Oligosaccharide compound as well as pharmaceutical composition and application thereof

Abstract

The invention relates to the technical field of biological medicine, and provides an oligosaccharide compound as well as a pharmaceutical composition and application thereof, the oligosaccharide compound comprises 5-6 sugar rings: alpha-L-sulfated fucosyl, alpha-L-4-deoxy-threo-hex-4-enuronic acid group or beta-D-glucuronic acid group, beta-D-2-deoxy-2-acetamido-4, 6-dihydroxy-beta-D-glucuronic acid group or beta-D-glucuronic acid group, beta-D-2-deoxy-2-acetamido-4, 6-dihydroxy-beta-D-glucuronic acid group or beta-D-glucuronic acid group The compound is selected from the group consisting of 1, 6-disulfated galactosyl, beta-D-3-sulfated glucuronyl, 2, 5-dehydrated talose or sugar alcohol or sugar amine thereof. The oligosaccharide compound is only pentasaccharide or hexasaccharide, but the activity of the oligosaccharide compound for inhibiting iXase is expected to be higher than or far higher than that of FG heptaose, octasaccharide, nonaose and decaose compounds reported at present, and the oligosaccharide compound has remarkable advantages and characteristics as an iXase inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an oligosaccharide compound, its pharmaceutical composition and application. Background Art

[0002] Venous thromboembolism (VTE) is one of the most major thrombotic diseases, and anticoagulant drugs are the cornerstone of the clinical treatment of VTE. However, classical anticoagulants such as heparin and coumarins can cause serious bleeding risks and have pharmacokinetic defects that increase the bleeding risk; although other clinical anticoagulant drugs such as low molecular weight heparin, hirudin, dabigatran, and rivaroxaban have made significant progress in predictable pharmacokinetic processes, the bleeding tendency and serious bleeding risks still exist.

[0003] In recent years, studies have shown that the intrinsic coagulation pathway is closely related to pathological thrombosis formation, but is not essential for hemostatic function, that is, selective inhibition of the intrinsic coagulation may have little or less impact on hemostatic function when producing an inhibitory effect on thrombus activity. Therefore, inhibitors of intrinsic coagulation factors have become the current international mainstream trend in the research and development of anticoagulant drugs with low bleeding tendency. The intrinsic coagulation factor X enzyme (IXa-VIIIa-PL-Ca 2+ complex, iXase) is the rate-limiting enzymatic active site on the intrinsic coagulation pathway, and thus is a promising target for novel anticoagulant drugs with the characteristic of low bleeding tendency.

[0004] Fucosylated glycosaminoglycan (FG) is a structurally special glycosaminoglycan derivative derived from animals of the class Holothuroidea. It has a backbone similar to chondroitin sulfate (CS) and contains a large number of sulfated fucosyl (L-fucosyl, Fuc) side chains. So far, through the structural analysis of a series of pure oligosaccharide fragments, the exact structures of dozens of natural FGs from sea cucumbers of different species have been confirmed. Comparing these FGs whose structures have been confirmed based on pure oligosaccharide structural fragments, the common structural characteristics known so far include that sulfate ester groups (-OSO3 - ) substitutions (GalNAc 4S6S ) exist at both the C4 and C6 positions of N-acetylgalactosamine (GalNAc) in the CS backbone, and side chain sulfated L-Fuc (L-FucS) substitutions exist at the C3 position of glucuronic acid (GlcA) contained in the backbone. The structural differences of FGs from different species mainly manifest as different sulfation forms of the side chain Fuc: three monosaccharide Fuc side chains with different sulfation forms have been found, single sulfation at the C4 position (L-Fuc 4S ) and disulfation at the C2 / 4 or C3 / 4 positions (L-Fuc 2S4S ) and L-Fuc 3S4S) In addition, it was found that D-α-galactosyl (Gal) or D-α-GalNAc substitution may further exist at the C2 position of the side-chain Fuc, that is, disaccharide side-chain substitution exists. At present, sulfate group substitution may exist at the C3 and / or C4 positions of Fuc in the side-chain disaccharide, while sulfate group substitution may also exist at the C4 and / or C6 positions of Gal and GalNAc.

[0005] Natural FG has strong anticoagulant activity that produces antithrombotic effects, but also has platelet activation and surface activation activities that may lead to thrombosis. The anticoagulant activity mechanism of natural FG is complex, and low-molecular-weight FG obtained by appropriate depolymerization can have the activity of inhibiting endogenous factor X enzyme (iXase) independent of antithrombin (AT) and the IIa inhibitory activity independent of heparin cofactor II (HC-II), etc. Analysis of the structure-activity relationship of pure FG oligosaccharides obtained by selective depolymerization of glycosidic bonds shows that when only containing a single Fuc sugar side chain, the nonasaccharide obtained by deamination depolymerization and the octasaccharide obtained by β-elimination depolymerization can both have strong and selective iXase inhibitory activities (Proc Natl Acad Sci USA, 2015; 112: 8284-9; J Biol Chem, 2018; 293: 14089-14099); in addition, it was found that the FG heptasaccharide containing a disaccharide side chain can sometimes also have strong iXase inhibitory activity (Biomacromolecules, 2021; 22: 1244-55; Carbohydr Polym, 2023; 321: 121304). In summary, among FG oligosaccharides, the smallest oligosaccharide structural fragment known to have strong inhibitory activity against iXase (IC50 < 1000 ng / mL) is the FG heptasaccharide (Biomacromolecules, 2021: 22(3): 1244-1255; Carbohydr Polym, 2023: 321: 121304). Therefore, obtaining FG oligosaccharide compounds with a smaller structure and strong inhibitory activity against iXase has important application value for the prevention and / or treatment of thrombotic diseases. Summary of the Invention

[0006] In view of this, the present invention provides an FG oligosaccharide compound with a smaller structure and strong inhibitory activity against iXase, its pharmaceutical composition and application.

[0007] Based on in-depth exploration of the selective depolymerization of FG glycosidic bonds, FG depolymerization products, and the essential structure for FG oligosaccharides to inhibit iXase activity, the inventors of the present invention have discovered a series of iXase inhibitors with novel structures and have applied for a series of intellectual property protections (CN2013100998009, 2013101274470, CN201410549676).

[0008] The present invention's recent research has for the first time discovered the presence of a C3-sulfated D-GlcA glycosyl group (GlcA 3S ) in FG oligosaccharides from a new species source, and has for the first time confirmed the presence of GlcA 3S in the natural FG backbone; further research has found that various FG pentasaccharides and hexasaccharides containing GlcA 3S have potent inhibitory activity against iXase, and the activity intensity is higher than that of the FG octasaccharides and nonasaccharides described above. These are the FG oligosaccharide compounds with the smallest degree of polymerization having potent inhibitory activity against iXase seen so far.

[0009] Research shows that the FG pentasaccharides and hexasaccharides containing GlcA 3S have anticoagulant and antithrombotic activities that depend on their potent inhibitory activity against iXase, and have the advantageous feature of a lower bleeding tendency compared to existing clinical anticoagulant drugs such as low molecular weight heparin. Obviously, considering that the oligosaccharides described in the present invention are oligosaccharides with a smaller structure and potent inhibitory activity against iXase, their antithrombotic activity feature with a lower bleeding tendency makes them have important application value in the prevention and / or treatment of thrombotic diseases.

[0010] The technical solution of the present invention is achieved as follows: In the first aspect, the present invention provides an oligosaccharide compound and a pharmaceutically acceptable salt thereof, and the oligosaccharide compound is a compound having the structure of formula (Ⅰ):

[0011]

[0012] The sugar ring A is an α-L-sulfated fucosyl group, the sugar ring B is an α-L-4-deoxy-threo-hex-4-enuronic acid group or a β-D-glucuronic acid group, the sugar ring C is a β-D-2-deoxy-2-acetamido-4,6-disulfated galactosyl group, and the sugar ring D is a β-D-3-sulfated glucuronic acid group;

[0013] R1 and R2 are independently -H or -SO 3- ;

[0014] R is optionally -H, -OH, or a β-D-2-deoxy-2-acetamido-4,6-disulfated galactosyl group;

[0015] R′ is optionally -OH, -R 12 , -OR 13 , a closed-ring D-2-deoxy-2-acetamido-4,6-disulfated galactose, an open-ring D-2-deoxy-2-acetamido-4,6-disulfated galactitol, a sugar amine or a derivative thereof, or 2,5-anhydrotalose or its sugar alcohol, sugar amine;

[0016] -R 12 , -OR 13are independently a substituted or unsubstituted C1-C6 straight-chain or branched-chain alkyl group, a C7-C12 saturated or unsaturated heterocyclic group containing N, O or S, or a substituted or unsubstituted C7-C12 aryl group.

[0017] Based on the above technical solutions, preferably, the sugar ring B is α-L-4-deoxy-threo-hex-4-enuronic acid group, R is -H, R' is a cyclic D-2-deoxy-2-acetamido-4,6-disulfated galactose (V), or an open-chain D-2-deoxy-2-acetamido-4,6-disulfated galactitol, sugar amine or its derivative (VI); the structural formula of the oligosaccharide compound is shown in formula (II):

[0018]

[0019] In formula (V), R3 is optionally -OH, -R5, -(R6)2 or -OR7; in formula (VI), R4 is optionally -OH, -NH2, -NHR8 or -N(R9)2; where R5, R6, R 7、 R8, R9 are independently a substituted or unsubstituted C1-C6 straight-chain or branched-chain alkyl group, a C7-C12 saturated or unsaturated heterocyclic alkyl group containing N, O or S, or a substituted or unsubstituted C7-C12 aryl group.

[0020] Based on the above technical solutions, preferably, the sugar ring B is β-D-glucuronic acid group, R is -OH, R' is 2,5-anhydrotalose or its sugar alcohol, sugar amine (VII); the structural formula of the oligosaccharide compound is shown in formula (III):

[0021]

[0022] In formula (VII), R 10 is optionally -CH=O, -CH(OH)2, -CH2OH, -CH2R 11 , -CH(R 12 )2, -CH2NH2, -CH2NHR 13 or -CH2N(R 14 )2; where R 11 , R 12 , R 13 , R 14 are independently a substituted or unsubstituted C1-C6 straight-chain or branched-chain alkyl group, a C7-C12 saturated or unsaturated heterocyclic group containing N, O or S, or a substituted or unsubstituted C7-C12 aryl group.

[0023] Based on the above technical solutions, preferably, the sugar ring B is β-D-glucuronic acid group, R is β-D-2-deoxy-2-acetamido-4,6-disulfated galactosyl group, R' is optionally -OH, -R12 , -OR 13 , the cyclic D-2-deoxy-2-acetamido-4,6-disulfogalactose (V), or the open-chain D-2-deoxy-2-acetamido-4,6-disulfogalactitol or glycosamine or its derivative (VI); the structural formula of the oligosaccharide compound is as shown in formula (IV):

[0024]

[0025] Based on the above technical solutions, preferably, the pharmaceutically acceptable salt is sodium salt, potassium salt or calcium salt.

[0026] In a second aspect, the present invention provides a method for preparing an oligosaccharide and a pharmaceutically acceptable salt thereof, the preparation method comprising the following steps:

[0027] S1, extracting a fucosylated glycosaminoglycan containing a β-D-3-sulfated glucuronic acid group fragment from the body wall and / or viscera of an animal of the class Holothuroidea of the phylum Echinodermata;

[0028] S2, treating the fucosylated glycosaminoglycan obtained in step S1 by a chemical depolymerization method to obtain a depolymerization product and separating a pure oligosaccharide compound from the depolymerization product by chromatography;

[0029] S3, performing terminal structure modification on the pure oligosaccharide compound obtained in step S2 to obtain an oligosaccharide compound.

[0030] Based on the above technical solutions, preferably, the animals of the class Holothuroidea of the phylum Echinodermata described in step 1 include but are not limited to: Cucumaria frondosa, Cucumaria japonica, Cucumaria curata, Cucumaria syracusana, Athyonidium chilensis, Cucumaria djakonovi, Cucumaria vegae, Cucumaria salma, Cucumaria pseudocurata, Cucumaria piperata, Cucumaria pallida, Cucumaria miniata, Cucumaria georgiana, Cucumaria echinata and Cucumaria dudexa.

[0031] Based on the above technical solutions, preferably, in step S2, the chemical depolymerization method is one of β-elimination depolymerization method, deacylation deamination depolymerization method, and unsaturated hexuronic acid cleavage method.

[0032] Based on the above technical solutions, preferably, the β-elimination depolymerization method: Under strong alkaline conditions, the glycosidic bond connecting to the C4 position of hexuronic acid in the main chain of fucosylated glycosaminoglycan is cleaved, and the non-reducing end hexuronic acid of the product has Δ 4,5 unsaturated double bonds. The main steps include but are not limited to:

[0033] (1) Quaternary ammonium salt conversion: Treat the aqueous solution of fucosylated glycosaminoglycan with a quaternary ammonium compound to convert it into a water-insoluble quaternary ammonium salt. The quaternary ammonium compounds include but are not limited to benzethonium chloride, benzyltriethylammonium chloride, and tetrabutylammonium sulfate.

[0034] (2) Carboxyl esterification: React the fucosylated glycosaminoglycan quaternary ammonium salt obtained in step 1 with a halogenated hydrocarbon in an organic solvent to esterify the carboxyl group on the main chain hexuronic acid and obtain a carboxyl esterification product. The halogenated hydrocarbons include but are not limited to benzyl chloride and bromoethane; the organic solvent is preferably N,N-dimethylformamide.

[0035] (3) β-elimination depolymerization: In an organic solvent, treat the carboxyl esterification product of fucosylated glycosaminoglycan obtained in step 2 with a strong base to cause a β-elimination reaction and depolymerize it; the reaction product removes the ester group on the carboxylic acid through post-treatment and is separated and purified to obtain a depolymerized product. The strong base is preferably sodium ethoxide prepared before use; the organic solvent is preferably N,N-dimethylformamide.

[0036] The basic steps of the β-elimination depolymerization are shown in Route 1:

[0037]

[0038]

[0039] In the figure, U and A are β-D-glucuronyl (β-D-GlcA) and β-D-2-deoxy-2-acetamido-4,6-disulfated galactosyl (β-D-Gal 4S6S ) respectively; dU is α-L-4-deoxy-threo-hex-4-enuronosyl (α-L-ΔU); Q is a quaternary ammonium; E is an ester group; R is sulfated L-fucose (α-L-FucS) or 2-glycosyl (sulfated α-D-galactose or galactosaminyl)ated α-L-FucS; R′ is H or D-GlcA. n is a natural number, usually 1-9.

[0040] Based on the above technical solutions, preferably, the deacylation and deamination depolymerization method: Use the hydrazine treatment method to partially remove the N-acetyl group on the aminohexose in the main chain of fucosylated glycosaminoglycan, and then use nitrous acid to cleave the glycosidic bond connecting to the C1 position of the deacetylated aminohexose, and the reducing end aminohexose of the product is converted into 2,5-anhydrohexose. The main steps include but are not limited to:

[0041] (1) Hydrazinolysis deacetylation: Treat fucosylated glycosaminoglycan with hydrazine or hydrazine hydrate with or without hydrazine sulfate, thereby partially removing the N-acetyl group on the aminohexose in the polysaccharide backbone and obtaining a partially deacetylated product. The deacetylation rate of the product is preferably 40%-75%.

[0042] (2) Deamination depolymerization: Treat the partially deacetylated product of the fucosylated glycosaminoglycan obtained in step 1 with a nitrous acid solution to cause a deamination depolymerization reaction, and the reaction solution is purified by post-treatment to obtain a depolymerized product. The pH value of the nitrous acid solution is preferably 2.0-5.0; the concentration of nitrous acid in the reaction solution is preferably 0.25-5.5 mol / L.

[0043] The basic steps of deacylation and deamination depolymerization are shown in Route 2:

[0044]

[0045] In the figure, the definitions of U, A, R, and n are the same as those in Route 1; R' is H or acetyl; T is D-4,6-disulfate-2,5-anhydrotalose (D-anTal 4S6S ).

[0046] On the basis of the above technical solutions, preferably, the unsaturated hexuronic acid cleavage method: Optionally treat the oligosaccharide compound prepared by the β-elimination depolymerization method with a mercury salt or ozone to cleave the glycosidic bond connected to the C1 position of the hexuronic acid containing a Δ4,5 unsaturated double bond, and obtain an oligosaccharide compound removing the unsaturated hexuronic acid and the fucosyl group of its side chain. The main steps include but are not limited to:

[0047] (1) Mercury salt treatment: Select the oligosaccharide compound obtained after β-elimination depolymerization of fucosylated glycosaminoglycan, and make it react with a mercury salt in an aqueous solution to cleave the glycosidic bond connected to the C1 position of the unsaturated hexuronic acid, and obtain an oligosaccharide compound removing the unsaturated aldehyde acid and the fucosyl group of its connected side chain. The mercury salt is preferably mercury acetate.

[0048] (2) Ozone treatment: Select the oligosaccharide compound obtained after β-elimination depolymerization of fucosylated glycosaminoglycan, and make it react with ozone in an aqueous solution, and then through post-treatment at a low pH value, thereby cleaving the glycosidic bond connected to the C1 position of the unsaturated hexuronic acid, and obtaining an oligosaccharide compound removing the unsaturated aldehyde acid and the fucosyl group of its connected side chain. The post-treatment at a low pH value means adjusting the pH of the reaction solution to 2.0-4.0 and continuing the reaction for 20-60 min.

[0049] The reaction process of the unsaturated hexuronic acid cleavage method can be briefly represented by Route 3:

[0050]

[0051] In the figure, dU, U, A, R, R′, and n are defined the same as in Route 1; X is a halogen atom or an acid radical.

[0052] Based on the above technical solutions, preferably, in step S3, the method for modifying the end group structure is to perform structure modification on the C1 aldehyde carbonyl group of the reducing end sugar group of the oligosaccharide compound or the aldehyde carbonyl group in the hemiacetal form, and the structure modification method is one of aldehyde carbonyl oxidation reaction, reduction reaction, reductive alkylation reaction, reductive amination reaction, and polymerization reaction.

[0053] For example, the oxidation reaction can convert the reducing end sugar group into a sugar acid, while the reduction reaction can convert it into a sugar alcohol; reacting with an alkylating reagent such as pyrazolone compounds can obtain a reducing end alkylated derivative; reacting with a compound containing an amino group can obtain an end group reductive amination derivative; and after the end group azidation transformation, a derivative containing a triazole structure can be obtained through the "click" reaction of azide and alkyne. Route 4 is a representative example of structure modification that is easy to implement, but it does not limit the scope of the structure modification described in the present invention.

[0054]

[0055]

[0056] In the figure, R is defined the same as in Route 1; R′ is a substituted or unsubstituted C1-C6 straight-chain or branched-chain alkyl group, a C7-C12 saturated or unsaturated heterocyclic group containing N, O, or S, or a substituted or unsubstituted C7-C12 aryl group.

[0057] In a third aspect, the present invention provides a pharmaceutical composition having antithrombotic activity, the pharmaceutical composition comprising the above-mentioned oligosaccharide compound and its pharmaceutically acceptable salt, as well as a pharmaceutical excipient.

[0058] Based on the above technical solutions, preferably, the dosage form of the pharmaceutical composition is an aqueous injection solution or a freeze-dried powder injection for injection.

[0059] Based on the above technical solutions, preferably, in the unit dose of the pharmaceutical composition of the present invention, the content of the oligosaccharide compound or its pharmaceutically acceptable salt is generally in the range of 30 mg to 200 mg, and the preferred content range is 50 mg to 120 mg.

[0060] The dosage form of the pharmaceutical composition of the present invention is an injection for parenteral administration, including but not limited to an aqueous injection solution and a freeze-dried powder injection for injection that is formulated into an aqueous solution with injection water before use.

[0061] Generally speaking, aqueous injection solutions are mostly sterilized aseptically by autoclaving. For active ingredients with good chemical stability under autoclaving conditions (116°C / 67 kPa × 30 min; 121°C / 97 kPa × 20 min or 126°C / 134 kPa × 15 min), aqueous injection solutions are preferably used; for active ingredients with poor chemical stability under autoclaving conditions, freeze-dried powder for injection that is formulated into an aqueous solution with injection water immediately before use is preferably used, and the latter usually uses ultrafiltration to remove bacteria.

[0062] Fourthly, the present invention provides the use of the above oligosaccharide compounds and their pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of thrombotic diseases, characterized in that the thrombotic disease is one of venous thrombosis, arterial embolism, ischemic heart disease and ischemic cerebrovascular disease.

[0063] An oligosaccharide compound, a pharmaceutical composition and an application thereof according to the present invention have the following

[0064] Advantages:

[0065] (1) The oligosaccharide compounds according to the present invention are shown in formulas (I) to (IV). Compared with other known oligosaccharide compounds derived from FG, they contain GlcA 3S glycosyl groups, and also contain GlcA glycosyl groups substituted by Fuc side chains at the C3 position, and are only pentasaccharides or hexasaccharides. The IC 50 (drug concentration required to reduce enzyme activity by 50%) of the oligosaccharide compounds or their pharmaceutically acceptable salts of the present invention to inhibit iXase is lower than about 100 nmol / L (mass concentration is lower than about 200 ng / mL), and their activity to inhibit iXase is unexpectedly stronger or much stronger than the reported FG heptasaccharide, octasaccharide, nonasaccharide and decasaccharide compounds, and has a strong activity to inhibit iXase.

[0066] (2) The present invention for the first time prepares and discovers FG oligosaccharides containing a chemically novel structural fragment of C3-sulfated-D-glucuronic acid group (D-GlcA 3S ), and for the first time confirms the existence of the D-GlcA 3S structure in natural FG through pure oligosaccharide structural fragments; in the presence of D-GlcA 3SIn the exploration of the activity of FG oligosaccharides of structural fragments, unexpectedly and pleasantly, it was found that some FG oligosaccharides with low degrees of polymerization (dp5-6) have the activity characteristics of strongly inhibiting iXase, and their activity far exceeds that of known FG oligosaccharides with the same degree of polymerization (dp 5-6) or even higher degrees of polymerization (dp 7-10). Since iXase inhibitors have important anticoagulant and antithrombotic activity characteristics with low bleeding tendency, the oligosaccharide compounds of the present invention are expected to be used as anticoagulants with low bleeding tendency for the clinical treatment and / or prevention of thrombotic diseases, and have important potential value in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0068] Figure 1 1H NMR spectrum of the compound prepared in Example 1 of the present invention; 1

[0069] Figure 2 13C NMR spectrum of the compound prepared in Example 1 of the present invention; 13

[0070] Figure 3 1H-13C HSQC spectrum of the compound prepared in Example 1 of the present invention; 1 H- 13

[0071] Figure 4 Q-TOF MS spectrum of the compound prepared in Example 1 of the present invention;

[0072] Figure 5 Doubled APTT prolonging activity of Compounds 1, 2, 3, and 5 prepared in the examples of the present invention;

[0073] Figure 6 Inhibitory activity of Compounds 1, 2, 3, and 5 prepared in the examples of the present invention against endogenous factor X enzyme;

[0074] Figure 7 Antithrombotic activity of the oligosaccharide compound shown in formula (II);

[0075] Figure 8 Effect of the oligosaccharide compound shown in formula (II) on bleeding. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0076] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0077] Example 1 Preparation of pentasaccharide compound 1

[0078] Materials: Dried body wall of Cucumaria japonica. Benzethonium chloride, benzyl chloride, N,N-dimethylformamide (DMF), sodium hydroxide, sodium chloride, ethanol and other reagents used are all commercially available analytical pure reagents. Sephadex G25, GE Healthcare; Bio-Gel P10, Bio-Rad; Dionex IonPac TM AS11-HC Semi-Prep Column, Thermo Scientific; Agilent 1200 / 1260 series high performance liquid chromatograph, Agilent.

[0079] The specific method is as follows:

[0080] Step 1: FG extraction and purification: According to the literature method (Lan et al., Carbohydr Polym, 2023, 321: 121304), it was extracted from 1.8 kg of dried body wall of Cucumaria japonica by the combined enzymatic-alkaline hydrolysis method and purified by the combined salting-out fractional alcohol precipitation and anion exchange column chromatography to obtain a pure polysaccharide; HPGPC analysis showed that it was a homogeneous polysaccharide component, and physicochemical analysis showed that it was fucosylated glycosaminoglycan (FG).

[0081] Step 2: Depolymerization of FG by β-elimination depolymerization method

[0082] (1) Quaternary ammonium salt conversion: Take 6 g of dry FG product and dissolve it in 90 mL of pure water; take another 15 g of benzethonium chloride and dissolve it in 240 mL of pure water. While stirring, slowly add the benzethonium chloride solution to the FG solution, further mix evenly, and then let it stand overnight at 4°C. The obtained solution was centrifuged (4000 rpm × 10 min), the supernatant was discarded, the precipitate was washed twice with pure water (30 mL) (centrifuged at 4000 rpm × 10 min to remove benzethonium chloride), and the obtained precipitate was vacuum dried at 40°C until constant weight to obtain 17.81 g of quaternary ammonium salt product.

[0083] (2) Carboxyl esterification: 89 mL of DMF was added to the quaternized FG precipitate (17.81 g). After it was completely dissolved, 7.22 mL of benzyl chloride was added, and the reaction was carried out at 35 °C for 24 h to benzyl-esterify the carboxyl group at the C6 position of D-GlcA contained in FG.

[0084] (3) β-Elimination depolymerization in the presence of a reducing agent: After the reaction solution in (2) was cooled to room temperature, 32.07 mL of sodium ethoxide / ethanol solution with a newly prepared concentration of 0.16 mol / L (containing 0.4 M reducing agent NaBH4) was added to make its final concentration 0.04 M (containing 0.1 M reducing agent NaBH4), and the alkaline hydrolysis reaction was carried out at room temperature for 30 min.

[0085] (4) Post-treatment: After the reaction in step (3) was completed, an equal volume of saturated sodium chloride (128 mL) was added, and it was shaken for 30 min to fully exchange the sodium salt and the quaternary ammonium salt. Then 1000 mL of absolute ethanol was added to make the final concentration 80%, and it was left to stand and centrifuged (4000 rpm × 10 min) to obtain a precipitate; the precipitate was exchanged with saturated sodium chloride again and repeated twice to obtain a precipitate.

[0086] (5) Hydrolysis of the benzyl ester of the carboxyl group: The precipitate obtained in step (4) was dissolved in 300 mL of water, and then 5 mL of 6 M NaOH was added to make the final concentration 0.1 M. The reaction was carried out at room temperature for 30 min. After the reaction was completed, it was adjusted to neutral with 6 M HCl, desalted by a Sephadex G25 chromatographic column and freeze-dried to obtain 2.5 g of the depolymerized product dFG containing a series of oligosaccharide homologues, and the yield was 42%.

[0087] Step 3: Purification of the pentasaccharide compound:

[0088] 2.5 g of dFG was dissolved and loaded onto the column in batches. It was separated by a Bio-Gel P10 gel column with a flow rate of about 10 mL / h, and the elution fractions were collected at 2.5 mL / tube. The elution fractions were detected by ultraviolet spectrophotometry (λ 232 ) and the elution curve was plotted, and selected points were subjected to HPGPC analysis (Superdex Peptide 10 / 300GL analytical column, isocratic elution with 0.2 M NaCl solution, detected by a differential refractive index detector combined with an ultraviolet detector). Using FG pentasaccharide as the standard product, it was repeatedly purified to a single chromatographic peak according to the retention time and the chromatographic peak shape. During the preparation of the pentasaccharide, it could be further purified by anion exchange chromatography. The obtained pentasaccharide was desalted by a Sephadex G25 gel column and freeze-dried.

[0089] Step 4: Spectral analysis of the pentasaccharide compound, nuclear magnetic resonance spectrum:

[0090] Approximately 10 mg of purified oligosaccharide was repeatedly exchanged with D2O three times, dissolved in 500 μL of D2O, and transferred to a 5-mm NMR tube. Its 1 1H NMR, 13 13C NMR, HMBC, and HSQC, 1 1H- 1 1H COSY, TOCSY, and ROESY spectra were measured. Mass spectrometry: Detection was performed using an ultra-high pressure liquid chromatography triple quadrupole tandem mass spectrometer, and data were collected in the negative ion mode. The sample concentration was approximately 0.2 mg / mL, and data analysis was performed using MassHunter software. The results are as follows:

[0091] According to Step 1, 14.3 g of FG was purified with a yield of 0.8%; HPGPC analysis showed it to be a homogeneous polysaccharide component, and physicochemical analysis showed it to be a fucosylated glycosaminoglycan. According to the methods described in Step 2 and Step 3, 150 mg of the compound was obtained.

[0092] Structural analysis of Compound 1: 1 The 1H NMR spectrum and its assignment are shown in the appendix Figure 1 as follows; 13 The 13C NMR spectrum and its assignment are shown in the appendix Figure 2 as follows; 1 1H- 13 13C HSQC spectrum and its assignment are shown in the appendix Figure 3 as follows; The ESI-Q-TOF MS spectrum and its assignment are shown in the appendix Figure 4 as follows; 1 1H / 13 13CNMR signal assignments are shown in Table 1. According to 1 1H- / 13 13C- and 2D NMR and ESI-Q-TOF MS analyses, the chemical structure of Compound 1 is L-Fuc 3S4S -α(1,3)-L-Δ 4,5 GlcA-α(1,3)-D-GalNAc 4S6S -β(1,4)-D-GlcA 3S -β(1,3)-D-GalNAc 4S6S -ol; Its structure is:

[0093]

[0094] Table 1 1H / 1 1H / 13 13C NMR signal assignments of pentasaccharide Compound 1

[0095]

[0096]

[0097] Glycosyl A is α-L-3,4-disulfated fucosyl; glycosyl B is α-L-4-deoxy-threo-hex-4-enuronosyl; glycosyl C is β-D-2-deoxy-2-acetamido-4,6-disulfated galactosyl; glycosyl D is β-D-3-sulfated glucuronosyl; glycosyl E is D-2-deoxy-2-acetamido-4,6-disulfated galactitol. In the appendix Figures 1 to 3 The labeling of each sugar residue is the same as that in Table 1.

[0098] Table 2 ESI-Q-TOF MS signal assignment of pentasaccharide compound 1

[0099]

[0100] Preparation of pentasaccharide compound 2 in Example 2

[0101] Materials: Fucosylated glycosaminoglycan (FG, sodium salt) from Cucumaria japonica, prepared in the same way as in Example 1. Reagents such as hydrazine hydrate, hydrazine sulfate, sodium nitrite, concentrated sulfuric acid, and ethanol are all commercially available analytical pure reagents. The materials and instruments required for oligosaccharide separation are the same as those in Example 1.

[0102] The specific method is as follows:

[0103] Step 1: Deacetylation by hydrazinolysis:

[0104] Place 5 g of natural FG in a 500 mL round-bottom reaction flask, add 1.25 g of hydrazine sulfate, then add 125 mL of hydrazine hydrate, protect with N2, and heat and stir at 90 °C for 24 h. After the reaction, add 500 mL of absolute ethanol to the reaction solution until the final ethanol concentration in the system is 80% (v / v), precipitate, and centrifuge to remove the supernatant. The obtained precipitate is dissolved in 125 mL of H2O, and then 500 mL of absolute ethanol (the resulting solution has a pure concentration of 80%, v / v) is added, and the alcohol precipitation is repeated 4 times. The precipitate is redissolved in water and dialyzed through a dialysis bag with a molecular weight cut-off of 3500 Da (product of Union Carbide Corporation, USA). The retentate is freeze-dried to obtain about 4 g of a partially deacetylated FG intermediate product, with a yield of about 80%.

[0105] Step 2: Depolymerization of partially deacetylated FG by nitrous acid treatment:

[0106] Dissolve the FG partial deacetylation intermediate obtained in Step 1 in 80 mL of H2O. Under ice bath conditions, add 160 mL of a 5.5 M nitrous acid solution with a pH of 4, stir and react for 10 - 20 min, then add 1 M NaOH to adjust the solution to neutrality to terminate the reaction. Dialyze the resulting reaction solution using a dialysis bag with a molecular weight cut-off of 500 Da, collect the retentate, and freeze-dry to obtain approximately 3 g of the depolymerized product with a yield of approximately 75%. The purification and spectral analysis of the pentasaccharide compound are the same as in Example 1.

[0107] Results: 260 mg of the compound was prepared according to the described method. Based on detailed spectral analysis, the chemical structure of Compound 2 was determined to be: L-Fuc 3S4S -α(1,3)-D-GlcA-β(1,3)-D-GalNAc 4S6S -β(1,4)-D-GlcA 3S -β(1,3)-D-anTal 4S6S ; The NMR spectral signals of Compound 2 were similar to those of Compound 1, but there were differences in the characteristic signals. A new anomeric and 4-H signal peak of the characteristic reducing end 2,5-anhydrotalose (an-Tal) appeared at approximately 5.0 - 5.1 ppm in Compound 2, while the 4-H signal of the unsaturated hexuronic acid at 5.6 - 5.7 ppm disappeared. The structure of Compound 2 is shown below. In the structural formula, glycosyl B is β-D-glucuronyl; glycosyl E is 4,6-disulfato-2,5-anhydrotalose, and the remaining sugar residues A, C, and D are defined as in Example 1.

[0108]

[0109] Example 3 Preparation of the hexasaccharide compound 3

[0110] Materials: Fucosylated glycosaminoglycan (FG, sodium salt) from Cucumaria japonica, prepared by the same method as in Step 1 of Example 1. Mercuric acetate is a commercially available analytical reagent. Cation exchange resin 50W×8 (H type), Alfa Aesar. The other materials and instruments required for the preparation process are the same as in Example 1.

[0111] The specific method is as follows:

[0112] (1) The preparation process of the oligomeric product is the same as in Step 2 of Example 1;

[0113] (2) Purification and preparation of the octasaccharide: Similar to the method in Step 3 of Example 1, the oligomeric product was separated and purified by gel column chromatography combined with anion exchange column chromatography to obtain approximately 100 mg of the pure octasaccharide;

[0114] (3) Mercuric hydrolysis reaction: Dissolve the octasaccharide described in (2) of this example in 5 mL of water, and adjust the pH to about 5 with acetic acid; then add an equal volume of mercuric acetate solution (70 mM, pH 5) to the oligosaccharide solution, stir and react at room temperature for 10 min. After the reaction, directly load the reaction solution onto a cation exchange chromatography column ( 50W×8), wash with 20 mL of water and collect the eluate. The obtained hexasaccharide is desalted by a Sephadex G25 gel column and freeze-dried.

[0115] (4) Structure analysis: The same as step 4 of Example 1.

[0116] Result: 70 mg of compound 3 was obtained by the method described. According to detailed spectroscopic analysis, the chemical structure of compound 3 was determined to be: D-GalNAc 4S6S -β(1,4)-[L-Fuc 3S4S -α(1,3)]-D-GlcA-β(1,3)-D-GalNAc 4S6S -β(1,4)-D-GlcA 3S -β(1,3)-D-GalNAc 4S6S -ol; The NMR spectral signals of compound 3 are similar to those of compound 1, but the hydrogen signal at the 4-position of the unsaturated hexuronic acid at 5.6 - 5.7 ppm disappears, which is consistent with the cleavage of the glycosidic bond connecting the C1 position of the unsaturated hexuronic acid by treatment with mercuric salt, thereby forming an oligosaccharide compound of de-unsaturated uronic acid and its attached side-chain fucose group; at the same time, a new set of sugar residue (D-GalNAc 4S6S ) signal peaks appear in compound 3. The structure of compound 3 is shown below. In the structural formula, sugar residue B is β-D-glucuronyl; sugar residue E is D-2-deoxy-2-acetamido-4,6-disulfogalactitol; sugar residue F is D-2-deoxy-2-acetamido-4,6-disulfogalactose; the remaining sugar residues A, C, and D are defined as in Example 1.

[0117]

[0118] Preparation of pentasaccharide compound 4 in Example 4

[0119] Materials: The same as in Example 3.

[0120] The specific method is as follows:

[0121] (1) The preparation process of the oligomeric product is similar to step 2 in Example 1; the only difference is that in process (3) of step 2, during the alkaline hydrolysis process, it is not necessary to add the reducing agent NaBH4. At this time, a "peeling reaction" occurs during the alkaline hydrolysis process, and the reducing-end N-acetylgalactosamine residue is lost;

[0122] (2) Purification and preparation of heptasaccharide: Similar to the method in step (6) of Example 1, the oligosaccharide product was separated and purified by gel chromatography combined with anion exchange column chromatography to obtain about 80 mg of pure heptasaccharide;

[0123] (3) Mercuric hydrolysis reaction: Similar to step (3) of Example 3, the unsaturated hexuronic acid (α-L-4-deoxy-threo-hex-4-enuronic acid) at the non-reducing end of the cleaved heptasaccharide and the sulfated fucosyl side chain linked to it were cleaved.

[0124] (4) The reaction product obtained in step (3) was separated by Bio-gel P10 gel column chromatography, and the target pentasaccharide was collected; the obtained pentasaccharide was desalted by Sephadex G25 gel column and freeze-dried.

[0125] (5) Structure analysis: Similar to step 4 of Example 1.

[0126] Result: 35 mg of compound 4 was obtained by the preparation method described above. According to detailed spectroscopic analysis, compound 4 was determined to be a pentasaccharide, and its chemical structural formula was: D-GalNAc 4S6S -β(1,4)-[L-Fuc 3S4S -α(1,3)]-D-GlcA-β(1,3)-D-GalNAc 4S6S -β(1,4)-D-GlcA 3S ; The structure of compound 4 was:

[0127]

[0128] In the structural formula, glycosyl A is α-L-3,4-disulfated fucosyl; glycosyl B is β-D-glucuronyl; glycosyl C and E are β-D-2-deoxy-2-acetamido-4,6-disulfated galactosyl; glycosyl D is β-D-3-sulfated glucuronyl.

[0129] Preparation of pentasaccharide derivative 5 in Example 5

[0130] Materials: Pentasaccharide compound 2, prepared in Example 2. 1-Phenyl-3-methyl-5-pyrazolone (PMP), biochemical reagent, purity 99%.

[0131] Method: 30 mg of the pentasaccharide obtained in Example 2 was dissolved in H2O (50 mg / mL), 1.5 mL of 0.5 M PMP methanol solution and 1 mL of 0.6 M NaOH solution were added, and the mixture was stirred at 50 °C for 90 min. After the reaction, the pH was adjusted to neutral. The obtained reaction product was desalted by Sephadex G25 gel column, and the sugar-containing part was combined and freeze-dried.

[0132] Result: 25 mg of compound pentasaccharide derivative 5 was obtained by preparation according to the described method. According to detailed spectroscopic analysis, compound 5 had sugar residue signals similar to those of compound 2. The difference was that a set of aromatic hydrogen signals from the benzene ring appeared in the low-field region of compound 5. At the same time, the characteristic signals of 2,5-anhydrotalose at the reducing end disappeared, and a set of methylene groups appeared at 2 ppm, indicating that compound 2 underwent anomeric alkylation. Through comprehensive judgment, the chemical structure of pentasaccharide derivative 5 was determined to be:

[0133]

[0134] Example 6 Preparation of Pentasaccharide Derivative 6

[0135] Materials: Pentasaccharide compound 4, prepared in Example 4. Reagents such as N-methylmorpholine, NaN3, 2-chloro-1,3-dimethylimidazolinium chloride, propargylamine, copper sulfate, and sodium ascorbate were all commercially available analytical pure reagents.

[0136] Method: Dissolve 30 mg of the pentasaccharide obtained in Example 2, 53 μL of N-methylmorpholine, and 70 mg of NaN3 in 300 μL of pure water. Place the reaction system in an ice-water bath, add 30 mg of 2-chloro-1,3-dimethylimidazolinium chloride, and after reacting for 15 min, adjust the system to room temperature and continue the reaction for 48 h. The reaction product was desalted to obtain 25 mg of the intermediate. Mix the reaction intermediate with 5 μL of propargylamine in a tetrahydrofuran / water (1:1, 100 μL) system, and add copper sulfate and sodium ascorbate to the reaction system. Stir at room temperature for 24 h for the reaction. After the reaction, the product was washed, extracted, and then desalted through a Sephadex G25 gel column, and the sugar-containing part was combined and freeze-dried.

[0137] Result: 15 mg of compound pentasaccharide derivative 6 was obtained by preparation according to the described method. Compared with pentasaccharide compound 4, 1 In the 1H NMR spectrum, two new sets of signals appeared at ~6.0 ppm and ~8.0 ppm for derivative 6, which were the H-1 of the reducing-end GlcA 3S and the triazole alkene hydrogen signal respectively. Through comprehensive analysis, the chemical structure of pentasaccharide derivative 6 was:

[0138]

[0139] Example 7 Analysis of the Anticoagulant Activity and Inhibitory Activity against Endogenous Factor iXase of a Series of Compounds Samples: Pentasaccharide compound 1, pentasaccharide compound 2, hexasaccharide compound 3, pentasaccharide derivative 5.

[0140] Reference substances: Enoxaparin sodium injection (LMWH, Mw ~ 4500 Da, Sanofi-Aventis); hs8, FG octasaccharide with fucose monosaccharide side chain (Yin et al., J Biol Chem, 2018, 293:14089-99), prepared in the early stage of the laboratory.

[0141] Reagents: Human coagulation quality control plasma, activated partial thromboplastin time (APTT) assay kit, products of TECO GmbH, Germany; Factor VIII detection kit, products of HYPHEN BioMed (France); Recombinant human factor VIII (FVIII) for injection, products of Bayer Healthcare LLC (Germany).

[0142] Instruments: MC-2000 coagulometer, TECO GmbH, Germany; VICTORNivo TM Multifunctional microplate reader, PerkinElmer; VortexGenie vortex oscillator, Scientific Industries, USA; XS105 electronic balance, FE20 pH meter, products of Mettler Toledo, USA.

[0143] Methods:

[0144] (1) Solution preparation

[0145] Preparation of human coagulation quality control plasma: According to the reagent instruction manual, add 1 mL of pure water to the freeze-dried powder of human coagulation quality control plasma, place it at room temperature for 15 min, and use it after it is completely dissolved.

[0146] Preparation of reference substance solution: The stock solution of low molecular weight heparin is 100 mg / mL, and it is dissolved with Tris-HCl buffer solution to prepare a stock solution of 1280 μg / mL, and then gradient dilution is carried out according to the experimental requirements.

[0147] Preparation of sample solution: Weigh accurately 1.28 mg of Compound 1, Compound 2, Compound 3, Compound 5 and reference substance hs8, add Tris-HCl buffer solution to dissolve and prepare a stock solution of 1280 μg / mL, and then gradient dilution is carried out according to the experimental requirements.

[0148] Factor VIII kit: Prepared according to the kit instruction method. Add 2.5 mL of pure water to R1, R2, and R3 and use it after it is completely dissolved.

[0149] (2) Anticoagulant activity detection: Take 5 μL of the reference substance or sample solution and add it to a colorimetric cup preheated at 37°C; add 45 μL of well-dissolved human coagulation quality control plasma and incubate at 37°C for 2 min; add 50 μL of APTT reagent preheated at 37°C and incubate the mixture at 37°C for 3 min; add 50 μL of preheated CaCl2 solution, start timing simultaneously and record the clotting time.

[0150] (3) Anticoagulant activity data processing: Plot the final concentration of the sample in plasma against the APTT time (the mean value of duplicate detections) and perform linear fitting. Calculate the final concentration of the sample required to double the APTT time according to the fitting equation (sample concentration - clotting time equation).

[0151] (4) iXase inhibitory activity analysis: Combine Factor VIII and Factor VIII detection kit, and perform the detection with reference to the kit instruction manual and literature methods. Specifically, add 30 μL of test article solution and reference substance solution (Tris-HCl buffer) with a series of concentrations to a 96-well plate respectively; add 30 μL of R2 (Activation Reagent), 30 μL of FVIII solution (2 IU / mL), mix well by shaking the plate at 37°C and incubate for 2 min; add 30 μL of R1 (FX), mix well by shaking the plate at 37°C and incubate for 1 min; add 30 μL of R3 (SXa-11), mix well by shaking the plate and then detect the absorbance value (OD 405 ) at 405 nm, and continuously analyze for 2 min at intervals of 15 s.

[0152] (5) iXase inhibitory activity data processing: Perform linear fitting of the mean value of duplicate OD 405 detections against time, and the slope (the change rate of absorbance value, OD 405 / min) is the activity of iXase. Take the iXase activity of the negative control (Tris-HCl buffer solution) well as 100%, and calculate the iXase activity (percentage) in the presence of the sample or positive control.

[0153] Results: The anticoagulant activity study showed that pentasaccharide compound 1, pentasaccharide compound 2, hexasaccharide compound 3, and pentasaccharide derivative 5 all had significant APTT prolonging activities. Among them, the concentration required for compound 1 to double APTT was 26.8 μg / mL, slightly stronger than the positive control hs8 (35.3 μg / mL). Figure 5 The results of doubling the prolonging activities of compound 1, compound 2, compound 3, and compound 5 are shown. The iXase inhibitory activity analysis showed that compound 1, compound 2, compound 3, and compound 5 all had potent iXase inhibitory activities, showing the influence of specific structural features on iXase; among them, the IC 50(111.5 ng / mL) is equivalent to the activity of LMWH and is approximately 6 times more active than the FG octasaccharide hs8. Figure 6 Shows the inhibitory activities of Compound 1, Compound 2, Compound 3, and Compound 5 prepared in the examples of this application against endogenous factor iXase.

[0154] Antithrombotic activity and bleeding effect of the pentasaccharide compound (Compound 1) in Example 8.

[0155] Materials: The sample is the pentasaccharide compound 1; the reference substance is Enoxaparin Sodium Injection; hs8, the same as in Example 5.

[0156] Reagents: Chloral hydrate, Tianjin Kemiou Chemical Reagent Co., Ltd.; 0.9% Sodium Chloride Injection, Guangxi Yuyuan Pharmaceutical Co., Ltd.; Purified water, Wahaha Group.

[0157] Experimental animals: SD rats, male, weighing 210 - 270 g, Hunan Slack Jingda Experimental Animal Co., Ltd.; KM mice, male, weighing 35 - 45 g, Hunan Slack Jingda Experimental Animal Co., Ltd.

[0158] Methods:

[0159] Analysis of antithrombotic activity: SD rats were randomly divided into groups, with 6 rats in each group, including: Control control group; LMWH group (2.38 mg / kg); hs8 group (4.94 mg / kg); Compound 1 group (2.79 mg / kg). Animals in each experimental group were administered by subcutaneous injection (Sc.) on the back, and the administration volume was 2 mL / kg for all. One hour after the rats were administered, a suspension of rabbit brain powder was injected intravenously to induce inferior vena cava thrombosis.

[0160] Preparation of the suspension of rabbit brain powder: Weigh a certain amount of rabbit brain powder accurately and dissolve it in physiological saline to prepare a 2% suspension. After vortex mixing for 30 min, centrifuge (200 g × 5 min), and take the supernatant for use.

[0161] Induction of inferior vena cava thrombosis with the extract of rabbit brain powder: SD rats were anesthetized intraperitoneally (0.3 ml / 100 g of 10% chloral hydrate). The abdominal cavity was opened longitudinally along the white line of the abdomen to expose the inferior vena cava. The inferior vena cava and its branches were separated, and a ligature was passed under the left renal vein of the inferior vena cava. The extract of rabbit brain powder (1.5 ml / kg of 2% suspension of rabbit brain powder) was injected through the femoral vein, and the injection was completed in 5 - 6 s. After circulating for 10 seconds, the blood vessels were ligated. Twenty minutes after ligation, the blood vessels were clamped with a hemostatic forceps 2 cm below the ligation site. The blood vessels were longitudinally dissected, and the thrombus was taken out. After drying at 50 °C for 24 h, the dry weight was weighed.

[0162] Analysis of bleeding effect: KM mice were randomly divided into groups of 8 each, including: Control control group; LMWH group (37 mg / kg); Compound 1 group (73 mg / kg). Animals in each experimental group were administered by subcutaneous injection (Sc.) on the back, and the administration volume was 0.1 mL / 10 g. Blood was collected from the tail after 1 h of injecting the drug. The specific operation is as follows: The mouse was placed in a fixator, and the tip of the mouse tail was cut off by 5 mm. The mouse tail was immersed in a beaker containing 40 mL of water (pre-warmed at 37 °C) and a stir bar, and the beaker was placed on a heating and constant temperature magnetic stirrer to maintain a constant temperature of 37 °C and stirred continuously. Timing started from the first drop of blood flowing out of the cut tail, and samples were collected after 1 h. After standing at room temperature for 1 h, the absorbance value (OD540) at 540 nm was measured using a UV spectrophotometer.

[0163] Another healthy mouse was placed in a fixator, and the tip of the mouse tail was cut off by 5 mm. 2.5, 5, 10, 20, 30, 40, 50, and 60 μL of blood were collected with a capillary tube and placed in 10 mL of water respectively. After standing at room temperature for 1 h, OD540 was measured using a UV spectrophotometer. A total of 5 mice were used as replicates. The average value of each concentration was taken to plot a volume-absorbance curve as the standard curve for calculating the blood loss. The results are as follows:

[0164] Antithrombotic activity: As shown in the appendix Figure 7 As shown, Compound 1 at the experimental dose has significant antithrombotic activity, and its thrombus formation inhibition rate can reach about 80%, which is equivalent to the activity of LMWH at the experimental dose.

[0165] Bleeding effect: As shown in the appendix Figure 8 As shown, at an equimultiple high dose of an equivalent antithrombotic dose, the blood loss of oligosaccharide Compound 1 is significantly lower than that of the LMWH administration group.

[0166] Comprehensive analysis shows that the compound prepared in this application has strong APTT prolonging activity and iXase inhibitory activity, showing strong antithrombotic activity and low bleeding tendency characteristics.

[0167] Preparation of freeze-dried powder injection of oligosaccharide compound in Example 9

[0168] Materials: Pentasaccharide Compound 1 prepared by the method of Example 1; Sterile injection water; 2 mL middle borosilicate glass control injection vial; Millipore Pellicon 2 ultrafiltration system (Merk Millipore), VirTis Ultra 35EL freeze dryer.

[0169] Prescription: 80 g of pentasaccharide Compound 1 and 500 mL of injection water, and a total of 1000 vials were prepared.

[0170] Preparation process: Weigh the prescribed amount of oligosaccharide compound 1, add water for injection to the full volume, stir until completely dissolved, and ultrafilter through a Millipore device to remove pyrogens. Under aseptic conditions, after filtering and sterilizing through a 0.22 μm membrane, fill it into vials with a capacity of 2 mL, 0.5 mL per vial. Monitor the filling volume during the filling process, semi-plug the vials, place them in the drying chamber of a pilot-scale freeze dryer, perform freeze-drying according to the set freeze-drying process, plug the vials, take them out of the chamber, crimp the caps, and inspect.

[0171] Freeze-drying process: Pre-cooling: Put the sample into the chamber, lower the temperature of the partition board to -25 °C, keep it for 1 h, then lower the temperature to -45 °C and keep it for 3 h; lower the temperature of the cold trap to -50 °C and start vacuum pumping to 40 Pa. Sublimation: Gradually raise the temperature to -30 °C at a uniform speed in 1 h and keep it for 2 h; gradually raise the temperature to -20 °C at a uniform speed in 2 h and keep it for 6 h, and keep the vacuum at 40 - 30 Pa. Drying: Raise the temperature to -5 °C in 2 h and keep it for 2 h, and keep the vacuum at 30 - 20 Pa; raise the temperature to 10 °C in 0.5 h and keep it for 3 h, and keep the vacuum at 30 - 20 Pa; raise the temperature to 40 °C in 0.5 h and keep it for 4 h, and pump the vacuum to the lowest level.

[0172] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An oligosaccharide compound and a pharmaceutically acceptable salt thereof, characterized in that: The oligosaccharide compound is a compound having the structure of formula (I): The sugar ring A is an α-L-sulfated fucosyl group, the sugar ring B is an α-L-4-deoxy-threo-hex-4-enuronic acid group or a β-D-glucuronic acid group, the sugar ring C is a β-D-2-deoxy-2-acetylamino-4,6-disulfated galactosyl group, and the sugar ring D is a β-D-3-sulfated glucuronic acid group; R1 and R2 are -H or -SO3 independently of each other - ; R is optionally -H, -OH, or β-D-2-deoxy-2-acetylamino-4,6-disulfated galactosyl; R' is optionally -OH, -R 12 , -OR 13 , closed-ring D-2-deoxy-2-acetylamino-4,6-disulfate galactose, open-ring D-2-deoxy-2-acetylamino-4,6-disulfate galactitol, sugar amine or its derivatives, or 2,5-anhydrotalose or its sugar alcohol, sugar amine; -R 12 , -OR 13 are independently substituted or unsubstituted C1-C6 straight or branched alkyl groups, C7-C12 saturated or unsaturated heterocyclic groups containing N, O or S, or substituted or unsubstituted C7-C12 aryl groups.

2. An oligosaccharide compound and a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The sugar ring B is α-L-4-deoxy-threo-hex-4-enuronic acid group, R is -H, and R′ is a closed-ring D-2-deoxy-2-acetylamino-4,6-disulfate galactose (V), or an open-ring D-2-deoxy-2-acetylamino-4,6-disulfate galactitol, sugar amine or its derivative (VI); the structural formula of the oligosaccharide compound is shown in formula (II): In formula (V), R3 is optionally -OH, -R5, -(R6)2 or -OR7; in formula (VI), R4 is optionally -OH, -NH2, -NHR8 or -N(R9)2; wherein R5, R6, R 7、 R8 and R9 are independently substituted or unsubstituted C1-C6 straight chain or branched alkyl groups, C7-C12 saturated or unsaturated heterocyclic alkyl groups containing N, O or S, or substituted or unsubstituted C7-C12 aryl groups.

3. An oligosaccharide compound and a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The sugar ring B is a β-D-glucuronic acid group, R is -OH, and R′ is 2,5-anhydrotalose or its sugar alcohol, sugar amine (VII); the structural formula of the oligosaccharide compound is shown in formula (III): In formula (VII), R 10 Optionally -CH=O, -CH(OH)2, -CH2OH, -CH2R 11 、-CH(R 12 )2, -CH2NH2, -CH2NHR 13 or -CH2N(R 14 )2; where R 11 , R 12 , R 13 , R 14 are independently substituted or unsubstituted C1-C6 straight or branched alkyl groups, C7-C12 saturated or unsaturated heterocyclic groups containing N, O or S, or substituted or unsubstituted C7-C12 aryl groups.

4. An oligosaccharide compound and a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The sugar ring B is a β-D-glucuronic acid group, R is a β-D-2-deoxy-2-acetylamino-4,6-disulfated galactosyl group, R′ is optionally -OH, -R 12 , -OR 13 , closed-ring D-2-deoxy-2-acetylamino-4,6-disulfate galactose (V), or open-ring D-2-deoxy-2-acetylamino-4,6-disulfate galactitol or sugar amine or its derivative (VI); the structural formula of the oligosaccharide compound is shown in formula (IV):

5. The oligosaccharide compound and a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: The pharmaceutically acceptable salt is a sodium salt, a potassium salt or a calcium salt.

6. The method for preparing the oligosaccharide and the pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: S1, fucosylated glycosaminoglycan containing β-D-3-sulfated glucuronyl fragments extracted from Holothuria, phylum Echinodermata; S2, treating the fucosylated glycosaminoglycan obtained in step S1 by chemical depolymerization to obtain a depolymerization product and separating a pure oligosaccharide compound therefrom; S3, modifying the terminal structure of the pure oligosaccharide compound obtained in step S2 to obtain an oligosaccharide compound.

7. The method for preparing the oligosaccharide and the pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: In step S2, the chemical depolymerization method is one of β-elimination depolymerization method, deacylation deamination depolymerization method, and unsaturated hexuronic acid cleavage method; In step S3, the method for modifying the terminal structure is to modify the C1 aldehyde carbonyl or the aldehyde carbonyl in the form of hemiacetal of the reducing end sugar group of the oligosaccharide compound, and the structural modification method is one of aldehyde carbonyl oxidation reaction, reduction reaction, reductive alkylation reaction, reductive amination reaction and polymerization reaction.

8. A pharmaceutical composition having antithrombotic activity, characterized in that: The pharmaceutical composition comprises the oligosaccharide compound according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

9. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the pharmaceutical composition is an aqueous solution for injection or a lyophilized powder for injection.

10. Use of the oligosaccharide compound and a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of a drug for preventing and / or treating thrombotic diseases, characterized in that: The thrombotic disease is one of venous thrombosis, arterial embolism, ischemic heart disease and ischemic cerebrovascular disease.

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