Oxidized cyclodextrin, preparation method and application of oxidized cyclodextrin in preparation of anticoagulant or thrombolytic drug

Abstract

The invention discloses oxidized cyclodextrin, a preparation method and application of the oxidized cyclodextrin in preparation of anticoagulants or thrombolytic drugs. The method comprises the following steps: under an ice bath condition, mixing an aqueous solution of cyclodextrin, TEMPO and potassium bromide with a sodium hypochlorite solution with the pH value of 10 + / -0.5 for reaction, continuously dropwise adding a NaOH solution, and keeping the pH value of the reaction solution at 10 + / -1 to obtain oxidized cyclodextrin. By controlling the proportion of the TEMPO, the potassium bromide, the sodium hypochlorite and the primary hydroxyl group in the cyclodextrin, the oxidized cyclodextrin with high carboxyl group content and oxygen content is obtained. In addition, the oxidized cyclodextrin prepared by the invention has excellent anticoagulation performance, and has potential application prospects in anticoagulation and thrombolytic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of cyclodextrin derivatives, and relates to an oxidized cyclodextrin, a preparation method thereof, and an application thereof in the preparation of anticoagulants or thrombolytic drugs. Background Art

[0002] Cyclodextrin (CD) is a general term for a class of cyclic oligosaccharides. Among them, those with application significance are mainly composed of 6, 7, and 8 glucose units, which are respectively called α-, β-, and γ-cyclodextrin. It is a compound with a circular ring shape and has the remarkable property of complexing with various guest molecules in the hydrophobic cavity. In an aqueous solution, compounds that are insoluble or poorly soluble in water can be embedded in their hydrophobic cavities to form inclusion complexes, changing the physical and chemical properties of the included substances. However, the hydrophobic region and catalytic activity of cyclodextrin are limited, and its application is restricted to a certain extent. In order to improve its inherent drawbacks, it can be chemically modified by oxidizing its hydroxyl groups. In recent years, cyclodextrin with a carboxylic acid functional group has attracted people's attention because it can react with other amine and alcohol compounds to synthesize new compounds (amides and esters).

[0003] In terms of the application of cyclodextrin oxidation derivatives, it can be combined with carboxymethyl chitosan to achieve hydrophilic modification of polyester fabrics (Chinese Patent Application CN107761383A). It can also be grafted and modified on wool fabrics to enhance their antibacterial properties and wash resistance (Chinese Patent Application CN105951436A). Reacting cyclodextrin with epichlorohydrin under alkaline catalysis to obtain a polyepoxidized cyclodextrin derivative, which has excellent water solubility and still maintains its original state after standing for 15 days and can effectively complex with perfume and drug molecules (Chinese Patent Application CN113321753A). Cyclodextrin oxidation derivatives can be used as the substrate of a non-ionic antibacterial material, which has good solubility, biocompatibility, and antibacterial properties (Chinese Patent Application CN116158438A). In addition, by controlling the oxidation degree of cyclodextrin, the disintegration time of its drug as a disintegrant can be controlled (Chinese Patent Application CN110859964A). Integrating cyclodextrin oxidation derivatives into a polymer skeleton, the prepared linear oxidized cyclodextrin copolymer can be used as a delivery carrier for various therapeutic agents (Chinese Patent CN100365024C).

[0004] VIGNON MICHEL et al. reported a method for the oxidation of α, β, γ-cyclodextrins, selectively oxidizing the primary hydroxyl groups of the glucopyranoside units of α-, β- or γ-cyclodextrins and controlling their degree of substitution. Specifically, 1-1.3 mol of sodium hypochlorite, 0.5 mol of sodium bromide and 1-10 mg of the catalyst 2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO) were added per 1 mol of primary hydroxyl group. Cyclodextrin, sodium hypochlorite, sodium bromide and TEMPO were all dissolved in 1-50 mL of deionized water, and the reaction was carried out at 0-5 °C and pH 9.5-10.5 to obtain a content of more than 25% of mono-6-deoxy-6-carboxy-α-, β- or γ-cyclodextrin (expressed as the sodium salt). And the obtained tri-(6-deoxy-6-carboxy)-α-, β- or γ-cyclodextrin can be used for the separation of isomers, enantiomers, carbohydrates and hydrophobic organic molecules, as well as the capture of toxic molecules in gas or liquid effluents and the fixation of metal cations such as heavy metals (Michel Vignon, Duval, Carole Fraschin. PROCEDE DE PREPARATION DEMONO-, DI-ET TRICARBOXY CYCLODEXTRINES PAR OXYDATION REGIOSELECTIVE ENPOSITION 6d'a ou βou y-CYCLODEXTRINES NATIVES[P]. France: FR2804437A1, 2000-01-31.). However, the preparation efficiency of this method is slow, and by analyzing the carboxyl content of the product and performing elemental analysis on the product, it is found that the degree of oxidation of the obtained oxidation product is relatively low. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide a method for oxidizing cyclodextrin, a preparation method thereof and its application in the preparation of anticoagulants or thrombolytic drugs. This method prepares a carboxyl oxidation derivative of cyclodextrin with a high degree of oxidation by efficiently oxidizing the primary hydroxyl groups of cyclodextrin.

[0006] The technical solution for achieving the object of the present invention is as follows:

[0007] A method for preparing oxidized cyclodextrin, comprising the following steps:

[0008] (1) Dissolve cyclodextrin, TEMPO and potassium bromide in water according to the molar ratio of TEMPO to the primary hydroxyl groups in cyclodextrin being 0.01:1 and the molar ratio of potassium bromide to the primary hydroxyl groups in cyclodextrin being 0.20-0.25:1, to obtain solution A, wherein the cyclodextrin is α-, β- or γ-cyclodextrin;

[0009] (2) Adjust the pH of the sodium hypochlorite solution to 10 ± 0.5 with hydrochloric acid to obtain solution B;

[0010] (3) Under ice bath conditions, mix solution A and solution B for reaction according to the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin being 5 - 6:1, and continuously add NaOH solution dropwise to maintain the pH of the reaction solution at 10 ± 1. After the reaction until the pH is stable, add ethanol to terminate the reaction, add hydrochloric acid dropwise to adjust the pH of the reaction solution to 3, then add ethanol again, filter to obtain the product. Dissolve the product in water, then add ethanol, centrifuge, and repeat the above extraction method three times to obtain oxidized cyclodextrin.

[0011] Specifically, every 1 mmol of α-, β-, and γ-cyclodextrins contain 6, 7, and 8 mmol of primary hydroxyl groups respectively.

[0012] Preferably, in step (1), the molar ratio of potassium bromide to primary hydroxyl groups in cyclodextrin is 0.23:1.

[0013] Preferably, in step (3), the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin is 5.6:1.

[0014] Preferably, in step (3), during filtration, the volume of ethanol is 3 times the volume of the reaction solution.

[0015] Preferably, in step (3), during extraction, the volume of ethanol is 3 times the volume of water.

[0016] The present invention also provides the oxidized cyclodextrin prepared by the above preparation method.

[0017] Furthermore, the present invention provides the application of the above oxidized cyclodextrin in the preparation of anticoagulants or thrombolytic drugs.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention is improved on the basis of the existing method. By controlling the ratio of TEMPO, potassium bromide, sodium hypochlorite to primary hydroxyl groups in cyclodextrin, the content of carboxyl groups and oxygen content in the product are higher than those of the products obtained by the existing methods. In addition, the oxidized cyclodextrin prepared by the present invention has excellent anticoagulant performance and has potential application prospects in anticoagulants and thrombolytic drugs. Description of the Drawings

[0020] Figure 1 It is the carboxyl group content of the oxidized β-cyclodextrin prepared in Example 1 and Comparative Example 1 determined by the potentiometric titration method.

[0021] Figure 2 It is the analysis result diagram of the C, H, N, and O contents in the oxidized β-cyclodextrin prepared in Example 1 and Comparative Example 1.

[0022] Figure 3Graph showing the blood coagulation test results of oxidized α-, β-, γ-cyclodextrins and β-cyclodextrin. Detailed implementation manners

[0023] The present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings.

[0024] Example 1

[0025] According to the molar ratio of TEMPO to primary hydroxyl groups in cyclodextrin being 0.01:1, the molar ratio of potassium bromide to primary hydroxyl groups in cyclodextrin being 0.23:1, and the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin being 5.6:1, β-cyclodextrin (3.24 g, 2.85 mmol), TEMPO (31 mg, 0.20 mmol), and potassium bromide (0.476 g, 4.6 mmol) were dissolved in 100 mL of deionized water to obtain solution A. 70 mL of sodium hypochlorite solution (112.8 mmol) was adjusted to pH 10 with hydrochloric acid to obtain solution B. Solutions A and B were placed in an ice bath. After the temperature dropped below 3°C, solutions A and B were mixed and reacted. During the reaction, the ice bath was maintained, and 0.5 M NaOH was continuously added dropwise to maintain the pH of the reaction solution at 10. After 9 hours, 20 mL of ethanol was added to terminate the reaction. Hydrochloric acid was added dropwise to adjust the pH of the reaction solution to 3. Then, ethanol with a volume three times that of the reaction solution was added, and the product was filtered. The product was dissolved in 30 mL of deionized water, and then 90 mL of ethanol was added, followed by centrifugation. Extraction was performed three times in this way, and finally oxidized β-cyclodextrin was obtained.

[0026] Example 2

[0027] This example is substantially the same as Example 1, except that the cyclodextrin used is α-cyclodextrin. Each 1 mmol of α-cyclodextrin contains 6 mmol of primary hydroxyl groups. Still, as in Example 1, the molar ratio of TEMPO to primary hydroxyl groups in cyclodextrin was controlled to be 0.01:1, the molar ratio of potassium bromide to primary hydroxyl groups in cyclodextrin was 0.23:1, and the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin was 5.6:1, to obtain oxidized α-cyclodextrin.

[0028] Example 3

[0029] This example is substantially the same as Example 1, except that the cyclodextrin used is γ-cyclodextrin. Each 1 mmol of γ-cyclodextrin contains 8 mmol of primary hydroxyl groups. Still, as in Example 1, the molar ratio of TEMPO to primary hydroxyl groups in cyclodextrin was controlled to be 0.01:1, the molar ratio of potassium bromide to primary hydroxyl groups in cyclodextrin was 0.23:1, and the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin was 5.6:1, to obtain oxidized γ-cyclodextrin.

[0030] Comparative Example 1

[0031] References (Michel Vignon, Duval, Carole Fraschin. PROCESS FOR PREPARING MONO-, DI- AND TRICARBOXY CYCLODEXTRINS BY REGIOSELECTIVE OXIDATION AT POSITION 6 OF α-, β- OR γ-CYCLODEXTRINS NATIVES[P]. France: FR2804437A1, 2000-01-31.) Preparation of oxidized β-cyclodextrin.

[0032] Examples of characterization and performance testing

[0033] 1. Determination of oxidation degree

[0034] (1) Determination of carboxyl group content:

[0035] The potentiometric titration method was used for determination. Specifically, 50 mg of the oxidized β-cyclodextrin prepared in Example 1 and Comparative Example 1 were respectively dissolved in 20 mL of deionized water, and then titrated with 0.04 M NaOH, and the conductivity change of the mixed solution was determined using a conductivity tester FE38-Standard (METTLER TOLEDO, China). The consumption of NaOH when the conductivity dropped to the plateau region was the carboxyl group content.

[0036] The results are as Figure 1 shown. By calculating the consumption of sodium hydroxide solution, the carboxyl group contents in the oxidized β-cyclodextrin prepared in Comparative Example 1 and Example 1 were 4.088 mmol·g -1 and 5.368 mmol·g -1 respectively. The carboxyl group content in the oxidized β-cyclodextrin prepared in Example 1 was significantly higher than that in Comparative Example 1.

[0037] (2) Determination of element content

[0038] 2 mg of the oxidized β-cyclodextrin prepared in Example 1 and Comparative Example 1 were respectively used for elemental analysis in C, H, N modes and O mode using an elementar vario Microcube.

[0039] The results are as Figure 2 shown in Figure (a) below. In the C, H, N modes, the H element content of the oxidized β-cyclodextrin prepared in Example 1 was 2.962%, and the H element content of the oxidized β-cyclodextrin prepared in Comparative Example 1 was 4.067%. There was no significant difference in the C and N element contents between the two, indicating that the oxidation degree of the oxidized β-cyclodextrin prepared in Example 1 might be higher than that in Comparative Example 1. As Figure 2As shown in Figure (b), under the O mode, the O element content of the oxidized β-cyclodextrin prepared in Example 1 is 54.314%, which is higher than that of the oxidized β-cyclodextrin prepared in Comparative Example 1 (52.628%). The above results prove that the oxidation degree of the oxidized β-cyclodextrin prepared in Example 1 is indeed higher than that of Comparative Example 1.

[0040] In summary, the carboxyl content and oxygen content of the oxidized β-cyclodextrin prepared by the method of the present invention are higher than those of the oxidized β-cyclodextrin prepared by the existing method in Comparative Example 1, indicating that the method of the present invention has a better oxidation effect.

[0041] 2. Anticoagulant properties

[0042] In order to study the anticoagulant properties of different oxidized cyclodextrins and cyclodextrins, the plasma activated partial thromboplastin time (APTT) was measured using an automatic coagulation analyzer CA-50 (Sysmex Co., Kobe, Japan). Fresh blood from healthy humans was collected venously in a special blood collection tube and ACD maintenance solution and anticoagulant were added. The anticoagulated whole blood was centrifuged to obtain the supernatant, and the process was repeated twice to prepare platelet-poor plasma (PPP). The plasma to be tested was preheated at 37°C. 50 μL of plasma, 50 μL of preheated APTT reagent, and 10 mg / mL of different cyclodextrin samples were fully mixed and incubated at 37°C for 3 minutes. Then, 50 μL of preheated 25 mM CaCl2 solution was added and the mixture was immediately placed in the coagulation analyzer for detection.

[0043] The results are as follows Figure 3 As shown, the oxidized α-, β-, and γ-cyclodextrin groups did not show obvious coagulation within 240 seconds of the test, while the β-cyclodextrin group showed coagulation after 73.2 seconds, and the blank control group showed coagulation after 36.9 seconds. The above results show that the oxidized cyclodextrin prepared by the method of the present invention has a significant anticoagulant effect, and its main anticoagulant mechanism may be: the oxidized cyclodextrin prepared by the method of the present invention combines with calcium ions in the blood to form a stable complex, thereby reducing the concentration of free calcium ions and inhibiting the activation of prothrombin and thrombosis.

[0044] In summary, the carboxyl content of the oxidized cyclodextrin prepared by the method of the present invention reaches 5.368 mmol·g -1 , which is higher than the carboxyl content of the product obtained by the existing method by 4.088mmol·g -1 The oxygen content of the oxidized cyclodextrin prepared by the method of the present invention reaches 54.314%, which is higher than 52.628% of the product obtained by the existing method. At the same time, the coagulation test results show that the oxidized cyclodextrin prepared by the method of the present invention does not coagulate within 240 seconds, and it has broad application prospects in the development of anticoagulant and thrombolytic drugs.

Claims

1. A method for preparing oxidized cyclodextrin, characterized in that, It includes the following steps: (1) Dissolve cyclodextrin, TEMPO and potassium bromide in water according to the molar ratio of TEMPO to primary hydroxyl groups in cyclodextrin being 0.01:1 and the molar ratio of potassium bromide to primary hydroxyl groups in cyclodextrin being 0.20 - 0.25:1 to obtain solution A, where the cyclodextrin is α-, β- or γ-cyclodextrin; (2) Adjust the pH of the sodium hypochlorite solution to 10 ± 0.5 with hydrochloric acid to obtain solution B; (3) Under ice bath conditions, mix and react solution A and solution B according to the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin being 5 - 6:1, and continuously add dropwise NaOH solution to keep the pH of the reaction solution at 10 ± 1. After the reaction until the pH is stable, add ethanol to terminate the reaction, add dropwise hydrochloric acid to adjust the pH of the reaction solution to 3, then add ethanol, filter to obtain the product. Dissolve the product in water, then add ethanol, centrifuge, and repeat the above extraction method three times to obtain oxidized cyclodextrin.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of potassium bromide to primary hydroxyl groups in cyclodextrin is 0.23:

1.

3. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of sodium hypochlorite to primary hydroxyl groups in cyclodextrin is 5.6:

1.

4. The preparation method according to claim 1, characterized in that, In step (3), during filtration, the volume of ethanol is 3 times the volume of the reaction solution.

5. The preparation method according to claim 1, characterized in that, In step (3), during extraction, the volume of ethanol is 3 times the volume of water.

6. Oxidized cyclodextrin prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the oxidized cyclodextrin according to claim 6 in the preparation of an anticoagulant or a thrombolytic drug.

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