Nano prodrug with broad-spectrum antibacterial activity as well as preparation method and application of nano prodrug

Abstract

The invention discloses a nano prodrug with broad-spectrum antibacterial activity as well as a preparation method and application of the nano prodrug, and relates to the technical field of nano medicines. A compound phenylboronic acid modified cibifloxacin is prepared by sequentially carrying out reactions with di-tert-butyl dicarbonate, 4-bromomethyl phenylboronic acid and hydrochloric acid, and the compound is subjected to a dialysis method to prepare the nano prodrug phenylboronic acid cibifloxacin (CPBP). In an infected microenvironment, CPBP can be activated by excessive hydrogen peroxide (H2O2) in the microenvironment to release an original drug cibifloxacin, broad-spectrum antibacterial activity is generated, and a specific bactericidal effect is achieved at an infected part.

Description

Technical Field

[0001] The present invention relates to the field of nano-medicine technology, and particularly relates to a nano-prodrug with broad-spectrum antibacterial activity, a preparation method thereof, and an application thereof. Background Art

[0002] With the development of nano-medicine, drug delivery strategies based on nano-carriers have shown good application prospects. The nano-drug delivery system constructed by using nano-technology can improve the drug solubility, prolong the drug half-life, increase its bioavailability, and is also expected to solve the drug resistance problem that appears in clinical treatment. However, the research on the pharmaceutic properties such as the effectiveness, safety, and pharmacokinetics of nano-carrier drugs is not deep enough and systematic enough.

[0003] In addition, nano-carrier drugs also face some "bottleneck" problems in future large-scale production applications, such as: ① low drug loading capacity of nano-carriers; ② complex preparation of carrier materials, poor controllability between batches, and high production cost; ③ carrier materials have potential systemic toxicity and immunogenicity. Therefore, the above problems have largely become the difficulties restricting the clinical transformation of nano-carrier drugs, and there are still major challenges in promoting the clinical transformation of nano-drugs. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems, and provide a nano-prodrug with broad-spectrum antibacterial activity, a preparation method thereof, and an application thereof.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] The present invention provides a nano-prodrug phenylboronic acid ciprofloxacin with broad-spectrum antibacterial activity, which is a compound prepared by sequentially reacting ciprofloxacin with broad-spectrum antibacterial activity as a starting material with di-tert-butyl dicarbonate, 4-bromomethylphenylboronic acid, and hydrochloric acid;

[0007] Its structural formula is as follows:

[0008]

[0009] Furthermore: the molecular weight of the nano-prodrug is 466.22.

[0010] A preparation method of a nano-prodrug with broad-spectrum antibacterial activity, characterized by comprising the following steps:

[0011] Step S1, under ice bath conditions, add di-tert-butyl dicarbonate to the tetrahydrofuran solution of ciprofloxacin, stir at room temperature for 4 h, add water, filter, and dry to obtain a white intermediate 1;

[0012]

[0013] Step S2: Stir the 1,4-dioxane mixture of intermediate 1, triethylamine, 2,4,6-trichlorobenzoyl chloride and DMAP at 25 °C for 15 min, add 4-hydroxymethylphenylboronic acid, stir at 25 °C for 1 h, then add brine to the mixture and extract with diethyl ether; after washing the organic layer with brine, dry it with Na2SO4 and concentrate it under vacuum; purify it by silica gel column to obtain intermediate 2 as a white solid;

[0014]

[0015] Step S3: Add 2M hydrochloric acid to intermediate 2 dissolved in THF and stir at room temperature for 2 h; evaporate THF under reduced pressure, extract 3 times with ethyl acetate, dry with Na2SO4, and evaporate to dryness under reduced pressure to obtain the product phenylboronic acid ciprofloxacin as a white solid;

[0016]

[0017] Step S4: Load the CPBP DMSO solution into a dialysis bag with a molecular weight cut-off of 100 and place the dialysis bag in a beaker containing a large amount of RO water for dialysis for 12 h, changing the RO water every hour; after dialysis, collect the solution in the dialysis bag to obtain the CPBP nano-prodrug aqueous solution.

[0018] Furthermore: In step S1, the molar ratio of ciprofloxacin to Boc2O is 2:3.

[0019] Furthermore: In step S2, the molar ratio of benzoic acid, triethylamine, 2,4,6-trichlorobenzoyl chloride and DMAP is 1.15:1.45:1.27:1.38, and 4-hydroxymethylphenylboronic acid is 20 mL.

[0020] The application of a nano-prodrug in broad-spectrum antibacterial.

[0021] Furthermore: The nano-prodrug improves the water solubility of the naked drug ciprofloxacin and effectively controls the broad-spectrum antibacterial activity of ciprofloxacin.

[0022] Compared with the prior art, the beneficial effects of this solution:

[0023] It improves the water solubility of naked ciprofloxacin and can effectively control the broad-spectrum antibacterial activity of ciprofloxacin. The principle is that the CPBP nano-prodrug can be activated by the excessive reactive oxygen species (H2O2) in the infected microenvironment to release the original drug. This mechanism of action can not only achieve the purpose of specific treatment of the infected site, but also effectively reduce the toxicity to normal tissues, and does not rely on external carrier materials, can achieve a higher drug loading, while avoiding the related toxicities and immunogenicity and other side effects brought by the carrier, and improves the biological safety of the drug; the preparation process is relatively simple, and a uniform and stable nanostructure can be formed by self-assembly, thereby reducing the complex preparation steps and preparation time of traditional carrier materials, which is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the nuclear magnetic resonance diagram of the CPBP molecule in the embodiment of the present invention, where A is 1 the nuclear magnetic resonance diagram of H, and B is 13 the nuclear magnetic resonance diagram of C;

[0025] Figure 2 is the scanning electron microscope diagram of the CPBP nano-prodrug in the embodiment of the present invention, where A is the scanning electron microscope diagram of the CPBP nano-prodrug, and B is the scanning electron microscope diagram after incubation in a medium containing 100 μM H2O2;

[0026] Figure 3 is the LB analysis result diagram of the killing activities of the CPBP nano-prodrug and the control group in the embodiment of the present invention against S. aureus (Staphylococcus aureus) and E. coli under different conditions;

[0027] Figure 4 is the live / dead staining analysis result diagram of the killing activities of the CPBP nano-prodrug and the control group in the embodiment of the present invention against S. aureus and E. coli under different conditions;

[0028] Figure 5 is the SEM analysis result diagram of the killing activities of the CPBP nano-prodrug and the control group in the embodiment of the present invention against S. aureus and E. coli under different conditions. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0030] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0031] Embodiment:

[0032] A nano - prodrug with broad - spectrum antibacterial activity, with a molecular weight of 466.22.

[0033] A compound prepared by sequentially reacting ciprofloxacin with broad - spectrum antibacterial activity as the starting material with di - tert - butyl dicarbonate, 4 - bromomethylphenylboronic acid, and hydrochloric acid.

[0034] Its structural formula is as follows:

[0035]

[0036] The synthesis of the prodrug molecule required for the nano - prodrug is carried out by the following steps:

[0037] First step: Under ice - bath conditions, add Boc2O (490 mg, 2.25 mmol) to a solution of ciprofloxacin (0.5 g, 1.5 mmol) in tetrahydrofuran (30 mL), and stir at room temperature for 4 h. Add 100 mL of water, filter, and dry to obtain 530 mg of white intermediate 1.

[0038] Second step: Stir a 1,4 - dioxane mixture (20 mL) of benzoic acid (500 mg, 1.15 mmol), triethylamine (150 mg, 1.45 mmol), 2,4,6 - trichlorobenzoyl chloride (300 mg, 1.27 mmol), and DMAP (170 mg, 1.38 mmol) at 25 °C for 15 min. Add 4 - hydroxymethylphenylboronic acid (210 mg, 1.38 mmol), stir at 25 °C for 1 h, then add brine (80 mL) to the mixture, and extract with ether (3×80 mL). Combine the organic layers, wash with brine (2×40 mL), dry with Na2SO4, and concentrate under vacuum. Purify by silica gel column to obtain intermediate 2 (380 mg, 70%), which is a white solid.

[0039] Third step: Add 2 M hydrochloric acid (5 mL, 10 mmol) to intermediate 2 (380 mg, 0.81 mmol) dissolved in THF (5 mL), and stir at room temperature for 2 h. Evaporate THF under reduced pressure, extract with ethyl acetate 3 times, dry with Na2SO4, and evaporate under reduced pressure to obtain 262 mg of the product nano - prodrug phenylboronic acid ciprofloxacin (CPBP) with a yield of 83%, which is a white solid.

[0040] The preparation method of the nano - prodrug of the present invention is as follows:

[0041] Load the CPBP DMSO solution (5 mg / mL) into a dialysis bag with a molecular weight cut-off of 100 and place the dialysis bag in a beaker filled with a large amount of RO water for dialysis for 12 h. Replace the RO water every hour. After dialysis, collect the solution in the dialysis bag and obtain the CPBP nano-prodrug after freeze-drying.

[0042] Summary

[0043] According to Figures 1-5 As shown, the CPBP nano-prodrug has a uniform spherical structure and has good H2O2 sensitivity. H2O2 can activate the CPBP nano-prodrug to produce antibacterial activity.

[0044] The above specific embodiments are only explanations of the present invention, and they do not limit the present invention. Those skilled in the art can make modifications to these embodiments without creative contributions according to their needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A nano-prodrug with broad-spectrum antibacterial activity, characterized in that: A compound prepared by using ciprofloxacin with broad-spectrum antibacterial activity as the starting material, and successively reacting with di-tert-butyl dicarbonate, 4-bromomethylphenylboronic acid, and hydrochloric acid; Its structural formula is as follows:

2. The nano prodrug with broad-spectrum antibacterial activity according to claim 1, characterized in that: The molecular weight of the nano-prodrug is 466.

22.

3. A preparation method of a nano prodrug with broad-spectrum antibacterial activity, characterized in that, It includes the following steps: Step S1: Under ice bath conditions, add di-tert-butyl dicarbonate to the tetrahydrofuran solution of ciprofloxacin, stir at room temperature for 4 h, add water, filter, and dry to obtain white intermediate 1; Step S2: Stir the mixture of intermediate 1, triethylamine, 2,4,6-trichlorobenzoyl chloride, DMAP, and 1,4-dioxane at 25 °C for 15 min, add 4-hydroxymethylphenylboronic acid, stir at 25 °C for 1 h, then add brine to the mixture and extract with ether; after washing the organic layer with brine, dry it with Na2SO4 and concentrate it under vacuum; purify it by silica gel column to obtain intermediate 2, which is a white solid; Step S3: Add 2 M hydrochloric acid to intermediate 2 dissolved in THF and stir at room temperature for 2 h; evaporate THF under reduced pressure, extract 3 times with ethyl acetate, dry with Na2SO4, and evaporate under reduced pressure to obtain the product phenylboronic acid ciprofloxacin, which is a white solid; Step S4: Load the DMSO solution of phenylboronic acid ciprofloxacin into a dialysis bag with a molecular weight of 100 and place the dialysis bag in a beaker filled with a large amount of RO water for dialysis for 12 h, changing the RO water every hour; after dialysis, collect the solution in the dialysis bag to obtain an aqueous solution of phenylboronic acid ciprofloxacin nano-prodrug.

4. The preparation method of a nano prodrug with broad-spectrum antibacterial activity as described in claim 3, characterized in that: In step S1, the molar ratio of ciprofloxacin to Boc2O is 2:

3.

5. The preparation method of a nano prodrug with broad-spectrum antibacterial activity as described in claim 3, characterized in that: In step S2, the molar ratio of intermediate 1, triethylamine, 2,4,6-trichlorobenzoyl chloride, and DMAP is 1.15:1.45:1.27:1.38, and the volume of 4-hydroxymethylphenylboronic acid is 20 mL.

6. Application of a nano-prodrug in broad-spectrum antibacterial.

7. The application according to claim 6, characterized in that: The nano-prodrug improves the water solubility of the naked drug ciprofloxacin and effectively controls the broad-spectrum antibacterial activity of ciprofloxacin.

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