Synthetic method of curcumin modified polymyxin B

Abstract

The invention discloses a synthetic method of curcumin modified polymyxin B. A synthesized medicine can be self-assembled into nano particles with the particle size of about 100 nm in water, the problem that curcumin is poor in water solubility is solved, the structure of the nano particles enables the polymyxin B to be more easily proportioned into an inhalation preparation, and the renal toxicity of the polymyxin B is reduced. The synthesis method comprises the following steps: (1) taking curcumin as an initial raw material, adding p-formylbenzoic acid, and carrying out esterification reaction to generate formyl-modified curcumin CF; (2) the obtained CF and polymyxin B are subjected to a Schiff base condensation reaction, and curcumin modified polymyxin B is synthesized; and (3) carrying out self-assembly on the product obtained in the step (2) to obtain the nanoparticles.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for synthesizing curcumin-modified polymyxin B. Background Art

[0002] Polymyxin B is a polypeptide antibiotic used to treat severe Gram-negative bacterial infections. Although it has significant efficacy against multidrug-resistant strains, it has some obvious shortcomings in practical applications. First, the toxicity of polymyxin B is prominent, especially its toxic side effects on the kidneys and nervous system. Secondly, the pharmacokinetic characteristics of polymyxin B are relatively complex and need to be administered by intravenous injection, which increases the inconvenience of treatment and may cause local injection site discomfort or allergic reactions. Thirdly, with the increase in clinical use, some pathogens have developed resistance to polymyxin B, weakening its antibacterial effect.

[0003] Curcumin (CUR), as a polyphenol compound extracted from turmeric, has multiple biological activities such as anti-inflammatory, antibacterial, and anticancer, but it has many deficiencies in practical applications, which limits its clinical promotion. First, the water solubility of curcumin is extremely low, resulting in poor absorption rate in the body, extremely limited bioavailability, and failure to achieve the desired therapeutic effect. Secondly, curcumin is easily metabolized and cleared rapidly in the gastrointestinal tract, further shortening its half-life in the body and limiting its sustained pharmacological effects. The chemical structure of curcumin is not stable enough, and it is easily decomposed by light, pH value and temperature, resulting in instability during its storage and use in the preparation. Thirdly, although curcumin is widely used in food and health products, its safety data still needs to be further evaluated when used at high doses for a long time. Therefore, it is urgent to improve the stability, bioavailability and efficacy of curcumin through technical means such as chemical modification and nanocarrier encapsulation to overcome its existing deficiencies and expand its application prospects in the medical field. Summary of the invention

[0004] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for synthesizing curcumin-modified polymyxin B. The synthesized drug can self-assemble into nanoparticles of about 100 nm in water, thereby improving the problem of poor water solubility of curcumin. In addition, the structure of the nanoparticles makes it easier to formulate polymyxin B into an inhalation preparation, thereby reducing the nephrotoxicity of polymyxin B.

[0005] The technical solution of the present invention is: a method for synthesizing curcumin-modified polymyxin B, which comprises the following steps:

[0006] (1) Synthesis of aldehyde-modified curcumin CF: Curcumin and p-formylbenzoic acid were mixed, and anhydrous tetrahydrofuran was added to generate a red solution; then, a dehydrating agent, dicyclohexylcarbodiimide, and a catalyst, 4-dimethylaminopyridine, were added, and the mixture was reacted at room temperature for 48 hours, and then impurities were removed, and silica gel powder was added and mixed evenly so that the sample was fully coated on the silica gel powder, and the organic solvent was removed by rotary evaporation to obtain red silica gel powder; the product was separated by column chromatography to obtain yellow powder CF;

[0007] (2) Synthesis of curcumin-modified polymyxin B: CF was dissolved in dimethyl sulfoxide and placed in a glass bottle. Polymyxin B sulfate was dissolved in deionized water and slowly added dropwise to the solution in the glass bottle. The mixture was stirred and reacted at room temperature for 36 hours. The reacted solution was dialyzed to remove dimethyl sulfoxide and unreacted small molecules. After 48 hours, a dialyzate was obtained and freeze-dried to obtain a yellow powder of curcumin-modified polymyxin B.

[0008] (3) Preparation of nanoparticles: The yellow powder of step (2) is dissolved in deionized water and shaken to dissolve to obtain curcumin-modified polymyxin B nanoparticles.

[0009] Compared with the prior art, the beneficial effects of the present invention are embodied in that: the curcumin-modified polymyxin B (CPB) synthesized by the present invention connects curcumin and polymyxin B through chemical reaction bonds through esterification reaction and Schiff base condensation reaction, and the synthesized CPB can self-assemble into nanoparticles (NPs) of about 100 nm in water, thereby improving the problem of poor water solubility of curcumin, and the structure of NPS can make polymyxin B easier to be formulated into an inhalation preparation, thereby reducing the nephrotoxicity of polymyxin B. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The synthetic route of aldehyde-modified curcumin (CF) is shown.

[0011] Figure 2 The synthetic route of curcumin-modified polymyxin B (CPB) is shown.

[0012] Figure 3 shows that CF is 6- H NMR spectrum of DMSO ( 1 1H NMR).

[0013] Figure 4 shows that CF is 6- NMR carbon spectrum in DMSO ( 13 C NMR).

[0014] Figure 5 shows that CPB in d 6- H NMR spectrum of DMSO ( 1 1H NMR).

[0015] Figure 6 X-ray photoelectron spectroscopy (XPS) of CPB is shown.

[0016] Figure 7 Dynamic light scattering (DLS) characterization of CPB nanoparticles is shown.

[0017] Figure 8 Transmission electron microscopy (TEM) images of CPB nanoparticles are shown. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of conflict with any incorporated document, the contents of this specification shall prevail. The experimental methods for which specific conditions are not indicated in the examples are selected according to conventional methods and conditions, or according to the product specifications.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The present invention provides a method for synthesizing curcumin-modified polymyxin B, which comprises the following steps:

[0024] (1) Synthesis of aldehyde-modified curcumin CF: Curcumin and p-formylbenzoic acid were mixed, and anhydrous tetrahydrofuran was added to generate a red solution; then, a dehydrating agent, dicyclohexylcarbodiimide, and a catalyst, 4-dimethylaminopyridine, were added, and the mixture was reacted at room temperature for 48 hours, and then impurities were removed, and silica gel powder was added and mixed evenly so that the sample was fully coated on the silica gel powder, and the organic solvent was removed by rotary evaporation to obtain red silica gel powder; the product was separated by column chromatography to obtain yellow powder CF;

[0025] (2) Synthesis of curcumin-modified polymyxin B: CF was dissolved in dimethyl sulfoxide and placed in a glass bottle. Polymyxin B sulfate was dissolved in deionized water and slowly added dropwise to the solution in the glass bottle. The mixture was stirred and reacted at room temperature for 36 hours. The reacted solution was dialyzed to remove dimethyl sulfoxide and unreacted small molecules. After 48 hours, a dialyzate was obtained and freeze-dried to obtain a yellow powder of curcumin-modified polymyxin B.

[0026] (3) Preparation of nanoparticles: The yellow powder of step (2) is dissolved in deionized water and shaken to dissolve to obtain curcumin-modified polymyxin B nanoparticles.

[0027] Compared with the prior art, the beneficial effects of the present invention are embodied in that: the curcumin-modified polymyxin B (CPB) synthesized by the present invention connects curcumin and polymyxin B through chemical reaction bonds through esterification reaction and Schiff base condensation reaction, and the synthesized CPB can self-assemble into nanoparticles (NPs) of about 100 nm in water, thereby improving the problem of poor water solubility of curcumin, and the structure of NPS can make polymyxin B easier to be formulated into an inhalation preparation, thereby reducing the nephrotoxicity of polymyxin B.

[0028] Preferably, in step (1), 5.075 mmol of curcumin and 2.03 mmol of p-formylbenzoic acid are placed in a 100 mL round-bottom flask, and 50 mL of anhydrous tetrahydrofuran (THF) is added; subsequently, 2.64 mmol of dicyclohexylcarbodiimide and 0.245 mmol of catalyst 4-dimethylaminopyridine are added.

[0029] Preferably, in step (1), after reacting at room temperature for 48 hours, the mixture is filtered twice to remove impurities, and 10 g of silica gel powder is added.

[0030] Preferably, in the step (1), in the column chromatography separation, the volume ratio of the eluent dichloromethane:ethyl acetate is 20:1.

[0031] Preferably, in step (2), 0.032 mmol CF is dissolved in 4 mL dimethyl sulfoxide and placed in a 20 mL glass bottle; 0.048 mmol polymyxin B sulfate is dissolved in 1 mL deionized water.

[0032] Preferably, in step (2), the molecular weight cut-off during dialysis is 3000.

[0033] Preferably, in the step (3), the size and morphology of the NPS are observed by dynamic light scattering and transmission electron microscopy, and curcumin-modified polymyxin B forms particles with a diameter of 100 nm in water.

[0034] Preferably, the molar ratio of curcumin: p-formylbenzoic acid: dicyclohexylcarbodiimide: 4-dimethylaminopyridine is controlled between 1.5:1:1:0.1 and 3:1:1.5:0.2.

[0035] Preferably, the molar ratio of curcumin: p-formylbenzoic acid is controlled between 1.5:1-3:1, and p-formylbenzoic acid: dicyclohexylcarbodiimide: 4-dimethylaminopyridine = 1:1.3:0.12.

[0036] The present invention is described in more detail below.

[0037] The synthetic method of the present invention is as follows:

[0038] 1. Synthesis of aldehyde-modified curcumin CF

[0039] Synthesis methods such as Figure 1 As shown. In detail, curcumin (CUR, 1.869g, 5.075mmol) and paraformylbenzoic acid (4-Form, 0.305g, 2.03mmol) were placed in a 100mL round-bottom flask, and 50mL of anhydrous tetrahydrofuran (THF) was added to generate a red solution. Subsequently, a dehydrating agent: dicyclohexylcarbodiimide (DCC, 0.545g, 2.64mmol) and a catalyst 4-dimethylaminopyridine (DMAP, 0.03g, 0.245mmol) were added, and after reacting at room temperature for 48 hours, the mixture was filtered twice to remove impurities, 10g of silica gel powder was added, mixed evenly, the sample was fully stained on the silica gel powder, and the organic solvent was removed by rotary evaporation to obtain red silica gel powder. Finally, the product was separated by column chromatography (volume ratio of dichloromethane: ethyl acetate = 20:1) to obtain a yellow powder CF with a yield of 80%.

[0040] 2. Synthesis of curcumin-modified polymyxin B (CPB)

[0041] Synthesis methods such as Figure 2As shown. In detail, CF (16 mg, 0.032 mmol) was dissolved in 4 mL of dimethyl sulfoxide (DMSO) and placed in a 20 mL glass bottle. Polymyxin B sulfate (PXB, 64 mg, 0.048 mmol) was weighed, dissolved in 1 mL of deionized water, slowly added dropwise to the CF / DMSO solution, stirred appropriately, and reacted at room temperature for 36 hours. The reacted solution was placed in a dialysis bag (molecular weight cutoff 3000) for dialyzing to remove DMSO and unreacted small molecules. After 48 hours, a dialysate was obtained, which was freeze-dried to obtain CPB yellow powder with a yield of 85%.

[0042] 3. Preparation and characterization of nanoparticles (CPB nps)

[0043] An appropriate amount of CPB powder was weighed, dissolved in an appropriate amount of deionized water, and shaken to dissolve to obtain CPB nps. The size and morphology of nps were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0044] Results and discussion:

[0045] (1) Synthesis of aldehyde-modified curcumin CF

[0046] In order to enable curcumin to react with polymyxin B in the subsequent reaction, aldehyde-modified curcumin CF was first synthesized by esterification reaction. 1 H NMR) and C NMR ( 13 C NMR) for the structure of CF, such as Figure 3 and Figure 4 shown. Figure 3 of 1 In the H NMR spectrum, the integrated areas of all peaks correspond to the number of hydrogen atoms in the CF structure, proving that the structure of CF was successfully synthesized. Figure 4 of 13 In the C NMR spectrum, the chemical shifts of all carbon atoms in the CF structure were clearly assigned, which further verified the successful synthesis of aldehyde-modified curcumin CF.

[0047] (2) Synthesis of curcumin-modified polymyxin B (CPB)

[0048] Furthermore, polymyxin B was linked to aldehyde-modified curcumin CF by Schiff base condensation reaction to form curcumin-modified polymyxin B (CPB). 1 H NMR) was used to characterize the structure of the synthesized CPB. Figure 5As shown, the peak at 8.09 ppm represents the formation of Schiff base bonds, which means that polymyxin B is successfully introduced into curcumin, indicating that the CPB molecule has been successfully synthesized. Further, the CPB molecule was analyzed by X-ray photoelectron spectroscopy (XPS), as shown in FIG. Figure 6 As shown, in the full XPS spectrum, obvious C1s, N 1s, and O 1s characteristic peaks were observed. The appearance of N 1s indicated that PXB was successfully introduced into CF, confirming the successful grafting of polymyxin B and curcumin.

[0049] (3) Preparation and characterization of nanoparticles (CPB NPs)

[0050] In the molecular structure of CPB, curcumin CUR is a hydrophobic molecule, while polymyxin B is a hydrophilic molecule. Therefore, through the hydrophilic and hydrophobic interaction, CPB molecules can easily form self-assemblies in water and can be detected by dynamic light scattering (DLS, Figure 7 ) and transmission electron microscopy (TEM, Figure 8 ) characterized its structure and the results showed that CPB can form particles with a diameter of about 100 nm in water, verifying its self-assembly ability and structural stability.

[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing curcumin-modified polymyxin B, characterized in that: It includes the following steps: (1) Synthesis of aldehyde-modified curcumin CF: Curcumin and p-formylbenzoic acid were mixed, and anhydrous tetrahydrofuran was added to generate a red solution; then, a dehydrating agent, dicyclohexylcarbodiimide, and a catalyst, 4-dimethylaminopyridine, were added, and the mixture was reacted at room temperature for 48 hours, and then impurities were removed, and silica gel powder was added and mixed evenly so that the sample was fully coated on the silica gel powder, and the organic solvent was removed by rotary evaporation to obtain red silica gel powder; the product was separated by column chromatography to obtain yellow powder CF; (2) Synthesis of curcumin-modified polymyxin B: CF was dissolved in dimethyl sulfoxide and placed in a glass bottle. Polymyxin B sulfate was dissolved in deionized water and slowly added dropwise to the solution in the glass bottle. The mixture was stirred and reacted at room temperature for 36 hours. The reacted solution was dialyzed to remove dimethyl sulfoxide and unreacted small molecules. After 48 hours, a dialyzate was obtained and freeze-dried to obtain a yellow powder of curcumin-modified polymyxin B. (3) Preparation of nanoparticles: The yellow powder of step (2) is dissolved in deionized water and shaken to dissolve to obtain curcumin-modified polymyxin B nanoparticles.

2. The method for synthesizing curcumin-modified polymyxin B according to claim 1, characterized in that: In the step (1), 5.075 mmol of curcumin and 2.03 mmol of p-formylbenzoic acid are placed in a 100 mL round-bottom flask, and 50 mL of anhydrous tetrahydrofuran (THF) is added; subsequently, 2.64 mmol of dicyclohexylcarbodiimide and 0.245 mmol of catalyst 4-dimethylaminopyridine are added.

3. The synthetic method of curcumin modified polymyxin B according to claim 2, characterized in that: In the step (1), after reacting at room temperature for 48 hours, the mixture was filtered twice to remove impurities, and 10 g of silica gel powder was added.

4. The method for synthesizing curcumin-modified polymyxin B according to claim 3, characterized in that: In the step (1), in the column chromatography separation, the volume ratio of dichloromethane:ethyl acetate is 20:

1.

5. The method for synthesizing curcumin-modified polymyxin B according to claim 4, characterized in that: In the step (2), 0.032 mmol CF was dissolved in 4 mL dimethyl sulfoxide and placed in a 20 mL glass bottle; 0.048 mmol polymyxin B sulfate was dissolved in 1 mL deionized water.

6. The method for synthesizing curcumin-modified polymyxin B according to claim 5, characterized in that: In the step (2), the molecular weight cut-off during dialysis is 3000.

7. The method for synthesizing curcumin-modified polymyxin B according to claim 1, characterized in that: In the step (3), the size and morphology of NPS were observed by dynamic light scattering and transmission electron microscopy, and curcumin-modified polymyxin B formed particles with a diameter of 100 nm in water.

8. The method for synthesizing curcumin-modified polymyxin B according to claim 1, characterized in that: The feeding molar ratio of curcumin: p-formylbenzoic acid: dicyclohexylcarbodiimide: 4-dimethylaminopyridine is controlled between 1.5:1:1:0.1 and 3:1:1.5:0.

2.

9. The method for synthesizing curcumin-modified polymyxin B according to claim 1, wherein the molar ratio of curcumin to p-formylbenzoic acid is controlled between 1.5:1 and 3:1, and the ratio of p-formylbenzoic acid to dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:1.3:0.12.

Images