CN117695290A - Preparation method and application of a copper complex with alkyl chain modification - Google Patents

Abstract

The invention belongs to the technical field of antibacterial medicines, and particularly relates to a preparation method and application of a copper complex with alkyl chain modification. The copper complex has a structure shown in a formula I. Compared with the traditional small organic molecules, the hydrophobic side chain of the copper complex provided by the invention can act with a bacterial cell membrane phospholipid bilayer, so that the transmembrane effect and the detention effect are enhanced, and the biological effect of the metal copper compound can completely enter the interior of bacteria by penetrating through a bacterial biological membrane barrier. According to the experiment of the embodiment of the invention, the copper complex with alkyl chain modification can effectively inhibit the growth of staphylococcus aureus and the formation of a biological film and the generation of alpha-hemolytic toxin thereof at the content of 0.39-1.56 mug/mL, and the copper complex does not trigger the drug resistance tendency of bacteria. Therefore, the alkyl chain modified copper complex provided by the invention has a certain potential in bacteriostasis.

Description

Preparation method and application of a copper complex with alkyl chain modification

Technical field

The invention belongs to the technical field of antibacterial medicine, and specifically relates to a preparation method and application of a copper complex with alkyl chain modification.

Background technique

Staphylococcus aureus ( S. aureus ) is one of the most common pathogens of clinical infectious diseases. It can cause infections in skin and soft tissues, blood system, lower respiratory tract and other parts of the body, leading to a series of diseases, such as pericarditis, pseudomycosis, etc. Membranous colitis, pneumonia and sepsis, etc. Treatment of these infections is challenging due to the emergence of drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), which are severely resistant to many antibiotics.

In addition, the development of biofilms in chronic infections is an important pathogenic factor involving multiple microorganisms, and therefore, biofilm-related infections also pose serious challenges. Generally, bacteria in biofilms are encapsulated in a hydrated matrix of extracellular polymeric substances (EPS) composed of polysaccharides, proteins, lipids, and nucleic acids, which provide mechanical stability and collectively provide immobilization of the biofilm-producing bacteria. cells, which enhance the adhesion of bacteria to surfaces, thereby forming stable three-dimensional (3D) connective tissue that protects microorganisms from external influences. Under normal circumstances, biofilms can resist the attack of most chemical bactericides, mainly phagocytic cells and neutrophils, and survive within them. It is precisely because of this resistance mechanism of bacteria that the bacteria are sensitive to antibiotics. The resistance is reduced, so how to eliminate the biofilm barrier has become one of the key points in antibiotic research and development.

Contents of the invention

The purpose of the present invention is to solve the deficiencies of the existing technology and provide a preparation method and application of a copper complex with alkyl chain modification, specifically adopting the following technical solutions:

A first aspect of the present invention provides a copper complex with alkyl chain modification. The copper complex with alkyl chain modification can be used in the preparation of drugs for inhibiting Staphylococcus aureus. The above-mentioned copper complex has formula I Structure shown:

Formula I; wherein n is selected from any positive integer from 3, 7, and 11.

Preferably, the structure of the above-mentioned copper complex is as shown in formula II:

Formula II.

The elemental and oxide forms of copper have been widely used as antibacterial reagents. The present invention obtains a metallic copper complex through multifunctional organic assembly. The complex can effectively resist bacterial drug resistance and inhibit bacterial biofilms, and experiments have proven that metallic copper has an antibacterial effect. It has good advantages in performance, low cost and can effectively inhibit the formation of bacterial biofilm. In the present invention, the hydrophobic alkyl side chain can be effectively inserted into the phospholipid bilayer of the bacterial cell membrane, and the metal copper complex causes intracellular substances to leak, leading to the death of the bacteria. Moreover, the multi-coordination configuration of the metal complex allows it to be modified with different ligands to achieve better biological activity. Therefore, by modifying metal copper complexes, bacterial biofilms can be destroyed or even eliminated, thereby killing drug-resistant strains.

A second aspect of the present invention also provides a method for preparing the above-mentioned copper complex with alkyl chain modification, including the following steps:

S1: Under argon conditions, heat the compound of formula I-a structure, the compound of formula I-d structure and potassium carbonate in acetonitrile to reflux. After cooling to room temperature, the solvent is evaporated under reduced pressure to obtain a crude product, which is purified to obtain the intermediate of formula I-e structure. body;

S2: Under argon conditions, heat the compound of formula I-b structure, the compound of formula I-d structure and potassium carbonate in acetonitrile to reflux. After cooling to room temperature, the solvent is evaporated under reduced pressure to obtain a crude product, which is purified to obtain the intermediate structure of formula I-f. body;

S3: Under argon conditions, heat the compound of the formula I-c structure, the compound of the formula I-d structure and potassium carbonate in acetonitrile to reflux. After cooling to room temperature, the solvent is evaporated under reduced pressure to obtain a crude product, which is purified to obtain the intermediate of the formula I-g structure. body;

S4: Dissolve the intermediate of the structure of formula I-e or the intermediate of the structure of formula I-f or the intermediate of the structure of formula I-g in an acetonitrile solution, then add an acetonitrile solution of copper bromide, react at room temperature for 6 hours, filter, wash, and dry to obtain a solution with Copper complexes modified with alkyl chains;

Among them, the molecular structural formulas of the compound of formula I-a structure, the compound of formula I-b structure, the compound of formula I-c structure, the compound of formula I-d structure, the intermediate of formula I-e structure, the intermediate of formula I-f structure and the intermediate of formula I-g structure are as follows Shown:

.

Preferably, in the above-mentioned steps S1, S2 and S3, the molar ratio of the compound of the formula I-a structure, the compound of the formula I-b structure and the compound of the formula I-c structure to the compound of the formula I-d structure is 1:1; the above-mentioned steps S1, S2 and S3 Medium purification is carried out on an alumina chromatography column using an eluent of methylene chloride/methanol = 200:1; the heating and refluxing temperature in the above steps S1, S2 and S3 is 85°C; the formula I-e structure in the above step S4 is The molar ratio of the intermediate, the intermediate of the formula I-f structure and the intermediate of the formula I-g structure to copper bromide is all 1:2.

A third aspect of the present invention also provides the use of the above-mentioned copper complex with alkyl chain modification in the preparation of drugs that inhibit α-hemolytic toxin.

The fourth aspect of the present invention also provides the use of the above-mentioned copper complex with alkyl chain modification in the preparation of bacteriostatic agents.

The beneficial effects of the present invention are: the present invention provides a copper complex with alkyl chain modification. The hydrophobic side chain of the copper complex can interact with the bacterial cell membrane phospholipid bilayer. Compared with traditional organic small molecules, In other words, the transmembrane effect and retention effect are enhanced, and the biological effect of the metallic copper compound can completely enter the interior of the bacteria by penetrating the bacterial biofilm barrier. According to the experiments of the embodiments of the present invention, the copper complex with alkyl chain modification of the present invention can effectively inhibit the growth of Staphylococcus aureus and the formation of biofilm and α- The production of hemolytic toxins, and the copper complexes have no tendency to trigger bacterial resistance. Therefore, the alkyl chain-modified copper complex provided by the present invention has certain potential in antibacterial activity.

Description of the drawings

Figure 1 shows the MIC measurement diagram of intermediate formula Ie, formula If, formula Ig and complexes C ₆ -Cu, C ₁₀ -Cu, C ₁₄ -Cu against Staphylococcus aureus;

Figure 2 shows the measurement chart of the biofilm inhibition of Staphylococcus aureus by complex C ₁₀ -Cu;

Figure 3 shows the measurement chart of the inhibitory effect of complex C ₁₀ -Cu on the α-hemolytic toxin of Staphylococcus aureus;

Figure 4 shows the resistance measurement chart of Staphylococcus aureus to the complex C ₁₀ -Cu;

Detailed ways

The following will give a clear and complete description of the concept, specific structure and technical effects of the present invention in conjunction with the embodiments and drawings, so as to fully understand the purpose, solutions and effects of the present invention. It should be noted that, as long as there is no conflict, the embodiments and features in the embodiments of this application can be combined with each other.

Example 1

A copper complex with alkyl chain modification, its preparation method includes the following steps:

The reaction is as shown in the following reaction route:

(1) Preparation of complex C ₆ -Cu

Step 1: Preparation of the intermediate of the formula I-e structure. The structural formula of the intermediate I-e is as follows:

The specific process is: add 1-bromohexane (0.5mmoL, 82.5mg), 2,2'-dipyridylmethylamine (0.5mmoL, 100mg), and potassium carbonate (1.5mmoL, 207mg) into a 25mL round-bottom reaction flask. , heated to reflux at 85°C in acetonitrile (2mL) solvent, and reacted for 24 hours under argon conditions. After the reaction was completed, cooled to room temperature, filtered to remove excess carbonate, and used dichloromethane-methanol mixture (200:1, v/ v) is the eluent, and the crude product is purified by column chromatography on neutral alumina. The solvent is removed under reduced pressure and dried under vacuum to obtain intermediate I-e as a yellow oil. Yield: 94 mg, 66.33%.

Step 2: Preparation of complex C ₆ -Cu. The structural formula of complex C ₆ -Cu is as follows:

The specific process is: weigh copper bromide (0.2mmoL, 45mg) into a 25mL round-bottomed reaction flask, add 2mL acetonitrile and stir thoroughly to obtain a copper bromide solution. Under stirring conditions, add intermediate Ie (0.1mmoL, 28mg) in acetonitrile. The solution was slowly dropped into the copper bromide solution, reacted at room temperature for 6 hours, filtered, collected the filter cake, washed with water three times, and dried under vacuum to obtain complex C ₆ -Cu as a green solid, yield: 28 mg, 55.6%.

The characterization results are as follows:

High-resolution mass spectrum (ESI) m/z: The theoretical value of C ₁₈ H ₂₅ BrCuN ₃ is 425.0528, and the calculated value of [M-Br] ⁺ is 425.0519. Elemental analysis (%): C ₁₈ H ₂₅ Br ₂ CuN ₃ (506.8), theoretical values C 42.66, H 4.97, N 8.29; measured values C 42.54, H 4.74, N 8.26.

(2) Preparation of complex C ₁₀ -Cu

Step 1: Preparation of the intermediate of the formula I-f structure. The structural formula of the intermediate I-f is as follows:

The specific process is: add 1-bromodecane (0.5mmoL, 118mg), 2,2'-dipyridylmethylamine (0.5mmoL, 100mg), and potassium carbonate (1.5mmoL, 207mg) into a 25mL round-bottom reaction bottle. Acetonitrile (2mL) solvent was heated to reflux at 85°C, and the reaction was carried out under argon conditions for 24 hours. After the reaction was completed, it was cooled to room temperature, filtered to remove excess carbonate, and replaced with dichloromethane-methanol mixture (200:1, v/v ) is the eluent, and the crude product is purified by column chromatography on neutral alumina. The solvent is removed under reduced pressure and dried under vacuum to obtain intermediate I-f as a yellow oil. Yield: 145.5 mg, 85.57%.

Step 2: Preparation of complex C ₁₀ -Cu. The structural formula of complex C ₁₀ -Cu is as follows:

The specific process is: weigh copper bromide (0.2mmoL, 45mg) into a 25mL round-bottomed reaction flask, add 2mL acetonitrile and stir thoroughly to obtain a copper bromide solution. Under stirring conditions, add intermediate If (0.1mmoL, 34mg) to acetonitrile. The solution was slowly dropped into the copper bromide solution, reacted at room temperature for 6 hours, filtered, collected the filter cake, washed with water three times, and dried under vacuum to obtain complex C ₁₀ -Cu as a green solid, yield: 33 mg, 58.9%.

The characterization results are as follows:

High-resolution mass spectrum (ESI) m/z: The theoretical value of C ₂₂ H ₃₃ BrCuN ₃ is 481.1154, and the calculated value of [M-Br] ⁺ is 481.1148. Elemental analysis (%): C ₂₂ H ₃₃ Br ₂ CuN ₃ (562.0), theoretical values C 46.94, H 5.91, N 7.47; measured values C 47.44, H 5.88, N 7.46.

(3) Preparation of complex C ₁₄ -Cu

Step 1: Preparation of the intermediate of the formula I-g structure. The structural formula of the intermediate I-g is as follows:

The specific process is: add 1-bromotetradecane (0.5mmoL, 140mg), 2,2'-dipyridylmethylamine (0.5mmoL, 100mg), and potassium carbonate (1.5mmoL, 207mg) into a 25mL round-bottomed reaction bottle. , heated to reflux at 85°C in acetonitrile (2mL) solvent, and reacted for 24 hours under argon conditions. After the reaction was completed, cooled to room temperature, filtered to remove excess carbonate, and used dichloromethane-methanol mixture (200:1, v/ v) is the eluent. The crude product is purified by column chromatography on neutral alumina. The solvent is removed under reduced pressure and dried under vacuum to obtain intermediate I-g as a yellow oil. Yield: 166.2 mg, 84.02%.

Step 2: Preparation of complex C ₁₄ -Cu. The structural formula of complex C ₁₄ -Cu is as follows:

The specific process is: weigh copper bromide (0.2mmoL, 45mg) into a 25mL round-bottomed reaction flask, add 2mL acetonitrile and stir thoroughly to obtain a copper bromide solution. Under stirring conditions, add intermediate Ig (0.1mmoL, 40mg) in acetonitrile. The solution was slowly dropped into the copper bromide solution, reacted at room temperature for 6 hours, filtered, collected the filter cake, washed with water three times, and dried under vacuum to obtain complex C ₁₄ -Cu as a green solid, yield: 34.1 mg, 55.4%.

The characterization results are as follows:

High-resolution mass spectrum (ESI) m/z: The theoretical value of C ₂₆ H ₄₁ BrCuN ₃ is 537.1780, and the calculated value of [M-Br] ⁺ is 537.1778. Elemental analysis (%): C ₂₆ H ₄₁ Br ₂ CuN ₃ (622.9, which contains 1% of C ₂₆ H ₄₁ N ₃ ), theoretical values C 50.63, H6.70, N 6.81; measured values C 51.12, H 6.66, N 6.71.

Example 2

In this example, the complex C ₆ -Cu, complex C ₁₀ -Cu and complex C ₁₄ -Cu prepared in Example 1 were subjected to an in vitro antibacterial activity test.

(1) Microdouble dilution method to determine MIC: Cultivate Staphylococcus aureus Newman strain in TSB medium until OD ₆₀₀ reaches 1; use fresh TSB medium to dilute the number of bacterial cells to approximately 5×10 ⁶ CFU/mL (OD ₆₀₀ =0.05); Subsequently, 50 µL of different concentrations of metal copper complexes or intermediates of formula Ie, formula If, and formula Ig were added to 200 µL of bacterial suspension. At this time, the final drug concentration in each well was from left to From the right: 200 µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.125 µg/mL, 1.56 µg/mL, 0.78 µg/mL, 0.39 µg/mL, 0.195µg/mL, in which 50 µL sterile water was added to the last well as a blank control; the mixture was placed into a 96-well plate and further incubated at 37°C for 20 hours before observing the results; observe the well plate In the case of turbidity, the lowest drug concentration corresponding to the clear administration hole is the MIC (minimum inhibitory concentration). The results are shown in Figure 1. Among them, rows A and H are at the edge, and no reagents are added to prevent contamination.

Figure 1 shows the MIC measurement chart of intermediate formula Ie, formula If, formula Ig and complexes C ₆ -Cu, C ₁₀ -Cu, C ₁₄ -Cu against Staphylococcus aureus; it can be judged by observing the turbidity of the well plate. The antibacterial effect of complexes C ₆ -Cu, C ₁₀ -Cu, and C ₁₄ -Cu on Staphylococcus aureus, the lowest drug concentration corresponding to the clear administration hole is the MIC (minimum inhibitory concentration); and by As shown in Figure 1, C ₆ -Cu, C ₁₀ -Cu, and C ₁₄ -Cu all have certain antibacterial effects. Among them, C ₆ -Cu, the complex with the shortest alkyl chain, has poor antibacterial activity. As the hydrophobic alkyl side chain As the growth rate increases, the antibacterial activity increases, but when the hydrophobic carbon chain length exceeds 10, the activity decreases to a certain extent. C ₁₀ -Cu shows the best antibacterial activity, and its MIC against Staphylococcus aureus=1.56 µg/mL.

(2) The effect of inhibiting biofilm was measured by biofilm experiment: Staphylococcus aureus Newman strain was cultured in TSB medium for 5 hours, and then the culture was diluted 1:200 with fresh sterile TSB medium containing 0.5%, A 24-well microtiter plate was filled with 2 mL aliquots of bacterial culture with or without metallic copper complexes; the plate was incubated at 37°C for 48 hours, then, washed three times with PBS, and the 24-well plate was incubated. Dry overnight at room temperature. The biofilm attached to the microtiter plate is soaked and incubated with 0.1% crystal violet solution for 15 minutes. Excess crystal violet is removed by washing with PBS. The crystal violet attached to the biofilm sample is dissolved with acetic acid and measured at 595 nm. The absorbance at to indicate the formation of biofilm, the results are shown in Figure 2.

Figure 2 shows the measurement chart of the biofilm inhibition of Staphylococcus aureus by complex C ₁₀ -Cu; the amount of biofilm after C ₁₀ -Cu acts on bacteria is judged by measuring the absorbance at OD ₅₉₅ ; and it can be seen from Figure 2 , C ₁₀ -Cu has a strong inhibitory effect on Staphylococcus aureus biofilm. At 0.39 µg/mL, the absorbance value decreased by 39% compared with the control group, while 0.78 µg/mL and 1.56 µg/mL showed respectively The cell membrane growth inhibition effects were 41.8% and 56.8%, indicating that C ₁₀ -Cu can inhibit the production of bacterial biofilm to a certain extent.

(3) The inhibitory effect of α-hemolytic toxin was measured by hemolysis rate of rabbit red blood cells: Staphylococcus aureus Newman strain was cultured in TSB medium for 5 hours. The bacteria were then diluted 1000-fold in fresh TSB medium. Fill a 24-well microtiter plate with 2 mL aliquots of bacterial culture with or without metallic copper complexes. The plate was incubated at 37°C for 10 hours. Subsequently, it was centrifuged and the bacterial supernatant was retained. Add 1 mL sterile PBS, 50 µL rabbit red blood cells, and 50 µL bacterial supernatant to a 1.5 mLep tube, and then incubate at 37°C for 30 minutes. After the incubation is completed, centrifuge (2000 rpm, 2 min), and measure the absorbance of the supernatant at OD ₅₄₀ nm. The results are shown in Figure 3.

Figure 3 shows the measurement chart of the inhibitory effect of complex C ₁₀ -Cu on the α-hemolytic toxin of Staphylococcus aureus; the production of bacterial α-hemolytic toxin after C ₁₀ -Cu treatment was determined by measuring the absorbance at OD ₅₄₀ nm. The inhibitory effect _of The inhibitory effect of α-hemolytic toxin is strong. At 0.39 µg/mL and 0.78 µg/mL, the absorbance value decreased by 42.1% and 45.7% compared with the control group. And 1.56 µg/mL showed an 86.3% inhibitory effect on the production of α-hemolytic toxin, indicating that C ₁₀ -Cu can inhibit the production of α-hemolytic toxin to a certain extent.

(4) Resistance study: First, measure the MIC of the complex C ₁₀ -Cu against Staphylococcus aureus. At this time, the MIC value is the value on day 0. The MIC value measurement method is determined by the aforementioned micro-two-fold dilution method; Then, transfer 5 µL of the bacterial culture solution of complex C ₁₀ -Cu at 1/2 × MIC concentration and re-culture it in 5 mL of fresh broth medium to the logarithmic phase, measure the MIC value again, and repeat the operation 20 times. , and record each MIC value. The MIC value of each day is divided by the MIC value of day 0 to obtain the multiple change value.

Figure 4 shows the resistance measurement chart of Staphylococcus aureus to the complex C10-Cu; by observing the changes in the MIC value of Staphylococcus aureus, we can observe whether resistance has developed. If resistance develops, the MIC of Staphylococcus aureus will become larger, indicating that the concentration at the first dose is no longer sufficient to inhibit the growth of Staphylococcus aureus. If resistance does not develop, the MIC value will not become larger. As can be seen from Figure 4, after 20 measurements, the MIC value of Staphylococcus aureus did not increase, indicating that Staphylococcus aureus did not develop drug resistance.

Although the present invention has been described in considerable detail and in particular to several of the described embodiments, it is not intended to be limited to any such details or embodiments or to any particular embodiment, but rather is to be considered by reference The appended claims are intended to provide the broadest possible interpretation of these claims, taking into account the prior art, to effectively cover the intended scope of the invention. In addition, the above description of the present invention is based on embodiments foreseeable by the inventor for the purpose of providing a useful description, and those non-substantive changes to the present invention that are not yet foreseen can still represent equivalent changes of the present invention.

Claims (9)

1. The application of a copper complex with alkyl chain modification in the preparation of a drug for inhibiting Staphylococcus aureus, characterized in that the copper complex with alkyl chain modification has the structure shown in formula I: Formula I; wherein n is selected from any positive integer from 3, 7, and 11. 2. Application according to claim 1, characterized in that the structure of the copper complex with alkyl chain modification is shown in Formula II: Formula II. 3. A method for preparing a copper complex with alkyl chain modification, characterized in that the copper complex with alkyl chain modification has the structure shown in formula I in claim 1, comprising the following steps: S1: Under argon conditions, heat the compound of formula I-a structure, the compound of formula I-d structure and potassium carbonate in acetonitrile to reflux. After cooling to room temperature, the solvent is evaporated under reduced pressure to obtain a crude product, which is purified to obtain the intermediate of formula I-e structure. body; S2: Under argon conditions, heat the compound of formula I-b structure, the compound of formula I-d structure and potassium carbonate in acetonitrile to reflux. After cooling to room temperature, the solvent is evaporated under reduced pressure to obtain a crude product, which is purified to obtain the intermediate structure of formula I-f. body; S3: Under argon conditions, heat the compound of the formula I-c structure, the compound of the formula I-d structure and potassium carbonate in acetonitrile to reflux. After cooling to room temperature, the solvent is evaporated under reduced pressure to obtain a crude product, which is purified to obtain the intermediate of the formula I-g structure. body; S4: Dissolve the intermediate of the structure of formula I-e or the intermediate of the structure of formula I-f or the intermediate of the structure of formula I-g in an acetonitrile solution, then add an acetonitrile solution of copper bromide, react at room temperature for 6 hours, filter, wash, and dry to obtain a solution with Copper complexes modified with alkyl chains; Among them, the molecular structural formulas of the compound of formula I-a structure, the compound of formula I-b structure, the compound of formula I-c structure, the compound of formula I-d structure, the intermediate of formula I-e structure, the intermediate of formula I-f structure and the intermediate of formula I-g structure are as follows Shown: . 4. The preparation method according to claim 3, characterized in that, in steps S1, S2 and S3, the molar ratios of the compound of formula I-a structure, the compound of formula I-b structure, the compound of formula I-c structure and the compound of formula I-d structure are all 1:1. 5. The preparation method according to claim 3, characterized in that the purification in steps S1, S2 and S3 is to elute and purify on an alumina chromatography column with an eluent of methylene chloride/methanol=200:1. . 6. The preparation method according to claim 3, characterized in that the heating and refluxing temperature in steps S1, S2 and S3 is 85°C. 7. The preparation method according to claim 3, characterized in that in step S4, the molar ratio of the intermediate of the formula I-e structure, the intermediate of the formula I-f structure and the intermediate of the formula I-g structure to copper bromide is 1:2. . 8. The application of a copper complex with alkyl chain modification in the preparation of drugs that inhibit α-hemolytic toxin, characterized in that the copper complex with alkyl chain modification is the structure shown in formula I in claim 1 . 9. The use of a copper complex with alkyl chain modification in the preparation of a bacteriostatic agent, characterized in that the copper complex with alkyl chain modification has a structure represented by formula I in claim 1.