Chemically modified glycosaminoglycans, methods of preparation and uses thereof

Abstract

According to the chemically modified glycosaminoglycan provided by the invention, on the basis of retaining a six-membered ring structure of part of glycosaminoglycan, an iduronic acid structure is introduced through a reaction, and the chemically modified glycosaminoglycan with good anti-tumor activity, good safety and excellent druggability is obtained by controlling a proper ring opening degree; the polymer has good physical / chemical stability. The chemically modified glycosaminoglycan has good anti-tumor activity, especially anti-tumor metastasis and anti-tumor growth activity, is good in stability, stable in metabolism, good in bioavailability, small in toxic and side effect and good in safety, and can be prepared into a proper preparation, so that the medicine safety is improved, and the patient compliance and the administration convenience are improved.

Description

Technical Field

[0001] The present invention relates to a chemically modified glycosaminoglycan (particularly, a glycosaminoglycan carboxylation derivative), a preparation method thereof, and use thereof for inhibiting tumor growth and / or metastasis. Background Art

[0002] With the rapid development of economy, the aggravation of environmental pollution and the change of people's lifestyle, malignant tumors have become a disease that seriously endangers the life and health of Chinese people and restricts the development of social economy. Among all causes of death, malignant tumors have become the first cause of death in my country in recent years.

[0003] In the development process of most malignant tumors, distant metastasis of tumors is the main cause of death in cancer patients. The steps of tumor metastasis mainly include tumor detachment from the primary site, vascular infiltration, intravascular transport, adhesion to vascular endothelium, extravasation of blood vessels and finally distant metastasis. Literature reports that vascular endothelial cells, fibroblasts, tumor-associated immune cells, extracellular matrix, inflammatory-related cytokines and other components in the tumor microenvironment play an important role in the invasion, metastasis and progression of tumors, but the specific mechanism has not yet been fully clarified.

[0004] Heparanase, also known as heparanase, is a β-endoglucuronidase that cleaves heparan sulfate (HS) in heparan sulfate proteoglycans, such as syndecan-1, thereby releasing growth factors bound to heparan sulfate.

[0005] Extensive research work over the past two decades has shown that heparanase expression is upregulated in a variety of human cancers, sarcomas, and hematological malignancies. Other studies have shown that elevated heparanase levels may be associated with late progression and metastasis in many tumor types. For example, high levels of heparanase are associated with shorter survival times after surgery. A direct role for heparanase in tumor metastasis has been demonstrated in the laboratories of Vlodavsky and Sanderson.

[0006] Heparin is a linear, polydisperse, sulfated polysaccharide in the glycosaminoglycan family that possesses anticoagulant and antithrombotic activities. The sugar chain of heparin is composed of alternating uronic acid and D-glucosamine, with the main repeating units being 2-O-sulfated L-iduronic acid (IdoA2S) and α(1→4)N-,6-O-disulfated D-glucosamine (GlcN6S); the minor components are non-sulfated L-iduronic acid and D-glucuronic acid, as well as N-acetyl D-glucosamine and N-,3-O-,6-O-trisulfated D-glucosamine (Casu B., 2005. "Structure and active domains of heparin." In: Chemistry and biology of heparin and heparan sulfate. Amsterdam: Elsevier. 1-28; Casu B. and Lindahl U., 2001, "Structure and biological interactions of heparin and heparansulfate." Adv Carbohydr Chem Biochem, 57: 159-206). Heparin can effectively inhibit heparanase, but due to its strong anticoagulant activity, it is impossible to use heparin at high doses as a tumor therapeutic agent. Bleeding risk is a necessary indicator for evaluating the safety of anticoagulant drugs, especially heparin analogs.

[0007] The prior art has disclosed a class of non-anticoagulant heparins that can be used as heparanase inhibitors, most of which contain structurally modified non-sulfated uronic acid residues. Such structures can be obtained by opening the glycoside ring by breaking the 2-3 carbon-carbon bond (diol cleavage) on the glycosaminoglycan residue. However, the efficacy or safety of the non-anticoagulant heparins in the prior art is relatively low, and there is still room for further improvement.

[0008] In summary, providing a drug with high safety and high tumor metastasis inhibitory activity is a technical problem that needs to be solved urgently. Summary of the invention

[0009] The present invention provides a chemically modified glycosaminoglycan having good anti-tumor activity, specifically, having good anti-tumor growth activity, anti-tumor metastasis activity and safety, especially having excellent anti-tumor metastasis activity and safety.

[0010] Chemically modified glycosaminoglycans

[0011] The present invention provides a chemically modified glycosaminoglycan, which comprises a structural unit of formula (I), a structural unit of formula (II), a structural unit of formula (III) and a structural unit of formula (IV):

[0012]

[0013] in:

[0014] R 1m , R 1n , R 1q Each occurrence is independently selected from H, -SO3 - and -(C=O)CH3, and R 1m and R 1n Preferably -SO3 at each occurrence - or -(C=O)CH3;

[0015] R 2m , R 2n , R 2q and R 2x Each occurrence is independently selected from H and -SO3 - ;

[0016] R 3n Each occurrence is independently selected from H and -SO3 - ;

[0017] R 4m and R 4n Each occurrence is independently selected from H and -SO3 - .

[0018] According to an embodiment of the present invention, the weight average molecular weight of the chemically modified glycosaminoglycan is 12000Da-20000Da, preferably 14000Da-19000Da, 14500Da-18800Da, 15000Da-18700Da, for example, 14500Da, 15000Da, 15200Da, 15500Da, 16000Da, 16200Da, 16500Da, 17000Da, 17500Da, 18000Da, 18500Da.

[0019] According to an embodiment of the present invention, the iduronic acid content of the chemically modified glycosaminoglycan is 10.0%-70.0%, preferably 20.0%-35.0%, preferably 21.0%-26.0%, for example 20.0%, 21.0%, 21.3%, 22.0%, 23.0%, 25.0%, 25.6%, 26.0%, 28.5%, 30%, 31.0%, 32.0%, 34.5%, 35.0%, 36.0%, 37.0%, 40.0%, 43.0%, 45.0%, 50.0%, 55.0%, 6.00%.

[0020] According to an embodiment of the present invention, the uronic acid ring-opening degree of the chemically modified glycosaminoglycan is 25%-45%, preferably 27%-44%, further preferably 35%-44%, for example 27.5%, 28.5%, 31%, 32%, 35%, 36%, 37%, 43%, 44%.

[0021] According to an embodiment of the present invention, the sulfonyl carboxyl ratio (SO3 - / COO - ) is 1.2-1.8, preferably 1.2-1.7, for example 1.2, 1.3, 1.4, 1.5, 1.6, 1.7.

[0022] According to an embodiment of the present invention, the carboxyl increment of the chemically modified glycosaminoglycan is 1.1-1.8, preferably 1.15-1.60, for example 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60.

[0023] According to an embodiment of the present invention, the polydispersity coefficient of the chemically modified glycosaminoglycan is 1.05-1.20, preferably 1.10-1.19, for example 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19.

[0024] According to an embodiment of the present invention, the molecular weight distribution of the chemically modified glycosaminoglycan is as follows:

[0025] Greater than 19000Da 20% to 38% (e.g., 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 35%);

[0026] 6000-19000 Da 62%-78% (e.g., 64%, 65%, 66%, 67%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%);

[0027] Less than 6000 Da 0% to 1.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%).

[0028] The sum of the above percentages is 100%.

[0029] According to an embodiment of the present invention, the molecular weight distribution of the chemically modified glycosaminoglycan is as follows:

[0030] Molecular weight range (Da) Ratio (weight %) More than 19000 21-37 6000-19000 64～78 Less than 6000 0～1.4 .

[0031] Preferably, the molecular weight distribution of the chemically modified glycosaminoglycan is as follows:

[0032] Molecular weight range (Da) Ratio (weight %) More than 19000 23-36 6000-19000 64-77 Less than 6000 0.1～1.2 .

[0033] According to an embodiment of the present invention, the structural unit of formula (I), the structural unit of formula (II), the structural unit of formula (III) and the structural unit of formula (IV) are statistically distributed along the polysaccharide chain.

[0034] According to an embodiment of the present invention, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (V):

[0035]

[0036] in:

[0037] R 2t and R 3t Each occurrence is independently selected from H and -SO3 - .

[0038] According to an embodiment of the present invention, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (VI):

[0039]

[0040]

[0041] in:

[0042] R 2p Each occurrence is independently selected from H and -SO3 - .

[0043] According to an embodiment of the present invention, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (VII):

[0044]

[0045] in:

[0046] R 2r and R 3r Each occurrence is independently selected from H and -SO3 - .

[0047] According to an embodiment of the present invention, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (VIII):

[0048]

[0049] in:

[0050] R 1s Each occurrence is independently selected from H, -SO3 - and -(C=O)CH3, and preferably -SO3 - or -(C=O)CH3;

[0051] R 2s and R 4s Each occurrence is independently selected from H and -SO3 - ;

[0052] R 3s -SO3 at each occurrence - .

[0053] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain m structural units of the formula (I), wherein m is selected from any integer of 1-20, including the end values; preferably selected from any integer of 1-15, including the end values.

[0054] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain n structural units of the formula (II), wherein n is selected from any integer of 1-10, including the end values; preferably selected from any integer of 1-8, including the end values.

[0055] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain q structural units of the formula (III), wherein q is selected from any integer from 1 to 15, including the end values; preferably selected from any integer from 1 to 10, including the end values.

[0056] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain x structural units of the formula (IV), wherein x is selected from any integer from 0 to 10, including the end values; preferably selected from any integer from 0 to 8, including the end values; more preferably selected from any integer from 1 to 8, including the end values.

[0057] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain t structural units of the formula (V), wherein t is selected from any integer of 0-6, including the end values; preferably selected from any integer of 0-4, including the end values; more preferably selected from any integer of 1-3, including the end values.

[0058] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain p structural units of the formula (VI), wherein p is selected from any integer of 0-8, including the end values; preferably selected from any integer of 0-4, including the end values; more preferably selected from any integer of 1-3, including the end values.

[0059] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain r structural units of the formula (VII), wherein r is selected from any integer of 0-8, including the end values; preferably selected from any integer of 0-4, including the end values.

[0060] Optionally, one or more polysaccharide chains of the chemically modified glycosaminoglycan may each contain s structural units of the formula (VIII), wherein s is selected from any integer of 1-25, including the end values; preferably selected from any integer of 1-15, including the end values.

[0061] In each polysaccharide chain of the chemically modified glycosaminoglycan of the present invention, the structural units are arranged in an arbitrary order.

[0062] According to an embodiment of the present invention, the number of structural units of formula (I) in the chemically modified glycosaminoglycan is greater than the sum of the numbers of structural units of formula (V) and formula (VI).

[0063] According to an embodiment of the present invention, the ratio of the number of structural units of formula (I) in the chemically modified glycosaminoglycan to the sum of the numbers of structural units of formula (I), formula (V) and formula (VI) is greater than 0.9.

[0064] According to an embodiment of the present invention, the ratio of the number of structural units of formula (I) to the sum of the number of structural units of formula (I) and formula (II) in the chemically modified glycosaminoglycan is 0.1-0.4.

[0065] According to an embodiment of the present invention, the ratio of the number of structural units of formula (IV) in the chemically modified glycosaminoglycan to the sum of the number of structural units of formula (I), formula (II), formula (III) and formula (IV) is less than 0.1.

[0066] According to an embodiment of the present invention, the uronic acid epoxidation degree is less than 25%, for example, the uronic acid epoxidation degree is 0.001%-10%, preferably 0.001%-1%, such as 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.010%, 0.015%, 0.018%.

[0067] The present invention covers any combination of the various embodiments.

[0068] The term "sulfation degree" herein refers to the sulfonate to carboxyl ratio (SO3- / COO - Molar ratio), which is determined by conductometric titration according to Casu B. and Gennaro U., 1975, Carbohydr Res 39, 168-176.

[0069] The term "carboxyl increment" herein refers to the ratio of the sulfation degree of the raw material to the sulfation degree of the carboxylated derivative. More specifically, the sulfation degree of the raw material is the sulfation degree measured after reducing a sample of the glycosaminoglycan intermediate obtained after the first oxidation step of the raw material with NaBH4.

[0070] The term "uronic acid ring opening degree" herein refers to the number of ring-opened uronic acid residues / total number of uronic acid residues, and the term "uronic acid epoxidation degree" refers to the ratio of uronic acid structural units with epoxy structures of formula (V) to total uronic acid residues. Their detection and calculation references Guerrini, M., Guglieri, S., Naggi, A., Sasisekharan, R., & Torri, G. (2007). Low molecular weight heparins: Structural differentiation by bidimentional nuclear magnetic resonance spectroscopy. Seminars in Thrombosis and Hemostasis, 33, 478–487 were used for nuclear magnetic resonance methods.

[0071] Preparation method of chemically modified glycosaminoglycan

[0072] The present invention provides a method for preparing the chemically modified glycosaminoglycan, which comprises the following steps:

[0073] a) optionally, using glycosaminoglycan as a starting material to prepare a glycosaminoglycan derivative containing a C2-hydroxyuronic acid residue; preferably, the glycosaminoglycan derivative is obtained by freeze-drying in the presence of a base (e.g., sodium hydroxide, potassium hydroxide) and a reducing agent (e.g., sodium borohydride);

[0074] b) oxidizing 25%-45% (preferably 30%-40%) of the 2-O- and optionally 2N-, 3-O- non-sulfated residues of the glycosaminoglycan under conditions effective to convert adjacent diols (vicinal diols) and optionally adjacent OH / NH2 to dialdehydes;

[0075] The oxidation is carried out in the presence of an oxidant, wherein the oxidant is selected from one of periodic acid and periodate (e.g. sodium periodate, potassium periodate); and

[0076] c) further oxidizing the product obtained from step b) under conditions effective to convert the dialdehyde into a carboxyl group and without nitrogen protection;

[0077] The further oxidation is preferably carried out by a chlorite (e.g. sodium chlorite, magnesium chlorite Mg(ClO2)2, barium chlorite Ba(ClO2)2);

[0078] d) separating and purifying the product of step c) through an anion exchange purification system to obtain the chemically modified glycosaminoglycan.

[0079] According to an embodiment of the present invention, the method for preparing chemically modified glycosaminoglycans uses glycosaminoglycans as the starting material, performs C2 desulfation under controlled conditions, keeps the configuration unchanged, effectively converts adjacent diols and optional adjacent OH / NH2 into aldehydes through a controlled vicinal diol cleavage reaction, and then converts the corresponding aldehyde groups into corresponding carboxyl groups under suitable oxidation conditions.

[0080] According to an embodiment of the present invention, the glycosaminoglycan is natural heparin or synthetic heparin (which is optionally chemically or enzymatically modified) from any animal and organ source, preferably, selected from optionally 2-O- and / or 2-N-desulfated heparin, unfractionated heparin (UFH), low molecular weight heparin (LMWH, whose molecular weight is 3,500-8,000 Da) and heparan sulfate (HS); the glycosaminoglycan has a sulfonyl carboxyl ratio of 0.8-2.8 (preferably 0.9-2.5); the glycosaminoglycan is more preferably selected from optionally 2-O- and / or 2-N-desulfated unfractionated heparin and low molecular weight heparin.

[0081] According to an embodiment of the present invention, the weight average molecular weight of the glycosaminoglycan is 10000Da to 30000Da, preferably 15000Da to 25000Da, for example 15000Da to 20000Da, 15000Da to 19000Da or 17000Da to 19000Da.

[0082] According to an embodiment of the present invention, the heparin chain may naturally contain about 5% to 35% of 2-O-non-sulfated uronic acid residues, 0% to 50% of N-acetylated glucosamine residues and about 0%-6% of N-unsubstituted (neither N-sulfated nor N-acetylated) glucosamine residues. The different sulfation degrees depend on the heparin source (animal species, organ source) and extraction operation.

[0083] Each 2-O- or 2-N-non-sulfated residue of glycosaminoglycans (without a 3-O-sulfate substituent) is susceptible to ring-opening (cleavage) reactions, and under oxidative conditions, the vicinal diols and OH / NH2 can undergo conversion to aldehydes.

[0084] According to an embodiment of the present invention, in step a) of the method, a freeze-drying reaction is carried out in the presence of a base (preferably sodium hydroxide, potassium hydroxide) and a reducing agent (preferably sodium borohydride) to remove the C2 sulfate group in the uronic acid structure of the glycosaminoglycan to obtain an iduronic acid structure.

[0085] According to an embodiment of the present invention, in step a), the ratio of the molar amount of the reducing agent to the mass of the starting material glycosaminoglycan is 1.0-20.0 mmol / g, preferably 2.0-10.0 mmol / g, for example, 1.0 mmol / g, 2.6 mmol / g, 3.0 mmol / g, 4.0 mmol / g, 5.0 mmol / g, 6.0 mmol / g, 7.0 mmol / g, 8.0 mmol / g, 9.0 mmol / g, 10.0 mmol / g, 13.0 mmol / g, 16.0 mmol / g.

[0086] According to an embodiment of the present invention, in step a), the ratio of the molar amount of the base to the mass of the starting material glycosaminoglycan is 5.0-25.0 mmol / g, preferably 8.0-19.0 mmol / g, for example 8.0 mmol / g, 9.0 mmol / g, 10.0 mmol / g, 11.0 mmol / g, 12.0 mmol / g, 13.0 mmol / g, 14.0 mmol / g, 15.0 mmol / g, 16.0 mmol / g, 17.0 mmol / g, 18.0 mmol / g, 19.0 mmol / g.

[0087] According to an embodiment of the present invention, in step a), freeze drying is carried out using a low-temperature vacuum freeze dryer (eg, LGJ-20F).

[0088] According to an embodiment of the present invention, in step a), the freeze-drying reaction includes: a pre-freezing step, a primary drying step, and a secondary drying step.

[0089] According to an embodiment of the present invention, the pre-freezing step is specifically as follows: lowering the plate temperature to -20°C to -80°C (e.g. -30 to -40°C) in 30-200 minutes and maintaining it for 30-300 minutes; for example, lowering the plate temperature to -40°C in 60 minutes and maintaining it for 120 minutes.

[0090] According to the implementation scheme of the present invention, the primary drying step is specifically as follows: raising the board temperature to -10°C to 15°C for 40-480 minutes, maintaining it for 720-4800 minutes, and the vacuum degree is 5-80Pa (preferably, the vacuum degree is 10-30Pa); for example, raising the board temperature to -5°C for 90 minutes, maintaining it for 720 minutes, and the vacuum degree is 25±5Pa.

[0091] According to an embodiment of the present invention, the secondary drying step is specifically as follows: raising the board temperature to 25-60°C for 30-240 minutes, maintaining it for 300-1200 minutes, and the vacuum degree is 5-50Pa (preferably, the vacuum degree is 5-25Pa); for example, raising the board temperature to 25°C for 30 minutes, maintaining it for 480 minutes, and the vacuum degree is 10±5Pa.

[0092] According to an embodiment of the present invention, in step a), the freeze-drying curve is as shown in Table 1:

[0093] Table 1

[0094]

[0095] According to an embodiment of the present invention, step a) specifically comprises the following steps:

[0096] a1) reacting an aqueous solution of a starting material glycosaminoglycan with a reducing agent (preferably sodium borohydride); preferably, reacting at room temperature; preferably, reacting for 10 h to 30 h; preferably, stirring the reaction for 12 h to 24 h;

[0097] a2) adding a base to the reaction solution of step a1) to adjust the pH to above 13.0, and then performing freeze-drying reaction;

[0098] Optionally, after the reaction is completed, the freeze-dried solid is dissolved with water, the pH is adjusted to 3.8-4.0, stirring is continued for 30 minutes, and the pH is adjusted back to 6.8-7.0; and ultrafiltration desalination and membrane filtration are optionally further performed.

[0099] According to an embodiment of the present invention, in step b), the ratio of the molar amount of the oxidant to the mass of the starting material glycosaminoglycan is 1.0-3.6 mmol / g; preferably 1.2-3.0 mmol / g, for example 1.2 mmol / g, 1.4 mmol / g, 1.6 mmol / g, 1.8 mmol / g, 2.0 mmol / g, 2.2 mmol / g, 2.4 mmol / g, 2.6 mmol / g, 2.8 mmol / g, 3.0 mmol / g.

[0100] According to an embodiment of the present invention, in step b), the reaction temperature is 0°C-20°C.

[0101] According to an embodiment of the present invention, in step b), the reaction time is 10 h-30 h, preferably 12 h-24 h, for example 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h.

[0102] According to an embodiment of the present invention, step b) is carried out under light-shielding conditions.

[0103] According to an embodiment of the present invention, in step b), after the reaction is completed, an excess of a terminating reagent is added to terminate the reaction; optionally, the reaction solution is further subjected to dialysis (for desalination) treatment.

[0104] According to an embodiment of the present invention, in step b), the termination reagent is selected from one or more of ethylene glycol, ethanolamine, glycerol, glucose, mannose, glyceraldehyde, threose, and xylose; preferably, the termination reagent is selected from one or more of ethylene glycol, ethanolamine, and glucose.

[0105] In step b) of the method, by controlling the cleavage process of the vicinal diol, the (non-sulfated) iduronic acid structural fragment can be specifically introduced into the final product. Acidic glycosaminoglycans containing non-sulfated iduronic acid structural fragments have good activity, safety and other related properties.

[0106] According to an embodiment of the present invention, the chemically modified glycosaminoglycan exhibits a carboxyl increment of 0.6 to 1.4, wherein the carboxyl increment is calculated as a ratio of the sulfation degree of the raw material to the sulfation degree of the glycosaminoglycan. More specifically, the sulfation degree of the raw material is the sulfation degree measured after reducing a sample of the glycosaminoglycan intermediate obtained after the first oxidation step (step b) of the raw material with NaBH4. The specific operation for calculating the carboxyl increment can be found in CN105744940A.

[0107] According to an embodiment of the present invention, in step c), the mass ratio of the molar amount of chlorite (e.g., sodium chlorite, magnesium chlorite Mg(ClO2)2, barium chlorite Ba(ClO2)2) to the starting material glycosaminoglycan is 4.0-13.0 mmol / g, preferably 6.5-9.0 mmol / g, for example, 4.0 mmol / g, 4.5 mmol / g, 5.0 mmol / g, 5.5 mmol / g, 6.0 mmol / g, 6.5 mmol / g, 7.0 mmol / g, 7.5 mmol / g, 8.0 mmol / g.

[0108] According to an embodiment of the present invention, in step c), the reaction temperature is 0°C-15°C.

[0109] According to an embodiment of the present invention, in step c), the reaction pH is 3.0-5.0, preferably 3.5-4.5, such as 3.5, 4.0, 4.5.

[0110] According to an embodiment of the present invention, in step c), the reaction time is 12h-36h, preferably 18h-30h, for example 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h.

[0111] According to an embodiment of the present invention, after the reaction in step c) is completed, the pH of the reaction solution is adjusted to neutral, ethanol is added, and the solution is allowed to stand to obtain a precipitate.

[0112] According to an embodiment of the present invention, in step c), the polydispersity coefficient of the precipitate is 1.20-1.40, preferably 1.22-1.30, for example 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29.

[0113] According to an embodiment of the present invention, in step d), the product of step c) is dissolved in an acidic aqueous solution, loaded, and separated and purified by an anion exchange purification system to obtain the chemically modified glycosaminoglycan.

[0114] According to an embodiment of the present invention, in step d), the acidic aqueous solution is selected from one or more of an acetic acid aqueous solution, a phosphoric acid aqueous solution, a citric acid aqueous solution, and a sodium dihydrogen phosphate aqueous solution; preferably, the concentration of the acidic aqueous solution is 0.05%-10% (w / w%), preferably, 0.2%-2% (w / w%).

[0115] According to an embodiment of the present invention, in step d), the anion exchange purification system is one of a weak anion exchange purification system and a strong anion exchange purification system; preferably, the anion exchange purification system is a weak anion exchange purification system; preferably, the chromatographic conditions of the anion exchange purification system include: the filler is selected from one of DEAE SepharoseFast Flow, ANX Sepharose 4FF, and DEAE Seplife FF.

[0116] According to an embodiment of the present invention, in step d), the chromatographic conditions of the anion exchange purification system include: mobile phase A is an acidic solution; preferably, the acid in the acidic solution is selected from one or more of acetic acid, phosphoric acid, citric acid, and sodium dihydrogen phosphate, preferably, the acid in the acidic solution is acetic acid; preferably, the concentration of the acidic solution is 0.05%-10% (w / w%), preferably, 0.2%-2% (w / w%), and further preferably, 0.1-0.5% acidic aqueous solution; for example, mobile phase A is 0.3% acetic acid aqueous solution.

[0117] According to an embodiment of the present invention, in step d), the chromatographic conditions of the anion exchange purification system include: mobile phase B is an acidic solution containing salt; preferably, the salt is selected from one or more of sodium chloride, sodium acetate, disodium hydrogen phosphate, and sodium perchlorate; preferably, the concentration of the salt in mobile phase B is 0.5-3 mol / L; preferably, the acid in the acidic solution is selected from one or more of acetic acid, phosphoric acid, citric acid, and sodium dihydrogen phosphate, preferably, the acid in the acidic solution is acetic acid; the concentration of the acidic solution is 0.05%-10% (w / w%), preferably, 0.2%-2% (w / w%), and further preferably, 0.1-0.5% acidic aqueous solution; for example, mobile phase B is a 0.1-0.5% (e.g., 0.3%) acetic acid aqueous solution containing 0.5-3 mol / L (e.g., 2 mol / L) sodium chloride.

[0118] According to an embodiment of the present invention, in step d), the chromatographic conditions of the anion exchange purification system include: the detector is selected from one of an ultraviolet detector and a conductivity detector.

[0119] According to an embodiment of the present invention, in step d), the chromatographic conditions of the anion exchange purification system include:

[0120] Preparative liquid chromatograph (eg SCG-030), UV detector (eg DAD 600EX UV detector).

[0121] According to an embodiment of the present invention, the chromatographic column filler is DEAE Sepharose Fast Flow, with a particle size of 90 μm and a size of 50 mm×100 mm;

[0122] Preferably, mobile phase A is a 0.1-0.5% (e.g. 0.3%) acetic acid aqueous solution, and mobile phase B is a 0.1-0.5% (e.g. 0.3%) acetic acid aqueous solution containing 0.5-3 mol / L (e.g. 2 mol / L) sodium chloride;

[0123] Preferably, the average linear velocity of the mobile phase is 0.1 to 5.0 cm / min; more preferably, the average linear velocity of the mobile phase is 0.5 to 2.0 cm / min; more preferably, the average linear velocity of the mobile phase is 0.8 to 1.2 cm / min (e.g., 1.0 cm / min);

[0124] Preferably, the sample is loaded at room temperature;

[0125] Preferably, the running time is 300 minutes;

[0126] Preferably, the gradient elution conditions are as shown in Table 2:

[0127] Table 2

[0128]

[0129] According to an embodiment of the present invention, in step d), in the elution process 2, the mobile phase B (%) is 25%, 26%, 27%, 28%, 29%, or 30%.

[0130] According to an embodiment of the present invention, in step d), in the elution process 3, the mobile phase B (%) is 50%, 51%, 52%, 53%, 54%, or 55%.

[0131] According to an embodiment of the present invention, in step d), the eluate of elution process 3 (210-300 min) is collected, the pH is adjusted to 6.0-7.0, desalted, concentrated, ethanol precipitated, dissolved in water and freeze-dried to obtain the chemically modified glycosaminoglycan.

[0132] The present invention also provides chemically modified glycosaminoglycan prepared by the above preparation method.

[0133] In the present invention, "the compound of the present invention", "chemically modified glycosaminoglycan", and "chemically modified glycosaminoglycan of the present invention" can be used interchangeably and all refer to the chemically modified glycosaminoglycan as described above.

[0134] The term "sulfation degree" herein refers to the sulfonate to carboxyl ratio (SO3 - / COO - Molar ratio), which is determined by conductometric titration according to Casu B. and Gennaro U., 1975, Carbohydr Res 39, 168-176.

[0135] The term "carboxyl increment" herein refers to the ratio of the sulfation degree of the raw material to the sulfation degree of the carboxylated derivative. More specifically, the sulfation degree of the raw material is the sulfation degree measured after reducing a sample of the glycosaminoglycan intermediate obtained after the first oxidation step of the raw material with NaBH4.

[0136] The term "uronic acid ring opening degree" herein refers to the number of ring-opened uronic acid residues / total number of uronic acid residues, and the term "uronic acid epoxidation degree" refers to the ratio of uronic acid structural units with epoxy structures of formula (V) to total uronic acid residues. Their detection and calculation references Guerrini, M., Guglieri, S., Naggi, A., Sasisekharan, R., & Torri, G. (2007). Low molecular weight heparins: Structural differentiation by bidimentional nuclear magnetic resonance spectroscopy. Seminars in Thrombosis and Hemostasis, 33, 478–487 were used for nuclear magnetic resonance methods.

[0137] Pharmaceutical compositions and methods of treatment

[0138] The present invention provides a pharmaceutical composition, which comprises a preventive or therapeutically effective amount of the glycosaminoglycan of the present invention and one or more pharmaceutically acceptable carriers; the pharmaceutical composition is preferably a solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.

[0139] In some embodiments, the pharmaceutical composition may further comprise one or more additional therapeutic agents.

[0140] The present invention also provides use of the glycosaminoglycan of the present invention or the pharmaceutical composition of the present invention in preparing a drug for inhibiting tumor growth and / or metastasis.

[0141] The present invention provides use of the chemically modified glycosaminoglycan or the pharmaceutical composition in preparing a drug for treating and / or preventing tumors / cancers.

[0142] The present invention provides the glycosaminoglycan of the present invention or the pharmaceutical composition of the present invention for use in inhibiting tumor growth and / or metastasis.

[0143] The present invention provides the chemically modified glycosaminoglycan or the pharmaceutical composition, which is used for treating and / or preventing tumors / cancers.

[0144] The present invention provides a method for inhibiting tumor growth and / or metastasis, which comprises administering an effective amount of the glycosaminoglycan of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

[0145] In some embodiments, the tumor is a solid tumor, a blood tumor, or a soft tissue tumor; preferably a solid tumor, such as breast cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, gastric cancer, or lung cancer.

[0146] The present invention provides a method for treating and / or preventing tumors / cancers, which comprises administering an effective amount of the glycosaminoglycan of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

[0147] In some embodiments, the tumor is a solid tumor, a blood tumor, or a soft tissue tumor; preferably a solid tumor, such as breast cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, gastric cancer, or lung cancer.

[0148] In the present invention, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle that is administered together with a therapeutic agent and is suitable for contact with the tissues of humans and / or other animals within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response or other problems or complications corresponding to a reasonable benefit / risk ratio.

[0149] Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition may also contain a small amount of wetting agent, emulsifier or pH buffer as needed. Oral formulations may contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

[0150] Pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, for example by injection (such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, local, in the form of ophthalmic preparations or by inhalation.

[0151] For these administration routes, the pharmaceutical composition of the present invention can be administered in suitable dosage forms.

[0152] The dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0153] As used herein, the term "effective amount" refers to that amount of a derivative which, when administered, will relieve to some extent one or more symptoms of the condition being treated.

[0154] The dosage regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is to be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the composition or supervising the administration of the composition.

[0155] The amount of the glycosaminoglycan of the present invention administered will depend on the individual being treated, the severity of the disease or condition, the rate of administration, the disposal of the glycosaminoglycan and the judgment of the prescribing physician. In general, the effective dose is about 0.0001 mg to about 50 mg per kg body weight per day, for example, about 0.01 mg / kg / day to about 10 mg / kg / day (single or divided administration). For a 70kg person, this will add up to about 0.007 mg / day to about 3500 mg / day, for example, about 0.7 mg / day to about 700 mg / day. In some cases, a dosage level not higher than the lower limit of the aforementioned range may be sufficient, and in other cases, a larger dose may still be used without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

[0156] The content or dosage of glycosaminoglycans of the present invention in the pharmaceutical composition can be about 0.01 mg to about 1000 mg, suitably 0.1 mg-500 mg, preferably 0.5 mg-300 mg, more preferably 1 mg-150 mg, particularly preferably 1-50 mg, for example 1.5 mg, 2 mg, 4 mg, 10 mg, 25 mg, etc.

[0157] As used herein, unless otherwise indicated, the terms "treat," ...

[0158] As used herein, "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) suffering from diseases (e.g., diseases described herein) or normal individuals. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0159] Beneficial Effects

[0160] The present invention provides a chemically modified glycosaminoglycan, which, on the basis of retaining a part of the six-membered ring structure of the glycosaminoglycan, introduces an iduronic acid structure through a reaction and obtains the chemically modified glycosaminoglycan of the present invention with good anti-tumor activity, good safety and excellent drugability by controlling the appropriate degree of ring opening, and has good physical / chemical stability. The chemically modified glycosaminoglycan of the present invention has good anti-tumor activity, especially anti-tumor metastasis and anti-tumor growth activity, has good stability, metabolic stability, good bioavailability, low toxic and side effects, good safety, can be made into a suitable preparation, improve drug safety, improve patient compliance and administration convenience. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 Molecular weight distribution diagram of intermediate 4j and sample J in Example 10;

[0162] Figure 2 Comparative Example 2 Overlay of the NMR HSQC spectra of intermediate 2j and unfractionated heparin sample; wherein the spectrum represented by blue is the NMR HSQC spectrum of intermediate 2j, and the spectrum represented by red is the NMR HSQC spectrum of unfractionated heparin (starting reactant);

[0163] Figure 3 NMR HSQC spectra of sample J in Comparative Example 3 and sample 1 in Comparative Example; Figure 3 a. Figure 3 b are the NMR HSQC spectra of sample J and the sample of comparative example 1, respectively; Figure 3 c is an overlay of the NMR HSQC spectra of sample J and the sample of comparative example 1, wherein the spectrum represented by red is the NMR HSQC spectrum of sample J of Example 10, and the spectrum represented by blue is the NMR HSQC spectrum of the sample of comparative example 1;

[0164] Figure 4 Bioluminescent imaging photos of each group of mice in the growth experiment of anti-MSTO-211H human pleural mesothelioma in Test Example 3 of the present application;

[0165] Figure 5 Tumor growth curve of each test sample in the experiment of Test Example 4 of the present application on subcutaneous transplanted tumor of human pancreatic cancer xenograft cell Mia PaCa-2;

[0166] Figure 6 The survival rate curve of each group of mice after subcutaneous injection of the compound of the present invention and the sample of Comparative Example 1 in the orthotopic 4T1 breast cancer lung metastasis animal experiment of Test Example 5 of the present application;

[0167] Figure 7The time-concentration curves of each drug in rat plasma after subcutaneous injection of the compound of the present invention and the sample of Comparative Example 1 in the experiment of Test Example 6 of the present application. DETAILED DESCRIPTION

[0168] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0169] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0170] Overview: Using unfractionated heparin (UFH) or low molecular weight heparin (LMWH) as the starting material, the C2 desulfurization group is carried out under controlled conditions to keep the stereo configuration unchanged to obtain glycosaminoglycans containing iduronic acid structure. Then, the adjacent diol and optional adjacent OH / NH2 are effectively converted into aldehydes through a controlled vicinal diol cleavage reaction, and then the corresponding aldehyde group is converted into the corresponding carboxyl group under appropriate oxidation conditions.

[0171] Glycosaminoglycans: Glycosaminoglycans are heteropolysaccharides, which are unbranched long-chain polymers composed of repeating complex units containing hexuronic acid and hexosamine components.

[0172] Unfractionated heparin (UFH), also known as ordinary heparin, is a sulfated glycosaminoglycan compound with a molecular weight of generally 3,000 to 30,000 Daltons (Da). It is a heterogeneous polysaccharide composed of disaccharide units formed by uronic acid (L-iduronic acid, IdoA and D-glucuronic acid, GlcA) and hexosamine (α-D-glucosamine, GlcN) and their derivatives (acetylated, sulfated).

[0173] Low molecular weight heparin (LMWH) is obtained from unfractionated heparin through different degradation processes. Its monosaccharide composition, substitution pattern and oligosaccharide sequence structure characteristics are similar to those of heparin, but its molecular weight is lower than that of ordinary heparin.

[0174] The freeze-drying equipment and process conditions used in the examples of the present invention are as follows:

[0175] Apparatus: Low temperature vacuum freeze dryer (LGJ-20F), freeze drying area 0.2 square meters.

[0176] The detailed parameters of the freeze-drying curve are shown in Table 1 below:

[0177] Table 1

[0178]

[0179] The chromatographic conditions (instrument and analysis conditions) of the weak anion exchange purification system used in the embodiment of the present invention are as follows:

[0180] Instrument: Preparative liquid chromatograph (SCG-030), UV detector (DAD 600EX UV detector).

[0181] Column specifications: Filler: DEAE Sepharose Fast Flow, particle size: 90 μm, size: 50 mm × 100 mm; mobile phase A is 0.3% acetic acid aqueous solution, mobile phase B is 0.3% acetic acid aqueous solution containing 2 mol / L sodium chloride; average linear velocity of mobile phase is 1.0 cm / min, sample loading at room temperature; running time is 300 minutes; gradient elution conditions are shown in Table 2:

[0182] Table 2

[0183]

[0184] Note: The sum of the proportions of mobile phase A and mobile phase B is 100%.

[0185] The polydispersity index (PDI) is a parameter that characterizes the dispersion of a substance. The polydispersity index measured by gel permeation chromatography (GPC) is the ratio of the weight average molecular weight to the number average molecular weight (i.e., PDI = Mw / Mn). The smaller the PDI value, the more uniform the molecular weight in the system, and the better the system quality; the PDI of an absolutely monodisperse system is equal to 1, and it is generally believed that a PDI less than 1.2 can be regarded as narrow dispersion.

[0186] The mean linear velocity of mobile phase is the average speed at which the mobile phase moves along the axial direction of the chromatographic column. It is the average length of the mobile phase flowing through the chromatographic column per unit time, and its unit is cm / min.

[0187] The apparatus and conditions of the gel permeation chromatography analysis method in the present invention are as follows:

[0188] EMPOWER workstation; ultra-high performance liquid chromatograph (ACQUITY UPLC), detector (differential detector), chromatographic column is ACQUITY APC AQ, 4.6mm X 150mm 2.5μm; injection volume is 10μl; mobile phase is 0.1M ammonium acetate solution containing 0.02% sodium azide, flow rate is 0.5ml / min, isocratic elution; acquisition time is 10min.

[0189] HSQC: heteronuclear singular quantum correlation (HSQC), is a type of NMR (nuclear magnetic resonance) CH COSY spectrum. HSQC provides information about directly connected carbon-hydrogen relationships, giving a one-key CH connection relationship. In this application, the HSQC method is used to measure the intensity of directly connected CH related signal peaks.

[0190] In the HSQC spectrum of the present application, the chemical shift value of the CH-related signal peak on the C2 acetyl group of N-acetylglucosamine is δ

[0191] (2.07±0.10, 104.8±1.0). The characteristic signal of sulfated iduronic acid is: the CH related signal peak at the C1 position of sulfated iduronic acid, and its chemical position value is δ(5.222±0.10, 101.97±1.0). The characteristic signal peak of iduronic acid is: the CH related signal peak at the C1 position of N-sulfonated glucosamine connected to iduronic acid through a C4 glycosidic bond, and its chemical shift value is δ(5.35±0.10, 98.2±1.0). The specific structure is shown in the following chemical structure formula:

[0192]

[0193] Taking pyranose glucose as an example, the numbering rule of C in the pyranose ring is shown in the following chemical structure formula. The numbering of C in uronic acid and glucosamine in the present invention is carried out with reference to the numbering rule of C in the pyranose ring of the following formula:

[0194]

[0195] In HSQC, the chemical shift of the NMR HSQC peak was assigned according to the reference "Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins" (Mauri, L., et al., Journal of Pharmaceutical and Biomedical Analysis, 2017, 136: 92-105).

[0196] Iduronic acid content, the absolute content of iduronic acid in this application is difficult to detect, and the amount of acetylated glucosamine remains basically unchanged during the reaction, so the CH-related signal on the acetyl group at the C2 position of N-acetylated glucosamine is used as the standard (i.e., internal standard) to characterize the relative content of iduronic acid. The calculation formula of iduronic acid content (relative content) is: iduronic acid content = (iduronic acid signal peak intensity / CH-related signal peak intensity on the acetyl group at the C2 position of N-acetylglucosamine) * 100%, and the HSQC method is used in this application to determine the intensity of each CH-related signal peak.

[0197] Example 1

[0198] (1) Weigh 5 g of unfractionated heparin and add it to 50 g of ultrapure water. Add sodium borohydride (5.0 mmol, 0.19 g) and stir to dissolve at room temperature.

[0199] (2) Add 1.6 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve (Table 1), and transfer the above solution to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0200] (3) Add sodium periodate solution (0.2 mol / L, 50 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 12 h. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 h, terminate the reaction, and dialyze for 24 h (to remove salt) to obtain a dialysate.

[0201] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 50 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 4.5, the reaction was continued with stirring for 12 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4a, and its polydispersity coefficient was 1.257.

[0202] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 25% of the eluent B, and the elution process 3 was 50% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample A. The yield was 68.5%.

[0203] Example 2

[0204] (1) Weigh 5 g of unfractionated heparin and add it to 120 g of ultrapure water. Add sodium borohydride (13.0 mmol, 0.49 g) and stir to dissolve at room temperature.

[0205] (2) Add 1.8 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0206] (3) Add sodium periodate solution (0.2 mol / L, 50 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 14 h. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 h, terminate the reaction, dialyze for 24 h (desalting), and obtain a dialysate.

[0207] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 50 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 4.0, the reaction was continued with stirring for 20 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4b, and its polydispersity coefficient was 1.283.

[0208] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein 30% of the eluent B was used in elution process 2, and 55% of the eluent B was used in elution process 3. The eluent was collected for 210 min to 300 min, and the pH of the eluent was adjusted to 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample B. The yield was 83.2%.

[0209] Example 3

[0210] (1) Weigh 5 g of unfractionated heparin and add it to 110 g of ultrapure water. Add sodium borohydride (21.0 mmol, 0.79 g) and stir to dissolve at room temperature.

[0211] (2) Add 2.1 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0212] (3) Add sodium periodate solution (0.2 mol / L, 50 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 18 hours. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 hour, terminate the reaction, dialyze for 24 hours (to remove salt), and obtain a dialysate.

[0213] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 50 mL) was added to the dialysate obtained in step (3), and glacial acetic acid was added to adjust the pH to about 4.0. The reaction was continued with stirring for 22 h, and the pH was adjusted to neutral with NaOH solution (30% w / v). Ethanol was added for precipitation, and the mixture was allowed to stand for 24 h. The supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4c, and its polydispersity coefficient was 1.275.

[0214] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 28% of the eluent B, and the elution process 3 was 53% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain a sample C. The yield was 76.9%.

[0215] Example 4

[0216] (1) Weigh 5 g of unfractionated heparin and add it to 40 g of ultrapure water. Add sodium borohydride (23.8 mmol, 0.90 g) and stir to dissolve at room temperature.

[0217] (2) Add 2.4 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0218] (3) Add sodium periodate solution (0.2 mol / L, 50 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 10 h. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 h, terminate the reaction, dialyze for 24 h (desalting), and obtain a dialysate.

[0219] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 50 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 4.0, the reaction was continued with stirring for 24 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4d, and its polydispersity coefficient was 1.239.

[0220] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 26% of the eluent B, and the elution process 3 was 51% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain a sample D. The yield was 70.7%.

[0221] Example 5

[0222] (1) Weigh 5 g of unfractionated heparin and add it to 100 g of ultrapure water. Add sodium borohydride (26.0 mmol, 0.98 g) and stir to dissolve at room temperature.

[0223] (2) Add 2.2 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0224] (3) Add sodium periodate solution (0.2 mol / L, 60 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 16 h. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 h, terminate the reaction, dialyze for 24 h (desalting), and obtain a dialysate.

[0225] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 60 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 4.0, the reaction was continued with stirring for 24 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4e, and its polydispersity coefficient was 1.253.

[0226] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 28% of the eluent B, and the elution process 3 was 53% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample E. The yield was 74.9%.

[0227] Example 6

[0228] (1) Weigh 5 g of unfractionated heparin and add it to 90 g of ultrapure water. Add sodium borohydride (27.0 mmol, 1.02 g) and stir to dissolve at room temperature.

[0229] (2) Add 1.9 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0230] (3) Add sodium periodate solution (0.2 mol / L, 60 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 18 h. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 h, terminate the reaction, dialyze for 24 h (desalting), and obtain a dialysate.

[0231] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 60 mL) was added to the dialysate obtained in step (3), and glacial acetic acid was added to adjust the pH to about 4.0. The reaction was continued with stirring for 22 h, and the pH was adjusted to neutral with NaOH solution (30% w / v). Ethanol was added for precipitation, and the mixture was allowed to stand for 24 h. The supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4f, and its polydispersity coefficient was 1.241.

[0232] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 26% of the eluent B, and the elution process 3 was 51% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample F. The yield was 70.1%.

[0233] Example 7

[0234] (1) Weigh 5 g of unfractionated heparin and add it to 100 g of ultrapure water. Add sodium borohydride (40.0 mmol, 1.51 g) and stir to dissolve at room temperature.

[0235] (2) Add 2.6 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0236] (3) Add sodium periodate solution (0.2 mol / L, 40 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 18 h. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 h, terminate the reaction, and dialyze for 24 h (to remove salt) to obtain a dialysate.

[0237] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 40 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 4.0, the reaction was continued with stirring for 26 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as 4 g of an intermediate, and its polydispersity coefficient was 1.278.

[0238] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above weak anion exchange purification system analysis method, wherein the elution process 2 was 28% of the eluent B, and the elution process 3 was 53% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample G. The yield was 76.0%.

[0239] Example 8

[0240] (1) Weigh 5 g of unfractionated heparin and add it to 70 g of ultrapure water. Add sodium borohydride (52.0 mmol, 1.97 g) and stir to dissolve at room temperature.

[0241] (2) Add 3.3 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0242] (3) Add sodium periodate solution (0.2 mol / L, 40 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 20 hours. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 hour, terminate the reaction, dialyze for 24 hours (to remove salt), and obtain a dialysate.

[0243] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 40 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 4.0, the reaction was continued with stirring for 30 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was counted as intermediate 4h, and its polydispersity coefficient was 1.248.

[0244] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 26% of the eluent B, and the elution process 3 was 51% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample H. The yield was 71.9%.

[0245] Example 9

[0246] (1) Weigh 5 g of unfractionated heparin and add it to 50 g of ultrapure water. Add sodium borohydride (66.0 mmol, 2.50 g) and stir to dissolve at room temperature.

[0247] (2) Add 3.5 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0248] (3) Add sodium periodate solution (0.2 mol / L, 30 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 22 hours. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 hour, terminate the reaction, dialyze for 24 hours (to remove salt), and obtain a dialysate.

[0249] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 30 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 3.0, the reaction was continued with stirring for 28 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4i, and its polydispersity coefficient was 1.281.

[0250] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 28% of the eluent B, and the elution process 3 was 53% of the eluent B. The eluent was collected for 210 min to 300 min, and the eluent was adjusted to pH 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample I. The yield was 77.5%.

[0251] Example 10

[0252] (1) Weigh 5 g of unfractionated heparin and add it to 60 g of ultrapure water. Add sodium borohydride (80.0 mmol, 3.03 g) and stir to dissolve at room temperature.

[0253] (2) Add 3.8 g of sodium hydroxide solid, stir evenly, measure the pH, the solution pH should be above 13.0, set the freeze-drying curve, and transfer the above solution to a freeze dryer for freeze-drying reaction. After freeze-drying, add 100 g of water to dissolve the solid, adjust the pH to 3.8-4.0 with 6 M hydrochloric acid, continue stirring for 30 min, adjust the pH to 6.8-7.0, remove salt by ultrafiltration, and filter with a 0.22 μm filter membrane.

[0254] (3) Add sodium periodate solution (0.2 mol / L, 30 mL) to the reaction solution obtained in step (2) at 0-20°C, protect from light, and stir for 24 hours. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 hour, terminate the reaction, dialyze for 24 hours (to remove salt), and obtain a dialysate.

[0255] (4) At 0-15°C, sodium chlorite solution (0.67 mol / L, 30 mL) was added to the dialysate obtained in step (3), glacial acetic acid was added to adjust the pH to about 3.5, the reaction was continued with stirring for 30 h, the pH was adjusted to neutral with NaOH solution (30% w / v), ethanol was added for precipitation, the mixture was allowed to stand for 24 h, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was calculated as intermediate 4j, and its polydispersity coefficient was 1.251.

[0256] (5) The precipitate obtained in step (4) was dissolved in mobile phase A, and 3 column volumes of the sample solution were quantitatively loaded, and separated and purified by a weak anion exchange purification system. The separation conditions were as described in the above-mentioned weak anion exchange purification system analysis method, wherein the elution process 2 was 26% of the eluent B, and the elution process 3 was 51% of the eluent B. The eluate was collected for 210 min to 300 min, and the pH of the eluate was adjusted to 6.0 to 7.0 with a NaOH solution (30% w / w) or a HCl solution (10% to 19%), desalted, concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain sample J with a yield of 72.8%.

[0257] The molecular weight distribution of intermediate 4j and sample J was measured by gel permeation chromatography (GPC). The molecular weight distribution of intermediate 4j and sample J is shown in the attached figure. Figure 1 The polydispersity coefficient of the refined sample J is 1.169, and the polydispersity coefficient of the intermediate 4j before purification is 1.251; after the purification step (5), the molecular weight distribution of the sample J is significantly narrowed, the uniformity of the product is higher, the polydispersity coefficient of the refined product is less than 1.2, and the product quality is better.

[0258] Comparative Example 1

[0259] The sample of Comparative Example 1 was prepared according to the method in Example 10 of WO2019149179A1, and the specific method is as follows:

[0260] 1) Weigh 5 g of unfractionated heparin (Mw=18306 Da) and dissolve it in 62.5 g of 1 mol / L NaOH solution. Heat the reaction solution to 60° C. and react for 30 min. After the reaction, cool the solution to room temperature and adjust the solution to neutrality with a 1:1 HCl solution (prepared with commercially available 36%-38% concentrated hydrochloric acid and purified water in a volume ratio of 1:1).

[0261] 2) The solution in step 1) was heated to 70°C and hydrolyzed under neutral conditions for 24 hours. After the reaction was completed, the temperature was cooled to room temperature.

[0262] 3) Sodium periodate oxidation: Add 100 g of sodium periodate solution (4.1%, w / w) to the reaction solution in step 2) under low temperature conditions, protect from light, control the temperature at 4°C, and stir for 16 hours. After the reaction is completed, add 10.0 mL of ethylene glycol, continue stirring for 1 hour, terminate the reaction, and dialyze the reaction solution.

[0263] 4) Add 100 g of 6.03% (m / v) NaClO2 solution to the reaction solution obtained in step 3) under low temperature conditions, adjust the pH to about 4.0 with glacial acetic acid, continue stirring and reacting for 24 hours, and adjust to neutral with 30% (m / v) NaOH solution. The reaction solution is dialyzed for 24 hours, and the dialyzate is concentrated by rotary evaporation and freeze-dried to obtain the sample of comparative example 1.

[0264] Comparative Example 2

[0265] The reaction was carried out according to the method described in step (1) and step (2) in Example 10, and the prepared reaction solution was concentrated, ethanol precipitated, dissolved in water, and freeze-dried to obtain intermediate 2j.

[0266] The prepared intermediate 2j and the unfractionated heparin sample (starting reactant) were subjected to NMR HSQC spectrum testing, and the NMR HSQC spectra of the two samples were superimposed. Figure 2 Attached Figure 2 The spectrum in blue is the NMR HSQC spectrum of intermediate 2j, and the spectrum in red is the NMR HSQC spectrum of unfractionated heparin (starting reactant).

[0267] By contrast, it can be clearly seen in the figure that there are a large number of characteristic signal peaks of iduronic acid in intermediate 2j (blue), and there is substantially no characteristic signal peak of sulfonated iduronic acid. It can be clearly seen in the figure that intermediate 2j (blue) is compared with the unclassified heparin (red) of starting material, and the non-sulfonated iduronic acid characteristic signal peak of intermediate 2j is significantly strengthened, and the sulfonated iduronic acid characteristic signal peak is significantly weakened; It is proved that there are a large number of non-sulfonated iduronic acid structures generated in the structure of intermediate 2j, and a large number of sulfonated iduronic acids are converted into non-sulfonated iduronic acid in the unclassified heparin structure of starting material. It is explained that through the reaction of embodiment 10 steps (1) and step (2), a large number of iduronic acid structures are produced.

[0268] Comparative Example 3

[0269] The nuclear magnetic resonance HSQC spectra of sample J in Example 10 and the sample in Comparative Example 1 were tested. The nuclear magnetic resonance HSQC spectra of sample J and the sample in Comparative Example 1 are shown in the attached Figure 3 a. Attachment Figure 3 b; Nuclear magnetic resonance The obtained nuclear magnetic resonance HSQC spectra of the two samples were superimposed, see attached Figure 3 c. Attachment Figure 3 The spectrum represented by red in c is the NMR HSQC spectrum of sample J in Example 10, and the spectrum represented by blue is the NMR HSQC spectrum of the sample in Comparative Example 1; Figure 3 It can be clearly seen in c that there are a large number of iduronic acid characteristic signal peaks (peak 1) in sample J, indicating that there are a large number of iduronic acid structures in sample J. The sample of comparative example 1 has almost no iduronic acid characteristic signal peaks, indicating that there is almost no iduronic acid structure in the sample of comparative example 1.

[0270] The preparation process of sample J in Example 10 and the sample in Comparative Example 1 does not involve changes in the acetyl group. Therefore, the integral value of the CH-related signal at position 1 of the N-sulfonated glucosamine connected with iduronic acid in the HSQC spectrum is A, and the integral value of the CH-related signal on the acetyl group of N-acetylated glucosamine in the HSQC spectrum is B. The ratio of A / B in each embodiment is used to quantitatively compare the remaining situation of the non-sulfonated iduronic acid structure, and the content of iduronic acid is defined as (%) = A / B*100%. The chemical shift attribution of the nuclear magnetic resonance HSQC peak is referenced to "Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins" (Mauri, L., et al., Journal of Pharmaceutical and Biomedical Analysis, 2017, 136: 92-105). The physicochemical property data of the products obtained in the above embodiments and Comparative Example 1 are summarized as shown in Table 3-4.

[0271] Table 3

[0272]

[0273] Table 4

[0274] serial number Step (4) Intermediate polydispersity coefficient Polydispersity coefficient of the final product Example 1 1.257 1.125 Example 2 1.283 1.185 Example 3 1.275 1.177 Example 4 1.239 1.152 Example 5 1.253 1.174 Example 6 1.241 1.144 Example 7 1.278 1.179 Example 8 1.248 1.142 Example 9 1.281 1.182 Example 10 1.251 1.169

[0275] By comparing the compound of the present invention with the sample of Comparative Example 1, the weight average molecular weight of the compound of the present invention is 14000-19000Da, the molecular weight distribution concentration of the compound of the present invention is high, the molecular weight is mainly distributed in the range of 6000-19000, the proportion of components with a molecular weight less than 6000 is less than <1.5%, and the proportion of components with a molecular weight greater than 19000 is 23-36%. The polydispersity coefficient of the compound of the present invention is good, and there is a significant difference in the molecular weight distribution of the sample of Comparative Example 1. The polydispersity coefficients of the compound products of the present invention are all lower than 1.2, and the polydispersity is good; the iduronic acid content of the product of the present invention is significantly higher than that of the sample of Comparative Example 1, which is 10 to 30.8 times that of the sample of Comparative Example 1.

[0276] In summary, there are significant differences in molecular weight distribution, iduronic acid content, polydispersity, etc. between the product of the present invention and the sample of Comparative Example 1.

[0277] Biological experiments

[0278] The prepared products were tested from the perspectives of in vitro heparanase inhibition, anticancer cell growth, anticancer cell metastasis, and acute toxicity.

[0279] Test Example 1: In vitro heparanase inhibition assay (Fondaparinux method, with Fondaparinux as substrate)

[0280] Referring to the method for in vitro detection of heparanase (heparanase-like) inhibitory activity in CN105744940A, the in vitro heparanase inhibitory activity of the product prepared in this application was detected. The results are shown in Table 5 below.

[0281] Table 5

[0282]

[0283]

[0284] As shown in Table 5, the compounds of the present invention have good heparanase inhibitory activity, and the heparanase inhibitory activity of the compounds of the present invention is better than that of the sample in Comparative Example 1.

[0285] Test Example 2 Isotope Method - Heparanase Inhibition Activity Detection

[0286] Use containing 35 Endothelial cells were cultured in medium containing S isotope, and after they were fully grown, the cells were removed, leaving only the extracellular matrix. 35 S-labeled endothelial extracellular matrix was coated on the surface of a 35 mm tissue culture dish and incubated with recombinant human heparanase (200 ng / ml) in the absence and presence of inhibitory molecules (5 h, 37 ° C, pH 6.0, final volume 1 ml). The reaction mixture contained: 50 mm NaCl, 1 mm DTT, 1 mm CaCl2, 10 mm phosphate-citrate buffer, pH 6.0. To assess the extent of extracellular matrix degradation, the culture medium was collected and gel filtered on a Sepharose 6B column. The fractions (0.2 ml) were eluted with PBS and the radioactivity was counted. The exclusion volume (Vo), elution volume (Ve) and total inclusion volume (Vt) were recorded, and the Kav value can be calculated, Kav = (Ve-Vo) / (Vt-Vo), where for the Sepharose 6B column, the products in the elution volume corresponding to Kav of 0.5 to 0.8 are the degradation fragments of the extracellular matrix, corresponding to fractions 20-35. By setting the experimental conditions, defining the heparanase activity corresponding to the radioactivity of fraction 20-35 when there is no heparanase as 0, and defining the heparanase activity corresponding to the radioactivity when there is no inhibitory molecule as 100%, the heparanase inhibitory activity corresponding to the inhibitory molecule at different concentrations can be obtained, and the IC of different inhibitory molecules can be obtained by calculation. 50 The specific values ​​are shown in Table 6 below.

[0287] Table 6

[0288] Sample No. <![CDATA[Heparanase inhibitory activity (IC 50 , ng / mL)]]> Example 1 Sample A 328.6 Example 2 Sample B 292.7 Example 3 Sample C 296.4 Example 4 Sample D 295.3 Example 5 Sample E 259.8 Example 6 Sample F 266.3 Example 7 Sample G 340.4 Example 8 Sample H 342.7 Example 9 Sample I 361.1 Example 10 Sample J 383.5 Comparative Example 1 Sample 538.7

[0289] It is well known to those skilled in the art that the isotope method-heparanase inhibition activity detection method used in this test example has significant advantages over the fondaparinux method-heparanase inhibition activity detection method in Test Example 1. The advantage of this test example method is that an isotope-labeled heparan sulfate substrate is used instead of fondaparinux sodium. The isotope-labeled heparan sulfate substrate is closer to the substrate of heparanase in the extracellular microenvironment of the organism, and can better characterize the heparanase inhibition activity of the compound of the present invention in the in vivo environment.

[0290] It can be seen from the results in Table 6 that the heparanase inhibitory activity of the compounds of the present application is significantly better than that of the sample in Comparative Example 1.

[0291] Test Example 3 Anti-MSTO-211H Human Pleural Mesothelioma Growth Experiment

[0292] Luciferase-labeled human pleural mesothelioma MSTO-211H cells (MSTO-211H / luciferase, purchased from Cyagen Biosciences Inc) were cultured in vitro in DMEM medium with 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. The cells were passaged three times a week. When the cells were in the exponential growth phase, the cells were harvested, counted, and diluted with PBS containing 5*10 6 Suspension of cells / mL. Take SPF-grade healthy NOD / SCID mice weighing 18-20g (6-7 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2021-0006), and inoculate luciferase-labeled MSTO-211H human mesothelioma cells into the peritoneum of NOD / SCID mice at a rate of 0.1mL / mouse by intraperitoneal injection. Taking cell inoculation as the first day, the mice were divided into 12 groups (5 mice in each group), which were set as model group and experimental group respectively. On the third day after cell inoculation, the experimental group was subcutaneously injected with the sample solution of Comparative Example 1 and the solution of the compound of the embodiment of the present invention, respectively, once a day, with a dose of 40mg / kg; the model group was subcutaneously injected with an equal volume of normal saline, once a day. Starting from the 10th day after cell inoculation, the small animal in vivo optical two-dimensional imaging platform ( Lumina) was used to give the animals luciferin substrates for imaging, and the bioluminescence of the tumor was observed to examine the tumor development. The experiment was terminated on the 31st day after cell inoculation. The bioluminescence imaging photos of each group on the 31st day are attached. Figure 4The fluorescence intensity results and tumor inhibition rate of each group are shown in Table 7. The calculation formula of MSTO-211H tumor inhibition rate is as follows: tumor inhibition rate = (average fluorescence intensity of model group - average fluorescence intensity of experimental group) / average fluorescence intensity of model group * 100%.

[0293] Table 7

[0294] Group Mean fluorescein intensity Mean standard error Tumor inhibition rate Model Group 1.72E+06 4.59E+05 N / A Comparative Example 1 Sample 4.26E+05 1.14E+05 75.2％ Example 1 Sample A 1.28E+05 3.97E+04 92.6％ Example 2 Sample B 5.31E+04 2.39E+04 96.9％ Example 3 Sample C 7.39E+04 2.23E+04 95.7％ Example 4 Sample D 8.01E+04 2.69E+04 95.3％ Example 5 Sample E 2.40E+04 5.10E+03 98.6％ Example 6 Sample F 3.41E+04 4.78E+03 98.0％ Example 7 Sample G 1.73E+05 8.45E+04 89.9％ Example 8 Sample H 1.46E+05 9.12E+04 91.5％ Example 9 Sample I 2.34E+05 1.13E+04 86.4％ Example 10 Sample J 2.79E+05 6.63E+04 83.8％

[0295] When the experimental mice were inoculated with human pleural mesothelioma MSTO-211H cells containing luciferase, the tumor cells would grow and metastasize. During bioluminescence imaging, the lower the intensity of luciferin, the lower the degree of growth and metastasis of the tumor cells in the mouse body, reflecting the stronger tumor inhibition ability of the corresponding compound. Figure 4 As shown, the compounds of the present invention all have good tumor inhibition rates, and the tumor inhibition activities of the compounds of the present invention are good; the tumor inhibition rates of the compounds of the present invention are all significantly higher than those of the samples in Comparative Example 1.

[0296] Test Example 4 Anti-Mia PaCa-2 human pancreatic cancer xenograft cell subcutaneous tumor growth experiment in nude mice

[0297] Experimental materials: female NPSG immunodeficient mice (6-7 weeks old, purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., production license number: SYXK (Shanghai) 2018-0023), experimental compounds (prepared according to the method of the embodiment of the present invention), comparative example 1 sample (prepared according to the method of comparative example 1 of the present invention), purified water, physiological saline (purchased from Guangdong Kelun Pharmaceutical Co., Ltd., batch number G22121402A), Mia PaCa-2 cells (purchased from the cell bank of the Chinese Academy of Sciences), DMEM medium (Gibco, Thermo Fisher Scientific (China) Co., Ltd.), fetal bovine serum (Gibco, Thermo Fisher Scientific (China) Co., Ltd.), horse serum (Gibco, Thermo Fisher Scientific (China) Co., Ltd.), trypsin (Gibco, Thermo Fisher Scientific (China) Co., Ltd.), and matrigel (Corning Incorporated).

[0298] Experimental method: Mia PaCa-2 cells were cultured in vitro in suspension in DMEM medium with 10% heat-inactivated fetal bovine serum and 2.5% horse serum at 37°C and 5% CO2. Cells were subcultured three times a week. When the cells were in the exponential growth phase, the cells were harvested and counted. 5×10 6 200 μL cell suspension of tumor cells (cells resuspended in 1:1 PBS and matrix gel) was inoculated into the subcutaneous tissue of the right anterior scapula of experimental nude mice to establish a mouse pancreatic cancer model. When the tumor grew to an average volume of about 90 mm 3The mice were randomly divided into groups according to the tumor volume, 10 mice / group. The model group was given normal saline, and the treatment group was subcutaneously injected with a solution of the compound of the present invention or the sample solution of Comparative Example 1, with a dose of 80 mg / kg, and subcutaneously injected once a week for continuous administration, and the first administration time was recorded as D1. The tumor diameter was measured with a vernier caliper twice a week, and the weight of the mice was measured. The T / C% or tumor inhibition rate TGI (%) calculated by the following formula was used to investigate the effect of the drug on tumor growth. After the experiment was completed (the experiment was completed on the 24th day after group administration), the animals were anesthetized with CO2, and then the tumors were dissected and photographed. The experimental results are shown in Table 8 below.

[0299] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 , where a and b represent the major diameter and minor diameter respectively;

[0300] T / C(%)=(T i -T0) / (C i -C0)×100, where T i , C i T0 is the average tumor volume of the treatment group and the isotype control group at the end of the experiment; T0 and C0 are the average tumor volumes of the treatment group and the isotype control group at the beginning of the experiment; tumor inhibition rate (TGI) (%) = 100-T / C.

[0301] Table 8 The efficacy of each test sample on subcutaneous transplanted tumors of human pancreatic cancer xenograft cells Mia PaCa-2

[0302]

[0303] The results are as follows Figure 5 As shown in Table 8, the tumor inhibition rates of sample E in Example 5 and sample F in Example 6 of the present invention were 53% and 44%, respectively, which were both higher than the tumor inhibition rate of 31% of the sample in Comparative Example 1 on Mia PaCa-2 subcutaneous transplanted tumors; indicating that the compound of the present invention has a significant inhibitory effect on the growth of Mia PaCa-2 human xenografted cells in nude mice subcutaneous tumors, and the inhibitory activity of the compound of the present invention on MiaPaCa-2 human xenografted cells in nude mice subcutaneous tumors is significantly better than that of the sample in Comparative Example 1. During the experiment, it was also observed that the tumor-bearing mice were well tolerated by the compound of the present invention, and no death or obvious weight loss occurred, indicating that the compound of the present invention has good safety.

[0304] Test Example 5: Animal experiment on in situ 4T1 breast cancer lung metastasis

[0305] 4T1 cells were cultured in vitro in suspension in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. Cells were subcultured three times a week. When the cells were in the exponential growth phase, the cells were harvested and counted.5 50 μL cell suspension of tumor cells (cells suspended in RPMI 1640 culture medium without fetal bovine serum) was inoculated into the fourth abdominal fat pad. The mice used were BALB / c mice, female, 6-8 weeks old, weighing 18-22 grams. They were randomly divided into groups according to body weight and tumor inoculation order, with 10 mice in each group. The test sample group began to be dosed on the second day of inoculation. Subcutaneous administration once a day was divided into 20 mg / kg dose group, 40 mg / kg dose group and 80 mg / kg dose group. The control group was given an equal amount of normal saline. The in situ tumor was surgically removed 12 days after tumor inoculation. No medication was given on the same day, and medication continued in the above manner on the second day after surgery.

[0306] Mouse survival: When each mouse reached the experimental endpoint (death or weight loss greater than 20%), it was euthanized and the survival time was recorded. The Kaplan-Meier survival curve was drawn. Figure 6 ; Calculate the median survival time and the median survival time extension percentage. The specific results are shown in Table 9 below. The calculation formula for the median survival time extension percentage is as follows.

[0307] Median survival extension percentage (%) = (median survival of the drug administration group - median survival of the negative control group) / median survival of the negative control group * 100%.

[0308] Table 9

[0309]

[0310] Note: “NA” means that the value cannot be calculated.

[0311] As shown in Table 9 above, the compounds of the present invention can significantly prolong the median survival of experimental subjects. The compounds of the present invention can prolong the median survival of model mice by 105%, 149%, and 222% respectively at low doses (20 mg / kg), medium doses (40 mg / kg), and high doses (80 mg / kg). At different doses, the median survival of model mice given the compounds of the present invention is much higher than that given to the sample group of Comparative Example 1, and the median survival extension percentage of the low dose (20 mg / kg) of the compounds of the present invention is higher than that of the high dose group (80 mg / kg) of the sample of Comparative Example 1. The above description shows that the compounds of the present invention have a stronger activity of inhibiting tumor metastasis and / or tumor growth relative to the sample of Comparative Example 1, and the compounds of the present invention can greatly prolong the survival of experimental subjects.

[0312] Test Example 6 Pharmacokinetic Test

[0313] The purpose of this experiment is to study the single subcutaneous injection of each compound solution of the present invention and the sample solution of Comparative Example 1 into SD rats, detect the active ingredients in plasma, and evaluate their pharmacokinetic (PK) properties in SD rats.

[0314] Experimental materials: SD rats (weighing 180-280 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2021-0006), experimental compounds (prepared according to the method of the embodiment of the present invention), comparative example 1 sample (prepared according to the method of comparative example 1 of the present invention), and purified water.

[0315] Experimental method: SD rats were randomly divided into groups (6 rats per group, half male and half female), and were allowed to drink water freely during the experiment. They were fasted for more than 12 hours before administration and fed 4 hours after administration. Subcutaneous injection was used to administer the sample E of Example or an equivalent amount of physiological saline solution at a dose of 50 mg / kg to each group of SD rats.

[0316] Blood samples were collected into K2EDTA anticoagulant tubes at 0 min before administration and 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after administration and stored on ice until centrifugation.

[0317] Plasma must be centrifuged within 60 minutes after blood collection (at 8000 rpm for 5 minutes at 2-8°C). After centrifugation, the plasma is transferred to a 96-well plate or centrifuge tube, transported in an ice box, and stored at ≤-15°C until LC-MS / MS detection. The drug concentration in SD rat plasma was detected by LC-MS / MS bioanalysis. The non-compartmental model was used to analyze the blood drug concentration-time data using WinNonlinTM (Version 8.3, Certara, USA) to evaluate its pharmacokinetic (PK) properties in SD rats. The data are shown in Table 10, and the pharmacokinetic curves are shown in the attached Figure 7 .

[0318] Table 10 Pharmacokinetic parameters in plasma of male rats after injection of the compounds of the present invention

[0319]

[0320] The above results show that the compounds of the present invention have significantly improved pharmacokinetic properties, in particular, the AUC and T 1 / 2 All of them have extremely significant improvements, indicating that the compound of the present invention has good bioavailability compared with the sample in Comparative Example 1, which is conducive to the preparation of sustained-release preparations or long-acting preparations for once-a-day administration or with longer dosing intervals, and can greatly improve patient compliance and the convenience of administration for medical workers.

[0321] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description, and such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including all patents, patent applications, journal articles, books and any other disclosures) is incorporated herein by reference in its entirety.

Claims

1. A chemically modified glycosaminoglycan comprising a structural unit of formula (I), a structural unit of formula (II), a structural unit of formula (III) and a structural unit of formula (IV): in: R 1m , R 1n , R 1q Each occurrence is independently selected from H, -SO3 - and -(C=O)CH3, and R 1m and R 1n Preferably -SO3 at each occurrence - or -(C=O)CH3; R 2m , R 2n , R 2q and R 2x Each occurrence is independently selected from H and -SO3 - ; R 3n Each occurrence is independently selected from H and -SO3 - ; R 4m and R 4n Each occurrence is independently selected from H and -SO3 - .

2. The chemically modified glycosaminoglycan according to claim 1, characterized in that The weight average molecular weight of the chemically modified glycosaminoglycan is 12000Da-20000Da, preferably 14000Da-19000Da, 14500Da-18800Da, 15000Da-18700Da; Preferably, the iduronic acid content of the chemically modified glycosaminoglycan is 10%-70%, preferably 20%-35%, preferably 21%-26%; Preferably, the uronic acid ring-opening degree of the chemically modified glycosaminoglycan is 25%-45%, preferably 27%-44%; Preferably, the sulfonyl carboxyl ratio (SO3 - / COO - ) is 1.2-1.8, preferably 1.2-1.7; Preferably, the carboxyl increment of the chemically modified glycosaminoglycan is 1.1-1.8, preferably 1.15-1.6; Preferably, the polydispersity coefficient of the chemically modified glycosaminoglycan is 1.05-1.20, preferably 1.12-1.19; Preferably, the molecular weight distribution of the chemically modified glycosaminoglycan is as follows: Greater than 19000Da 20%~38% 6000-19000Da 62%~78% Less than 6000Da 0%~1.5%; Preferably, the epoxidation degree of uronic acid is less than 25%, and the epoxidation degree of uronic acid is 0.001%-10%.

3. The chemically modified glycosaminoglycan according to claim 1 or 2, characterized in that The chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (V): in: R 2t and R 3t Each occurrence is independently selected from H and -SO3 - ; Preferably, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (VI): in: R 2p Each occurrence is independently selected from H and -SO3 - ; Preferably, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (VII): in: R 2r and R 3r Each occurrence is independently selected from H and -SO3 - ; Preferably, the chemically modified glycosaminoglycan optionally further comprises a structural unit of formula (VIII): in: R 1s Each occurrence is independently selected from H, -SO3 - and -(C=O)CH3, and preferably -SO3 - or -(C=O)CH3; R 2s and R 4s Each occurrence is independently selected from H and -SO3 - ; R 3s -SO3 at each occurrence - .

4. A method for preparing the chemically modified glycosaminoglycan according to any one of claims 1 to 3, comprising the following steps: a) optionally, using glycosaminoglycan as a starting material to prepare a glycosaminoglycan derivative containing a C2-hydroxyuronic acid residue; preferably, the glycosaminoglycan derivative is obtained by freeze-drying in the presence of a base and a reducing agent; b) oxidizing 25%-45% (preferably 30%-40%) of the 2-O- and optionally 2N-, 3-O- non-sulfated residues of the glycosaminoglycan under conditions effective to convert adjacent diols (vicinal diols) and optionally adjacent OH / NH2 to dialdehydes; The oxidation is carried out in the presence of an oxidant, wherein the oxidant is selected from one of periodic acid and periodate (e.g. sodium periodate, potassium periodate); and c) further oxidizing the product obtained from step b) under conditions effective to convert the dialdehyde into a carboxyl group and without nitrogen protection; The further oxidation is preferably carried out by a chlorite (e.g. sodium chlorite, magnesium chlorite Mg(ClO2)2, barium chlorite Ba(ClO2)2); d) separating and purifying the product of step c) through an anion exchange purification system to obtain the chemically modified glycosaminoglycan.

5. The preparation method according to claim 4, characterized in that: In step a), the ratio of the molar amount of the reducing agent to the mass of the starting material glycosaminoglycan is 1.0-20.0 mmol / g, preferably 2.0-10.0 mmol / g; Preferably, in step a), the ratio of the molar amount of the base to the mass of the starting material glycosaminoglycan is 5.0-25.0 mmol / g, preferably 8.0-19.0 mmol / g; Preferably, in step a), the freeze-drying reaction comprises: a pre-freezing step, a primary drying step, and a secondary drying step; Preferably, the pre-freezing step is specifically as follows: lowering the plate temperature to -20°C to -80°C for 30-200 minutes and maintaining it for 30-300 minutes; for example, lowering the plate temperature to -40°C for 60 minutes and maintaining it for 120 minutes; Preferably, the primary drying step is as follows: raising the temperature of the board layer to -10-15°C for 40-480 minutes, maintaining it for 720-4800 minutes, and the vacuum degree is 5-80Pa (preferably, the vacuum degree is 10-30Pa); for example, raising the temperature of the board layer to -5°C for 90 minutes, maintaining it for 720 minutes, and the vacuum degree is 25±5Pa; Preferably, the secondary drying step is specifically as follows: raising the temperature of the board layer to 25-60°C for 30-240 minutes, maintaining it for 300-1200 minutes, and the vacuum degree is 5-50Pa (preferably, the vacuum degree is 5-25Pa); for example, raising the temperature of the board layer to 25°C for 30 minutes, maintaining it for 480 minutes, and the vacuum degree is 10±5Pa; Preferably, step a) specifically comprises the following steps: a1) reacting an aqueous solution of a starting material glycosaminoglycan with a reducing agent (preferably sodium borohydride); preferably, reacting at room temperature; preferably, reacting for 10-30 hours; preferably, stirring the reaction for 12-24 hours; a2) adding a base to the reaction solution of step a1) to adjust the pH to above 13.0, and then performing freeze-drying reaction; Optionally, after the reaction is completed, the freeze-dried solid is dissolved with water, the pH is adjusted to 3.8-4.0, stirring is continued for 30 minutes, and the pH is adjusted back to 6.8-7.0; optionally, ultrafiltration desalination and membrane filtration are further performed; Preferably, in step b), the ratio of the molar amount of the oxidant to the mass of the starting material glycosaminoglycan is 1-3.6 mmol / g; preferably 1.2-3.0 mmol / g; Preferably, in step b), the reaction temperature is 0°C-20°C; Preferably, in step b), the reaction time is 10h-30h, preferably 12h-24h; Preferably, step b) is reacted under light-proof conditions; Preferably, in step b), after the reaction is completed, an excess of a terminating reagent is added to terminate the reaction; optionally, the reaction solution is further dialyzed (for desalination); Preferably, in step b), the termination reagent is selected from one or more of ethylene glycol, ethanolamine, glycerol, glucose, mannose, glyceraldehyde, threose, and xylose; preferably, the termination reagent is selected from one or more of ethylene glycol, ethanolamine, and glucose; Preferably, in step c), the ratio of the molar amount of chlorite to the mass of the starting material glycosaminoglycan is 4.0-13.0 mmol / g, preferably 6.5-9.0 mmol / g; Preferably, in step c), the reaction temperature is 0°C-15°C; Preferably, in step c), the reaction pH is 3.0-5.0, preferably 3.5-4.5; Preferably, in step c), the reaction time is 12h-36h, preferably 18h-30h; Preferably, after the reaction in step c) is completed, the pH of the reaction solution is adjusted to neutral, ethanol is added, and the mixture is allowed to stand to obtain a precipitate; Preferably, in step d), the product of step c) is dissolved in an acidic aqueous solution, loaded, and separated and purified by an anion exchange purification system to obtain the chemically modified glycosaminoglycan; preferably, the acidic aqueous solution is selected from one or more of an acetic acid aqueous solution, a phosphoric acid aqueous solution, a citric acid aqueous solution, and a sodium dihydrogen phosphate aqueous solution; preferably, the concentration of the acidic aqueous solution is 0.05%-10% (w / w%); Preferably, the anion exchange purification system is one of a weak anion exchange purification system and a strong anion exchange purification system; Preferably, the chromatographic conditions of the anion exchange purification system include: the filler is selected from one of DEAE Sepharose FastFlow, ANX Sepharose 4FF, and DEAE Seplife FF; Preferably, the chromatographic conditions of the anion exchange purification system include: mobile phase A is an acidic solution; preferably, the acid in the acidic solution is selected from one or more of acetic acid, phosphoric acid, citric acid, and sodium dihydrogen phosphate; preferably, the concentration of the acidic solution is 0.05%-10% (w / w%); Preferably, the chromatographic conditions of the anion exchange purification system include: mobile phase B is an acidic solution containing salt; preferably, the salt is selected from one or more of sodium chloride, sodium acetate, disodium hydrogen phosphate, and sodium perchlorate; preferably, the concentration of the salt in mobile phase B is 0.5-3 mol / L; preferably, the acid in the acidic solution is selected from one or more of acetic acid, phosphoric acid, citric acid, and sodium dihydrogen phosphate; preferably, the concentration of the acidic solution is 0.05%-10% (w / w%); Preferably, in step d), the chromatographic conditions of the anion exchange purification system include: Preparative liquid chromatograph (e.g. SCG-030), UV detector (e.g. DAD 600EX UV detector); Preferably, the chromatographic column filler is DEAE Sepharose Fast Flow, with a particle size of 90 μm and a size of 50 mm × 100 mm; Preferably, mobile phase A is a 0.1-0.5% (e.g. 0.3%) acetic acid aqueous solution, and mobile phase B is a 0.1-0.5% (e.g. 0.3%) acetic acid aqueous solution containing 0.5-3 mol / L (e.g. 2 mol / L) sodium chloride; Preferably, the average linear velocity of the mobile phase is 0.1 to 5.0 cm / min; further preferably, the average linear velocity of the mobile phase is 0.5 to 2.0 cm / min; more preferably, the average linear velocity of the mobile phase is 0.8 to 1.2 cm / min (e.g., 1.0 cm / min); Preferably, the sample is loaded at room temperature; Preferably, the running time is 300 minutes; Preferably, the gradient elution conditions are as shown in Table 2: Table 2 Preferably, in step d), in the elution process 2, the mobile phase B (%) is 25%, 26%, 27%, 28%, 29%, 30%; Preferably, in step d), in the elution process 3, the mobile phase B (%) is 50%, 51%, 52%, 53%, 54%, 55%; Preferably, in step d), the eluate from the elution process 3 (210-300 min) is collected, the pH is adjusted to 6.0-7.0, desalted, concentrated, ethanol precipitated, dissolved in water and freeze-dried to obtain the chemically modified glycosaminoglycan.

6. Chemically modified glycosaminoglycan prepared by the preparation method according to claim 4 or 5.

7. A pharmaceutical composition comprising a preventive or therapeutically effective amount of the chemically modified glycosaminoglycan according to any one of claims 1-3 and 6 and one or more pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, wherein The pharmaceutical composition is a solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.

9. The pharmaceutical composition according to claim 7 or 8, wherein The pharmaceutical composition comprises one or more additional therapeutic agents.

10. Use of the chemically modified glycosaminoglycan according to any one of claims 1-3 and 6 or the pharmaceutical composition according to any one of claims 7-9 in the preparation of a drug for inhibiting tumor growth and / or metastasis, or use of the chemically modified glycosaminoglycan according to any one of claims 1-3 and 6 or the pharmaceutical composition according to any one of claims 7-9 in the preparation of a drug for treating and / or preventing tumors / cancers; Preferably, the tumor is a solid tumor, a blood tumor or a soft tissue tumor; preferably a solid tumor, such as breast cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, gastric cancer or lung cancer.

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