CN120173029A - Oligosaccharide compound and its pharmaceutical composition and application - Google Patents

Abstract

The invention relates to the technical field of biological medicines, and provides an oligosaccharide compound, a pharmaceutical composition and application thereof, wherein the oligosaccharide compound comprises 5-6 sugar rings, namely alpha-L-sulfated fucosyl, alpha-L-4-deoxy-threo-hex-4-enealdonic acid group or beta-D-glucuronic acid group, beta-D-2-deoxy-2-acetamido-4, 6-disulfated galactose group, beta-D-3-sulfated glucuronic acid group, 2, 5-anhydrotalose 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 unexpectedly stronger or far stronger than that of FG heptasaccharide, octasaccharide, nonasaccharide and decasaccharide compounds which are reported at present, and the oligosaccharide compound has the remarkable advantage characteristic of serving as iXase inhibitor.

Description

Oligosaccharide compound, and pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an oligosaccharide compound, a pharmaceutical composition and application thereof.

Background

Venous Thromboembolism (VTE) is one of the most prominent thrombotic diseases, and anticoagulants are the cornerstone of clinical treatment for VTE. However, classical anticoagulant heparins and coumarins can cause severe bleeding risk and have pharmacokinetic defects that increase bleeding risk, while other clinical anticoagulant drugs such as low molecular weight heparins, hirudins, sarbans, garbans, etc. have seen significant advances in predictable pharmacokinetic processes, bleeding tendencies and severe bleeding risk remain.

Recent studies have shown that the intrinsic coagulation pathway is closely related to pathological thrombosis but is not required for hemostatic function, i.e. selective inhibition of intrinsic coagulation may not or less affect hemostatic function in producing an inhibitory effect on thrombotic activity, whereby inhibitors of intrinsic coagulation factors have become the current international mainstream trend in the development of anticoagulant drugs with low bleeding tendency. Endogenous factor X enzyme (IXa-VIIIa-PL-Ca ²⁺ complex, iXase) is the rate-limiting enzyme active site on the endogenous blood coagulation pathway and is therefore the target of action of novel anticoagulant drugs with promising low bleeding tendency characteristics.

Fucoidan (FG) is a structurally specific glycosaminoglycan derivative derived from sea cucumber animals, having a main chain resembling Chondroitin Sulfate (CS) and containing a large number of sulfated fucosyl (L-fucosyl, fuc) side chains. To date, by structural analysis based on a series of pure oligosaccharide fragments, the exact structure of natural FG from several tens of different species of sea cucumber has been confirmed. Comparing FG based on the confirmation structure of the pure oligosaccharide structural fragment, the common structural features of the present invention include that sulfate group (-OSO ₃ ^-) substitution (GalNAc _4S6S) exists at both C4 and C6 positions of acetamido galactose (GalNAc) in CS main chain, and that side chain sulfated L-Fuc (L-FucS) substitution exists at both C3 positions of glucuronic acid (GlcA) contained in main chain. The structural differences in FG from different species are mainly represented by the difference in the sulfated form of the side chain Fuc, the three monosaccharides Fuc side chains having been found to differ in the sulfated form, monosulfated at the C4 position (L-Fuc _4S) and disulfated at the C2/4 or C3/4 positions (L-Fuc _2S4S and L-Fuc _3S4S), and furthermore, the D-alpha-galactosyl (Gal) or D-alpha-GalNAc substitution at the C2 position of the side chain Fuc, that is, the disaccharide side chain substitution, was found. Sulfate group substitutions may be present at the C3 and/or C4 position of Fuc in the side chain disaccharides currently seen, while sulfate group substitutions may also be present at the C4 and/or C6 positions of Gal and GalNAc.

Natural FG has potent anticoagulant activity that produces antithrombotic effects, but also has platelet activation and surface activation activity that may lead to thrombosis. The anticoagulant activity mechanism of natural FG is complex, and low molecular weight FG obtained by proper depolymerization can have the activity of inhibiting endogenous factor X enzyme (iXase) independent of Antithrombin (AT), IIa inhibiting activity independent of heparin cofactor II (HC-II), and the like. Analysis of the structure-activity relationship of FG oligosaccharides obtained from selective depolymerization of glycosidic bonds shows that both the nine saccharides obtained from deamination depolymerization and the octasaccharides obtained from beta-elimination depolymerization can have potent and selective iXase inhibitory activity when only Fuc monosaccharide side chains are contained (ProcNatlAcadSci USA,2015;112:8284-9;JBiol Chem,2018;293:14089-14099), and that FG heptasaccharides containing disaccharide side chains can sometimes have stronger iXase inhibitory activity (Biomacromolecules, 2021;22:1244-55; carbohydro polym,2023; 321:121304). In summary, among FG oligosaccharides, the smallest oligosaccharide structural fragment known to be having potent inhibitory iXase activity (IC 50<1000 ng/mL) is FG heptasaccharide (Biomacro-molecules, 2021:22 (3): 1244-1255;Carbohydr Polym,2023:321:121304). Therefore, FG oligosaccharide compound with smaller structure and strong iXase inhibiting activity has important application value for preventing and/or treating thrombotic diseases.

Disclosure of Invention

In view of this, the present invention proposes a FG oligosaccharide compound having a smaller structure and potent inhibitory activity iXase, and its pharmaceutical compositions and uses.

Based on intensive studies on selective depolymerization of FG glycosidic bonds, FG depolymerization products and the structures necessary to inhibit the activity iXase of FG oligosaccharides, the present inventors have discovered a series of novel structure iXase inhibitors and filed a series of intellectual property protections (CN 2013100998009,2013101274470, CN 201410549676).

The present invention newly studied to find that the D-GlcA glycosyl (GlcA _3S) which is sulfated at the C3 position exists in FG oligosaccharides derived from new species for the first time, and confirms that GlcA _3S exists in a natural FG main chain for the first time, and further studied to find that various FG pentasaccharides and hexasaccharides containing GlcA _3S have strong inhibitory iXase activity and higher activity intensity than the FG octasaccharides and nine saccharides described above, which are FG oligosaccharide compounds with the least polymerization degree and the strong inhibitory iXase activity which have been found so far.

Studies show that FG pentasaccharide and hexasaccharide containing GlcA _3S have anticoagulant antithrombotic activity dependent on potent inhibitory activity iXase, and have the advantage of low bleeding tendency relative to existing clinical anticoagulant drugs such as low molecular heparin. Obviously, the oligosaccharide has smaller structure and strong activity of iXase inhibition, and the antithrombotic activity characteristic of low bleeding tendency makes the oligosaccharide have important application value in preventing and/or treating thrombotic diseases.

The technical scheme of the invention is realized in that in the first aspect, the invention provides an oligosaccharide compound and pharmaceutically acceptable salt thereof, wherein the oligosaccharide compound is a compound with a structure shown in a formula (I):

the sugar ring A is alpha-L-sulfated fucosyl, the sugar ring B is alpha-L-4-deoxidized-threo-hex-4-enadienoic acid group or beta-D-glucuronic acid group, the sugar ring C is beta-D-2-deoxidized-2-acetamido-4, 6-disulfated galactosyl, and the sugar ring D is beta-D-3-sulfated glucuronic acid group;

R ₁ and R ₂ are independently-H or-SO _3-;

R is optionally-H, -OH, or β -D-2-deoxy-2-acetamido-4, 6-disulfated galactosyl;

R' is optionally-OH, -R ₁₂,-OR₁₃, ring-closed D-2-deoxy-2-acetamido-4, 6-disulfate galactose, ring-opened D-2-deoxy-2-acetamido-4, 6-disulfate galactitol, sugar amine or a derivative thereof, or 2, 5-anhydrotalose or sugar alcohol, sugar amine thereof;

R ₁₂,-OR₁₃ is independently of one another a substituted or unsubstituted C1-C6 linear or branched alkyl radical, a C7-C12 saturated or unsaturated heterocyclic radical containing N, O or S, or a substituted or unsubstituted C7-C12 aryl radical.

Based on the above technical scheme, preferably, the sugar ring B is alpha-L-4-deoxidization-threo-hex-4-ene aldonic acid group, R is-H, R' is ring-closed D-2-deoxidization-2-acetamido-4, 6-disulfate galactose (V), or ring-opened D-2-deoxidization-2-acetamido-4, 6-disulfate galactose alcohol, sugar amine or derivative (VI), and the structural formula of the oligosaccharide compound is shown as formula (II):

In the formula (V), R ₃ is selected from-OH, -R ₅,-(R₆)₂ OR-OR ₇, in the formula (VI), R ₄ is selected from-OH, -NH ₂、-NHR₈ OR-N (R ₉)₂; wherein R ₅、R₆、R_7、R₈、R₉ is independently substituted OR unsubstituted C1-C6 straight-chain OR branched alkyl, C7-C12 saturated OR unsaturated heterocyclic alkyl containing N, O OR S, OR substituted OR unsubstituted C7-C12 aryl.

Based on the technical scheme, preferably, the sugar ring B is beta-D-glucuronyl, R is-OH, R' is 2, 5-anhydrotalose or sugar alcohol and sugar amine (VII) thereof, and the structural formula of the oligosaccharide compound is shown as formula (III):

In the formula (VII), R ₁₀ is -CH＝O、-CH(OH)₂、-CH₂OH、-CH₂R₁₁、-CH(R₁₂)₂、-CH₂NH₂、-CH₂NHR₁₃ or-CH ₂N(R₁₄)₂, wherein R ₁₁、R₁₂、R₁₃、R₁₄ is independently substituted or unsubstituted C1-C6 straight-chain or branched alkyl, C7-C12 saturated or unsaturated heterocyclic group containing N, O or S, or substituted or unsubstituted C7-C12 aryl.

Based on the above technical scheme, preferably, the sugar ring B is beta-D-glucuronyl, R is beta-D-2-deoxy-2-acetamido-4, 6-disulfated galactosyl, R' is optionally-OH, -R ₁₂,-OR₁₃, closed-loop D-2-deoxy-2-acetamido-4, 6-disulfated galactose (V), or open-loop D-2-deoxy-2-acetamido-4, 6-disulfated galactose alcohol or sugar amine or derivative (VI), and the structural formula of the oligosaccharide compound is shown as formula (IV):

on the basis of the above technical scheme, preferably, the pharmaceutically acceptable salt is sodium salt, potassium salt or calcium salt.

In a second aspect, the present invention provides a process for the preparation of oligosaccharides and pharmaceutically acceptable salts thereof, said process comprising the steps of:

S1, extracting fucosylated glycosaminoglycan containing beta-D-3-sulfated glucuronyl fragments from body walls and/or viscera of a body of a sea cucumber of the phylum Echinodermata;

s2, treating the fucosylated glycosaminoglycan obtained in the step S1 by adopting a chemical depolymerization method to obtain a depolymerized product, and separating a pure oligosaccharide compound from the depolymerized product by using a chromatographic method;

s3, carrying out end group structure modification on the pure oligosaccharide compound obtained in the step S2 to obtain the oligosaccharide compound.

Based on the above technical scheme, preferably, the echinoderm trepanda of step 1 includes, but is not limited to ：Cucumariafrondosa、Cucumariajaponica、Cucumaria curata、Cucumaria syracusana、Athyonidium chilensis、Cucumaria djakonovi、Cucumaria vegae、Cucumaria salma、Cucumariapseudocurata、Cucumariapiperata、Cucumaria pallida、Cucumaria miniata、Cucumaria georgiana、Cucumaria echinata and Cucumaria dudexa.

In addition to the above, in step S2, the chemical depolymerization method is preferably one of a β -elimination depolymerization method, a deamination depolymerization method, and an unsaturated hexuronic acid cleavage method.

Based on the above technical scheme, the beta-elimination depolymerization method is preferred, namely, the glycosidic bond connected to the C4 position of the hexuronic acid in the main chain of the fucosylated glycosaminoglycan is cracked under the condition of strong alkali, and the product of the beta-elimination depolymerization method has delta _4,5 unsaturated double bond at the non-reducing end of the hexuronic acid. The main steps include, but are not limited to:

(1) Quaternary ammonium salt conversion-quaternary ammonium compounds treat aqueous solutions of fucosylated glycosaminoglycans, converting them into water insoluble quaternary ammonium salts. Such quaternary ammonium compounds include, but are not limited to, benzethonium chloride, and sulfated tetrabutylammonium.

(2) Carboxyl esterification, namely reacting the fucosylated glycosaminoglycan quaternary ammonium salt obtained in the step 1 with halohydrocarbon in an organic solvent, thereby esterifying carboxyl on the main chain hexuronic acid to obtain a carboxyl esterification product. The halogenated hydrocarbon comprises, but is not limited to, benzyl chloride and bromoethane, and the organic solvent is preferably N, N-dimethylformamide.

(3) And (2) beta-elimination and depolymerization, namely treating the carboxyl esterification product of the fucosylated glycosaminoglycan obtained in the step (2) with strong alkali in an organic solvent to enable the carboxyl esterification product to undergo beta-elimination reaction and depolymerization, and removing ester groups on carboxylic acid by post-treatment of the reaction product, and separating and purifying to obtain a depolymerization product. The strong base is preferably sodium ethoxide prepared immediately before use, and the organic solvent is preferably N, N-dimethylformamide.

The basic steps of the beta-elimination depolymerization are shown in scheme 1:

In the figure, U, A is beta-D-glucuronyl (beta-D-GlcA) and beta-D-2-deoxy-2-acetamido-4, 6-disulfated galactosyl (beta-D-Gal _4S6S), dU is alpha-L-4-deoxy-thre-hex-4-enoluronate (alpha-L-DeltaU), Q is quaternary ammonium, E is ester group, R is sulfated L-fucose (alpha-L-FucS) or 2-glycosyl (sulfated alpha-D-galactose or amino galactose group) and R' is H or D-GlcA. n is a natural number, usually 1 to 9.

Based on the technical scheme, the deacylation deamination depolymerization method is preferred, namely, a hydrazine treatment method is adopted to partially remove N-acetyl groups on the amino hexose of the fucosylated glycosaminoglycan main chain, then nitrous acid treatment is adopted to crack the glycosidic bond connected with the C1 position of the deacetylated amino hexose, and the product reduction terminal amino hexose is converted into 2, 5-anhydrohexose. The main steps include, but are not limited to:

(1) Hydrazine method deacetylation by treating fucosylated glycosaminoglycan with hydrazine or hydrazine hydrate with or without hydrazine sulfate, thereby partially removing N-acetyl groups on hexosamines in the polysaccharide backbone, and obtaining a partially deacetylated product. The deacetylation rate of the product is preferably 40% to 75%.

(2) Deamination and depolymerization, namely treating the partially deacetylated product of the fucosylated glycosaminoglycan obtained in the step 1 by using nitrous acid solution to cause deamination and depolymerization reaction, and purifying the reaction solution to obtain a depolymerization product. The pH value of the nitrous acid solution is preferably 2.0-5.0, and the nitrous acid concentration in the reaction solution is preferably 0.25-5.5mol/L.

The basic steps of deacylation deamination depolymerization are shown in scheme 2:

In the figure, U, A, R and n are defined as the same route 1;R' is H or acetyl, and T is D-4, 6-disulfate-2, 5-anhydrotalose (D-anTal _4S6S).

On the basis of the technical scheme, the unsaturated hexuronic acid cracking method is preferred, namely the oligosaccharide compound prepared by the beta-elimination depolymerization method is optionally treated with mercury salt or ozone to crack the glycosidic bond connected with the C1 position of the hexuronic acid containing delta 4,5 unsaturated double bond, so as to obtain the oligosaccharide compound with unsaturated hexuronic acid and side chain fucosyl removed. The main steps include, but are not limited to:

(1) And (3) mercury salt treatment, namely selecting an oligosaccharide compound obtained by beta-elimination and depolymerization of the fucosylated glycosaminoglycan, enabling the oligosaccharide compound to react with mercury salt in an aqueous solution, and cracking a glycosidic bond connected with the C1 position of unsaturated hexuronic acid to obtain the oligosaccharide compound of the desaturated aldehyde acid and the side chain fucosyl connected with the desaturated aldehyde acid. The mercury salt is preferably mercury acetate.

(2) Ozone treatment, namely selecting an oligosaccharide compound obtained by beta-elimination and depolymerization of fucosylated glycosaminoglycan, enabling the oligosaccharide compound to react with ozone in an aqueous solution, and then performing low-pH post-treatment to crack a glycosidic bond connected with the C1 position of unsaturated hexuronic acid, thereby obtaining the oligosaccharide compound of desaturated aldehyde acid and a side chain fucosyl connected with the desaturated aldehyde acid. The low pH value post-treatment means that the pH value of the reaction liquid is adjusted to 2.0-4.0 and the reaction is continued for 20-60 min.

The course of the unsaturated hexuronic acid cleavage reaction can be briefly represented by scheme 3:

in the figure, dU, U, A, R, R' n are defined as the same route 1;X as halogen atoms or acid groups.

Based on the above technical scheme, preferably, in step S3, the terminal structure modification method is structural modification of an aldehyde carbonyl group existing in a form of a C1 aldehyde carbonyl group or a hemiacetal of the oligosaccharide reducing terminal glycosyl, and the structural modification method is one of aldehyde carbonyl oxidation reaction, reduction reaction, reductive alkylation reaction, reductive amination reaction and polymerization reaction.

For example, oxidation may convert a reducing terminal sugar group to a sugar acid, which may be converted to a sugar alcohol, reaction with an alkylating agent such as pyrazolone compounds to give a reducing terminal alkylated derivative, reaction with an amino compound to give a terminal reductive amination derivative, and post-terminal azide conversion may give a triazole-containing derivative by a "click" reaction of azide and alkyne. Route 4 is an example of a representative structural modification that is easy to implement, but does not limit the scope of the structural modification described in the present invention.

Wherein R is as defined in the same way 1;R' is a substituted or unsubstituted C1-C6 straight or branched alkyl group, a C7-C12 saturated or unsaturated heterocyclic group containing N, O or S, or a substituted or unsubstituted C7-C12 aryl group.

In a third aspect, the present invention provides a pharmaceutical composition having antithrombotic activity, comprising an oligosaccharide compound as described above and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient.

Based on the technical scheme, preferably, the dosage form of the pharmaceutical composition is water solution for injection or freeze-dried powder injection for injection.

On the basis of the above technical scheme, preferably, in the unit dose of the pharmaceutical composition of the invention, the content of the oligosaccharide compound or the pharmaceutically acceptable salt thereof is generally in the range of 30 mg-200 mg, and the preferred content is in the range of 50 mg-120 mg.

The dosage form of the pharmaceutical composition is an injection for parenteral administration, including but not limited to an aqueous solution for injection, and a freeze-dried powder injection for injection prepared into an aqueous solution by using water for injection before use.

Generally, the aqueous solution for injection is sterilized by autoclaving, and the aqueous solution for injection is preferably used for the active ingredient having good chemical stability under autoclaving conditions (116 ℃ C./67 kPa X30 min;121 ℃ C./97 kPa X20 min or 126 ℃ C./134 kPa X15 min), and the lyophilized powder for injection prepared into an aqueous solution with water for injection immediately before use is preferably used for the active ingredient having poor chemical stability under autoclaving conditions, and the latter is usually sterilized by ultrafiltration.

In a fourth aspect, the present invention provides the use of the above oligosaccharide compound and a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prophylaxis and/or treatment of thrombotic diseases, characterized in that the thrombotic diseases are one of venous thrombosis, arterial embolism, ischemic heart disease and ischemic cerebrovascular disease.

The oligosaccharide compound, the pharmaceutical composition and the application thereof of the invention have the following compared with the prior art

The beneficial effects are that:

(1) The oligosaccharide compound disclosed by the invention is shown in the formulas (I) - (IV), and compared with other known oligosaccharide compounds from FG sources, the oligosaccharide compound contains GlcA _3S glycosyl, and also contains GlcA glycosyl with a C3 side chain substituted by Fuc, and is only pentasaccharide or hexasaccharide. The oligosaccharide compound or pharmaceutically acceptable salt thereof inhibits IC ₅₀ of iXase (the concentration of the drug required for reducing the enzyme activity by 50%) to be lower than about 100nmol/L (the mass concentration is lower than about 200 ng/mL), and the activity of inhibiting iXase is unexpectedly stronger or far stronger than that of FG heptasaccharide, octasaccharide, nine-saccharide and decasaccharide compounds which are reported at present, and has the activity of strongly inhibiting iXase.

(2) The FG oligosaccharide containing the C3-sulfated-D-glucuronyl (D-GlcA _3S) structural fragment with novel chemical structure is prepared and discovered for the first time, the existence of the D-GlcA _3S structure in natural FG is confirmed through the pure oligosaccharide structural fragment, and in the activity exploration of FG oligosaccharide containing the D-GlcA _3S structural fragment, it is unexpectedly and surprisingly found that FG oligosaccharides with low polymerization degree (dp 5-6) have the characteristic of strong inhibition iXase activity, and the activity of the FG oligosaccharide is far superior to that of the known FG oligosaccharide with the same polymerization degree (dp 5-6) and even higher polymerization degree (dp 7-10). Because iXase inhibitors have important anticoagulation antithrombotic activity characteristics with low bleeding tendency, the oligosaccharide compound is expected to be applied to clinical treatment and/or prevention of thrombotic diseases as anticoagulants with low bleeding tendency, and has important potential value in clinical application.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a ¹ H NMR spectrum of a compound prepared in example 1 of the present invention;

FIG. 2 is a ¹³ C NMR spectrum of the compound prepared in example 1 of the present invention;

FIG. 3 is a ¹H-¹³ C HSQC spectrum of the compound prepared in example 1 of the present invention;

FIG. 4 is a chart of Q-TOF MS of the compound prepared in example 1 of the present invention;

FIG. 5 shows the multiplied APTT prolongation activities of Compound 1, compound 2, compound 3, compound 5 prepared in the examples of the present invention;

FIG. 6 shows the inhibitory activity of Compound 1, compound 2, compound 3, compound 5 on endogenous factor X enzyme prepared in the examples of the present invention;

FIG. 7 shows antithrombotic activity of oligosaccharide compounds of formula (II);

FIG. 8 shows the bleeding effect of an oligosaccharide compound of formula (II).

Detailed Description

The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

EXAMPLE 1 preparation of pentasaccharide Compound 1

The material is dried sea cucumber Cucumariajaponica body wall. The reagents used in benzethonium chloride, benzyl chloride, N-Dimethylformamide (DMF), sodium hydroxide, sodium chloride, ethanol and the like are all commercial analytical pure reagents .Sephadex G25,GE Healthcare;Bio-Gel P10,Bio-Rad;Dionex IonPac^TMAS11-HC Semi-Prep Column,Thermo Scientific; Agilent 1200/1260 series high performance liquid chromatograph, agilent.

The specific method comprises the following steps:

FG extraction and purification according to literature methods (Lan et al, carbohydrPolym,2023, 321:121304), from 1.8kg of dried body wall of sea cucumber Cucumariajaponica by enzymolysis-alkaline hydrolysis combined extraction and purification by salting-out fractionation and alcohol precipitation combined with anion exchange column chromatography to obtain pure polysaccharide, HPGPC analysis shows that it is a homogeneous polysaccharide component, and physicochemical analysis shows that it is Fucosylated Glycosaminoglycan (FG).

Step 2 depolymerizing FG by beta-elimination depolymerization

(1) The quaternary ammonium salt is converted by taking 6g of FG dried product and dissolving in 90mL of pure water, and taking 15g of benzethonium chloride and dissolving in 240mL of pure water. While stirring, the benzethonium chloride solution was slowly added to the FG solution, and after further mixing well, it was allowed to stand overnight at 4 ℃. The resulting solution was centrifuged (4000 rpm. Times.10 min), the supernatant was discarded, and the precipitate was washed twice with pure water (30 mL) (4000 rpm. Times.10 min for removal of benzethonium chloride) and the resulting precipitate was dried under vacuum at 40℃to constant weight to give 17.81g of the quaternized product.

(2) Carboxyesterification 89mL of DMF was added to the quaternized FG precipitate (17.81 g) and after complete dissolution 7.22mL of benzyl chloride was added and reacted at 35℃for 24h to benzyl esterify the C6-carboxyl group of D-GlcA contained in FG.

(3) Beta-elimination depolymerization in the presence of reducing agent after the reaction solution of (2) is cooled to room temperature, 32.07mL of newly prepared sodium ethoxide/ethanol solution with the concentration of 0.16mol/L (containing 0.4M reducing agent NaBH ₄) is added to make the final concentration of 0.04M (containing 0.1M reducing agent NaBH ₄), and alkaline hydrolysis reaction is carried out at room temperature for 30min.

(4) And (3) after the reaction of the step (3) is finished, adding an equal volume of saturated sodium chloride (128 mL), oscillating for 30min to enable sodium salt and quaternary ammonium salt to be fully exchanged, adding 1000mL of absolute ethyl alcohol to reach a final concentration of 80%, standing, centrifuging (4000 rpm multiplied by 10 min) to obtain a precipitate, and exchanging the precipitate with the saturated sodium chloride again, and repeating the steps for two times to obtain the precipitate.

(5) And (3) hydrolyzing benzyl ester of carboxyl, namely dissolving the precipitate obtained in the step (4) by 300mL of water, adding 5mL of 6M NaOH to a final concentration of 0.1M, reacting for 30min at room temperature, regulating to be neutral by using 6M HCl after the reaction is finished, desalting by using a Sephadex G25 chromatographic column, and freeze-drying to obtain dFG 2.5.5G of depolymerized product containing a series of oligosaccharide homologs, wherein the yield is 42%.

Step3, purification of pentasaccharide compound:

2.5g dFG dissolved, loaded in portions, separated by Bio-Gel P10 Gel column at a flow rate of about 10mL/h and 2.5 mL/tube to collect the eluted fraction. Detecting the eluted fraction by ultraviolet spectrophotometry (lambda ₂₃₂), drawing an elution curve, selecting points for HPGPC analysis (Superdex Peptide 10/300GL analytical column, 0.2MNaCl solution isocratic elution, differential refraction detector combined detection by ultraviolet detector), taking FG pentasaccharide as standard substance, repeatedly purifying to single chromatographic peak according to retention time and chromatographic peak shape, and further purifying by anion exchange chromatography in the pentasaccharide preparation process. The resulting pentasaccharide was desalted by Sephadex G25 gel column and lyophilized.

Step 4, spectrum analysis of pentasaccharide compound, nuclear magnetic resonance spectrum:

About 10mg of purified oligosaccharides were repeatedly exchanged 3 times with D ₂ O, dissolved in 500. Mu. L D ₂ O and transferred to a nuclear magnetic tube 5mm in diameter, and their ¹H NMR、¹³ C NMR, HMBC and HSQC, ¹H-¹ H COSY, TOCSY and ROESY spectra were determined on a ADVANCE III MHz nuclear magnetic resonance apparatus. And mass spectrum, namely detecting by adopting an ultrahigh pressure liquid chromatography triple quadrupole tandem mass spectrometer, and collecting in a negative ion mode. The sample concentration was about 0.2mg/mL and the data analysis was performed using MassHunter software. The results were as follows:

purification according to step 1 gave FG 14.3g with a yield of 0.8%, HPGPC analysis of the homogeneous polysaccharide component, physicochemical analysis showed it to be fucosylated glycosaminoglycan. 150mg of compound was obtained as described in step 2 and step 3.

The structural analysis of the compound 1 is shown in figure 1 for ¹ H NMR spectrum and attribution, figure 2 for ¹³ C NMR spectrum and attribution, figure 3 for ¹H-¹³ C HSQC spectrum and attribution, figure 4 for ESI-Q-TOF MS spectrum and attribution, and figure 1 for ¹H/¹³ C NMR signal attribution. According to ¹H-/¹³ C-and 2D NMR and ESI-Q-TOF MS analyses, compound 1 had the chemical structure L-Fuc_3S4S-α(1,3)-L-Δ^4,5GlcA-α(1,3)-D-GalNAc_4S6S-β(1,4)-D-GlcA_3S-β(1,3)-D-Gal NAc_4S6S-ol; with the structure:

TABLE 1 assignment of ¹H/¹³ C NMR signals for pentasaccharide Compound 1

Glycosyl A is alpha-L-3, 4-disulfated fucosyl, glycosyl B is alpha-L-4-deoxy-threo-hex-4-enealdonic acid group, glycosyl C is beta-D-2-deoxy-2-acetamido-4, 6-disulfated galactosyl group, glycosyl D is beta-D-3-sulfated glucuronic acid group, and glycosyl E is D-2-deoxy-2-acetamido-4, 6-disulfated galactitol. The sugar residues in the figures 1-3 are marked in the table 1.

TABLE 2 ESI-Q-TOF MS Signal assignment for pentasaccharide Compound 1

EXAMPLE 2 preparation of pentasaccharide Compound 2

Cucumariajaponica source fucosylated glycosaminoglycan (FG, sodium salt) was prepared as in example 1. The reagents used for hydrazine hydrate, hydrazine sulfate, sodium nitrite, concentrated sulfuric acid, ethanol and the like are all commercially available analytically pure reagents. The materials and apparatus required for the separation of the oligosaccharides are the same as in example 1.

The specific method comprises the following steps:

step 1, deacetylation by hydrazine method:

5g of natural FG was placed in a 500mL round bottom reaction flask, 1.25g of hydrazine sulfate was added, then 125mL of hydrazine hydrate was added, and the reaction was heated and stirred at 90℃for 24h. After the reaction is finished, 500mL of absolute ethyl alcohol is added into the reaction solution until the final concentration of the system ethyl alcohol is 80% (v/v), precipitation is carried out, and the supernatant is removed by centrifugation. After the obtained precipitate was dissolved in 125mL of H ₂ O, 500mL of absolute ethanol (the pure concentration of the obtained solution was 80%, v/v) was added, and the ethanol precipitation was repeated 4 times. The precipitate was redissolved in water and dialyzed against 3500Da dialysis bag (U.S. Co., ltd.) to obtain a partially deacetylated FG intermediate product of about 4g in a yield of about 80%.

Step2, depolymerizing part of deacetylated FG by nitrous acid treatment:

And (3) dissolving the FG partial deacetylation intermediate product obtained in the step (1) in 80mL of H ₂ O, adding 160mL of 5.5M nitrous acid solution with the pH of 4 under the ice bath condition, stirring and reacting for 10-20min, and then adding 1M NaOH to adjust the solution to be neutral, and stopping the reaction. The obtained reaction solution was dialyzed with a dialysis bag having a molecular weight cut-off of 500Da, and the cut-off was collected, and freeze-dried to obtain about 3g of depolymerized product, with a yield of about 75%. Purification and spectroscopic analysis of the pentasaccharide compound were carried out as in example 1.

As a result, 260mg of the obtained compound was produced according to the method. From the detailed spectrum analysis, it was confirmed that the chemical structure of the compound 2 was ：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; that the NMR spectrum signals of the compound 2 and the compound 1 were similar, but there was a difference in characteristic signals, new end groups and 4-hydrogen signal peaks from the characteristic reducing end 2, 5-anhydrotalose (an-Tal) appeared at about 5.0 to 5.1ppm in the compound 2, and the 4-hydrogen signal of unsaturated hexuronic acid disappeared at 5.6 to 5.7 ppm. The structure of compound 2 is shown below, in the structural formula, glycosyl B is beta-D-glucuronyl, glycosyl E is 4, 6-disulfate-2, 5-anhydrotalose, and the rest of sugar residues A, C and D are defined as in example 1.

EXAMPLE 3 preparation of hexasaccharide 3

Cucumariajaponica source fucosylated glycosaminoglycan (FG, sodium salt) preparation method was the same as in example 1, step 1. Mercury acetate is a commercially available analytically pure reagent. Cation exchange resin [ ]50 W.times.8 (H type), alfaAesar. The materials and instruments required for the rest of the preparation process are the same as in example 1.

The specific method comprises the following steps:

(1) The oligomerization product was prepared in the same manner as in step 2 of example 1;

(2) Purification preparation of octasaccharide the oligomeric product was isolated and purified by gel column chromatography combined with anion exchange column chromatography in a similar manner to step 3 of example 1 to give pure octasaccharide about 100mg;

(3) Mercury decomposition reaction, namely dissolving the octasaccharide in 5mL of water, adjusting the pH to about 5 by acetic acid, adding an equal volume of mercury acetate solution (70 mM, pH 5) into the oligosaccharide solution, stirring at room temperature for 10min, and directly loading the reaction solution on a cation exchange chromatographic column after the reaction is finished 50 W.times.8), 20mL of water was eluted, and the resulting hexasaccharide was desalted by Sephadex G25 gel column and lyophilized.

(4) Structural analysis is the same as in step 4 of example 1.

As a result, compound 3 was obtained as 70mg by the method. According to detailed spectrum analysis, the chemical structure of the compound 3 is 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 spectrum signals of the compound 3 and the compound 1 are approximate, the 4-hydrogen signal of the unsaturated hexuronic acid at 5.6-5.7 ppm is disappeared, the compound is consistent with the oligosaccharide compound which is formed by splitting unsaturated hexuronic acid C1 through mercury salt treatment to form unsaturated aldehyde acid and linked side chain fucosyl, meanwhile, a group of signal peaks of sugar residues (D-GalNAc _4S6S) appear in the compound 3, the structure of the compound 3 is shown as follows, in the structural formula, the sugar group B is beta-D-glucuronyl, the sugar group E is D-2-deoxy-2-acetamido-4, 6-disulfate galactitol, the sugar group F is D-2-deoxy-2-acetamido-4, 6-disulfate galactose, and the rest sugar residues A, C and D are defined in the example 1.

EXAMPLE 4 preparation of pentasaccharide Compound 4

The material was the same as in example 3.

The specific method comprises the following steps:

(1) The process for preparing the oligomerization product is similar to step 2 of example 1, the only difference is that in the alkaline hydrolysis of step 2, step (3), no reducing agent NaBH ₄ is added, and the reaction product undergoes a peeling reaction in the alkaline hydrolysis process, so that the residue of the reduced-end acetamido galactose is lost;

(2) Purification of heptasaccharide the oligomeric product was isolated and purified by gel chromatography combined with anion exchange column chromatography in a similar manner to step (6) of example 1 to yield pure heptasaccharide about 80mg;

(3) Mercerization reaction in the same manner as in step (3) of example 3 to cleave heptasaccharide non-reducing terminal unsaturated hexuronic acid (α -L-4-deoxy-threo-hex-4-eneuronic acid) and side chain sulfated fucosyl group attached thereto.

(4) Separating the reaction product obtained in the step (3) by adopting a Bio-gel P10 gel column chromatography, collecting target pentasaccharide, desalting the obtained pentasaccharide by using a Sephadex G25 gel column, and freeze-drying.

(5) Structural analysis is the same as in step 4 of example 1.

As a result, 35mg of Compound 4 was obtained by the method. From the detailed spectrum analysis, it was determined that compound 4 was pentasaccharide, and compound 4 having the chemical structural formula ：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; had the structure:

In the structural formula, glycosyl A is alpha-L-3, 4-disulfated fucosyl, glycosyl B is beta-D-glucuronyl, glycosyl C and glycosyl E are beta-D-2-deoxy-2-acetamido-4, 6-disulfated galactosyl, and glycosyl D is beta-D-3-sulfated glucuronyl.

EXAMPLE 5 preparation of pentasaccharide derivative 5

Material pentasaccharide compound 2, example 2. 1-phenyl-3-methyl-5-pyrazolone (PMP), biochemical reagent, purity 99%.

30Mg of pentasaccharide obtained in example 2 was dissolved in H ₂ O (50 mg/mL), 1.5mL of 0.5M PMP methanol solution and 1mL of 0.6M NaOH solution were added thereto, and the mixture was stirred at 50℃for 90 minutes to thereby effect a reaction, and the pH was adjusted to be neutral after the completion of the reaction. The resulting reaction product was desalted using a Sephadex G25 gel column, and the sugar-containing fractions were combined and lyophilized.

As a result, the pentasaccharide derivative 5 was prepared according to the method to give 25mg. According to careful spectroscopic analysis, compound 5 had a similar sugar residue signal to compound 2, except that the low field region of compound 5 exhibited a set of aromatic hydrogen signals derived from benzene rings, while the characteristic signal of reduced-end 2, 5-anhydrotalose disappeared, and a set of methylene groups appeared at 2ppm, and it was seen that end group alkylation of compound 2 occurred. The chemical structure of the pentasaccharide derivative 5 can be determined by comprehensive judgment:

EXAMPLE 6 preparation of pentasaccharide derivative 6

Material pentasaccharide compound 4, example 4. The reagents used in N-methylmorpholine, naN ₃, 2-chloro-1, 3-dimethylimidazoline chloride, propargylamine, copper sulfate, sodium ascorbate and the like are all commercially available analytically pure reagents.

30Mg of pentasaccharide obtained in example 2, 53 mu L of N-methylmorpholine and 53 mu L of NaN ₃ and mg are dissolved in 300 mu L of pure water, the reaction system is placed in an ice-water bath, 30mg of 2-chloro-1, 3-dimethylimidazolidine chloride is added, the reaction is continued for 48 hours after 15 minutes at room temperature, and 25mg of intermediate is obtained after desalting the reaction product. The reaction intermediate and propargylamine 5 mu L are mixed in a tetrahydrofuran/water (1:1, 100 mu L) system, copper sulfate and sodium ascorbate are added into the reaction system, stirring is carried out for 24 hours at room temperature, the reaction product is washed and extracted after the reaction is finished, and then the product is desalted through a Sephadex G25 gel column, and sugar-containing parts are combined and freeze-dried.

As a result, compound pentasaccharide derivative 6 was prepared according to the method described to give 15mg. Compared with the pentasaccharide compound 4, the ¹ H NMR spectrum shows that two groups of signals newly appear at the positions of 6.0ppm to 8.0ppm of the derivative 6, which are H-1 and triazolene hydrogen signals of a reducing end GlcA _3S respectively, and the chemical structure of the pentasaccharide derivative 6 is comprehensively analyzed as follows:

Example 7 analysis of anticoagulant Activity of series of Compounds and inhibitory Activity against endogenous factor iXase samples were pentasaccharide compound 1, pentasaccharide compound 2, hexasaccharide compound 3, pentasaccharide derivative 5.

Control, enoxaparin sodium injection (LMWH, mw-4500 Da, sanofi-Aventis Co.), hs8, FG octasaccharide of fucose side chain (Yin et al, jbiol Chem,2018, 293:14089-99), prepared in the early stage of the laboratory.

Reagent, human coagulation quality control plasma, activated Partial Thromboplastin Time (APTT) determination kit, germany TECO GmbH company product, factor VIII detection kit, HYPHEN BioMed company (France) product, recombinant human Factor VIII (FVIII) for injection, bayer HEALTHCARE LLC company (Germany) product.

The instrument comprises MC-2000 coagulometer, germany TECO GmbH, VICTORNivo ^TM multifunctional enzyme-labeled instrument, perkinelmer, vortexGenie vortex oscillator, SCIENTIFIC INDUSTRIES, XS105 electronic balance, FE20 pH meter, mettler Toledo, U.S.A.

The method comprises the following steps:

(1) Solution preparation

The preparation of the human coagulation quality control plasma comprises the steps of adding 1mL of purified water into the human coagulation quality control plasma freeze-dried powder according to a reagent instruction, standing for 15min at room temperature, and using after the purified water is completely dissolved.

Preparing reference substance solution, namely dissolving mother solution of low molecular heparin at 100mg/mL with Tris-HCl buffer solution to prepare 1280 mug/mL stock solution, and carrying out gradient dilution according to experimental requirements.

Sample solution preparation, namely precisely weighing 1.28mg of compound 1, compound 2, compound 3, compound 5 and reference hs8, adding Tris-HCl buffer solution to dissolve and prepare 1280 mug/mL mother liquor, and carrying out gradient dilution according to experimental requirements.

The factor VIII kit is prepared according to the method of kit description, and 2.5mL of purified water is added into R1, R2 and R3 for use after the purified water is completely dissolved.

(2) Anticoagulation activity detection, which is to take 5. Mu.L of control substance or sample solution, add the control substance or sample solution into a 37 ℃ preheated cuvette, add 45. Mu.L of dissolved human coagulation quality control plasma, incubate for 2min at 37 ℃, add 50. Mu.L of 37 ℃ preheated APTT reagent, incubate the mixture for 3min at 37 ℃, add 50. Mu.L of preheated CaCl ₂ solution, start timing and record coagulation time.

(3) And (3) anticoagulation activity data processing, namely mapping the final concentration of the sample in plasma and APTT time (average value of multi-well detection), performing linear fitting, and calculating according to a fitting equation (sample concentration-clotting time equation), so that the APTT time is prolonged by 1 time to obtain the required final concentration of the sample.

(4) IXase inhibition activity assay by combining factor VIII with a factor VIII detection kit, and detecting by referring to kit instructions and literature methods. Specifically, 30. Mu.L of test and control solutions (Tris-HCl buffer) were added to 96-well plates at a serial concentration, 30. Mu. L R2 (Activation Reagent), 30. Mu.L of FVIII solution (2 IU/mL), shaking plates at 37℃for 2min, 30. Mu.LR 1 (FX), shaking plates at 37℃for 1min, 30. Mu.LR 3 (SXa-11) was added, and absorbance at 405nm (OD ₄₀₅) was detected after shaking plates were mixed and analyzed continuously at 15s intervals for 2min.

(5) IXase inhibition activity data processing, namely linear fitting is carried out on time by using a detection mean value of a compound hole OD ₄₀₅, and the slope (the change rate of the light absorption value, OD ₄₀₅/min) is the activity of iXase. The iXase activity (percent) in the presence of the sample or positive control was calculated as iXase activity of the negative control (Tris-HCl buffer) wells as 100%.

As a result, anticoagulation activity studies show that each of the pentasaccharide compound 1, the pentasaccharide compound 2, the hexasaccharide compound 3 and the pentasaccharide derivative 5 has remarkable APTT prolongation activity, wherein the concentration required for doubling the APTT of the compound 1 is 26.8 mug/mL, and the concentration is slightly stronger than that of a positive control hs8 (35.3 mug/mL). Figure 5 shows the results of compound 1, compound 2, compound 3, compound 5 multiplied by the extended activity. Analysis of iXase inhibition activity showed that compound 1, compound 2, compound 3, and compound 5 all had potent iXase inhibition activity, showing the effect of specific structural features on iXase, wherein IC ₅₀ (111.5 ng/mL) of compound 1 was comparable to LMWH activity, about 6-fold stronger than FG octasaccharide hs8 activity. FIG. 6 shows the inhibitory activity of endogenous factors iXase, compound 1, compound 2, compound 3, and compound 5 prepared in the examples of the present application.

Example 8 antithrombotic activity of pentasaccharide compound (compound 1) and bleeding effect.

The materials are pentasaccharide compound 1 as sample, enoxaparin sodium injection as reference, hs8 as example 5.

The reagent comprises chloral hydrate, miou chemical reagent Co., ltd, 0.9% sodium chloride injection, guangxi Yuyuan pharmaceutical Co., ltd, purified water, baby haha group.

The experimental animals comprise SD rats, male, weight 210-270 g, hunan Laickida experimental animals Limited, KM mice, male, weight 35-45g, hunan Laickida experimental animals Limited.

The method comprises the following steps:

The anti-thrombotic activity assay was that SD rats were randomly divided into 6 groups, each including Control group, LMWH group (2.38 mg/kg), hs8 group (4.94 mg/kg), and Compound 1 group (2.79 mg/kg). Animals of each experimental group were dosed with subcutaneous injection (Sc.) at a volume of 2mL/kg. Intravenous injection of the rabbit brain powder suspension induced inferior vena cava thrombosis 1 hour after rat dosing.

The preparation method of the rabbit brain powder suspension comprises accurately weighing a certain amount of rabbit brain powder, dissolving in physiological saline to prepare 2% suspension, mixing by vortex for 30min, centrifuging (200 g×5 min), and collecting supernatant for use.

The rabbit brain powder leaching solution induces the thrombus formation of the inferior vena cava, namely, SD rat is anesthetized (0.3 ml/100g 10% chloral hydrate), the abdominal cavity is opened longitudinally along the abdominal white line, the inferior vena cava is exposed, the inferior vena cava and branches thereof are separated, and a ligature is passed through the inferior border of the left renal vein of the inferior vena cava. The rabbit brain powder leaching solution (1.5 ml/kg 2% rabbit brain powder suspension) is injected from femoral vein, after 5-6s injection is completed, blood vessels are ligated after 10 seconds of circulation. After ligation for 20 minutes, the vessel was clamped with a hemostat at a position 2cm below the ligature, the vessel was dissected longitudinally, the thrombus was removed, and the dry weight was weighed after drying at 50 ℃ for 24 hours.

Bleeding effect analysis KM mice were randomly grouped, 8 per group, including Control, LMWH (37 mg/kg), compound 1 (73 mg/kg). Animals of each experimental group were dosed with back subcutaneous injection (Sc.) at a volume of 0.1mL/10g. Tail breaking and blood taking are carried out after 1h of medicine injection. The specific operation is that the mice are placed in a fixer, 5mm tail tips are cut off, the tail is immersed in a beaker containing 40mL of water (pre-temperature of 37 ℃) and a stirrer, and the beaker is placed on a heating constant-temperature magnetic stirrer to be kept at a constant temperature of 37 ℃ and stirred continuously. Starting from the flow of 1 st drop of blood from the cut tail, the sample was collected after 1h, and after 1h standing at room temperature, the absorbance at 540nm (OD 540) was measured with an ultraviolet spectrophotometer.

The healthy mice were placed in a holder, 5mm tail tips were cut off, capillary blood was collected by 2.5, 5, 10, 20, 30, 40, 50 and 60 μl, each placed in 10mL water, and after 1h at room temperature, OD540 was detected by uv spectrophotometry. A total of 5 mice were taken as replicates. The average value of each concentration was taken to draw a volume-absorbance curve as a standard curve for calculating the amount of bleeding. The results were as follows:

Antithrombotic Activity As shown in FIG. 7, compound 1 has significant antithrombotic activity at experimental doses, which can reach a thrombosis inhibition of about 80%, comparable to LMWH activity at experimental doses.

Bleeding effects as shown in figure 8, the amount of bleeding of oligosaccharide compound 1 was significantly lower than in LMWH-administered group at an equivalent high dose of the equivalent antithrombotic dose.

Comprehensive analysis shows that the compound prepared by the application has strong APTT prolongation activity and iXase inhibition activity, and shows strong antithrombotic activity and low bleeding tendency characteristics.

EXAMPLE 9 preparation of lyophilized powder for injection of oligosaccharide Compound

The material comprises pentasaccharide compound 1 prepared by the method of example 1, sterile water for injection, 2mL borosilicate glass tube injection bottle, millipore Pellicon Ultra-filtration system (Merk Millipore), virTis Ultra 35EL freeze dryer.

Prescription of five sugar compound 180g and water for injection 500mL, 1000 pieces are prepared.

The preparation method comprises weighing oligosaccharide compound 1 in prescribed amount, adding injectable water to full amount, stirring to dissolve completely, and ultrafiltering with Millipore device to remove pyrogen. Under the aseptic environment, after 0.22 mu m membrane filtration and sterilization, filling in penicillin bottles with the capacity of 2mL, 0.5mL of each bottle, monitoring the filling amount in the filling process, half-pressing the plugs, putting the bottles into a drying box of a pilot-type freeze dryer, freeze-drying according to the set freeze-drying process, pressing the plugs, taking out the box, rolling the caps and checking.

And the freeze-drying process comprises the steps of pre-cooling, namely, feeding a sample into a box, cooling a partition plate to-25 ℃, keeping the temperature for 1h, cooling to-45 ℃, keeping the temperature for 3h, cooling a cold trap to-50 ℃, and starting vacuumizing to 40Pa. Sublimation, namely heating to-30 ℃ at a constant speed for 1h, keeping for 2h, heating to-20 ℃ at a constant speed for 2h, keeping for 6h, and keeping the vacuum at 40-30 Pa. Drying, namely heating to-5 ℃ for 2 hours, maintaining the vacuum at 30-20 Pa, heating to 10 ℃ for 0.5 hour, maintaining for 3 hours, maintaining the vacuum at 30-20 Pa, heating to 40 ℃ for 0.5 hour, maintaining for 4 hours, and vacuumizing to the lowest.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (10)

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; R ₁ and R ₂ are independently -H or -SO ₃ ^- ; 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 derivative, or 2,5-anhydrotalose or its sugar alcohol, sugar amine; -R ₁₂ and -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), R ₃ is optionally -OH, -R ₅ , -(R ₆ ) ₂ or -OR ₇ ; in formula (VI), R ₄ is optionally -OH, -NH ₂ , -NHR ₈ or -N(R ₉ ) ₂ ; wherein R ₅ , R ₆ , R _7, R ₈ and R ₉ are independently substituted or unsubstituted C1-C6 straight chain or branched alkyl, C7-C12 saturated or unsaturated heterocyclic alkyl containing N, O or S, or substituted or unsubstituted C7-C12 aryl. 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 is optionally -CH=O, -CH(OH) ₂ , -CH ₂ OH, -CH ₂ R ₁₁ , -CH(R ₁₂ ) ₂ , -CH ₂ NH ₂ , -CH ₂ NHR ₁₃ or -CH ₂ N(R ₁₄ ) ₂ ; wherein R ₁₁ , R ₁₂ , R ₁₃ and R ₁₄ are independently substituted or unsubstituted C1-C6 straight chain or branched alkyl, C7-C12 saturated or unsaturated heterocyclic group containing N, O or S, or substituted or unsubstituted C7-C12 aryl group. 4. An oligosaccharide compound and a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: the sugar ring B is β-D-glucuronic acid group, R is 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-disulfated galactose (V), or open-ring D-2-deoxy-2-acetylamino-4,6-disulfated 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 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 pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of a drug 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.