A method for synthesizing curcumin-modified polymyxin B

Abstract

The application discloses a synthesis method of curcumin modified polymyxin B. The synthesized drug can self-assemble into nanoparticles with a size of about 100 nm in water, improves the poor water solubility of curcumin, and makes polymyxin B easier to be proportioned into an inhalation preparation, and reduces the nephrotoxicity of polymyxin B. The synthesis method comprises the following steps: (1) taking curcumin as a starting material, adding p-aldehyde benzene acid, and generating aldehyde-modified curcumin CF through esterification; (2) synthesizing curcumin modified polymyxin B through Schiff base condensation reaction of the obtained CF and polymyxin B; and (3) performing self-assembly on the product obtained in the step (2) to obtain nanoparticles.

Description

Technical Field

[0001] This invention relates to the technical field of pharmaceuticals, specifically to a method for synthesizing curcumin-modified polymyxin B. Background Technology

[0002] Polymyxin B is a polypeptide antibiotic used to treat severe Gram-negative bacterial infections. Although it is highly effective against multidrug-resistant strains, its practical application has several significant limitations. First, polymyxin B has prominent toxicity issues, particularly significant renal and nervous system toxicity. Second, its pharmacokinetic characteristics are complex, requiring intravenous administration, which increases treatment inconvenience and may cause local injection site discomfort or allergic reactions. Third, with increased clinical use, some pathogens have developed resistance to polymyxin B, weakening its antibacterial efficacy.

[0003] Curcumin (CUR), a polyphenolic compound extracted from turmeric, possesses various biological activities such as anti-inflammatory, antibacterial, and anticancer properties. However, its practical application suffers from several shortcomings, limiting its clinical application. First, curcumin's extremely low water solubility leads to poor absorption and limited bioavailability in the body, failing to achieve ideal therapeutic effects. Second, curcumin is rapidly metabolized and eliminated in the gastrointestinal tract, further shortening its half-life and limiting its sustained pharmacological effects. The chemical structure of curcumin is not sufficiently stable, easily decomposing under the influence of light, pH, and temperature, resulting in instability during its storage and use in formulations. Third, although curcumin is widely used in food and health products, its safety data requires further evaluation for long-term use at high doses. Therefore, it is urgent to improve the stability, bioavailability, and efficacy of curcumin through chemical modification, nanocarrier encapsulation, and other technologies to overcome its current shortcomings and expand its application prospects in the pharmaceutical field. Summary of the Invention

[0004] To overcome the shortcomings 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, which improves the problem of poor water solubility of curcumin. Furthermore, the structure of the nanoparticles makes it easier to formulate polymyxin B into an inhalation preparation and reduces the nephrotoxicity of polymyxin B.

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

[0006] (1) Synthesis of aldehyde-modified curcumin CF: Curcumin and p-aldehyde benzoic acid were mixed and anhydrous tetrahydrofuran was added to generate a red solution; then, dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added as dehydrating agents and catalysts, and the mixture was reacted at room temperature for 48 hours. After removing impurities, silica gel powder was added and mixed evenly to ensure that the sample was fully coated on the silica gel powder. 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 solution was dialyzed to remove dimethyl sulfoxide and unreacted small molecules. After 48 hours, the dialysate was obtained and freeze-dried to obtain a yellow powder of curcumin-modified polymyxin B.

[0008] (3) Preparation of nanoparticles: Dissolve the yellow powder from step (2) in deionized water and shake to dissolve, thereby obtaining curcumin-modified polymyxin B nanoparticles.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: The curcumin-modified polymyxin B (CPB) synthesized by the present invention links curcumin and polymyxin B through chemical reaction bonds via esterification and Schiff base condensation reactions. The synthesized CPB can self-assemble into nanoparticles (NPS) of about 100 nm in water, which improves the problem of poor water solubility of curcumin. Furthermore, the structure of NPS makes it easier to formulate polymyxin B into inhalation formulations and reduces the nephrotoxicity of polymyxin B. Attached Figure Description

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

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

[0012] Figure 3 It shows CF in d 6- 1H NMR spectrum in dmso 1 H NMR).

[0013] Figure 4 It shows CF in d 6- Carbon NMR spectrum in dmso 13 (C NMR).

[0014] Figure 5 The CPB in d is shown 6- 1H NMR spectrum in dmso 1 H NMR).

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

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

[0017] Figure 8 The image shows a transmission electron microscope (TEM) image of CPB nanoparticles. Detailed Implementation

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

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail. Experimental methods not specifically described in the examples are performed according to conventional methods and conditions, or as selected according to the trade instructions.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

[0024] (1) Synthesis of aldehyde-modified curcumin CF: Curcumin and p-aldehyde benzoic acid were mixed and anhydrous tetrahydrofuran was added to generate a red solution; then, dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added as dehydrating agents and catalysts, and the mixture was reacted at room temperature for 48 hours. After removing impurities, silica gel powder was added and mixed evenly to ensure that the sample was fully coated on the silica gel powder. 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 solution was dialyzed to remove dimethyl sulfoxide and unreacted small molecules. After 48 hours, the dialysate was obtained and freeze-dried to obtain a yellow powder of curcumin-modified polymyxin B.

[0026] (3) Preparation of nanoparticles: Dissolve the yellow powder from step (2) in deionized water and shake to dissolve, thereby obtaining curcumin-modified polymyxin B nanoparticles.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The curcumin-modified polymyxin B (CPB) synthesized by the present invention links curcumin and polymyxin B through chemical reaction bonds via esterification and Schiff base condensation reactions. The synthesized CPB can self-assemble into nanoparticles (NPS) of about 100 nm in water, which improves the problem of poor water solubility of curcumin. Furthermore, the structure of NPS makes it easier to formulate polymyxin B into inhalation formulations and reduces the nephrotoxicity of polymyxin B.

[0028] Preferably, in step (1), 5.075 mmol curcumin and 2.03 mmol p-aldehyde benzoic acid are placed in a 100 mL round-bottom flask, and 50 mL of anhydrous tetrahydrofuran (THF) is added; subsequently, 2.64 mmol dicyclohexylcarbodiimide and 0.245 mmol 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 10g of silica powder is added.

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

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

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

[0033] Preferably, in step (3), the size and morphology of 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-aldehyde benzoic 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 to p-aldehyde benzoic acid is controlled between 1.5:1 and 3:1, and the ratio of p-aldehyde benzoic acid to dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:1.3:0.12.

[0036] The present invention will now be described in more detail.

[0037] The synthesis 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. Specifically, curcumin (CUR, 1.869 g, 5.075 mmol) and p-formaldehyde benzoic acid (4-Form, 0.305 g, 2.03 mmol) were placed in a 100 mL round-bottom flask, and 50 mL of anhydrous tetrahydrofuran (THF) was added, producing a red solution. Subsequently, a dehydrating agent, dicyclohexylcarbodiimide (DCC, 0.545 g, 2.64 mmol), and a catalyst, 4-dimethylaminopyridine (DMAP, 0.03 g, 0.245 mmol), were added. After reacting at room temperature for 48 hours, the mixture was filtered twice to remove impurities. 10 g of silica gel powder was added, and the mixture was thoroughly mixed to ensure the sample was fully coated onto the silica gel. The organic solvent was removed by rotary evaporation, yielding a red silica gel powder. Finally, the product was separated by column chromatography (dichloromethane:ethyl acetate, volume ratio = 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. Specifically, 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, and slowly added dropwise to the CF / DMSO solution. The mixture was stirred appropriately and reacted at room temperature for 36 hours. The resulting solution was then dialyzed in a dialysis bag (molecular weight cutoff 3000) to remove DMSO and unreacted small molecules. After 48 hours, the dialysate was obtained, freeze-dried, and a yellow CPB powder was obtained with a yield of 85%.

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

[0043] Weigh an appropriate amount of CPB powder, dissolve it in an appropriate amount of deionized water, and shake to dissolve, obtaining CPB nps. Observe the size and morphology of the nps by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0044] Results analysis and discussion:

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

[0046] To enable curcumin to undergo a subsequent reaction with polymyxin B, aldehyde-modified curcumin CF was first synthesized via esterification. The reaction was then analyzed using 1H NMR spectroscopy. 1 H NMR) and carbon NMR ( 13 The structure of CF by C NMR, such as Figure 3 and Figure 4 As 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 CF structure 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 via a Schiff base condensation reaction to form curcumin-modified polymyxin B (CPB). The results were analyzed by 1H NMR spectroscopy. 1 The synthesized CPB was characterized by ¹H NMR, and the results are as follows: Figure 5As shown, the peak at 8.09 ppm represents the formation of a Schiff base bond, indicating the successful introduction of polymyxin B into curcumin, signifying the successful synthesis of the CPB molecule. Further, elemental analysis of the CPB molecule was performed using X-ray photoelectron spectroscopy (XPS), such as... Figure 6 As shown, obvious C1s, N1s, and O1s characteristic peaks were observed in the XPS full spectrum. The appearance of N1s indicates 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 hydrophilic-hydrophobic interactions, CPB molecules readily form self-assemblies in water and undergo dynamic light scattering (DLS). Figure 7 ) and transmission electron microscopy (TEM) Figure 8 Its structure was characterized, 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 merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope 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-aldehyde benzoic acid were mixed and anhydrous tetrahydrofuran was added to generate a red solution; then, dicyclohexylcarbodiimide as a dehydrating agent and 4-dimethylaminopyridine as a catalyst were added, and after reacting at room temperature for 48 hours, impurities were removed, silica gel powder was added, and the mixture was mixed evenly to ensure that the sample was fully coated on the silica gel powder. 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. The structural formula of CF is as follows: ； (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, the dialysate was obtained and freeze-dried to obtain curcumin-modified polymyxin B yellow powder CPB. The structural formula of CPB is as follows: ； (3) Preparation of nanoparticles: Dissolve the yellow powder CPB from step (2) in deionized water and shake to dissolve, thereby obtaining curcumin-modified polymyxin B nanoparticles.

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

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

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

1.

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

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

7. The method for synthesizing curcumin-modified polymyxin B according to claim 1, characterized in that: In step (3), the size and morphology of the nanoparticles are observed by dynamic light scattering and transmission electron microscopy. Curcumin-modified polymyxin B forms 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 molar ratio of curcumin, p-aldehyde benzoic acid, dicyclohexylcarbodiimide, and 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, characterized in that: the molar ratio of curcumin to p-aldehyde benzoic acid is controlled between 1.5:1 and 3:1, and the ratio of p-aldehyde benzoic acid to dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:1.3:0.12.

Images