Cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis as well as preparation method and application of cinnamaldehyde-modified polypyridine ruthenium complex

Abstract

The invention belongs to the technical field of antibacterial medicines, and particularly relates to a cinnamyl aldehyde modified polypyridine ruthenium complex for inhibiting hemolysis as well as a preparation method and application of the cinnamyl aldehyde modified polypyridine ruthenium complex. The cinnamyl aldehyde modified ruthenium polypyridine complex disclosed by the invention has a structure as shown in a formula I; in the formula I, the # imgabs0 # has a structure as shown in a formula I1, a formula I2 or a formula I3; the polypyridine ruthenium complex with the cinnamyl aldehyde structure can effectively reduce the hemolysis effect of toxin released by staphylococcus aureus on red blood cells, and the ruthenium complex does not have the tendency of inducing drug resistance of bacteria and can remarkably improve the sensitivity of staphylococcus aureus to aminoglycoside antibiotics. # imgabs1 #

Description

Technical Field

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

[0002] The escape of hemoglobin is called erythrocyte lysis, or hemolysis for short. Hemolysis can be caused by toxins, most of which are produced by Staphylococcus aureus. This is because the α-hemolytic toxin of Staphylococcus aureus can affect the permeability of vascular endothelial cells and interact with various cells of the host. It is the main virulence factor that causes skin necrosis and severe infection.

[0003] However, with the abuse of antibiotics, more and more methicillin-resistant Staphylococcus aureus have been detected, resulting in resistance to multiple antibiotics, which aggravates the difficulty of clinical treatment of this infection. Summary of the invention

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

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

[0006]

[0007] In Formula I, Having formula I 1 , Formula I 2 Or Formula I 3 The structure shown:

[0008]

[0009] It should be noted that the cinnamic acid-modified ruthenium polypyridine complex of the present invention contains ruthenium ions, and the ruthenium ions and the compound of formula I 1 , Formula I 2 Or Formula I 3 At the same time, ruthenium ions carry a positive charge, while bacterial cell membranes usually carry a negative charge. Positively charged organic molecules can interact with the phospholipid bilayer of bacterial cell membranes through electrostatic interaction, leading to the destruction of cell membranes, leakage of intracellular substances, and the inability of bacteria to produce toxins, thereby inhibiting bacterial growth. At the same time, ruthenium complexes have a multi-coordinated configuration, which allows them to be modified with different ligands, thereby making them more biologically active and less toxic.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned cinnamaldehyde-modified ruthenium polypyridine complex, comprising the following steps:

[0011] The main ligand shown in formula a and the auxiliary ligand shown in formula b are dissolved in a solvent, and under a protective atmosphere, the N atom of the pyridine group in the main ligand and the Cl atom in the auxiliary ligand undergo a coordination substitution reaction to introduce the main ligand into the auxiliary ligand to obtain a cinnamaldehyde-modified ruthenium polypyridine complex shown in formula I; the synthesis route is shown in reaction formula 1:

[0012]

[0013] Reaction formula 1.

[0014] It should be noted that the present invention uses ruthenium complex as an auxiliary ligand. Ruthenium belongs to transition metals. Ruthenium complex has good biocompatibility, electrochemical and photophysical properties, and can form small molecule compounds and nanomaterials. In addition, the rigid octahedral geometric structure of the ruthenium complex is easy to structurally modify and has very high plasticity. Ruthenium element has a variable valence state 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 polypyridine ruthenium complex enhances its ability to penetrate bacterial cell membranes and retention effect. Therefore, the present invention can achieve the destruction of bacterial membranes by modifying the polypyridine ruthenium complex, thereby inhibiting 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 Formula I 1 When the structure shown in the formula b-1 is shown in the formula b-2, the auxiliary ligand has the structure shown in the formula b-1, and the chemical formula is Ru(bpy)2Cl2·2H2O; when Formula I 2 When the structure shown in the formula b-2 is shown in the formula b-3, the auxiliary ligand has the structure shown in the formula b-2, and the chemical formula is Ru(dmb)2Cl2·2H2O; when Formula I 3 When the structure shown in the formula is, the auxiliary ligand has the structure shown in 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° C. to 160° C., and the time is 6 h to 10 h.

[0019] Preferably, the solvent is ethylene glycol.

[0020] It should also be noted that the main ligand of the present invention is prepared by the following steps: 1,10-phenanthroline-5,6-dione, α-amylcinnamaldehyde and ammonium acetate are mixed, and under acid catalysis, the carbonyl provided by 1,10-phenanthroline-5,6-dione and the aldehyde provided by α-amylcinnamaldehyde undergo a ketoaldehyde condensation reaction to form a main ligand containing cinnamaldehyde. During the reaction, ammonium acetate will undergo a certain degree of dissociation to produce ammonium ions and undergo a nucleophilic addition reaction with α-amylcinnamaldehyde, and form an imine product through a dehydration reaction. At the same time, the nitrogen atom of the imine will attack the carbonyl carbon of 1,10-phenanthroline-5,6-dione to form a new negatively charged oxygen intermediate. The negatively charged oxygen intermediate can react with another carbonyl group of 1,10-phenanthroline-5,6-dione through intramolecular electron transfer and proton transfer processes to form a new carbon-oxygen bond, and a double bond is formed between the nitrogen atom and another carbonyl carbon, and an intramolecular cyclization reaction occurs to generate a product containing a heterocyclic structure, thereby obtaining a main ligand containing cinnamaldehyde. Preferably, the molar ratio of 1,10-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° C. to 140° C., and the synthesis time is 2 h to 4 h.

[0022] Preferably, the α-amylcinnamaldehyde is prepared by the following steps: benzylidene (amyl)-λ 3 -Alkyl oxide and 2-(triphenyl-λ 5 -phosphite) acetaldehyde undergoes ylide reaction in toluene, benzylidene (pentyl)-λ 3 -Alkyl oxide and 2-(triphenyl-λ 5 -phosphite) acetaldehyde in toluene to form a phosphorus ylide intermediate. The negatively charged carbon of the intermediate attacks the benzylidene (pentyl) -λ 3 -Oxidation of the carbonyl-like carbon of the alkane forms a four-membered ring intermediate, which then undergoes ring cleavage to eliminate triphenylphosphine oxide and form a carbon-carbon double bond to give α-pentylcinnamaldehyde.

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

[0024] The third object of the present invention is to provide the use of the above-mentioned cinnamaldehyde-modified ruthenium polypyridine complex in the preparation of antibacterial drugs.

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

[0026] The fourth object of the present invention is to provide the use of the above-mentioned cinnamaldehyde-modified ruthenium polypyridine complex in the preparation of a drug for reducing hemolysis of red blood cells.

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

[0028] The present invention provides a polypyridine ruthenium complex modified with a cinnamaldehyde structure. Since ruthenium is a transition metal and carries a positive charge, and bacterial cell membranes are usually negatively charged, positively charged organic molecules can interact with the phospholipid bilayer of bacterial cell membranes through electrostatic interaction, resulting in the destruction of cell membranes, thereby inhibiting bacterial growth; at the same time, compared with traditional organic small molecules, the polypyridine ruthenium complex modified with a cinnamaldehyde structure of the present invention enhances its ability to penetrate bacterial cell membranes and retention effect. Since the ruthenium complex has a rigid octahedral geometric structure and is easier to modify, it is easier to optimize the binding affinity of cell targets than organic drugs; the polypyridine structure is a typical nitrogen-containing heterocyclic compound, which allows it to have more targets, thereby destroying bacterial cell membranes, thereby inhibiting the production of bacterial toxins.

[0029] The present invention utilizes 1,10-phenanthroline-5,6-dione and α-pentylcinnamaldehyde to carry out ketoaldehyde condensation reaction to form a main ligand containing cinnamaldehyde; then the main ligand containing the cinnamaldehyde small molecule is introduced into the auxiliary ligand containing the ruthenium element to synthesize three polypyridine ruthenium complexes with different auxiliary ligands. The polypyridine ruthenium complex of the present invention can effectively reduce the hemolytic effect of the toxins released by Staphylococcus aureus on red blood cells, and the polypyridine ruthenium complex has no tendency to induce bacterial resistance, and can significantly increase the sensitivity of Staphylococcus aureus to aminoglycoside antibiotics. It can be seen that the polypyridine ruthenium complex with a cinnamaldehyde structure provided by the present invention has certain potential in inhibiting hemolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a diagram showing the hemolytic effect of the Ru-3 complex at different concentrations of the present invention on Staphylococcus aureus; from left to right, they are 0.25×MIC, 0.5×MIC, 1×MIC, CTRL, PBS and Culture medium.

[0031] Figure 2 It is the hemolysis rate of the Ru-3 complex at different concentrations of the present invention on Staphylococcus aureus. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0033] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0034] Example 1

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

[0036] (1) Preparation of intermediates:

[0037] 177.27 mg of benzylidene (pentyl)-λ 3 - alkyl oxide and 204.33 mg of 2-(triphenyl-λ 5 -phosphite) acetaldehyde is dissolved in toluene and reacted at 80°C for 1h; the product obtained by the reaction is extracted and separated with ethyl acetate and aqueous solution to obtain α-amylcinnamaldehyde, i.e., the intermediate, with a yield of 78%; its preparation reaction route is shown in Reaction Formula 2:

[0038]

[0039] Reaction formula 2.

[0040] (2) Preparation of primary ligand:

[0041] 210.0 mg of 1,10-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 bottle, and 60 ml of acetic acid was added, and the mixture was refluxed and stirred at 130° C. for 3 h; after the reaction solution was cooled, water was added to dilute it, and the pH was adjusted to neutral with ammonia water to precipitate a yellow precipitate; the precipitate was filtered under reduced pressure, and then separated and purified by silica gel column chromatography using ethanol as an eluent to obtain a pure product, and then vacuum dried to obtain (E)-2-(1-phenylhept-1-en-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline as shown in formula a, i.e., the main ligand, with a yield of 67%; its preparation reaction route is shown in Reaction Formula 3:

[0042]

[0043] Reaction formula 3.

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

[0045] 117.75 mg of the main ligand shown in formula a and 158.7 mg of Ru(bpy)2Cl2·2H2O were added to a 50 mL three-necked flask and dissolved in ethylene glycol; under the protection of argon, the mixture was stirred and refluxed at 150°C for 8 h. After the reaction was completed, the mixture was cooled to room temperature, and a saturated potassium hexafluorophosphate solution was added to precipitate a large amount of red-brown solid; 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 using a mixed solution of xylene and acetonitrile as a concentration gradient eluent; wherein the volume ratio of toluene to acetonitrile was 10:1, and a cinnamaldehyde-modified polypyridine ruthenium complex was obtained, which was recorded as Ru-1, and the yield was 72%; its preparation reaction route is shown in Reaction Formula 4:

[0046]

[0047] Reaction formula 4.

[0048] Example 2

[0049] This embodiment provides a method for preparing a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis.

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

[0051]

[0052] Reaction formula 5.

[0053] Example 3

[0054] This embodiment provides a method for preparing a cinnamaldehyde-modified polypyridine ruthenium complex for inhibiting hemolysis.

[0055] 117.75 mg of the main ligand shown in formula a and 212.6 mg of Ru(dtb)2Cl2·2H2O were added to a 50 mL three-necked flask and dissolved in ethylene glycol; under the protection of argon, the mixture was stirred and refluxed at 150°C for 8 h. After the reaction was completed, the mixture was cooled to room temperature, and a large amount of red-brown solid was precipitated after adding a saturated potassium hexafluorophosphate solution; the precipitate was filtered and collected, and a crude product was obtained by vacuum drying; the crude product was separated and purified by a neutral alumina column using a mixed solution of xylene and acetonitrile as a concentration gradient eluent; wherein the volume ratio of toluene to acetonitrile was 10:1, and a cinnamaldehyde-modified polypyridine ruthenium complex was obtained, which was recorded as Ru-3, and the yield was 65%; its preparation reaction route is shown in Reaction Formula 6:

[0056]

[0057] Reaction formula 6.

[0058] Experiment on inhibiting secretion of bacterial hemolytic toxins:

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

[0060] Figure 1The figure is the effect diagram of the hemolytic effect of Ru-3 complexes at different concentrations on Staphylococcus aureus; from left to right, they are 0.25×MIC, 0.5×MIC, 1×MIC, CTRL, PBS and Culture medium. The MIC values ​​of Ru-1 to Ru-3 were determined experimentally, and the MIC values ​​of Ru-1, Ru-2 and Ru-3 were 3.12μg / mL, 12.5μg / mL and 1.56μg / mL respectively. The MIC value of Ru-3 was the smallest, and Ru-3 had the best antibacterial activity. The hemolysis test showed that the clearer the color of the supernatant, the stronger the inhibitory effect on bacterial toxins. The test results show that Ru-3 has a strong inhibitory effect on bacterial toxins at a value of 1×MIC, thereby inhibiting bacterial hemolysis. The blank control test of PBS and culture medium showed that PBS and culture medium did not cause rabbit red blood cell rupture. This shows that the cinnamaldehyde-modified polypyridyl ruthenium complex prepared by the present invention can significantly reduce the secretion of hemolytic toxins of Staphylococcus aureus.

[0061] The incubated suspension was centrifuged at 2000 rpm for 2 min, and the supernatant was taken to measure the absorbance at 543 nm to quantify the rupture of red blood cells and calculate the hemolysis rate.

[0062] Figure 2 is the hemolysis rate of Ru-3 complex at different concentrations. Figure 2 As shown, the hemolytic rates of 0.25×MIC, 0.5×MIC and 1×MIC were quantitatively detected by OD543nm absorbance. The hemolytic toxin secretion of Ru-3 complex at concentrations of 0.25×MIC, 0.5×MIC and 1×MIC was reduced by 29.33%, 77.10% and 93.4%, respectively.

[0063] The polypyridyl ruthenium complex of the present invention can effectively reduce the hemolytic effect of toxins released by Staphylococcus aureus on red blood cells, and the polypyridyl ruthenium complex has no tendency to induce bacterial resistance and can significantly increase the sensitivity of Staphylococcus aureus to aminoglycoside antibiotics.

[0064] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic creative concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0065] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.

Claims

1. A cinnamaldehyde-modified ruthenium polypyridine complex for inhibiting hemolysis, characterized in that: The cinnamaldehyde-modified ruthenium polypyridine complex has a structure as shown in Formula I: In Formula I, Having 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: The following steps are involved: The main ligand shown in formula a and the auxiliary ligand shown in formula b are dissolved in a solvent, and under a protective atmosphere, the N atom of the pyridine group in the main ligand and the Cl atom in the auxiliary ligand undergo a coordination substitution reaction to introduce the main ligand into the auxiliary ligand to obtain a cinnamaldehyde-modified ruthenium polypyridine complex shown in formula I; Its synthetic route is shown in reaction formula 1:

3. The method for preparing the cinnamaldehyde-modified ruthenium polypyridine complex 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 the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 2, characterized in that: The temperature of the coordination substitution reaction is 140° C. to 160° C., and the time is 6 h to 10 h.

5. The method for preparing the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 2, characterized in that: when Formula I 1 When the structure shown in formula b-1 is shown in formula b-2, the auxiliary ligand has a structure as shown in formula b-3; when Formula I 2 When the structure shown in formula b-2 is shown, the auxiliary ligand has a structure as shown in formula b-3; when Formula I 3 When the structure shown in formula b-3 is shown in formula b-4, the auxiliary ligand has a structure as shown in formula b-5:

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

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

8. The method for preparing the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 6, characterized in that: The temperature of the ketoaldehyde condensation reaction is 120° C. to 140° C., and the time is 2 h to 4 h.

9. Use of the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 1 in the preparation of antibacterial drugs, characterized in that: The antibacterial activity is the inhibition of Staphylococcus aureus.

10. Use of the cinnamaldehyde-modified ruthenium polypyridine complex according to claim 1 in preparing a drug for reducing hemolysis of red blood cells.

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