sample solutions. PBS and culture medium were set as blank control groups; the rabbit red blood cells were washed with PBS buffer repeatedly for three times as the PBS blank control group, denoted as PBS; the rabbit red blood cells were added with culture medium as the culture medium blank control group, denoted as Culture medium; a positive experimental group with only bacteria and rabbit red blood cells was set, denoted as CTRL; three samples were taken for each group.

[0060] Figure 1The hemolysis effect diagram of Ru-3 complex under different concentrations on Staphylococcus aureus; from left to right, 0.25* MIC, 0.5* MIC, 1* MIC, CTRL, PBS and Culture medium. The MIC values of Ru-1 to Ru-3 are determined by experiments, and the MIC values of Ru-1, Ru-2 and Ru-3 are 3.12 μg / mL, 12.5 μg / mL and 1.56 μg / mL respectively, the MIC value of Ru-3 is the smallest, and the antibacterial activity of Ru-3 is the best. Through the hemolysis test, the clearer the supernatant color is, the stronger the inhibition of bacterial toxin is. The test results show that Ru-3 has a strong inhibition of bacterial toxin at 1* MIC value, thereby inhibiting bacterial hemolysis. The blank control test of PBS and culture medium shows that PBS and culture medium do not cause rabbit red blood cell rupture. Therefore, the cinnamaldehyde modified polypyridine ruthenium complex prepared in the application can significantly reduce the secretion of Staphylococcus aureus hemolysin.

[0061] The incubated suspension is centrifuged at a high speed of 2000 rpm for 2 min, the supernatant is taken, the absorbance at 543 nm is determined, the rupture of red blood cells is quantified, and the hemolysis rate is calculated.

[0062] Figure 2 The hemolysis rate of Ru-3 complex under different concentrations is shown in the following table. Figure 2 As shown in the table, the hemolysis rates of 0.25* MIC, 0.5* MIC and 1* MIC are quantitatively detected by OD543 nm absorbance, and the hemolysin secretion of Ru-3 complex under 0.25* MIC, 0.5* MIC and 1* MIC is reduced by 29.33%, 77.10% and 93.4% respectively.

[0063] The polypyridine ruthenium complex of the application can effectively reduce the hemolysis of red blood cells caused by the toxin released by Staphylococcus aureus, and the polypyridine ruthenium complex does not induce the drug resistance tendency of bacteria, and can significantly increase the sensitivity of Staphylococcus aureus to aminoglycoside antibiotics.

[0064] It should be noted that when the numerical range is involved in the application, the two endpoints of each numerical range and any number between the two endpoints can be selected. Since the same steps and examples are used, in order to prevent repetition, the preferred examples are described in the application. Although the preferred examples of the application have been described, those skilled in the art can make additional changes and modifications to these examples once they know the basic creative concept, and these changes and modifications all fall within the scope of the application.

[0065] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Such modifications and variations are considered to be within the scope of the application.

Claims

1. A cinnamaldehyde-modified ruthenium polypyridine complex for inhibiting hemolysis, characterized in that, The cinnamaldehyde-modified ruthenium polypyridine complex has the structure shown in Formula I: ； In formula I, Having as shown in formula I 1 , Formula I 2 or formula I 3 The structure shown: 。 2. A method for preparing the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 1, characterized in that, Includes the following steps: The main ligand shown in Formula a and the auxiliary ligand shown in Formula b are dissolved in a solvent. Under a protective atmosphere, the N atom of the pyridine group in the main ligand undergoes a coordination substitution reaction with the Cl atom in the auxiliary ligand, so that the main ligand is introduced into the auxiliary ligand, and the ruthenium polypyridine complex modified with cinnamaldehyde is obtained as shown in Formula I. Its synthetic route is shown in reaction formula 1: Reaction 1.

3. The method for preparing cinnamaldehyde-modified ruthenium polypyridine complexes according to claim 2, characterized in that, The molar ratio of the main ligand to the auxiliary ligand is 1:0.9~1.

1.

4. The method for preparing cinnamaldehyde-modified ruthenium polypyridine complexes according to claim 2, characterized in that, The coordination substitution reaction was carried out at a temperature of 140℃~160℃ for 6h~10h.

5. The method for preparing cinnamaldehyde-modified ruthenium polypyridine complexes according to claim 2, characterized in that, The main ligand is prepared by the following steps: 1,10-o-phenanthroline-5,6-dione, α-pentylcinnamaldehyde and ammonium acetate are mixed and, under acid catalysis, the carbonyl group provided by 1,10-o-phenanthroline-5,6-dione undergoes a ketaldehyde condensation reaction with the aldehyde group provided by α-pentylcinnamaldehyde to form a main ligand containing cinnamaldehyde.

6. The method for preparing cinnamaldehyde-modified ruthenium polypyridine complexes according to claim 5, characterized in that, The molar ratio of 1,10-o-phenanthroline-5,6-dione, α-pentylcinnamaldehyde, and ammonium acetate is 1:1:29~31.

7. The method for preparing cinnamaldehyde-modified ruthenium polypyridine complexes according to claim 5, characterized in that, The ketaldehyde condensation reaction was carried out at a temperature of 120℃~140℃ for 2h~4h.

8. The use of the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 1 in the preparation of antibacterial drugs, characterized in that, The antibacterial effect is the inhibition of Staphylococcus aureus.

9. The use of the cinnamaldehyde-modified ruthenium polypyridine complex of claim 1 in the preparation of a drug to reduce erythrocyte hemolysis.

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