阅读说明：本技术 一种寡糖化合物及其药学上可接受的盐、制备方法及应用 (Oligosaccharide compound and pharmaceutically acceptable salt thereof, preparation method and application ) 是由 赵金华 尹荣华 高娜 周路坦 刘吉开 于 2020-05-14 设计创作，主要内容包括：本申请涉及药物领域,具体而言,涉及一种寡糖化合物及其药学上可接受的盐、制备方法及应用。寡糖化合物是由D-α-硫酸化半乳糖、L-α-硫酸化岩藻糖、L-α-Δ<Sup>4,5</Sup>葡萄糖醛酸和/或D-β-葡萄糖醛酸或其糖醇、D-β-2-脱氧-2-乙酰氨基-硫酸化半乳糖以及任选含有2,5-脱水塔罗糖组成。本申请提供的寡糖化合物及其药物组合物具有强效因子X酶抑制活性及血栓形成抑制活性,可用于制备抗血栓药物。(The application relates to the field of medicines, in particular to an oligosaccharide compound and pharmaceutically acceptable salts, a preparation method and application thereof, wherein the oligosaccharide compound is prepared from D- α -sulfated galactose, L- α -sulfated fucose, L- α -delta 4,5 The oligosaccharide compound and the pharmaceutical composition thereof have strong factor X enzyme inhibiting activity and thrombosis inhibiting activity and can be used for preparing antithrombotic medicaments.)

1. An oligosaccharide compound and pharmaceutically acceptable salts thereof, wherein the oligosaccharide compound has the chemical structure shown in formula (I):

in formula (I):

glycosyl A is D- α -4, 6-disulfated galactosyl;

glycosyl B is L- α -3-sulfated fucosyl;

glycosyl C is D- β -glucuronyl or L- α -4-deoxy-threo-hex-4-enouronyl;

glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl;

glycosyl E is D- β -glucuronyl;

glycosyl F is L- α -2, 4-disulfated fucosyl;

wherein when C is L- α -4-deoxy-threo-hex-4-enouronyl, R is₁Is composed ofR₂is-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Any one of (a); wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl

When C is D- β -glucuronyl, R₁Is composed ofR₂is-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Any one of (a); wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl.

2. The oligosaccharide compound of claim 1, wherein the oligosaccharide compound has the chemical structure according to formula (II):

in the formula (II):

glycosyl A is D- α -4, 6-disulfated galactosyl;

glycosyl B is L- α -3-sulfated fucosyl;

glycosyl C is L- α -4-deoxy-threo-hex-4-enalate;

glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl;

glycosyl E is D- β -glucuronyl;

glycosyl F is L- α -2, 4-disulfated fucosyl;

glycosyl G is D- α -2-deoxy-2-acetamido-4, 6-galactose disulphate or sugar alcohol or sugar amine thereof;

R₂selected from-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl; and when R is₂In the case of-CH ═ O, this terminal aldehyde group forms a hemiacetal six-membered sugar ring structure with the hydroxyl group at C5 in the sugar ring.

3. The oligosaccharide compound of claim 1, wherein the oligosaccharide compound has the chemical structure according to formula (III):

in the formula (III):

glycosyl A is D- α -4, 6-disulfated galactosyl;

glycosyl B is L- α -3-sulfated fucosyl;

glycosyl C is D- β -glucuronyl;

glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl;

glycosyl E is D- β -glucuronyl;

glycosyl F is L- α -2, 4-disulfated fucosyl;

the glycosyl G is 2, 5-anhydrotalose or sugar alcohol or sugar amine thereof;

R₂selected from-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl.

4. The oligosaccharide compound as claimed in claim 2, wherein the structural formula of the formula (II):

R₂is selected from-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl;

alternatively, R₂is-CH ═ O or-CH (OH)₂；

Alternatively, R₂is-CH₂OH。

5. The oligosaccharide compound as claimed in claim 3, wherein the structural formula of the formula (III) is:

R₂is selected from-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl;

alternatively, R₂is-CH ═ O or-CH (OH)₂；

Alternatively, R₂is-CH₂OH。

6. A process for the preparation of an oligosaccharide compound of formula (II) as claimed in claim 2, which comprises:

carrying out quaternary ammonium salinization and benzyl esterification on fucosylated glycosaminoglycan extracted and prepared from the body wall of Thelenota ananas, and then carrying out β -elimination reaction on benzyl esterified carboxylate to break β 1,4 glycosidic bonds connecting D-GalNAc and D-GlcUA in the backbone of the quaternary ammonium salinized fucosylated glycosaminoglycan, so as to obtain a depolymerization product with a non-reducing end of L- α -4-deoxy-threo-hex-4-enosyluronate;

separating and purifying the depolymerized product by adopting a chromatography to obtain purified oligosaccharide, and then carrying out terminal structure modification; or modifying the end structure of the depolymerized product and separating and purifying by adopting a chromatography;

wherein the step of separating and purifying by chromatography comprises the following steps:

to mark withComparing the retention time of hexasaccharide and octasaccharide, performing HPGPC analysis and GPC separation, purifying heptasaccharide component with ion type semi-preparative column or preparative column under the condition that the eluate is separated from H within 0-120min₂Changing the O gradient to 2mol/l NaCl, and the pH value of the buffer solution is 3-4; the obtained oligosaccharide was desalted by gel column.

7. The method for preparing oligosaccharide compound according to claim 6, wherein the quaternary ammonium salt of the quaternized fucosylated glycosaminoglycan is benzalkonium salt (N, N-dimethyl-N- [2- [2- [4(1,1,3, 3-tetramethylbutyl) phenoxy ] ethoxy ] ethyl ] benzalkonium salt) and the carboxylic acid ester is benzyl ester.

8. A process for the preparation of an oligosaccharide compound of formula (iii) as claimed in claim 3, which comprises:

extracting fucosylated glycosaminoglycan from body wall of Thelenota ananas (Thelenota ananas), and removing part of acetyl groups contained in the fucosylated glycosaminoglycan by hydrazine and/or hydrazine sulfate treatment; followed by treatment with nitrous acid to cleave the D-GalNH linkages in the backbone₂And β 1,4 glycosidic linkages of D-GlcUA to obtain a deamidated depolymerization product with reducing end of 2, 5-anhydrotalose;

separating and purifying the deamination depolymerization product by adopting a chromatography to obtain purified oligosaccharide, and then carrying out end structure modification; or modifying the tail end structure of the deamination depolymerization product and then separating and purifying by adopting a chromatography;

the steps of separating and purifying by chromatography comprise:

taking the retention time of hexasaccharide and octasaccharide as reference, carrying out HPGPC analysis and GPC separation, purifying deamination depolymerization product by adopting an ionic semi-preparative column or preparative column, and eluting under the following conditions: eluting with H within 0-120min₂Changing the O gradient to 2mol/l NaCl, and the pH value of the buffer solution is 3-4; the obtained oligosaccharide was desalted by gel column.

9. Use of an oligosaccharide compound as claimed in any of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the activity of an endogenous factor X enzyme.

10. Use of an oligosaccharide compound as claimed in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of a thrombotic disorder; optionally, the thrombotic disease is venous thrombosis, arterial thrombosis or ischemic cardiovascular and cerebrovascular disease.

11. A medicament for inhibiting the activity of an endogenous factor X enzyme, comprising an excipient and the oligosaccharide compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof.

12. The agent for inhibiting the activity of an endogenous factor X enzyme according to claim 11, wherein the agent is administered parenterally.

Technical Field

The application relates to the field of medicines, in particular to an oligosaccharide compound and pharmaceutically acceptable salts, a preparation method and application thereof.

Background

Fucosylated Glycosaminoglycan (FG) derived from echinodermata of the class holothuria has potent anticoagulant activity, and its anticoagulant activity is mainly associated with potent inhibition of the activity of endogenous factor X enzyme (iXase).

The research on the structure-effect relationship of FG oligosaccharide in inhibiting iXase and the anticoagulant antithrombotic activity shows that in the series of FG deamination depolymerization products with fucoidan as the side chain, the nonaose (nonasaccharoide) is the smallest structural fragment with the strong inhibition activity of iXase, while in the β -series depolymerization products with elimination of depolymerization sources, the octaose (octasaccharoide) has the similar intensity of iXase inhibition activity to the nonaose, which is the FG oligosaccharide with the smallest structure and capable of strongly inhibiting iXase so far.

Disclosure of Invention

The embodiment of the application aims to provide an oligosaccharide compound and pharmaceutically acceptable salts thereof, a preparation method and application thereof, and aims to provide an oligosaccharide (heptasaccharide) compound which has the characteristics of strong inhibition of iXase, obvious anticoagulation and antithrombotic activity and low bleeding tendency and pharmaceutically acceptable salts thereof.

In a first aspect, the present application provides an oligosaccharide compound, and pharmaceutically acceptable salts thereof, having the chemical structure according to formula (I):

in formula (I):

glycosyl A is D- α -4, 6-disulfated galactosyl;

glycosyl B is L- α -3-sulfated fucosyl;

glycosyl C is D- β -glucuronyl or L- α -4-deoxy-threo-hex-4-enouronyl;

glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl;

glycosyl E is D- β -glucuronyl;

glycosyl F is L- α -2, 4-disulfated fucosyl;

wherein, when C is L- α -4-deoxy-threo-hex-4-In the case of alkenouronic acid radicals, R₁Is composed ofR₂is-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Any one of (a); wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl

When C is D- β -glucuronyl, R₁Is composed ofR₂is-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Any one of (a); wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl.

The oligosaccharide compound shown in the formula (I) and the pharmaceutically acceptable salt thereof can inhibit the activity of endogenous factor X enzyme, have remarkable antithrombotic activity with low bleeding tendency, and are glycosaminoglycan-derived oligosaccharide compounds which can strongly inhibit the polymerization degree of iXase at the minimum. Heptasaccharide is used as oligosaccharide with smaller structure and strong inhibition of iXase activity, and has important application value for preventing and/or treating thrombotic diseases due to the antithrombotic activity characteristic of low bleeding tendency.

In a second aspect, the present application provides a process for the preparation of an oligosaccharide compound of formula (II), comprising:

carrying out quaternary ammonium salinization and benzyl esterification on fucosylated glycosaminoglycan extracted and prepared from the body wall of Thelenota ananas, and then carrying out β -elimination reaction on benzyl esterified carboxylic ester to break β 1,4 glycosidic bonds connecting D-GalNAc and D-GlcUA in FG main chain, so as to obtain a depolymerization product with a non-reducing end of L- α -4-deoxy-threo-hex-4-ene uronic acid group.

Separating and purifying the depolymerized product by adopting a chromatography to obtain purified oligosaccharide, and then carrying out terminal structure modification; or modifying the end structure of the depolymerized product and separating and purifying by adopting a chromatography.

Wherein the step of separating and purifying by chromatography comprises the following steps:

HPGPC analysis was performed with reference to retention times of the standard hexa-and octa-saccharides, with GPC separation of the oligo FG mixture followed by purification of the heptasaccharide fraction using an ionic semi-preparative or preparative column, elution conditions were: eluting with H within 0-120min₂Changing the O gradient to 2mol/l NaCl, and the pH value of the buffer solution is 3-4; the obtained oligosaccharide was desalted by gel column.

In a third aspect, the present application provides a process for producing an oligosaccharide compound represented by formula (iii), comprising:

extracting fucosylated glycosaminoglycan from body wall of Thelenota ananas (Thelenota ananas), and removing part of acetyl groups contained in the fucosylated glycosaminoglycan by hydrazine and/or hydrazine sulfate treatment; followed by treatment with nitrous acid to cleave the D-GalNH linkages in the FG backbone₂And β 1,4 glycosidic linkages of D-GlcUA to obtain a deamidated depolymerization product with reducing end of 2, 5-anhydrotalose;

separating and purifying the deamination depolymerization product by adopting a chromatography to obtain purified oligosaccharide, and then carrying out end structure modification; or modifying the end structure of the deamination depolymerization product and then separating and purifying by chromatography.

The steps of separating and purifying by chromatography comprise:

using the retention time of hexasaccharide and octasaccharide as reference, carrying out HPGPC analysis, separating deamination depolymerization product by GPC, and purifying heptasaccharide component by ion type semi-preparative column or preparative column, wherein the elution conditions are as follows: eluting with H within 0-120min₂Changing the O gradient to 2mol/l NaCl, and the pH value of the buffer solution is 3-4; the obtained oligosaccharide was desalted by gel column.

In a fourth aspect, the present application provides an oligosaccharide compound as shown above and the use of a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity of an endogenous factor X enzyme.

In a fifth aspect, the present application provides a use of the oligosaccharide compound and the pharmaceutically acceptable salt thereof, which are used for preparing a medicament for treating and/or preventing thrombotic diseases; optionally, the thrombotic disease is venous thrombosis, arterial thrombosis or ischemic cardiovascular and cerebrovascular disease.

In a sixth aspect, the present application provides a medicament for inhibiting the activity of an endogenous factor X enzyme, the medicament comprising an adjuvant and the oligosaccharide compound and pharmaceutically acceptable salts thereof as described above.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 shows the compound prepared in example 1 of the present application¹H NMR spectrum.

FIG. 2 shows the compound prepared in example 1 of the present application¹³C NMR spectrum.

FIG. 3 shows the compound prepared in example 1 of the present application¹H-¹³C HSQC spectrum.

FIG. 4 shows the Q-TOF MS spectrum and assignment of the compound prepared in example 1 of the present application.

Figure 5 shows the doubling of APTT-extending activity of the compound prepared in example 1 of the present application.

FIG. 6 shows the inhibitory activity of the compound prepared in example 1 of the present application against endogenous factor X enzyme.

FIG. 7 shows the antithrombotic activity of the oligosaccharide compound represented by the formula (II).

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

In FIGS. 5-8, "1" represents the experimental group of heptasaccharide Compound 1 and "L MWH" represents the experimental group of enoxaparin sodium injection.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The oligosaccharide compounds of the examples of the present application and pharmaceutically acceptable salts thereof are specifically described below.

The application provides an oligosaccharide compound and pharmaceutically acceptable salts thereof, the chemical structure of the oligosaccharide compound is shown as a formula (I),

in formula (I):

glycosyl A is D- α -4, 6-disulfated galactosyl;

glycosyl B is L- α -3-sulfated fucosyl;

glycosyl C is D- β -glucuronyl or L- α -4-deoxy-threo-hex-4-enouronyl;

glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl;

glycosyl E is D- β -glucuronyl;

glycosyl F is L- α -2, 4-disulfated fucosyl;

wherein when C is L- α -4-deoxy-threo-hex-4-enouronyl, R is₁Is composed ofR₂is-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Any one of (a); wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl

When C is D- β -glucuronyl, R₁Is composed ofR₂is-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Any one of (a); wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl.

In detail, the present application mainly provides two types of oligosaccharide compounds and pharmaceutically acceptable salts thereof.

The first chemical structure is shown as formula (II):

in the formula (II):

glycosyl A is D- α -4, 6-disulfated galactosyl, glycosyl B is L- α -3-sulfated fucosyl, glycosyl C is L- α -4-deoxy-threo-hex-4-enosyluronyl, glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl, glycosyl E is D- β -glucuronyl, glycosyl F is L- α -2, 4-disulfated fucosyl, glycosyl G is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactose or sugar alcohol or sugar amine thereof.

R₂Selected from-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl; and when R is₂When the end aldehyde group is-CH ═ O, the end aldehyde group and the hydroxyl group at the C5 position in the sugar ring form a hemiacetal six-membered sugar ring structure;

as exemplary, R₂Can be-CH ═ O, -CH (OH)₂、-CH₂OH or-CH₂NH₂。

Further, R₂Can be-CH₂NHR' or-CH₂N(R’)₂(ii) a Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl; for example, R' is substituted or unsubstituted C6A linear or branched alkyl group; r' is substituted or unsubstituted C2 or C3 alkyl; r' is a substituted or unsubstituted C4, C5 or C6 straight or branched chain alkyl; r' is a substituted or unsubstituted C7-C12 aryl group, and the substituent may be-CH ═ O, -OH, or the like.

When R is₂In the case of-CH ═ O, this terminal aldehyde group forms a hemiacetal six-membered sugar ring structure with the hydroxyl group at C5 in the sugar ring.

In the present application, the oligosaccharide compound represented by the formula (II) can be prepared by the following method:

fucosylated glycosaminoglycan extracted from the body wall of Thelenota ananas is quaternized and benzylated, and then is further subjected to β -elimination reaction to break β 1,4 glycosidic bonds connecting D-GalNAc and D-GlcUA in the backbone of the quaternized fucosylated glycosaminoglycan, so as to obtain a depolymerization product with a non-reducing end of L- α -4-deoxy-threo-hex-4-enouronyl, and a depolymerization product with a non-reducing end of L- α -4-deoxy-threo-hex-4-enouronyl is also called an oligomeric mixture.

Illustratively, carboxylic acid esters of quaternized fucosylated glycosaminoglycans can be prepared by the following method:

conversion of quaternary ammonium salt: fucosylated Glycosaminoglycans (FG) from Thelenota ananas are used as reaction starting materials, and an excess of organic ammonium salt compound is added to an aqueous solution of alkali metal or alkaline earth metal salt of FG, whereby a water-insoluble FG quaternary ammonium salt is formed which can be easily precipitated from the aqueous solution. Alternatively, FG quaternary ammonium salts can also be obtained by exchanging alkali metal or alkaline earth metal salts of FG for H-type FG using ion exchange resins, followed by neutralization of the H-type FG with basic organic ammonium.

Illustratively, the quaternary ammonium salt is a benzethonium salt (N, N-dimethyl-N- [2- [2- [ 4- (1,1,3, 3-tetramethylbutyl) phenoxy ] ethoxy ] ethyl ] benzylammonium salt). The quaternary ammonium salt may be in the form of other quaternary ammonium salts.

And (3) carboxyl esterification, namely, completely or partially converting the carboxyl on the D- β -glucuronyl group in the FG quaternary ammonium salt obtained above into carboxylic ester, wherein the carboxylic ester can be benzyl ester as an example, and the carboxylic ester can be other in other embodiments of the application.

For example, FG quaternary ammonium salt carboxyl esterification reaction may be carried out in Dimethylformamide (DMF) or a mixed solvent of DMF and a lower alcohol, ketone and/or ether, reacting the carboxyl groups in FG with a stoichiometric amount of a halogenated hydrocarbon; the halogenated hydrocarbon can be a C1-C6 straight chain or branched chain, saturated or unsaturated, substituted or unsubstituted aliphatic hydrocarbon group; or a substituted or unsubstituted C7-C12 aromatic hydrocarbon group, and the like.

The carboxylate ester of the quaternized fucosylated glycosaminoglycan is then subjected to β -elimination reaction.

The carboxylic acid ester of the quaternized fucosylated glycosaminoglycan is cleaved by β -elimination reaction of the carboxylic acid ester group in the backbone of the quaternized fucosylated glycosaminoglycan by the presence of an alkaline agent such as sodium alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo or a mixture thereof, which may be NaOH, KOH, C1-C4, a depolymerization product (oligomeric FG mixture) having a non-reducing end of L- α -4-deoxy-threo-hex-4-enal acid group, which is obtained by cleaving β 1,4 glycosidic bond of D-GlcUA linking D-GalNAc and D-GlcUA in the backbone chain of the quaternized fucosylated glycosaminoglycan by the β -elimination reaction of the carboxylic acid ester group.

Purifying the obtained depolymerized product (oligomeric FG mixture) by chromatography to obtain purified oligosaccharide, and modifying the terminal structure; or the end structure of the depolymerized product (oligomeric FG mixture) is modified and then separated and purified by chromatography. The order of isolation and purification and modification of the terminal structure can be adjusted according to the condition of the target compound. For example, when the target compound in the form of a sugar alcohol is produced, the depolymerization reaction may be followed by a reduction reaction, followed by purification of the oligosaccharide.

The steps of separation and purification comprise: HPGPC analysis was performed with reference to retention times of the standard hexa-and octasaccharides, and the oligo FG mixture was separated by GPC followed by purification using an ionic semi-preparative or preparative column, elution conditions were: eluting with H within 0-120min₂Changing the O gradient to 2mol/l NaCl, and the pH value of the buffer solution is 3-4; coagulating the obtained oligosaccharideAnd (5) desalting by using a gel column.

Illustratively, the separation and purification can be optionally combined with techniques such as ultrafiltration, salting-out, etc. to improve the efficiency of the separation and purification.

The step of modifying the terminal structure of the reducing end glycosyl comprises the following steps: reducing the aldehyde group into an alcoholic hydroxyl group, reducing the aldehyde group into an alkyl derivative through a reductive alkylation reaction or reducing the aldehyde group into an amino derivative through a reductive amination reaction and the like according to the reducibility of the aldehyde group at the end of the reducing glycosyl group and a target product.

For example: with NaBH under alkaline conditions₄Reaction, reducing the reducing end glycosyl into sugar alcohol; reacting with 1-phenyl-3-methyl-5-pyrazolone (PMP) under alkaline condition to generate oligosaccharide-PMP or oligosaccharide-2 PMP derivative; the 1-position aldehyde group of the reducing end glycosyl is reacted in the presence of organic amine to generate Schiff base, and then reduced into secondary amine in the presence of reducing agent, so that the reducing end glycosyl can be reduced into sugar amine or sugar amine derivative.

R can be obtained by modifying the end structure of reducing end glycosyl₂is-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 linear or branched alkyl group, or a substituted or unsubstituted C7-C12 aryl group.

The application also provides an oligosaccharide compound with a chemical structure shown as a formula (III).

In the formula (III):

glycosyl A is D- α -4, 6-disulfated galactosyl, glycosyl B is L- α -3-sulfated fucosyl, glycosyl C is D- β -glucuronyl, glycosyl D is D- α -2-deoxy-2-acetamido-4, 6-disulfated galactosyl, glycosyl E is D- β -glucuronyl, glycosyl F is L- α -2, 4-disulfated fucosyl, and glycosyl G is 2, 5-anhydrotalose or sugar alcohol or sugar amine thereof.

R₂Selected from-CH ═ O, -CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl.

As exemplary, R₂Can be-CH ═ O, -CH (OH)₂、-CH₂OH or-CH₂NH₂。

Further, R₂Can be-CH₂NHR' or-CH₂N(R’)₂(ii) a Wherein R' is a substituted or unsubstituted C1-C6 straight or branched chain alkyl, substituted or unsubstituted C7-C12 aryl; for example, R' is a substituted or unsubstituted C6 straight or branched chain alkyl; r' is substituted or unsubstituted C2 or C3 alkyl; r' is a substituted or unsubstituted C4, C5 or C6 straight or branched chain alkyl; r' is a substituted or unsubstituted C7-C12 aryl group, and the substituent may be-CH ═ O, -OH, or the like.

When R is₂In the case of-CH ═ O, this terminal aldehyde group forms a hemiacetal six-membered sugar ring structure with the hydroxyl group at C5 in the sugar ring.

In the present application, the oligosaccharide compound represented by the formula (III) can be prepared by the following method:

extracting fucosylated glycosaminoglycan from body wall of Thelenota ananas (Thelenota ananas), and removing part of acetyl groups contained in the fucosylated glycosaminoglycan by hydrazine and/or hydrazine sulfate treatment; followed by treatment with nitrous acid to cleave the D-GalNH linkages in the backbone₂And β 1,4 glycosidic linkages of D-GlcUA to give a deaminized depolymerization product of 2, 5-anhydrotalose with reducing end

Illustratively, the nitrous acid depolymerization product of fucosylated glycosaminoglycan extracted from the body wall of thelenota ananas can be prepared by the following method:

FG partial deacetylation, namely removing partial acetyl of D- α -2-deoxy-2-acetamido-4, 6-disulfuric acid galactosyl in FG by hydrazine treatment by using Fucosylated Glycosaminoglycan (FG) derived from Thelenota ananas as a reaction starting material, illustratively, adding anhydrous hydrazine or hydrazine hydrate solution into FG, and reacting for 2-14 h at the temperature of 75-125 ℃ while stirring.

FG deamination depolymerization: treating the obtained partially deacetylated FG intermediate product with nitrous acid to generate deamination depolymerization, and connecting D-GalNH in a fracture main chain₂And β 1,4 glycosidic bonds of D-GlcUA to obtain a depolymerization product (oligomerization FG mixture) with a reducing end of 2, 5-anhydrotalose, wherein the nitrous acid treatment conditions include, for example, adding 4-6 mol/L of nitrous acid solution (pH 1-5) to the partial deacetylation product obtained by the hydrazinolysis treatment in an ice bath or at room temperature, and after reacting for 5-60 min, adjusting the pH of the alkaline solution to 8 or more to terminate the reaction.

Separating and purifying the depolymerized product (oligomeric FG mixture) by adopting a chromatography method to obtain purified oligosaccharide, and then carrying out terminal structure modification; alternatively, the depolymerized product (oligomeric FG mixture) is end-structure modified and then purified by chromatography.

The order of isolation and purification and modification of the terminal structure can be adjusted according to the condition of the target compound. For example, when the target compound in the form of a sugar alcohol is produced, the depolymerization reaction may be followed by a reduction reaction, followed by purification of the oligosaccharide.

Wherein, the step of separating and purifying by adopting a chromatography comprises the following steps:

HPGPC analysis was performed with reference to retention times of the standard hexa-and octasaccharides, and the oligo FG mixture was separated by GPC followed by purification using an ionic semi-preparative or preparative column, elution conditions were: eluting with H within 0-120min₂Changing the O gradient to 2mol/l NaCl, and the pH value of the buffer solution is 3-4; the obtained oligosaccharide was desalted by gel column.

The separation and purification can be optionally combined with technical methods such as ultrafiltration, salting-out method and the like to improve the efficiency of the separation and purification.

The step of modifying the terminal structure of the reducing end glycosyl comprises the following steps: reducing the aldehyde group into an alcoholic hydroxyl group, reducing the aldehyde group into an alkyl derivative through a reductive alkylation reaction or reducing the aldehyde group into an amino derivative through a reductive amination reaction and the like according to the reducibility of the aldehyde group at the end of the reducing glycosyl group and a target product.

For example: with NaBH under alkaline conditions₄Reaction, reducing the reducing end glycosyl into sugar alcohol; reacting with 1-phenyl-3-methyl-5-pyrazolone (PMP) under alkaline condition to generate oligosaccharide-PMP or oligosaccharide-2 PMP derivative; the 1-position aldehyde group of the reducing end glycosyl is reacted in the presence of organic amine to generate Schiff base, and then reduced into secondary amine in the presence of reducing agent, so that the reducing end glycosyl can be reduced into sugar amine or sugar amine derivative.

R can be obtained by modifying the end structure of reducing end glycosyl₂is-CH₂OH、-CH₂NH₂、-CH₂NHR' or-CH₂N(R’)₂Wherein R' is a substituted or unsubstituted C1-C6 linear or branched alkyl group, or a substituted or unsubstituted C7-C12 aryl group.

In conclusion, the oligosaccharide compound shown in the formula (II) or the formula (III) can be obtained by different preparation methods.

Correspondingly, the pharmaceutically acceptable salt of the oligosaccharide compound can be prepared by adopting the preparation method of the corresponding salt by adopting the oligosaccharide compound shown in the formula (II) or the formula (III) as a reactant.

Illustratively, the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, or an organic ammonium salt.

The oligosaccharide compound shown in the formula (II) or the formula (III) and the pharmaceutically acceptable salt thereof have strong activity of endogenous factor X enzyme. IC for inhibiting human endogenous factor X enzyme in vitro₅₀The value is in the range of 200-500 nmol/L, and has no or weak influence on other coagulation factors, coagulation cofactors, antithrombin (AT-III) and the like in the coagulation waterfall.

In vitro anticoagulant activity analysis shows that the oligosaccharide compound shown in the formula (II) or the formula (III) can obviously prolong the Activated Partial Thrombin Time (APTT) value of human plasma, the drug concentration required for multiplying the APTT of the human plasma is generally in the range of 60-120 mu g/m L, and the oligosaccharide compound has no obvious influence or weak influence on the Prothrombin Time (PT) and the Thrombin Time (TT) of the human plasma, which indicates that the oligosaccharide compound shown in the formula (II) or the formula (III) can strongly inhibit the intrinsic coagulation pathway, and has no obvious influence or small influence on the extrinsic coagulation pathway and the common coagulation pathway.

In a pathological model of tissue factor-induced deep vein thrombosis of rats, the oligosaccharide compound shown in the formula (II) or the formula (III) can obviously inhibit the deep vein thrombosis. The inhibition rate of 4.7 mg/kg-13.6 mg/kg subcutaneous injection (sc.) of oligosaccharide compound shown in formula (II) or formula (III) on deep vein thrombosis of rats induced by vascular ligation and thromboplastin can reach 83% -93% by weight of thrombus, and under the equivalent antithrombotic agent amount, the influence of heptasaccharide compound shown in formula (II) or formula (III) on bleeding amount can be obviously lower than that of low molecular weight heparin medicines used clinically.

The inventors have found in their studies that, in the compounds represented by the formula (II) or the formula (iii), when the sugar group a is linked to the sugar group F, the heptasaccharide compound thereof does not exhibit a potent iXase inhibitory activity; the potent inhibitory activity of iXase is only exhibited when the sugar group a in the heptasaccharide compound is linked to the sugar group B (i.e., the oligosaccharide compound represented by formula (II) or formula (iii)).

The previous study reported that Thelenota ananas FG contains Fuc_2S4S、Fuc_4SAnd Fuc_3SThe inventors surmised that the possible reasons for this include, on the one hand, that the previous samples for structural studies were polysaccharide or oligosaccharide fractions in the form of mixtures of proto-polysaccharide, partial acid hydrolysate and oxidative depolymerization product, and that the analysis of the spectral structure of these samples was technically difficult, while several easily recognizable spectral signals from D- α -GalS in the disaccharide side chain were easily confused with the signal from L- α -FucS, and on the other hand, that the structure of the saccharide fractions contained in the partial acid hydrolysate and oxidative depolymerization product was not sufficiently regular, and it was difficult to separate and purify pure saccharide compounds therefromA sexual structure; however, there has been no report of a glycosaminoglycan having a disaccharide side chain which is found in natural GAGs for the first time.

In the application, the oligosaccharide compound shown in the formula (II) or the formula (III) is obtained by analyzing the structure of the oligosaccharide separated and purified from a depolymerized product, wherein the oligosaccharide compound shown in the formula (II) or the formula (III) contains a special D-sulfated galactose (GalS) - α 1, 2-L-FucS- α 1- "disaccharide side chain, the FG heptasaccharide compound with a special structure and containing D-GalS- α 1, 2-L-FucS- α 1-side chain disaccharide at the non-reducing end has strong inhibition activity on iXase and remarkable antithrombotic activity with low bleeding tendency, and the FG heptasaccharide is an oligosaccharide compound which is known to be from glycosaminoglycan and can strongly inhibit the polymerization degree of the iXase and has the smallest structure, has strong inhibition activity on the iXase, and the antithrombotic activity characteristic of low bleeding tendency makes the FG heptasaccharide have important application value for preventing and/or treating thrombotic diseases.

In addition, for thelenota ananas FG oligosaccharide with the polymerization degree of more than or equal to 8, the existence of a disaccharide side chain has no significant influence on the inhibition of the iXase and the anticoagulant and antithrombotic activity, for the octasaccharide containing the disaccharide side chain (D-GalS- α 1, 2-L- α -FucS- α 1-) and the FG oligosaccharide compounds above, the existence of D- α -GalS in the disaccharide side chain does not exist, and the compounds have no significant difference on the iXase inhibition activity, and in the oligosaccharide compounds shown in the formula (II) or the formula (III), the D- α -GalS glycosyl connected to the non-reducing end has special contribution and effect on the iXase inhibition activity of the compounds.

In summary, it can be seen that: the oligosaccharide compound shown in the formula (II) or the formula (III) can inhibit the activity of the iXase, and the oligosaccharide compound and the pharmaceutically acceptable salt thereof can be used for preparing the medicine for inhibiting the activity of the iXase.

Furthermore, the oligosaccharide compound and the pharmaceutically acceptable salt thereof can be used for preparing medicaments for treating and/or preventing thrombotic diseases; for example, the thrombotic disease is venous thrombosis, arterial thrombosis or ischemic cardiovascular and cerebrovascular diseases.

The application also provides a medicine for inhibiting the activity of endogenous factor X enzyme, which comprises auxiliary materials and the oligosaccharide compound and pharmaceutically acceptable salts thereof.

Furthermore, the pharmaceutical unit preparation can contain 50mg to 200mg of the oligosaccharide compound or the pharmaceutically acceptable salt thereof.

The oligosaccharide compounds of formula (II) or (III) have very limited bioavailability when administered parenterally and, therefore, the pharmaceutical dosage form is parenteral. Such as intravenous formulations.

The features and properties of the present application are described in further detail below with reference to examples.