A method for preparing a polysulfated mucopolysaccharide

Abstract

This invention provides a method for preparing polysulfated mucopolysaccharides, using chondroitin A sodium salt as the starting material, through a two-step reaction to obtain polysulfated mucopolysaccharides. The preparation method of this invention features high yield, precise controllable molecular weight, and product sulfonation degree meeting requirements. Furthermore, the entire process does not require freeze-drying, and the reaction conditions are mild, making it suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a method for preparing polysulfated mucopolysaccharides, and more particularly to a method for preparing polysulfated mucopolysaccharides with high yield, controllable molecular weight, and suitable for industrial production. Background Technology

[0002] Mucopolysaccharide polysulfate (MPS) is a large glycosaminoglycan drug composed of several disaccharide units consisting of sulfonated D-glucuronic acid and N-acetyl-galactosamine linked together, and has the following chemical structural formula: .

[0003] Studies by Gorog et al. (effect of a mucopolysaccharide polysulfate after systemic, topical and percutaneous application, Arzneimittel-Forschung. (1987), 37(3), 342-5)) have shown that mucopolysaccharide polysulfates have antithrombotic effects by acting on the blood coagulation and fibrinolytic systems; at the same time, they have anti-inflammatory effects by inhibiting various enzymes involved in catabolism and affecting the prostaglandin and complement systems. Kumokawa et al. (Satoshi, Dermal absorption of mucopolysaccharide polysulfate (heparinoid) in human and minipig, Arzneimittel-Forschung (2011), 61(2), 85-91) have also shown that mucopolysaccharide polysulfates can promote the regeneration of connective tissue by promoting the synthesis of mesenchymal cells and restoring the ability of intercellular substances to retain water. Therefore, polysulfated mucopolysaccharides are widely used clinically to prevent the formation of superficial thrombi, promote thrombus absorption, inhibit the development of local inflammation, and accelerate hematoma absorption.

[0004] The known methods for preparing polysulfated mucopolysaccharides mainly include the following techniques or routes: CN109970882A discloses a method for preparing polysulfated chondroitin sulfate, which involves sulfating chondroitin sulfate with chlorosulfonic acid, followed by multiple dissolution processes and freeze-drying. This method uses highly corrosive chlorosulfonic acid and employs a freeze-drying process, resulting in poor atom economy and high energy consumption, making it unsuitable for industrial production.

[0005] CN118994438A discloses a method for first degrading chondroitin sulfate A with hydrogen peroxide, followed by freeze-drying to obtain degraded polysaccharides; then sulfonating with chlorosulfonic acid formamide, followed by dialysis and freeze-drying to obtain MPS. Although this method achieves the separation of degradation and sulfonation, it still has the following shortcomings: (1) Hydrogen peroxide is a chemical that is easily explosive, and its large-scale use poses a safety hazard; (2) Chlorosulfonic acid is still a highly corrosive reagent; (3) The yield is low and each step of the reaction depends on the freeze-drying process, resulting in high energy consumption and making it unsuitable for industrial scale-up.

[0006] CN121226582A discloses a method that uses pyridine sulfur trioxide complex as a sulfonating agent and controls the molecular weight of the product by adjusting the ratio of DMF / DMSO mixed solvent. Although this method avoids the use of chlorosulfonic acid, the reaction process is slow, and the solution obtained after the depolymerization of chondroitin sulfate sodium is a mixture, making it difficult to guarantee the molecular weight; moreover, dialysis is only performed in the last step, making it difficult to control impurities and molecular weight. It has the following shortcomings: (1) The one-step direct sulfonation method starts with a molecular weight of 19,000 Da, and the reduction of the product molecular weight depends on the degradation side reaction during the reaction process, which is difficult to control; (2) The yield is low and it depends on the freeze-drying process, which consumes a lot of energy and is not suitable for industrial production.

[0007] CN1789287A discloses a method for sulfonating chondroitin sulfate using a mixture of chlorosulfonic acid and concentrated sulfuric acid, followed by gel chromatography and lyophilization to obtain polysulfated chondroitin sulfate. The drawbacks of this method are: (1) the use of concentrated sulfuric acid, which is highly corrosive, poses a high operational risk, and requires stringent equipment; (2) sulfonation and degradation occur simultaneously, making it difficult to precisely control the molecular weight; and (3) the product yield is low, and the post-processing is complex.

[0008] EP1634893A1 discloses the sulfonation of chondroitin sulfate in formamide using chlorosulfonic acid, achieving synergistic control of degradation and sulfonation by controlling the reaction temperature (15-40℃). Although the process is relatively simple, it still has the following shortcomings: (1) The literature clearly states that if the reaction temperature is below 15℃, degradation cannot be effective, and if it is above 40℃, the sulfonation efficiency will decrease, resulting in a narrow operating window; (2) The highly corrosive chlorosulfonic acid is used, and the post-treatment involves acetone precipitation and ion exchange, without disclosing a lyophilization alternative.

[0009] In summary, there is currently a lack of a green preparation method for polysulfonic acid mucopolysaccharides that can simultaneously achieve high yield, precise control of molecular weight, eliminate the need for freeze drying, and be suitable for industrial production. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a method for preparing polysulfonic acid mucopolysaccharides to overcome at least one of the following problems in the prior art: (1) the molecular weight is difficult to control precisely during the degradation process; (2) the sulfonation reaction is inefficient and the conversion rate is insufficient; (3) the post-processing depends on the freeze-drying process, which consumes a lot of energy, requires a large investment in equipment, and is not suitable for industrial scale-up; (4) the overall yield is low.

[0011] Specifically, the present invention aims to provide a method for preparing polysulfonic acid mucopolysaccharides with high yield, controllable molecular weight, no need for freeze drying, and suitable for industrial production.

[0012] To achieve the above objectives, the present invention provides a method for preparing polysulfonic acid mucopolysaccharides, the chemical reaction equation of which is as follows:

[0013] The preparation method includes the following steps: Step (1): Chondroitin A sodium salt (compound I) is subjected to a pyrolysis reaction in the presence of a pyrolysis agent and a catalyst, and then post-processed to obtain compound II; Step (2): Compound II is subjected to sulfonation reaction under the action of sulfonating agent and oxygenation agent, and then post-processed to obtain polysulfated mucopolysaccharide (compound III).

[0014] Preferably, the preparation method includes the following steps: Step (1): Chondroitin A sodium salt (compound I) is subjected to a pyrolysis reaction in the presence of a pyrolysis agent and a catalyst, and then post-treated with a salting-out agent to obtain compound II; Step (2): Compound II is subjected to sulfonation reaction under the action of sulfonating agent and oxygenation agent, and then post-processed to obtain polysulfated mucopolysaccharide (compound III).

[0015] More preferably, the preparation method includes the following steps: Step (1): Dissolve chondroitin sulfate A sodium salt (compound I) in a solvent and carry out a pyrolysis reaction under the action of a pyrolysis agent and a catalyst. After the reaction is completed, add a salting-out agent to adjust the pH to 6.3-6.7. After alcohol precipitation, filtration and drying, compound II is obtained. Step (2): Dissolve compound II in a solvent, add an oxygenating agent and stir, then add a sulfonating agent to carry out a sulfonation reaction. After the reaction is completed, the mixture is cooled and quenched, crystallized, filtered, dissolved, adsorbed by anion exchange resin, pH adjusted to 7.2-7.8, dialyzed with a 3500Da dialysis bag, precipitated with alcohol, filtered, and vacuum dried to obtain compound III (polysulfonic acid mucopolysaccharide).

[0016] More preferably, in step (1), the post-treatment includes: after the pyrolysis reaction is completed, adding a salting-out agent and adjusting the pH of the solution to 6.3-6.7, adding the reaction solution to ethanol for alcohol precipitation, filtration, and drying to obtain compound II; in step (2), the post-treatment includes: after the sulfonation reaction is completed, cooling and quenching the reaction solution, crystallizing, filtering, dissolving, and adsorbing with anion exchange resin to obtain an eluent, adjusting the pH of the eluent to 7.2-7.8 with a pH adjuster, dialyzing through a dialysis bag with a molecular weight cutoff of 3500 Da, adding the dialysate to ethanol for alcohol precipitation, filtration, and drying to obtain compound III; the pH adjuster is selected from one or more of sodium carbonate, sodium bicarbonate, and sodium hydroxide. The drying described above is vacuum drying.

[0017] In the preparation method described above, the pyrolysis agent in step (1) is selected from one or more of cerium ammonium nitrate, ammonium nitrate, and ammonium perchlorate; the catalyst is selected from one or more of tetraamminecopper(II) sulfate monohydrate, sodium ethylenediaminetetraacetate, and 8-hydroxyquinoline copper; and the salting-out agent is selected from one or more of sodium acetate, potassium acetate, and ammonium acetate.

[0018] In the preparation method described above, the sulfonating agent in step (2) is selected from one or more of sulfur trioxide pyridine, sulfur trioxide-triethylamine complex, and trimethylammonium sulfur trioxide copolymer; the oxygen-providing agent is selected from one or more of acetylacetone vanadium oxyacetate, vanadium sulfate, and vanadium oxalate.

[0019] In the preparation method described above, the weight ratio of compound I: cracking agent: catalyst: salting-out agent in step (1) is 1:(0.15~0.5):(0.02~0.06):(0.1~0.5); the temperature of the cracking reaction is 10~40℃.

[0020] In the preparation method described above, in step (2), compound II and oxygen-providing agent are added and stirred first, and then sulfonating agent is added; the molar ratio of compound II: sulfonating agent: oxygen-providing agent is 1: (12-20): (0.05-0.2); the temperature of the sulfonation reaction is 30-50℃.

[0021] In the preparation method described above, the weight-average molecular weight of compound II should be 8,000 to 12,000 Da; the specific rotation should be -25° to -32°, preferably -28° to -29°.

[0022] In the preparation method described above, the anion exchange resin in step (2) is preferably D218 macroporous anion exchange resin (Cl⁻ type) with a crosslinking degree of 10% and an adsorption temperature of 20-30℃. The adsorption includes dynamic adsorption and static adsorption. Dynamic adsorption involves stirring the resin in the solution at a speed generally of 200-600 r / min. Static adsorption involves immersing the resin in the filtrate for static adsorption, with an immersion time generally of 30 min-4 h. The preferred weight ratio of resin to solution is 1:(0.5-1).

[0023] A polysulfonic acid mucopolysaccharide, characterized in that it is prepared by the above-described preparation method and has a weight-average molecular weight of 5,000 to 15,000 Da; preferably, the weight-average molecular weight is 8,000 to 12,000 Da; more preferably, the weight-average molecular weight is 9,000 to 11,000 Da.

[0024] The reaction pathway described in this invention is as follows: Compound I (sodium chondroitin sulfate, degree of polymerization n2) → pyrolysis reaction (pyrolysis agent + catalyst) → Compound II (degree of polymerization n1, n1 < 0.5n2) → sulfonation reaction (sulfonating agent + oxygenation agent) → Compound III (polysulfonic acid mucopolysaccharide).

[0025] The polysulfated mucopolysaccharide obtained by the method of this invention can be used in the preparation of drugs for treating thrombotic diseases; the drug is selected from creams, gels, patches, liniments, microneedles, etc.; the thrombotic disease is varicose phlebitis, thrombophlebitis, or deep vein thrombosis. In this invention, compound I is chondroitin A sodium salt with a molecular weight of 50,000-80,000 Da, which is commercially available. Compound III is a polysulfated mucopolysaccharide with a molecular weight of 5,000-15,000 Da.

[0026] The 'pyrolysis agent' described in this invention refers to an oxidizing compound capable of selectively cleaving the glycosidic bonds of polysaccharides, and the 'oxygen-supporting agent' refers to a metal complex capable of assisting SO3 in the directional attack of hydroxyl groups during sulfonation reactions. The salting-out agent is added to the solution after the pyrolysis reaction to promote the precipitation and separation of pyrolysis products.

[0027] In this invention, the pyrolysis agent (such as cerium ammonium nitrate) acts as an oxidant to initiate the cleavage of glycosidic bonds, and the copper complex catalyst enhances the selective oxidation ability of the pyrolysis agent through coordination, thereby achieving precise control of molecular weight.

[0028] In the preparation of compound II, this invention generates compound II with a molecular weight matching that of polysulfonic acid mucopolysaccharide by adding a pyrolysis agent to compound I; at the same time, the reaction conversion rate can be improved by adding a catalyst.

[0029] In this invention, vanadium oxyacetylacetonate complexes (such as vanadium oxyacetylacetonate) act as oxygen-promoting agents, improving the selectivity and conversion rate of the sulfonation reaction by forming active intermediates with sulfonating agents or promoting the directional attack of SO3.

[0030] In the preparation of compound III, this invention involves first adding compound II and an oxygen-providing agent, followed by the addition of a sulfonating agent. This improves the conversion rate of the sulfonation reaction, resulting in a more complete reaction. Simultaneously, post-treatment processes such as ion exchange and pH adjustment effectively enhance product quality and simplify the operation.

[0031] In this invention, "ethanol precipitation" refers to the operation of precipitating the target product by adding ethanol to a solution or adding a solution to ethanol. The ethanol can be 75% (v / v) or higher, preferably 95% (v / v) or higher, and more preferably anhydrous ethanol.

[0032] In this invention, the molecular weights of compounds II and III can be determined using the following methods or methods reported in pharmacopoeias, quality standards, or literature: Determination conditions and limits for the molecular weight of compound II: The SEC-MALLS method was used (column: TSK gelG3000SWXL 7.8×300mm; mobile phase: 0.2mol / L Na2SO4 (containing 0.02% NaN3); flow rate: 0.5mL / min; detector: DAWN HELLOS-Ⅱ 18-angle laser light scattering detector and Optilab rEX differential detector in series; column temperature: 30℃; injection volume: 100μL). The weight-average molecular weight should be 8,000~12,000Da. The specific rotation should be -25°~-32° (refer to the detection method in the Chinese Pharmacopoeia).

[0033] The molecular weight determination of compound III can be referred to the literature Topical mucopolysaccharide polysulfate (MPS) in the treatment of thrombophlebitis (DOI: 10.1055 / s-0037-1617306), and the weight-average molecular weight should be 5,000-15,000 Da.

[0034] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improved yield: The mass yield of compound II in step (1) of this invention can reach 87.5% to 88.5%, and the mass yield of compound III in step (2) can reach 68.5% to 69.6%, which is much higher than that of the prior art; (2) Molecular weight is precisely controllable: The weight average molecular weight of compound II obtained by this invention is 9,920 to 10,580 Da, which meets the target range of 8,000 to 12,000 Da; the weight average molecular weight of compound III is 9,760 to 9,870 Da, which meets the target range of 5,000 to 15,000 Da, and the molecular weight distribution is narrow, the control precision is high, and the product uniformity is good. (3) High degree of sulfonation and good product quality: The organic sulfate ion of the compound obtained by this invention reaches 27.5%, the product is highly sulfonated, and meets the quality standards of polysulfonic acid mucopolysaccharide; (4) Simplified post-processing and low energy consumption: This invention adopts conventional post-processing methods such as alcohol precipitation, filtration, and vacuum drying, without using freeze drying, which significantly reduces equipment investment and production energy consumption, making it more suitable for industrial production; (5) The reaction conditions are mild and the operation is safe. It does not use hydrogen peroxide, which is easy to generate explosions, or chlorosulfonic acid, which is highly corrosive, so the operation is safe. Attached Figure Description

[0035] Figure 1 : 1H-NMR and HSQC spectra of compound III (Example 1 polysulfonic acid mucopolysaccharide). Detailed Implementation

[0036] The technical solution of the present invention will now be further described with reference to specific embodiments. These embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It is neither necessary nor possible for those skilled in the art to exhaustively describe all implementation methods. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solution of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional conditions or according to the conditions recommended by the manufacturer.

[0037] Note: Compound I is sodium chondroitin sulfate A (CAS: 39455-18-0), with a molecular weight of 50,000-80,000 Da, and is commercially available. The yield calculation method is as follows: (1) Compound II: yield of Compound II / amount of Compound I × 100%; (2) Compound III: yield of Compound III / amount of Compound II × 100%. In the chemical structure of Compound II, it is calculated as a tetrasaccharide unit. R in the tetrasaccharide unit indicates the presence of 2 SO3⁻ and 2 H atoms, and the molecular weight is calculated as 931.77.

[0038] Example 1 Compound I (30.0 g) was added to water / acetonitrile (330 mL, V:V = 10:1) and dissolved by stirring at 10 °C. Tetraamminecopper(II) sulfate monohydrate (1.8 g) was added, and the mixture was stirred for 30 minutes. Cerium ammonium nitrate (4.5 g) was then added, and the mixture was stirred at this temperature for 3 hours. Sodium acetate (3.0 g) was added, and the pH of the solution was adjusted to 6.3 with acetic acid. The solution was added dropwise to ethanol (150 mL), and the mixture was stirred at 0–10 °C for 2 hours to induce crystallization. The crystals were filtered, and the filter cake was added to ethanol (90 mL) and stirred at 0–10 °C. After filtration, the mixture was dried under vacuum at 40 °C to obtain compound II (26.37 g), with a yield of 87.9%. The weight-average molecular weight was 10,016 Da, and the specific rotation was -29°.

[0039] Compound II (20.0 g, 0.02146 mol) and acetylacetone vanadium oxide (0.001073 mol, 0.285 g) were added to formamide (160 mL) and stirred at 30 °C for 1 h. Then, pyridine sulfur trioxide (0.2576 mol, 41.0 g) was added, and the reaction was continued at 30 °C for 5 h. The reaction solution was cooled to 0–10 °C and quenched dropwise in a 10% sodium carbonate aqueous solution (500 mL). Crystallization was carried out at 0–10 °C for 1 h, filtered, and the filter cake was dissolved in water (80 mL). The solution was filtered, and the filtrate was dynamically adsorbed twice by D218 macroporous anion exchange resin, followed by static adsorption for 3 h to obtain the eluent. Sodium carbonate was added to the eluent to adjust the pH to 7.2, and the solution was added to a dialysis bag (molecular weight cutoff 3500 Da) for dialysis for 12 h (the dialysate was discarded every 3 h, and 80 mL of purified water was added) to obtain the dialysate. The dialysate was dropped into ethanol (160 mL) for 2 hours to crystallize. The mixture was filtered, and the filter cake was washed with ethanol (80 mL) successively. After filtration, the mixture was dried under vacuum at 50 °C to give compound III (13.76 g), with a yield of 68.8%. The weight-average molecular weight was 9,870 Da.

[0040] Example 2 Compound I (30.0 g) was added to water / acetonitrile (330 mL, V:V = 10:1) and dissolved by stirring at 40 °C. Sodium copper ethylenediaminetetraacetate (0.6 g) was added and stirred for 30 minutes, followed by ammonium nitrate (15.0 g) and stirring at this temperature for 3 hours. Potassium acetate (15.0 g) was added, and the pH of the solution was adjusted to 6.7 with acetic acid. The solution was added dropwise to ethanol (150 mL), and the mixture was stirred at 0–10 °C for 2 hours to induce crystallization. The crystals were filtered, and the filter cake was added to ethanol (90 mL) and slurried at 0–10 °C. After filtration, the mixture was dried under vacuum at 40 °C to obtain compound II (26.55 g), with a yield of 88.5%. The weight-average molecular weight was 10,580 Da, and the specific rotation was -28°.

[0041] Compound II (20.0 g, 0.02146 mol) and vanadium oxysulfate (0.004293 mol, 0.699 g) were added to formamide (160 mL) and stirred at 50 °C for 1 h. Then, sulfur trioxide-triethylamine complex (0.4293 mol, 77.8 g) was added, and the reaction was continued at 50 °C for 5 h. The reaction solution was cooled to 0–10 °C and quenched dropwise in 10% sodium carbonate aqueous solution (500 mL). Crystallization was carried out at 0–10 °C for 1 h, filtered, and the filter cake was dissolved in water (80 mL). The solution was filtered, and the filtrate was subjected to dynamic adsorption twice with D218 macroporous anion exchange resin, followed by static adsorption for 3 h to obtain the eluent. Sodium bicarbonate was added to the eluent to adjust the pH to 7.8. The eluent was then added to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed for 12 hours (the dialysate was discarded every 3 hours, and 80 mL of purified water was added). The dialysate was then added dropwise to 160 mL of ethanol for crystallization over 2 hours. The crystals were filtered, and the filter cake was washed successively with 80 mL of ethanol, filtered again, and dried under vacuum at 50 °C to obtain compound III (13.92 g), with a yield of 69.6%. The weight-average molecular weight was 9,760 Da.

[0042] Example 3 Compound I (30.0 g) was added to water / acetonitrile (330 mL, V:V = 10:1) and dissolved by stirring at 25 °C. 1.2 g of 8-hydroxyquinoline copper was added and stirred for 30 minutes, followed by ammonium perchlorate (9.0 g) and stirring at this temperature for 3 hours. Ammonium acetate (9.0 g) was added, and the pH of the solution was adjusted to 6.5 with acetic acid. The solution was added dropwise to 150 mL of ethanol, and the mixture was stirred at 0–10 °C for 2 hours to induce crystallization. The crystals were filtered, and the filter cake was added to 90 mL of ethanol and stirred at 0–10 °C. After filtration, the mixture was dried under vacuum at 40 °C to obtain compound II (26.25 g), with a yield of 87.5%. The weight-average molecular weight was 9,920 Da, and the specific rotation was -29°.

[0043] Compound II (20.0 g, 0.02146 mol) and vanadium oxalate (0.002146 mol, 0.333 g) were added to formamide (160 mL) and stirred at 40 °C for 1 h. Then, trimethylammonium sulfur trioxide copolymer (0.3434 mol, 47.79 g) was added, and the reaction was continued at 40 °C for 5 h. The reaction solution was cooled to 0–10 °C and quenched dropwise in a 10% sodium carbonate aqueous solution (500 mL). Crystallization was carried out at 0–10 °C for 1 h, filtered, and the filter cake was dissolved in water (80 mL). The solution was filtered, and the filtrate was subjected to dynamic adsorption twice with D218 macroporous anion exchange resin, followed by static adsorption for 3 h to obtain the eluent. Sodium hydroxide was added to the eluent to adjust the pH to 7.5. The eluent was then added to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed for 12 hours (the dialysate was discarded every 3 hours, and 80 mL of purified water was added). The dialysate was then added dropwise to 160 mL of ethanol for crystallization over 2 hours. The crystals were filtered, and the filter cake was washed successively with 80 mL of ethanol, filtered again, and dried under vacuum at 50 °C to obtain compound III (13.70 g), with a yield of 68.5%. The weight-average molecular weight was 9,830 Da.

[0044] Comparative Example 1 Compound I (30.0 g) was added to water / acetonitrile (330 mL, V:V = 10:1) and dissolved by stirring at 25 °C. Ammonium perchlorate (9.0 g) was then added, and the mixture was stirred at this temperature for 3 h. Ammonium acetate (9.0 g) was added, and the pH of the solution was adjusted to 6.5 with acetic acid. The solution was added dropwise to ethanol (150 mL), and the mixture was stirred at 0–10 °C for 2 h to induce crystallization. The crystals were filtered, and the filter cake was added to ethanol (90 mL) and slurried at 0–10 °C. After filtration, the mixture was dried under vacuum at 40 °C to obtain compound II (23.07 g), with a yield of 76.9%. The weight-average molecular weight was 32,460 Da, and the specific rotation was -40°.

[0045] Compound II (20.0 g, 0.02146 mol) was added to formamide (160 mL) and stirred at 40 °C for 1 h. Then, trimethylammonium sulfur trioxide copolymer (0.3434 mol, 47.79 g) was added, and the reaction was continued at 40 °C for 5 h. The reaction solution was cooled to 0–10 °C and quenched dropwise in a 10% sodium carbonate aqueous solution (500 mL). Crystallization was carried out at 0–10 °C for 1 h, filtered, and the filter cake was dissolved in water (80 mL). The solution was filtered, and the filtrate was dynamically adsorbed twice by D218 macroporous anion exchange resin, followed by static adsorption for 3 h to obtain the eluent. Sodium hydroxide was added to the eluent to adjust the pH to 7.5, and the solution was added to a dialysis bag (molecular weight cutoff 3500 Da) for dialysis for 12 h (the dialysate was discarded every 3 h, and 80 mL of purified water was added) to obtain the dialysate. The dialysate was dropped into 160 mL of ethanol for 2 hours to crystallize. The mixture was filtered, and the filter cake was washed with 80 mL of ethanol, filtered, and dried under vacuum at 50 °C to give compound III (10.22 g), with a yield of 51.1%. The weight-average molecular weight was 28,550 Da.

[0046] Comparative Example 1 shows that when there is no catalyst in the pyrolysis reaction, the yield of compound II decreased from 87.5% to 76.9%, and the yield of compound III decreased from 68.5% to 51.1%, and the molecular weight (28,550 Da) deviated significantly from the target range (5,000-15,000 Da). This shows that the catalyst is crucial for accurately controlling the molecular weight and improving the yield.

[0047] Comparative Example 2 Compound I (30.0 g) was added to water / acetonitrile (330 mL, V:V = 10:1) and dissolved by stirring at 50 °C. 8-Hydroxyquinoline copper (1.2 g) was added and stirred for 30 minutes, followed by ammonium perchlorate (9.0 g), and the mixture was kept warm and stirred for 3 hours. The pH of the solution was adjusted to 6.0 with acetic acid. The solution was added dropwise to ethanol (150 mL), and crystallization was carried out by stirring at 0–10 °C (crystallization was difficult and the amount precipitated was small). The mixture was filtered, and the filter cake was added to ethanol (90 mL) and slurried at 0–10 °C. After filtration, the mixture was dried under vacuum at 40 °C to obtain compound II (20.39 g), with a yield of 68.0%. The weight-average molecular weight was 18,670 Da, and the specific rotation was -36°.

[0048] Compound II (20.0 g, 0.02146 mol) and trimethylammonium sulfur trioxide copolymer (0.3434 mol, 47.79 g) were added to formamide (160 mL) and stirred at 40 °C for 1 h. Vanadyl oxalate (0.002146 mol, 0.333 g) was then added, and the reaction was continued at 40 °C for 5 h. The reaction solution was cooled to 0–10 °C and quenched dropwise in a 10% sodium carbonate aqueous solution (500 mL). Crystallization was carried out at 0–10 °C for 1 h, filtered, and the filter cake was dissolved in water (80 mL). The solution was filtered, and the filtrate was subjected to dynamic adsorption twice with D218 macroporous anion exchange resin, followed by static adsorption for 3 h to obtain the eluent. Sodium hydroxide was added to the eluent to adjust the pH to 8.0. The eluent was then added to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed for 12 hours (the dialysate was discarded every 3 hours, and 80 mL of purified water was added). The dialysate was then added dropwise to 160 mL of ethanol for 2 hours to crystallize. The crystals were filtered, and the filter cake was washed successively with 80 mL of ethanol, filtered again, and dried under vacuum at 50 °C to give compound III (9.84 g), with a yield of 32.8%. The weight-average molecular weight was 17,930 Da.

[0049] Comparative Example 2 shows that when the pyrolysis temperature is increased to 50°C, there is a lack of salting-out agent, improper pH control, improper feeding sequence in the sulfonation step, and pH adjustment is out of range, the yield and molecular weight of Compound II and Compound III are severely deteriorated, proving that the synergistic control of each process parameter is crucial.

[0050] Comparative Example 3 Compound I (30.0 g) was added to water / acetonitrile (330 mL, V:V = 10:1) and dissolved by stirring at 25 °C. 8-Hydroxyquinoline copper (1.2 g) was added and stirred for 30 minutes, followed by ammonium perchlorate (9.0 g) and stirring at this temperature for 3 hours. Ammonium acetate (9.0 g) was then added. The solution was added dropwise to ethanol (150 mL), and the mixture was stirred at 0–10 °C for 2 hours to induce crystallization. The crystals were filtered, and the filter cake was added to ethanol (90 mL) and slurried at 0–10 °C. After filtration, the mixture was dried under vacuum at 40 °C to obtain compound II (22.68 g), with a yield of 75.6%. The weight-average molecular weight was 17,800 Da, and the specific rotation was -34°.

[0051] Compound II (calculated as a tetrasaccharide unit, where R in the tetrasaccharide unit indicates the presence of 2 SO₃⁻ and 2 H atoms, with a molecular weight of 931.77; 20.0 g, 0.02146 mol), vanadium oxalate (0.002146 mol, 0.333 g), and trimethylammonium sulfur trioxide copolymer (0.3434 mol, 47.79 g) were added to formamide (160 mL), and the mixture was stirred at 40 °C for 6 h. The reaction solution was cooled to 0–10 °C and quenched dropwise in a 10% sodium carbonate aqueous solution (500 mL). Crystallization was carried out at 0–10 °C for 1 h, and the mixture was filtered. The filter cake was dissolved in water (80 mL). The solution was filtered, and the filtrate was subjected to dynamic adsorption twice with D218 macroporous anion exchange resin, followed by static adsorption for 3 h to obtain the eluent. The eluent was added to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed for 12 h (the dialysate was discarded every 3 h, and 80 mL of purified water was added), yielding the dialysate. The dialysate was added dropwise to 160 mL of ethanol for 2 h to crystallize. The crystals were filtered, and the filter cake was washed successively with 80 mL of ethanol, filtered, and dried under vacuum at 50 °C to give compound III (12.18 g), with a yield of 60.9%. The weight-average molecular weight was 18,350 Da.

[0052] Comparative Example 3 shows that when Compound II, the oxygen-providing agent, and the sulfonating agent are added simultaneously in the sulfonation step (instead of adding the oxygen-providing agent first and then the sulfonating agent) and the pH adjustment step is omitted, the yield of Compound III drops to 60.9%, and the molecular weight deviates from the target range, proving that the order of addition and pH adjustment are indispensable for obtaining high-quality products.

[0053] Comparative Example 4: Refer to Example 1 of CN109970882A Chondroitin sulfate (50.0 g) was added to formamide (200.0 g), and then slowly added to a chlorosulfonic acid-formamide solution (a mixture of chlorosulfonic acid (75.0 g) and formamide (150.0 g)) with stirring. The mixture was stirred at 30°C for 5 hours, then heated to 60°C and stirred for 4 hours. 200 ml of 95% ethanol was added, the mixture was allowed to stand, and then filtered to obtain wet chondroitin sulfate polysulfate. The wet product was dissolved in 500 ml of water, and the pH was adjusted to 9.0 with sodium hydroxide solution. 2000 ml of 95% ethanol was then added, and the mixture was allowed to stand for 12 hours. The precipitate was dissolved in 500 ml of water, and the pH was adjusted to 9.0 with sodium hydroxide solution. The mixture was heated to 60°C and stirred for 3 hours, then filtered. 2000 ml of 95% ethanol was added to the filtrate, and the mixture was allowed to stand for 12 hours. Filter the solution, dissolve the filter cake in 500ml of water, then add 5.0g of sodium chloride, and continuously add 30% hydrogen peroxide solution until the hydrogen peroxide concentration reaches 0.5%. Adjust the pH to 10 with 20% sodium hydroxide solution. Stir at 30℃, maintain a vacuum of 0.05~0.10MPa for 12 hours, maintain the pH at 10, and then let it stand at 25℃ under normal pressure for 12 hours for decolorization. Filter through a 0.45μm filter membrane, adjust the pH to 8.0 with 3M hydrochloric acid, add 2000ml of 95% ethanol, and let it stand for 12 hours. Filter the solution again, dissolve the filter cake in 500ml of water, then add 5.0g of sodium chloride and adjust the pH to 7.0. The filter cake was obtained by filtration through a 0.45 μm filter membrane. The filter cake was dissolved in water (500 ml), and the pH was adjusted to 6.0~7.0. After filtration through a 0.45 μm filter membrane, the chondroitin sulfate polysulfate was freeze-dried (10.35 g, yield 20.7%, weight average molecular weight 54260 Da).

[0054] Comparative Example 5: CN118994438A Example 1 Chondroitin sulfate A (40.0 g) was added to a hydrogen peroxide solution (700 ml deionized water + 30% hydrogen peroxide (300 ml)) and stirred at 70°C for 6 h. The pH was adjusted to 3-4 with acetic acid, and the solution was concentrated under reduced pressure. The concentrate was added to a 3500 Da dialysis bag and dialyzed with deionized water for 48 h. The dialysate was lyophilized to obtain degraded polysaccharide (22.0 g, yield 55.0%, weight average molecular weight 28670 Da). The degraded polysaccharide (20.0 g) was added to formamide (3000 ml) and dissolved by stirring at 60 °C. The resulting solution was added to a mixed acid solution (chlorosulfonic acid was slowly added to the formamide under ice bath conditions) and stirred at 50 °C for 4 h. The pH of the solution was adjusted to 7-7.5 with 4M sodium hydroxide solution. The reaction solution was added to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 30 h (the external dialysate was changed every 4 h). The internal dialysate was concentrated to a small volume and then lyophilized to obtain polysulfated mucopolysaccharide (6.58 g, yield 32.9%, weight average molecular weight 23500 Da).

[0055] Comparative Example 6: CN121226582A Example 1 Chondroitin sulfate sodium (20.0 g, weight-average molecular weight 19000 Da) and pyridine sulfur trioxide (100.0 g) were added to N,N-dimethylformamide (400 ml) and stirred at 50 °C for 24 h. The reaction solution was added to 0 °C saturated sodium bicarbonate solution (1600 ml), and then 95% ethanol (8000 ml) was added to crystallize a wet product of polysulfonic acid mucopolysaccharide. The wet product was added to 95% ethanol (800 ml) and stirred for 1 h, filtered, and this operation was repeated until the formamide concentration in the waste ethanol was <0.1 wt%. The product was filtered to obtain the initial polysulfonic acid mucopolysaccharide product. The initial product was dissolved in water (800 ml) and added to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed for 24 h, and the dialysate was lyophilized to obtain polysulfonic acid mucopolysaccharide (7.64 g, yield 38.2%, weight-average molecular weight 17620 Da).

[0056] Experimental Example 1: The products prepared in Examples 1-3 and Comparative Examples 1-3 were tested, and the results are shown in Table 1.

[0057] Table 1. Comparison of product quality between Examples 1-3 and Comparative Examples 1-3

[0058] Results Analysis: All indicators of Examples 1-3 met the target requirements: the Mw of compound II was 9,920-10,580 Da (target 8,000-12,000 Da), and the specific rotation was -28° to -29° (target -25° to -32°); the Mw of compound III was 9,760-9,870 Da (target 5,000-15,000 Da), and the yield was 68.5%-69.6%.

[0059] In Comparative Example 1, no catalyst was used in the pyrolysis step, resulting in a molecular weight (Mw) of 32,460 Da for compound II, far exceeding the target range. Compound III's Mw was 28,550 Da, also deviating from the target, demonstrating the crucial role of catalysts in precise molecular weight control. Inappropriate reaction temperature, feeding sequence, and pH in Comparative Example 2, and inappropriate feeding sequence and pH adjustment in Comparative Example 3, also caused the product molecular weights to deviate from the target range, resulting in a significant decrease in yield.

[0060] The above comparative data fully demonstrates that the key technical features of this invention—the use of catalysts, the control of pyrolysis temperature, the optimization of sulfonation feeding sequence, and the precise adjustment of pH—are indispensable for achieving high yield and precise control of molecular weight in the preparation of polysulfonic acid mucopolysaccharides.

[0061] Experimental Example 2: Determination of Organic Sulfate in Compound III Determination method: Sulfate ion chromatography (Chinese Pharmacopoeia General Chapter 0513, 2025 edition) was used for determination.

[0062] After hydrolysis, accurately weigh approximately 20 mg of the sample and place it in a 50 ml round-bottom flask. Accurately add 20 ml of 2 mol / L trifluoroacetic acid solution, seal the flask, and hydrolyze by stirring in an oil bath at 100°C for 7 hours. Remove the flask and allow it to cool to room temperature. Transfer the entire hydrolysate to a 100 ml volumetric flask, dilute with water to the mark, shake well, filter, and collect the filtrate.

[0063] Accurately weigh approximately 10 mg of the unhydrolyzed test sample into a 10 ml volumetric flask, dissolve and dilute with water to the mark, shake well, filter, and collect the filtrate to obtain the final solution.

[0064] The standard curve solution is prepared by taking potassium sulfate standard solution (1000 μg / ml), diluting it stepwise with water to prepare solutions containing 10 μg, 40 μg, 100 μg, 200 μg, and 400 μg of potassium sulfate per ml.

[0065] Chromatographic conditions using an anion exchange column (Dionex IonPac) TM AS11-HC, 4.0 mm × 250 mm, 4 μm or equivalent column; using 15 mmol / L potassium hydroxide solution as the mobile phase, flow rate of 1.0 ml / min, conductivity detector, detection mode of suppression detector, column temperature of 30 °C, and injection volume of 10 μl.

[0066] The assay involves precisely measuring the hydrolyzed test solution, the unhydrolyzed test solution, and the standard curve solution, injecting them separately into an ion chromatograph, and recording the chromatograms.

[0067] The limit is calculated using the standard curve method, based on the peak area. The sulfate content of this product is obtained by subtracting the unhydrolyzed sulfate content from the sulfate content after hydrolysis.

[0068] Standard: It should be 25.8%-37.3%.

[0069] Measurement results:

[0070] Results Analysis: The sulfate ions in Compound III obtained in Examples 1-3 were all within the standard range, indicating that the polysulfated mucopolysaccharide prepared by this invention has a high degree of sulfonation, which meets the standard. High sulfonation is the structural basis for the excellent anticoagulant and anti-inflammatory activities of polysulfated mucopolysaccharide, and also proves that the sulfonation reaction in the preparation method of this invention is relatively complete and efficient.

[0071] Experimental Example 3: Structural Confirmation of Compound III Assay method: Weigh about 30 mg of compound III obtained in Example 1, dissolve it in 0.5 mL D2O, lyophilize and then redissolve it in 0.5 mL D2O. Use acetone as an internal standard for ¹H-NMR, ¹³C-NMR and HSQC tests.

[0072] Based on the combined ¹H-NMR, ¹³C-NMR, and HSQC spectral analyses, compound III obtained in this invention can be confirmed as a polysulfated mucopolysaccharide (MPS). In its structure, both the C-4 and C-6 positions of galactosamine are sulfonated (the C-6 position is almost completely sulfonated), and the C-2 and C-3 positions of glucuronic acid are also partially sulfonated, exhibiting a highly sulfonated structural characteristic. This structural feature is consistent with the structures of polysulfated mucopolysaccharides reported in the literature.

[0073] Experimental Example 4: Stability Study of Compound III Measurement method: Compound III obtained in Example 1 was placed under accelerated conditions of 40℃±2℃ / 75%RH±5%RH for 3 months. Samples were taken at 0, 1, 2 and 3 months to detect weight-average molecular weight, organic sulfate and purity (HPLC area normalization method) to examine the stability of the product.

[0074] Measurement results:

[0075] Results Analysis: After 3 months of accelerated storage, the weight-average molecular weight and organic sulfate content of compound III decreased slightly, and the HPLC purity decreased from 98.5% to 97.2%, all remaining within acceptable ranges. These results demonstrate that the polysulfated mucopolysaccharide prepared in this invention exhibits good stability, further proving the reliability of the post-processing method and its suitability for storage and formulation development as a raw material.

Claims

1. A method for preparing a polysulfated mucopolysaccharide, characterized in that, Includes the following steps: ， Step (1): Chondroitin A sodium salt (compound I) is subjected to a pyrolysis reaction in the presence of a pyrolysis agent and a catalyst, and then post-processed to obtain compound II; Step (2): Compound II is subjected to sulfonation reaction under the action of sulfonating agent and oxygenation agent, and after post-treatment, polysulfated mucopolysaccharide (compound III) is obtained.

2. The production method according to claim 1, characterized by, The pyrolysis agent is selected from one or more of cerium ammonium nitrate, ammonium nitrate, and ammonium perchlorate; the catalyst is selected from one or more of tetraamminecopper(II) sulfate monohydrate, sodium ethylenediaminetetraacetate copper, and 8-hydroxyquinoline copper; the sulfonating agent is selected from one or more of sulfur trioxide pyridine, sulfur trioxide-triethylamine complex, and trimethylammonium sulfur trioxide copolymer; and the oxygen-providing agent is selected from one or more of acetylacetonate vanadyl, vanadyl sulfate, and vanadyl oxalate.

3. The production method according to claim 1 or 2, characterized by, In step (1), the post-processing includes: adding a salting-out agent after the pyrolysis reaction is completed, adjusting the pH of the solution to 6.3-6.7, adding the reaction solution to ethanol for alcohol precipitation, filtering, and drying to obtain compound II; the salting-out agent is selected from one or more of sodium acetate, potassium acetate, and ammonium acetate.

4. The production method according to claim 1 or 2, characterized by, In step (1), the weight ratio of compound I: cracking agent: catalyst: salting-out agent is 1:(0.15~0.5):(0.02~0.06):(0.1~0.5), and the temperature of the cracking reaction is 10~40℃.

5. The production method according to claim 1 or 2, characterized by, In step (2), compound II and oxygen-providing agent are added and stirred first, and then sulfonating agent is added; the molar ratio of compound II: sulfonating agent: oxygen-providing agent is 1: (12-20): (0.05-0.2); the temperature of the sulfonation reaction is 30-50℃.

6. The preparation method according to claim 1 or 2, characterized in that, In step (2), the post-processing includes: after the sulfonation reaction is completed, the reaction solution is cooled, quenched, crystallized, filtered, and dissolved, and the eluent is obtained by adsorption through anion exchange resin; the pH of the eluent is adjusted to 7.2-7.8 by adding a pH adjuster, and dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da; the dialysate is added to ethanol for alcohol precipitation, filtered, and dried to obtain compound III; preferably, the pH adjuster is selected from one or more of sodium carbonate, sodium bicarbonate, and sodium hydroxide.

7. The production method according to claim 3 or 6, characterized by, The drying process is vacuum drying.

8. The method of claim 1, wherein, The weight-average molecular weight of compound II is 8,000 to 12,000 Da; the specific rotation of compound II is -25° to -32°; preferably, the specific rotation of compound II is -28° to -29°.

9. A polysulfated mucopolysaccharide characterized by, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The polysulfated mucopolysaccharide of claim 9, wherein, The weight-average molecular weight of the polysulfated mucopolysaccharide is 5,000 to 15,000 Da; preferably, the weight-average molecular weight of the polysulfated mucopolysaccharide is 8,000 to 12,000 Da; more preferably, the weight-average molecular weight of the polysulfated mucopolysaccharide is 9,000 to 11,000 Da.

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