Metal-based nitroimidazole derivative nano-enzyme as well as preparation method and application thereof
By preparing copper or manganese-based nitroimidazole derivative nanozymes, the problems of drug resistance and poor solubility of nitroimidazole antibacterial drugs were solved, efficient bacterial targeting and bactericidal effects were achieved, and wound healing was promoted.
Patent Information
- Application Number
- CN202510869261.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-26
- Publication Date
- 2025-09-26
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of metal nanozymes, and in particular to a metal-based nitroimidazole derivative nanozyme and a preparation method and application thereof. Background Art
[0002] Bacterial infections remain a major global public health challenge. Although antibiotic therapy remains the dominant treatment for these diseases, long-term, excessive, and unregulated use has led to the rapid evolution and spread of drug-resistant superbugs. The development of antibiotic resistance has made addressing bacterial infections increasingly challenging. Nitroimidazole antimicrobials (such as metronidazole, tinidazole, and ornidazole) are broad-spectrum anti-anaerobic and antiprotozoal drugs widely used in the clinical treatment of infectious diseases. However, these drugs suffer from poor water solubility, uneven tissue distribution, a high incidence of adverse reactions, and the potential for bacterial resistance with long-term use. Further research and development efforts are needed to expand the nitroimidazole family. In recent years, research has focused on addressing drug resistance and solubility issues, developing new dosage forms, and modifying the nitroimidazole backbone. Phenylboronic acid and its derivatives can form reversible covalent bonds with peptidoglycan disaccharides (such as MurNAc) in bacterial cell walls or cis-diols in lipopolysaccharide (LPS), increasing the local concentration of the drug on the bacterial surface, enhancing bacterial targeting, and reducing the development of drug resistance. The commonly used method at present is to functionalize nanoparticles with phenylboronic acid, but due to the tolerance of nanoparticles, the types and methods of functionalization are very limited.
[0003] Metal-based nanoparticles (NPs) have demonstrated unique abilities to target diverse biomolecules and bacteria, posing a threat not only to the development of drug-resistant strains but also to bacterial toxicity through diverse mechanisms, including the generation of reactive oxygen species (ROS), including H₂O₂, hydroxyl radicals, and singlet oxygen, as well as ion leaching. These NPs have largely addressed the challenge of drug resistance stemming from overuse of antibiotics and provided therapeutic strategies for bacterial wound infections. Fighting bacterial infection is a dynamic process, requiring ROS generation to kill bacteria during the initial infection phase and scavenging them to promote wound healing during the later stages. Therefore, the development of nanozymes with varying activity levels tailored to specific needs is crucial.
[0004] In response to the above situation, the present invention proposes a method for preparing a copper or manganese-based nitroimidazolephenylboronic acid (dopamine) derivative nanozyme with POD-like or CAT-like activity. Summary of the Invention
[0005] The problem existing in the prior art is: based on the problem pointed out in the background technology section that the currently used nitroimidazole antibacterial drugs are prone to bacterial resistance and poor solubility, the present invention uses the nitroimidazole skeleton as raw material, modifies it through a series of reactions to obtain a new nitroimidazole derivative, enhances its coordination ability with metals, and then prepares a copper or manganese-based nitroimidazole derivative nanozyme with POD-like or CAT-like activity with copper salts or manganese salts through self-assembly and applies it to antibacterial research.
[0006] The present invention provides a metal-based nitroimidazole derivative nanozyme, which is obtained by coordination self-assembly of a metal salt and a nitroimidazole derivative, wherein the metal salt is a soluble copper salt or manganese salt; the structural formula of the nitroimidazole derivative is: ; where R 1 Any one of H, Cl, Br; When X is O, R 2 is ethyl; or when X is NH, R 2 for 、 .
[0007] Furthermore, when the metal in the metal-based nitroimidazole derivative nanozyme is copper, the nitroimidazole derivative X is O, R 2 is ethyl; or X is NH, R 2 for .
[0008] When the metal in the metal-based nitroimidazole derivative nanozyme is manganese, the nitroimidazole derivative X is NH, R 2 for .
[0009] The present invention also provides a preparation method of the novel copper or manganese-based nitroimidazole derivative nanozyme as follows: dissolving the nitroimidazole derivative in anhydrous N,N-dimethylformamide (DMF) or methanol solution to obtain solution A; dissolving the metal salt (anhydrous copper chloride or manganese chloride tetrahydrate) solid in a mixed solution of deionized water and acetonitrile or a methanol solution, adding triethylamine aqueous solution dropwise, and stirring with a magnetic stirrer to obtain solution B; adding solution A dropwise to solution B and stirring, placing the obtained colloidal solution in a centrifuge tube, centrifuging to remove the supernatant, adding methanol for redissolution, centrifuging again and removing the supernatant to obtain a solid precipitate, washing the obtained solid precipitate with deionized water multiple times, adding deionized water for redissolution, and then freeze-drying to obtain the copper or manganese-based nitroimidazole derivative nanozyme.
[0010] Preferably, the concentration of the triethylamine aqueous solution is 0.07 M; and the molar ratio of the metallic copper or manganese to triethylamine is 1:1 to 1.5.
[0011] Preferably, the molar ratio of metallic copper or manganese to the nitroimidazole derivative is 1:1.
[0012] Preferably, the stirring time is 5 min.
[0013] Preferably, the stirring temperature is room temperature.
[0014] Preferably, the synthetic route of nitroimidazole derivatives is as follows:
[0015] Among them, R 1 is any one of H, Cl, Br; R 2 for 、 .
[0016] The synthesis method of the nitroimidazole derivatives: The nitroimidazole skeleton undergoes a substitution reaction with tert-butyl 2-bromoethylcarbamate to generate intermediate S1, which is then deprotected under the action of trifluoroacetic acid to obtain intermediate S2, which then reacts with monoethyl oxalyl chloride to generate nitroimidazole derivative 1, and finally undergoes an ester exchange reaction with different amines to obtain nitroimidazole derivative 2.
[0017] Multiple characterizations have confirmed that the average particle size of the copper-based nitroimidazole derivative nanozyme prepared by this method is about 200 nm; the average particle size of the manganese-based nitroimidazole derivative nanozyme is about 600-900 nm. Both are nanoscale materials, can enter bacteria well and can be slowly degraded into corresponding copper ions and manganese ions under acidic conditions, and have high biocompatibility.
[0018] Another object of the present invention is to propose the application of copper- or manganese-based nitroimidazole derivative nanozymes prepared by the above method in the antibacterial field. The copper-based nitroimidazole derivative nanozyme has POD-like activity but no CAT-like activity; the manganese-based nitroimidazole derivative nanozyme has CAT-like activity but no POD-like activity. Both nitroimidazole derivative nanozymes have good activity in inhibiting Staphylococcus aureus.
[0019] The present invention has the following beneficial effects: The present invention introduces phenylboronic acid to target bacterial cell walls. Meanwhile, the copper-nitroimidazole nanoparticles dissociate in weakly acidic infected wounds, releasing copper ions and nitroimidazole, reducing the development of drug resistance. Unmodified nitroimidazole and manganese ions cannot form stable nanoparticles. Ketoamide and the hydroxyl groups on dopamine coordinate with the manganese ions to form a stable nanostructure.
[0020] Particle size is a key parameter for the antibacterial effect of nano-antimicrobial materials. Smaller nanoparticles have a significantly larger specific surface area, exposing more active sites and enhancing contact efficiency with bacteria. The nanozymes prepared in this invention have a smaller average particle size and a more uniform particle size distribution, which is more conducive to their antibacterial effect.
[0021] The copper or manganese-based nitroimidazole derivative nanozymes of the present invention can enhance antibacterial activity through a dual mechanism: on the one hand, metal ions promote the reduction activation of nitro groups, enhancing the bactericidal effect of nitroimidazole derivatives; on the other hand, the released metal ions directly destroy the bacterial membrane, stimulate POD-like or CAT-like activity, and thus induce bacterial death.
[0022] The copper- or manganese-based nitroimidazole derivative nanozyme of the present invention is simple to prepare and easy to operate. The raw materials used are non-toxic, environmentally friendly, and low-cost, and the preparation process is environmentally friendly. The copper-based nitroimidazole derivative nanozyme obtained by the present invention has POD-like activity but no CAT-like activity. It generates ROS without scavenging ROS, which can enhance the antibacterial activity of nitroimidazole compounds. The manganese-based nitroimidazole derivative nanozyme has CAT-like activity but no POD-like activity. It can release oxygen to alleviate the hypoxic environment of infected wounds, reduce inflammatory reactions, promote angiogenesis in the wound, and is more conducive to the late healing of bacterial-infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 TEM image of Cu-2NIE NPs.
[0024] Figure 2 Particle size distribution diagram of Cu-2NIE NPs and Mn-2BrNI NPs.
[0025] Figure 3 Figure 2 shows the POD activity test of Cu-2NIE NPs and Cu-2NIB NPs.
[0026] Figure 4 CAT activity test diagram of Mn-2NI NPs and Mn-2BrNI NPs.
[0027] Figure 5 The diagram shows the bactericidal effect of Cu-2NIE NPs and Cu-2NIB NPs.
[0028] Figure 6 The diagram shows the bactericidal effect of Mn-2NI NPs and Mn-2BrNI NPs. DETAILED DESCRIPTION
[0029] The present invention will be described in detail below with reference to the following examples. However, it should be understood that the following examples are merely illustrative of the embodiments of the present invention and are not intended to limit the scope of the present invention.
[0030] The synthetic routes of the nitroimidazole derivatives described in the following examples of the present invention are:
[0031] where R 1 is any one of H, Cl, Br; R 2 for 、 .
[0032] Synthesis of nitroimidazole derivatives: The nitroimidazole skeleton undergoes a substitution reaction with tert-butyl 2-bromoethylcarbamate to generate intermediate S1, which is then deprotected under the action of trifluoroacetic acid to obtain intermediate S2, which is then reacted with ethyl oxalyl chloride to generate nitroimidazole derivative 1 (ethyl 2-((2-(2-nitro-1H-imidazol-1-yl)ethyl)amino)-2-oxoacetate), and finally undergoes an ester exchange reaction with different amines (4-aminomethylphenylboronic acid, dihydroxyamphetamine) to obtain the target product nitroimidazole. Oxazole derivatives 2 (N1-(2-(2-nitro-1H-imidazol-1-yl)ethyl)-N2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxamide, N1-(3,4-dihydroxyphenethyl)-N2-(2-(2-nitro-1H-imidazol-1-yl)ethyl)oxamide, N1-(2-(2-bromo-4-nitro-1H-imidazol-1-yl)ethyl)-N2-(3,4-dihydroxyphenethyl)oxamide).
[0033] The nitroimidazole derivatives synthesized by the invention are all insoluble in water.
[0034] Example 1
[0035] 25.6 mg of ethyl 2-((2-(2-nitro-1H-imidazol-1-yl)ethyl)amino)-2-oxoacetate was weighed and dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) to obtain solution A. 13.4 mg of anhydrous copper chloride solid was dissolved in a mixture of 15 mL of deionized water and 5 mL of acetonitrile. 2 mL of 0.07 M triethylamine aqueous solution was added dropwise, and the mixture was stirred on a magnetic stirrer for 5 min to obtain solution B. Solution A was added dropwise to solution B with stirring. The resulting colloidal solution was placed in a centrifuge tube and centrifuged at 12,000 rpm for 10 min. The supernatant was removed and methanol was added for reconstitution. After centrifugation, the supernatant was removed again to obtain a solid precipitate. The obtained solid precipitate was washed with deionized water several times, reconstituted with deionized water, and then freeze-dried to obtain copper or manganese-based nitroimidazole derivative nanozymes, which were designated as Cu-2NIE NPs.
[0036] The TEM image of Cu-2NIE NPs obtained in Example 1 is shown in the attached specification. Figure 1 As shown in the figure, the morphology is approximately spherical, the particle size is about 100 nm, and the distribution is uniform.
[0037] The particle size distribution of Cu-2NIE NPs obtained in Example 1 is shown in the attached specification. Figure 2 As shown in the figure, the average particle size of the obtained Cu-2NIE NPs is 202.6 nm and the PDI value is 0.285.
[0038] The POD activity of Cu-2NIE NPs obtained in Example 1 is shown in the attached manual. Figure 3 As shown in the figure, it can be seen that Cu-2NIE NPs have good POD activity.
[0039] The CAT activity test results of the Cu-2NIE NPs obtained in Example 1 showed that they had no CAT activity.
[0040] The bactericidal effect of Cu-2NIE NPs obtained in Example 1 is as shown in the attached manual. Figure 5 As shown in the image, Cu-2NIENPs has a good bactericidal effect.
[0041] Example 2
[0042] 44.3 mg of N1-(2-(2-nitro-1H-imidazol-1-yl)ethyl)-N2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxamide was weighed and dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) to obtain solution A. 13.4 mg of anhydrous copper chloride solid was dissolved in a mixture of 15 mL of deionized water and 5 mL of acetonitrile, and 2 mL of 0.07 M triethylamine aqueous solution was added dropwise. The mixture was stirred on a magnetic stirrer for 5 min to obtain solution B. Solution A was added dropwise to solution B with stirring. The resulting colloidal solution was placed in a centrifuge tube, centrifuged to remove the supernatant, and then re-dissolved in methanol. After centrifugation, the supernatant was removed again to obtain a solid precipitate. The obtained solid precipitate was washed with deionized water several times, re-dissolved in deionized water, and then freeze-dried to obtain a copper or manganese-based nitroimidazole derivative nanozyme, designated as Cu-2NIB. NPs.
[0043] The POD activity of Cu-2NIB NPs obtained in Example 2 is shown in the attached manual. Figure 3 As shown in the figure, it can be seen that Cu-2NIB NPs have better POD activity, but the POD activity of Cu-2NIB NPs is slightly lower than that of Cu-2NIE NPs.
[0044] The CAT activity test results of the Cu-2NIE NPs obtained in Example 2 showed that they had no CAT activity.
[0045] Example 3
[0046] Referring to the method of Example 1, 13.4 mg of anhydrous copper chloride solid was replaced with 19.8 mg of manganese chloride tetrahydrate solid, but no stable colloidal solution was obtained.
[0047] Example 4
[0048] Referring to the method of Example 3, the solvent of solution A was replaced with methanol, but no stable colloidal solution was obtained.
[0049] Example 5
[0050] Referring to the method of Example 4, the solvent of solution B was replaced with methanol, but no stable colloidal solution was obtained.
[0051] Example 6
[0052] Referring to the method of Example 5, 25.6 mg of ethyl 2-((2-(2-nitro-1H-imidazol-1-yl)ethyl)amino)-2-oxoacetate was replaced with 36.6 mg of N1-(3,4-dihydroxyphenethyl)-N2-(2-(2-nitro-1H-imidazol-1-yl)ethyl)oxamide to obtain stable manganese-based nitroimidazole derivative nanozymes, which were designated as Mn-2NI NPs.
[0053] The CAT activity of Mn-2NI NPs obtained in Example 6 is as shown in the attached manual. Figure 4 As shown in the figure, Mn-2NI NPs have good CAT activity. The results of POD activity test show that they have no POD activity.
[0054] Example 7
[0055] Referring to the method of Example 6, 36.6 mg of N1-(3,4-dihydroxyphenethyl)-N2-(2-(2-nitro-1H-imidazol-1-yl)ethyl)oxalamide was replaced with 44.1 mg of N1-(2-(2-bromo-4-nitro-1H-imidazol-1-yl)ethyl)-N2-(3,4-dihydroxyphenethyl)oxalamide to obtain stable manganese-nitroimidazole derivative nanozymes, recorded as Mn-2BrNI NPs.
[0056] The CAT activity of Mn-2BrNI NPs obtained in Example 7 is as shown in the attached manual. Figure 4 As shown in the figure, Mn-2BrNINPs have almost no CAT activity. The results of POD activity assay showed that they have no POD activity.
[0057] Comparative Example 1
[0058] Referring to the method of Example 1, 25.6 mg of ethyl 2-((2-(2-nitro-1H-imidazol-1-yl)ethyl)amino)-2-oxoacetate was replaced with 11.3 mg of 2-nitroimidazole to obtain copper nitroimidazole nanoparticles, designated as Cu-2NI NPs, for antibacterial activity comparison.
[0059] The metal nanozymes obtained in Examples 1, 2, 6, and 7 were tested for POD activity and CAT activity, respectively. The specific test results are shown in Table 1.
[0060] in, The test method for catalase (CAT) activity is as follows: The metal nanozymes obtained in Examples 1, 2, 6, and 7 of the present invention were immersed in the same volume of a 100 mM H2O2 aqueous solution to observe whether bubbles were generated in the solution. If no bubbles were generated, it was proved that the material had no CAT activity. On the contrary, the more bubbles generated in the solution, the stronger the ability of the material to catalyze H2O2 to produce O2, and the stronger the CAT activity of the material.
[0061] The test method for peroxidase (POD) activity is as follows: The metal nanozymes obtained in Examples 1, 2, 6, and 7 of the present invention were added to the same volume of 100 mM H2O2 aqueous solution to obtain 6 test solutions. The same mass of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each test solution. After standing at room temperature for 20 min, the 6 test solutions were tested for the presence of full-wavelength ultraviolet absorption at 650 nm.
[0062] The testing principle is as follows: If the material has POD activity, it will catalyze H2O2 to produce a large number of hydroxyl radicals in a short period of time. 3,3',5,5'-tetramethylbenzidine (TMB) in the test solution combines with the hydroxyl radicals and is oxidized to ox-TMB, which exhibits a characteristic UV absorption peak at 650 nm. If the material lacks POD activity, it cannot catalyze H2O2 to produce a large number of hydroxyl radicals. No ox-TMB will be generated in the test solution, and no characteristic UV absorption peak will appear at 650 nm.
[0063] The antibacterial activity of the nanozymes obtained in the above examples and comparative examples against Staphylococcus aureus was tested using an antibacterial plate test. The results are shown in Figure 5 and Figure 6 .
[0064] Table 1
[0065] With the above-described preferred embodiments of the present invention as a guide, and with reference to the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of this invention. The technical scope of this invention is not limited to the contents of the specification and must be determined according to the scope of the claims.
Claims
1. A metal-based nitroimidazole derivative nanozyme, characterized in that: It is obtained by self-assembly of a metal salt and a nitroimidazole derivative, wherein the metal salt is a soluble copper salt or manganese salt; the structural formula of the nitroimidazole derivative is: ; where R 1 is any one of H, Cl, Br; when X is O, R 2 is ethyl; or when X is NH, R 2 for 、 .
2. The metal-based nitroimidazole derivative nanozyme according to claim 1, characterized in that When the metal in the metal-based nitroimidazole derivative nanozyme is copper, the nitroimidazole derivative X is O, R 2 is ethyl; or X is NH, R 2 for ; The metal-based nitroimidazole derivative nanozyme has peroxidase-like activity but no catalase-like activity.
3. The metal-based nitroimidazole derivative nanozyme according to claim 1, characterized in that When the metal in the metal-based nitroimidazole derivative nanozyme is manganese, the nitroimidazole derivative X is NH, R 2 for ; The metal-based nitroimidazole derivative nanozyme has catalase-like activity but no peroxidase-like activity.
4. The metal-based nitroimidazole derivative nanozyme according to claim 1, characterized in that The preparation method of the nitroimidazole derivative involves a substitution reaction between the nitroimidazole skeleton and tert-butyl 2-bromoethylcarbamate to produce intermediate S1, followed by removal of the protecting group under the action of trifluoroacetic acid to produce intermediate S2, which is then reacted with monoethyl oxalyl chloride to produce nitroimidazole derivative 1, and finally undergoes an ester exchange reaction with different amines to produce nitroimidazole derivative 2.
5. The method for preparing the metal-based nitroimidazole derivative nanozyme according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: dissolving a nitroimidazole derivative in an organic solvent I to obtain a solution A; dissolving a metal salt solid in an organic solvent II, adding a triethylamine aqueous solution dropwise, and stirring the mixture to obtain a solution B; and adding the solution A dropwise to the solution B and stirring the mixture to obtain a colloidal solution. The solid precipitate is collected by centrifugation, washed, redissolved in water, and freeze-dried to obtain the metal-based nitroimidazole derivative nanozyme.
6. The method for preparing the metal-based nitroimidazole derivative nanozyme according to claim 5, characterized in that: The molar ratio of the metal ion in the metal salt to the nitroimidazole derivative is 1:
1.
7. The method for preparing the metal-based nitroimidazole derivative nanozyme according to claim 5, characterized in that: The molar ratio of the metal salt to triethylamine is 1:1 to 1.
5.
8. The method for preparing the metal-based nitroimidazole derivative nanozyme according to claim 5, characterized in that: The metal salt is anhydrous copper chloride or manganese chloride tetrahydrate; the organic solvent I is anhydrous N,N-dimethylformamide or methanol; and the organic solvent II is a mixed solution of deionized water and acetonitrile or methanol.
9. Use of the metal-based nitroimidazole derivative nanozyme according to any one of claims 1 to 3 in the preparation of antibacterial drugs.