A bisboronic acid bridged oligoxin-5-aminosalicylic acid conjugate, preparation method and application thereof

Abstract

This invention discloses a bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate, its preparation method, and its applications, belonging to the field of medicinal chemistry. In the conjugate, xylooligosaccharide is connected to one end of a bis(o-o)diol bridging molecule via a borate ester bond, and the other end of the bis(o-o)diol bridging molecule is connected to a hydroxyl group via a borate ester bond. Furthermore, 5-aminosalicylic acid is covalently linked to both bis(o-o)diol units of the bis(o-o)diol bridging molecule. The bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate prepared by this invention uses gallic acid dimer as the bridging core. A stable bisboronic acid ester bridging structure is constructed by forming borate ester bonds with xylooligosaccharide and gallic acid respectively, achieving colon-targeted delivery of 5-aminosalicylic acid and excellent pH-gated colon-targeted release effects. Simultaneously, it significantly reduces the drug's toxicity to normal cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to a diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate, its preparation method, and its application. Background Technology

[0002] 5-Aminosalicylic acid (5-ASA), also known as mesalazine, is a first-line drug for the clinical treatment of ulcerative colitis (UC). Its mechanism of action is mainly through local inhibition of the inflammatory response of the colonic mucosa and scavenging of oxygen free radicals, thereby relieving symptoms such as abdominal pain, diarrhea, and rectal bleeding in UC patients. However, after oral administration, 5-ASA is rapidly absorbed primarily in the stomach and small intestine, with only a small amount reaching the lesion site in the colon, resulting in insufficient local drug concentration and poor efficacy. Simultaneously, the 5-ASA absorbed into the bloodstream distributes throughout the body, causing systemic side effects such as nausea, headache, and liver and kidney damage, severely impacting patient medication adherence and quality of life.

[0003] To improve the colonic targeting of 5-ASA, existing technologies covalently link 5-ASA to polysaccharide carriers, leveraging the property of polysaccharides to be degraded by specific enzymes in the colon for targeted drug release. Commonly used polysaccharide carriers include chitosan, pectin, dextran, and alginate, which possess good biocompatibility and colonic enzyme degradation properties. The connection methods between 5-ASA and polysaccharides mainly include azo bond, ester bond, and amide bond. The synthesis of azo bonds typically requires reagents such as nitrites, resulting in harsh reaction conditions, and the introduction of azo bonds may affect the solubility and biocompatibility of the polysaccharide. Ester bonds exhibit poor stability in gastric and intestinal fluids, easily undergoing premature hydrolysis, leading to a large release of the drug before reaching the colon. Amide bonds formed by the amino group of 5-ASA and the carboxyl group of the polysaccharide generally result in lower drug loading due to the limited number of reactant carboxyl sites in the polysaccharide. Summary of the Invention

[0004] To address the issues of poor colonic targeting and insufficient stability of linkages in existing oral 5-ASA formulations, this invention aims to provide a diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: First, this invention provides a diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate having the following structure: ; In the formula, R1 is xylooligosaccharide, and its structural formula is: , R2 is 5-aminosalicylic acid, and its structural formula is: .

[0006] In one embodiment, m = 1~3, n = 1~6.

[0007] In one embodiment, the structural formula of the conjugate is: , n = 1~6.

[0008] Secondly, the present invention also provides a method for preparing a bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate, which includes the following steps: (1) Reaction of gallic acid with ethylenediamine produces gallic acid-ethylenediamine-gallic acid dimer; (2) The gallic acid-ethylenediamine-gallic acid dimer is reacted with boric acid to generate a diboronic acid ester intermediate; (3) React 5-aminosalicylic acid with the diboronic acid ester intermediate, and graft 5-aminosalicylic acid onto the diboronic acid ester intermediate through free phenolic hydroxyl groups to generate 5-aminosalicylic acid-diboronic acid intermediate; (4) Graft the 5-aminosalicylic acid-diboronic acid intermediate with xylooligosaccharide to prepare xylooligosaccharide-5-aminosalicylic acid conjugate.

[0009] In one embodiment, step (1) includes: Gallic acid was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and then reacted with ethylenediamine to obtain gallic acid-ethylenediamine monoamide. The gallic acid-ethylenediamine monoamide was then activated again and reacted with a second molecule of gallic acid to obtain gallic acid-ethylenediamine-gallic acid dimer.

[0010] In one embodiment, the reaction solvent in step (2) is sodium bicarbonate buffer solution, and the pH of the reaction solvent is 8.2.

[0011] In one embodiment, step (3) includes: After the free phenolic hydroxyl group in the diboronic ester intermediate is activated by formaldehyde hydroxymethylation, it undergoes a condensation reaction with the amino group of 5-aminosalicylic acid to form a Schiff base bond, thus completing the grafting of 5-aminosalicylic acid.

[0012] In one embodiment, in step (4), the molar ratio of the 5-aminosalicylic acid-diboronic acid intermediate to the xylooligosaccharide is 1:0.8~1.2.

[0013] Finally, this invention also provides the application of a diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate in a drug for treating ulcerative colitis. The conjugate achieves pH-gated release through a diboronic acid-bridged structure, is stable in the stomach and small intestine, and releases 5-aminosalicylic acid in the colon.

[0014] The bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate prepared in this invention uses gallic acid dimer as the bridging core. By forming borate ester bonds with xylooligosaccharide and gallic acid respectively, a stable bisboronic ester bridging structure is constructed, which realizes colon-targeted delivery of 5-aminosalicylic acid and excellent pH-gated colon-targeted release effect. At the same time, it also significantly reduces the toxicity of the drug to normal cells.

[0015] To make the above-mentioned features and advantages of the invention more apparent and understandable, specific embodiments are described below, and detailed descriptions are provided in conjunction with the accompanying drawings. Attached Figure Description

[0016] Figure 1 This is a reaction route diagram of the present invention. Detailed Implementation

[0017] To make the objectives and technical solutions of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0018] Example 1 This embodiment aims to prepare xylooligosaccharide-5-aminosalicylic acid conjugate, including the following steps: S1, synthesizes gallic acid dimer; 1.70 g of gallic acid was weighed and dissolved in 30 mL of dimethylformamide (DMF) to form a mixed solution. 1.86 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 1.38 g of N-hydroxysuccinimide (NHS) were added to the mixed solution in sequence. The mixture was stirred at room temperature for 30 min to activate the solution and obtain activated gallic acid ester.

[0019] Weigh 0.30 g of ethylenediamine and dissolve it in 10 mL of DMF. Slowly add the solution of the activated gallic acid ester to the solution, adjust the pH to 8.0, and react at room temperature for 5 h. After the reaction is complete, evaporate the DMF solvent by rotary evaporation. Extract the residue with ethyl acetate, wash with water, and dry to obtain crude gallic acid-ethylenediamine monoamide. The reaction formula is as follows: Figure 1 As shown in Chinese (1).

[0020] 1.50 g of the crude gallic acid-ethylenediamine monoamide was dissolved in 20 mL of DFM to form a mixed solution. 0.93 g of EDC and 0.69 g of NHS were added sequentially to the mixed solution. After activation for 30 min, 0.85 g of gallic acid was added, and the reaction was carried out at room temperature for 5 h. After the reaction was completed, the reaction solution was concentrated, purified by silica gel column chromatography, concentrated under reduced pressure, and dried to obtain 1.58 g of white solid, thus preparing the gallic acid dimer. The reaction formula is as follows: Figure 1 As shown in Chinese (2).

[0021] S2, preparation of diboronic acid intermediate; 0.44 g of the gallic acid dimer was dissolved in 50 mL of sodium bicarbonate buffer solution (pH 8.2), and 0.13 g of boric acid was added. The mixture was heated to 50 °C and stirred for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the pH was adjusted to 5.0 with dilute hydrochloric acid. The precipitate was collected by centrifugation, and the solid was freeze-dried to obtain 0.48 g of a yellow solid, which is the diboronic acid intermediate. The reaction formula is as follows: Figure 1 As shown in Chinese formula (3).

[0022] S3,5-Aminosalicylic acid grafted with diboronic acid intermediate; Dissolve 0.48 g of the diboronic acid intermediate in 20 ml of PBS buffer, add 0.1 mL of formaldehyde solution, and stir at room temperature for 30 min to carry out the hydroxymethylation reaction, as shown in the reaction formula. Figure 1 As shown in Chinese formula (4).

[0023] After the reaction was complete, 0.15 g of 5-aminosalicylic acid (5-ASA) was added, and the reaction was continued with stirring for 5 h. After the reaction was complete, the mixture was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 500 Da. The solution was then freeze-dried to obtain 0.41 g of the 5-ASA-diboronic acid intermediate. The reaction formula is as follows: Figure 1 As shown in Chinese formula (5).

[0024] S4, xylooligosaccharide-grafted 5-ASA-bisboronic acid intermediate; The 5-ASA-diboronic acid intermediate was dissolved in sodium bicarbonate buffer at pH 7.8, and xylooligosaccharide (XOS, degree of polymerization 2-7) with a molar ratio of 1:1 to the 5-ASA-diboronic acid intermediate was added. The mixture was stirred at room temperature for 2.5 h. After the reaction, the mixture was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1000 Da. The product was then freeze-dried to obtain 0.52 g of the target product, namely the diboronic acid-bridged XOS-5-ASA conjugate, designated as sample 1. The reaction formula is as follows: Figure 1 As shown in Chinese formula (6).

[0025] Example 2 This embodiment aims to prepare xylooligosaccharide-5-aminosalicylic acid conjugate. The only difference between this embodiment and Example 1 is that the molar ratio of the 5-ASA-bisboronic acid intermediate to the xylooligosaccharide is 1:0.5 and 1:2, respectively. The products obtained are referred to as Sample 2 and Sample 3, respectively.

[0026] Comparative Example 1 0.15 g of 5-ASA and 0.35 g of XOS (degree of polymerization 2~7) were simply mixed and ground evenly to prepare reference standard 1.

[0027] Comparative Example 2 This comparative example aims to prepare a monoboronic acid bridged XOS-5-ASA conjugate, including the following steps: 1 mmol XOS was dissolved in 20 mL of sodium bicarbonate buffer at pH 8.0, and 0.062 g of boric acid was added. The mixture was reacted at 50 °C for 2 h to prepare the boric acid-XOS intermediate.

[0028] 1 mmol of 5-ASA was dissolved in 10 mL of DMF, followed by the addition of 0.19 g of EDC and 0.14 g of NHS. After activation for 30 min, the boric acid-XOS intermediate was added, and the reaction was carried out at room temperature for 18 h. After the reaction was completed, the mixture was dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 500 Da. After freeze-drying, the monoboronic acid bridged-XOS-5-ASA conjugate was obtained, designated as reference standard 2.

[0029] Verification Example 1 This validation example is an in vitro drug release validation experiment. It includes the following steps: Prepare a simulated gastric juice solution: Dissolve 2.0 g of sodium chloride in water, add 7.0 mL of hydrochloric acid, dilute with water to 1000 mL, shake well, and adjust the pH to 1.2. Add 0.1% pepsin just before use.

[0030] Prepare intestinal fluid simulation solution: Weigh 6.8 g potassium dihydrogen phosphate, add 1000 mL of water to dissolve, adjust the pH to 6.8, and add 0.1% trypsin before use.

[0031] Prepare colonic fluid simulation solution: Weigh 6.8 g potassium dihydrogen phosphate, add 1000 mL of water to dissolve, adjust the pH to 7.4, and add 0.1% azo reductase before use.

[0032] Comparative Example 1, Reference 1, and Reference 2 were placed in corresponding gastric juice, intestinal juice, and colonic juice simulation solutions, respectively, and shaken at 37°C. Samples were taken at 2 h, 6 h, 12 h, and 24 h, respectively, and the content of 5-ASA was determined by HPLC, and the release rate (%) of 5-ASA was calculated. The results are shown in Table 1. Table 1 Results of in vitro drug release validation experiments As shown in Table 1, the 5-ASA in control 1, a physical mixture of 5-ASA and XOS, was almost completely dissolved within 2 hours, exhibiting no pH responsiveness. Control 2, a monoboric acid bridged conjugate, showed slow release in gastric and intestinal fluids, with cumulative release rates of 18.2% and 25.3% after 24 hours, respectively. The release rate was significantly accelerated in intestinal fluid, reaching 15.3% after 2 hours, potentially leading to premature drug release before reaching the colon. Sample 1, prepared in this invention, is a diboric acid bridged conjugate. Its release in gastric and intestinal fluids was relatively slow, with cumulative release rates of only 10.5% and 18.5% after 24 hours, respectively, significantly better than control 2, indicating that the double-bridged structure effectively enhances the stability of borate ester bonds under low pH conditions. In colonic fluid, the 2-hour release rate was only 5.2%, significantly lower than the 15.3% of control 2, indicating that the double-bridged structure effectively delays the initial release of the drug in a weakly alkaline environment; the 24-hour release rate reached 82.4%, ensuring sufficient drug release in the colon.

[0033] Verification Example 2 This validation example is a cytotoxicity experiment.

[0034] The cytotoxicity of sample 1, free 5-ASA, and control 2 against normal human colonic epithelial cells NCM460 was detected using the CCK-8 assay. NCM460 cells were cultured at 1×10⁻⁶ cells / cells. 4 Cells were seeded per well in 96-well plates and cultured for 24 hours. Then, different concentrations of the test sample (0 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL) were added, and the plates were cultured for another 48 hours. CCK-8 reagent was added, and after incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader. Cell viability (%) was calculated. The experimental results are shown in Table 2. Table 2 Cytotoxicity test data Table 2 shows that the attenuation effect of the conjugate of the present invention is particularly significant at high concentrations. The survival rate of the product of Example 1 against normal colonic epithelial cells was significantly higher than that of free 5-ASA at all concentrations, reaching as high as 92.8% at 100 μg / mL, while that of free 5-ASA was only 65.2%, indicating that the conjugate of the present invention can effectively reduce the systemic cytotoxicity of 5-ASA. The survival rate of the product of Example 1 at all concentrations was higher than that of the product of Comparative Example 2, demonstrating that the diboronic acid bridge structure has better biocompatibility than the monoboronic acid bridge structure. This is because, under normal physiological conditions, the diboronic acid bridge structure is more stable, and 5-ASA is less likely to dissociate from the xylooligosaccharide carrier, further delaying the release of 5-ASA at non-target sites, thus achieving better safety and lower direct toxicity to normal cells. Furthermore, xylooligosaccharide, as a hydrophilic polysaccharide carrier, can reduce the direct contact between 5-ASA and the cell membrane, reducing its cellular uptake and intracellular toxicity.

[0035] In summary, the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate prepared in this invention uses gallic acid dimer as the bridging core. By forming borate ester bonds with xylooligosaccharide and gallic acid respectively, a stable diboronic ester bridging structure is constructed, achieving colon-targeted delivery of 5-ASA and excellent pH-gated colon-targeted release effect. At the same time, it also significantly reduces the toxicity of the drug to normal cells.

[0036] Although the invention has been described and illustrated in detail with reference to preferred embodiments, the invention is not limited to the disclosed examples, and those skilled in the art can derive other variations therefrom without departing from the scope of protection of the invention.

Claims

1. A bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate, characterized in that, It has the following structure: ； In the formula, R1 is xylooligosaccharide, and its structural formula is: ， R2 is 5-aminosalicylic acid, and its structural formula is: 。 2. The xylooligosaccharide-5-aminosalicylic acid conjugate bridged by bisboronic acid according to claim 1, characterized in that, m=1~3, n=1~6.

3. A method for preparing a bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate, used to prepare the bisboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Reaction of gallic acid with ethylenediamine produces gallic acid-ethylenediamine-gallic acid dimer; (2) The gallic acid-ethylenediamine-gallic acid dimer is reacted with boric acid to generate a diboronic acid ester intermediate; (3) React 5-aminosalicylic acid with the diboronic acid ester intermediate, and graft 5-aminosalicylic acid onto the diboronic acid ester intermediate through free phenolic hydroxyl groups to generate 5-aminosalicylic acid-diboronic acid intermediate; (4) Graft the 5-aminosalicylic acid-diboronic acid intermediate with xylooligosaccharide to prepare xylooligosaccharide-5-aminosalicylic acid conjugate.

4. The method for preparing the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate according to claim 3, characterized in that, Step (1) includes: Gallic acid was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and then reacted with ethylenediamine to obtain gallic acid-ethylenediamine monoamide. The gallic acid-ethylenediamine monoamide was then activated again and reacted with a second molecule of gallic acid to obtain gallic acid-ethylenediamine-gallic acid dimer.

5. The method for preparing the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate according to claim 3, characterized in that, In step (2), the reaction solvent is sodium bicarbonate buffer solution with a pH of 8.

2.

6. The method for preparing the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate according to claim 3, characterized in that, Step (3) includes: After the free phenolic hydroxyl group in the diboronic ester intermediate is activated by formaldehyde hydroxymethylation, it undergoes a condensation reaction with the amino group of 5-aminosalicylic acid to form a Schiff base bond, thus completing the grafting of 5-aminosalicylic acid.

7. The method for preparing the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate according to claim 3, characterized in that, In step (4), the molar ratio of the 5-aminosalicylic acid-diboronic acid intermediate to the xylooligosaccharide is 1:0.8~1.

2.

8. The use of the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate as described in claim 1 or 2 in a medicament for treating ulcerative colitis.

9. The application of the diboronic acid-bridged xylooligosaccharide-5-aminosalicylic acid conjugate according to claim 8, characterized in that, The conjugate achieves pH-gated release via a bisboronic acid bridging structure, is stable in the stomach and small intestine, and releases 5-aminosalicylic acid in the colon.

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