CN111423523B

CN111423523B - Oligosaccharide compound and pharmaceutically acceptable salt thereof, preparation method and application

Info

Publication number: CN111423523B
Application number: CN202010410013.1A
Authority: CN
Inventors: 赵金华; 尹荣华; 高娜; 周路坦; 刘吉开
Original assignee: South Central University for Nationalities
Current assignee: Endian Science & Technology Development Of Yunnan Co ltd
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2021-10-26
Anticipated expiration: 2040-05-14
Also published as: CN111423523A

Abstract

The present application relates to the field of medicine, in particular, to an oligosaccharide compound and a pharmaceutically acceptable salt thereof, a preparation method and an application thereof. The oligosaccharide compound is composed of D-α-sulfated galactose, L-α-sulfated fucose, L-α-Δ ^4,5 glucuronic acid and/or D-β-glucuronic acid or its sugar alcohol, D-β-2-deoxy-2-acetamido-sulfated galactose and optionally 2,5-anhydrotalose. The oligosaccharide compound and the pharmaceutical composition thereof provided by the present application have potent factor X enzyme inhibitory activity and thrombosis inhibitory activity, and can be used for the preparation of antithrombotic drugs.

Description

Oligosaccharide compound and pharmaceutically acceptable salt thereof, preparation method and application

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 echinoderm, holothurian, has potent anticoagulant activity, and its anticoagulant activity is mainly associated with potent inhibition of the activity of endogenous factor X enzyme (iXase).

Research on structure-effect relationship of FG oligosaccharide in inhibiting iXase and anticoagulant and antithrombotic activity of the iXase shows that in series FG deamination depolymerization products with fucomonosaccharide as a side chain, nonaose (nonasaccharoide) is the smallest structural fragment with strong inhibition of the iXase activity; in the series of depolymerization products derived from β -elimination depolymerization, octasaccharide (octasaccharide) has an iXase inhibitory activity of similar intensity to the aforementioned nine saccharide, which is the FG oligosaccharide having the smallest structure so far found that can strongly inhibit iXase.

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-alpha-4, 6-disulfated galactosyl;

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

glycosyl C is D-beta-glucuronyl or L-alpha-4-deoxy-threo-hex-4-enouronyl;

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

glycosyl E is D-beta-glucuronyl;

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

wherein, when C is L-alpha-4-deoxy-threo-hex-4-enouronyl, R is₁Is composed of

R₂is-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-beta-glucuronyl, R₁Is composed of

R₂is-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:

performing quaternary ammonium salinization and benzyl esterification on fucosylated glycosaminoglycan extracted and prepared from the body wall of Thelenota ananas, and then performing beta-elimination reaction on benzyl esterified carboxylate to break

beta

1,4 glycosidic bonds connecting D-GalNAc and D-GlcUA in FG main chain to obtain a depolymerization product with a non-reducing end of L-alpha-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:

fucosylated glycosaminoglycan is extracted from body wall of Thelenota ananas (Thelenota ananas), and is extracted with hydrazine and/or hydrazine sulfateTreating to remove part of acetyl groups contained in the fucosylated glycosaminoglycan; followed by treatment with nitrous acid to cleave the D-GalNH linkages in the FG backbone₂And the

beta

1,4 glycosidic bond of D-GlcUA to obtain a deamidated depolymerization product with a 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 group of heptasaccharide Compound 1 and "LMWH" represents the 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-alpha-4, 6-disulfated galactosyl;

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

glycosyl C is D-beta-glucuronyl or L-alpha-4-deoxy-threo-hex-4-enouronyl;

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

glycosyl E is D-beta-glucuronyl;

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

wherein, when C is L-alpha-4-deoxy-threo-hex-4-enouronyl, R is₁Is composed of

When C is D-beta-glucuronyl, R₁Is composed of

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-alpha-4, 6-disulfated galactosyl; glycosyl B is L-alpha-3-sulfated fucosyl; glycosyl C is L-alpha-4-deoxy-threo-hex-4-enal; glycosyl D is D-alpha-2-deoxy-2-acetamido-4, 6-disulfated galactosyl; glycosyl E is D-beta-glucuronyl; glycosyl F is L-alpha-2, 4-disulfated fucosyl; the glycosyl G is D-alpha-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 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 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 (II) can be prepared by the following method:

after the fucosylated glycosaminoglycan extracted and prepared from the body wall of Thelenota ananas is quaternized and benzylated, beta-elimination reaction is further carried out to break the

beta

1,4 glycosidic bond connecting D-GalNAc and D-GlcUA in the backbone of the quaternized fucosylated glycosaminoglycan, and a depolymerization product with the non-reductive end being L-alpha-4-deoxy-threo-hex-4-ene uronic acid group is obtained. The depolymerization product with a non-reducing end group of L- α -4-deoxy-threo-hex-4-enouronyl is also known as oligomeric FG 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.

Esterification of carboxyl groups: the carboxyl group on the D- β -glucuronic acid group in the FG quaternary ammonium salt obtained above is converted, in whole or in part, into a carboxylic acid ester, which may be, for example, a benzyl ester, and in other embodiments of the present application, may be other.

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 a β -elimination reaction. For example, the following method can be employed:

a carboxylic acid ester of a quaternized fucosylated glycosaminoglycan is cleaved by a β -elimination reaction of the carboxylic acid ester group in a nonaqueous solvent in the presence of an alkaline agent to cleave a

β

1,4 glycosidic bond connecting D-GalNAc and D-GlcUA in the backbone of the quaternized fucosylated glycosaminoglycan, to obtain a depolymerization product (oligomeric FG mixture) having a non-reducing terminal L- α -4-deoxy-threo-hex-4-enalate group. The non-aqueous solvent may be, for example, Dimethylformamide (DMF) or a mixed solvent of DMF and a lower alcohol, ketone and/or ether, and the alkaline agent may be, for example, NaOH, KOH, sodium alkoxide of C1-C4, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo-ring or a mixture thereof.

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; the obtained oligosaccharide was desalted by 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 derivativeAn organism; 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-alpha-4, 6-disulfated galactosyl; glycosyl B is L-alpha-3-sulfated fucosyl; glycosyl C is D-beta-glucuronyl; glycosyl D is D-alpha-2-deoxy-2-acetamido-4, 6-disulfated galactosyl; glycosyl E is D-beta-glucuronyl; glycosyl F is L-alpha-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.

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 unsubstitutedC2 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.

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 the

beta

1,4 glycosidic bond of D-GlcUA to obtain a deaminized depolymerization product of 2, 5-anhydrotalose at the 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: fucosylated Glycosaminoglycan (FG) derived from Thelenota ananas is used as a reaction starting material, and a part of acetyl groups of D-alpha-2-deoxy-2-acetamido-4, 6-disulfated galactosyl groups in FG are removed by hydrazine treatment. Illustratively, adding anhydrous hydrazine or hydrazine hydrate solution into FG, and reacting for 2-14 h at the temperature of 75-125 ℃ while stirring. In some embodiments, the reaction may be catalyzed in a catalyst such as hydrazine sulfate, hydrazine hydrochloride, and the like.

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 the

beta

1,4 glycosidic bond of D-GlcUA, to give a depolymerized product of 2, 5-anhydrotalose with reducing ends (oligomeric FG mixture). As an example, the conditions of the nitrous acid treatment include: under the condition of ice bath or room temperature, adding 4-6 mol/L of nitrous acid solution (pH 1-5) into a partial deacetylation product obtained by hydrazinolysis treatment, reacting for 5-60 min, and adjusting the pH of an alkali solution to be 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.

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’)₂An oligosaccharide compound represented by the formula (III), whereinR' is substituted or unsubstituted C1-C6 straight chain or branched chain alkyl, and substituted or unsubstituted C7-C12 aryl.

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 obvious influence or weak influence on other blood coagulation factors, blood coagulation cofactors, antithrombin (AT-III) and the like in the blood 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/mL, 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 effectively 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_3SEtc. which could not be found to contain disaccharide side chains, the inventors hypothesized possible reasons for this including: first, the samples for structural studies have been polysaccharide or oligosaccharide fractions in the form of mixtures of prototype polysaccharides, partial acid hydrolysates, and oxidative depolymerization products, and the analysis of the spectral structure of these samples has been technically difficult, whereas several easily recognizable spectral signals from D- α -GalS in the disaccharide side chain are easily confused with the signal from L- α -FucS; secondly, the structures of the carbohydrate components contained in the partial acid hydrolysate and the oxidative depolymerization product are not regular enough, and it is difficult to separate and purify the pure carbohydrate compounds therefrom. In fact, because glycosaminoglycans in the animal body assume many important and fundamental physiological functions, the basic structure of glycosaminoglycans is highly conserved even during the long-term evolution in the animal kingdom, with the exception of FG, which contains a large number of FucS side chain glycosyl groups, and the common GAG-like components mostly being simple linear structures; 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 present application, the oligosaccharide compound represented by the formula (II) or (III) is obtained by analyzing the structure of the oligosaccharide separated and purified from the depolymerization product. The oligosaccharide compound shown in the formula (II) or the formula (III) contains a special disaccharide side chain of 'D-sulfated galactose (GalS) -alpha 1, 2-L-FucS-alpha 1-'. FG heptasaccharide compound with a special structure in which D-GalS-alpha 1, 2-L-FucS-alpha 1-side chain disaccharide exists at the non-reducing end has strong inhibition activity on iXase and has remarkable antithrombotic activity with low bleeding tendency, which is glycosaminoglycan-derived oligosaccharide compound with the smallest polymerization degree capable of strongly inhibiting iXase so far. Heptasaccharide is used as oligosaccharide with smaller structure and strong inhibition of iXase activity, and the antithrombotic activity characteristic of low bleeding tendency of heptasaccharide ensures that heptasaccharide has important application value for preventing and/or treating thrombotic diseases.

In addition, for thelenota ananas FG oligosaccharide with a degree of polymerization greater than or equal to 8, the presence of the disaccharide side chain has no significant effect on its iXase inhibiting and anticoagulant antithrombotic activities. For octasaccharide and FG oligosaccharide compounds containing the disaccharide side chain described herein (D-GalS- α 1,2-L- α -FucS- α 1-) there is no significant difference in iXase inhibitory activity between the compounds with or without the presence of D- α -GalS in the disaccharide side chain. In the oligosaccharide compound represented by the formula (II) or the formula (III), the D-alpha-GalS glycosyl group connected to the non-reducing end has special contribution and effect on the iXase inhibition activity of the compound.

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.

Example 1

This example provides an oligosaccharide compound prepared by the following method:

material

The reagents used, such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride and ethanol, are all commercially available analytical reagents.

Sephadex G25，medium(50-100μm)，GE Healthcare；Bio-Gel P10,medium(90-180μm)，Bio-Rad；Dionex IonPac^TMAS11-HC Semi-Prep Column (9 mm. times.250 mm), Thermo Scientific; HPLC chromatograph, Agilent 1200/1260 series chromatograph.

Preparation method

(1) Quaternary ammonium salt conversion of TaFG: 25g benzethonium chloride was dissolved in 400mL H₂O and slowly added dropwise to 10g of TaFG (dissolved in 150mL of H)₂O), a white precipitate was formed, left to stand overnight, centrifuged at 4000 rpm. times.15 min, and the precipitate was washed with 300mL of H₂O was washed three times repeatedly to remove excess benzethonium chloride, and the resulting precipitate was dried to constant weight at 40 ℃ with phosphorus pentoxide as a desiccant to give 25.306g of FG-quaternary ammonium salt.

(2) Carboxyl esterification of TaFG: dissolving TaFG quaternary ammonium salt obtained in the step (1) in 127mL of DMF, adding 12mL of benzyl chloride, performing benzyl esterification at 35 ℃ for 24h, and cooling the system to 25 ℃ after the reaction is finished.

(3) Beta-elimination depolymerization in the presence of a reducing agent: adding 46.3mL of the 0.08M EtONa/EtOH solution (containing 0.4M NaBH) prepared in the prior art into the reaction solution in the step (2)₄) To a final concentration of 0.02M EtONa/EtOH solution (containing 0.1M NaBH)₄) The reaction was stirred for 30 min.

(4) Sodium salt conversion and carboxylic ester hydrolysis of the depolymerization product: adding 215mL of saturated NaCl solution into the reaction solution obtained in the step (3) for exchange, adding absolute ethyl alcohol until the absolute ethyl alcohol is precipitated by 80 percent, centrifuging at 4000rpm multiplied by 10min to obtain a precipitate, repeating the process for three times, and using 520mL of H to obtain the precipitate₂O dissolved and 4.37mL of fresh 6M NaOH solution (containing 12M NaBH) was added₄) To a final concentration of 0.05M (containing 0.1M NaBH)₄) And stirring and reacting for 30min at 25 ℃, after the reaction is finished, neutralizing the reaction system by using 6M HCl, desalting and freeze-drying the neutralized solution by Sephadex G-25 to obtain 6.8G of depolymerized product dTaFG, wherein the yield is 68%.

(5) Separation and purification of heptasaccharide: 6.8g of dTaFG was dissolved in a predetermined amount of 0.2M NaCl, and the solution was applied to a Bio-Gel P10 Gel column in portions, eluted with 0.2M NaCl at a flow rate of 10mL/h, and the eluted fractions were collected in 2.5 mL/tube. Detecting the eluate by ultraviolet spectrophotometry, drawing elution curve, mixing the eluates with the same components, purifying the unpurified sample with Bio-Gel P-10 Gel column, comparing with retention time of hexasaccharide and octasaccharide, performing HPGPC analysis (Superdex Peptide 10/300 GL column, isocratic elution with 0.4M NaCl solution, and detection with refractometric detector and ultraviolet detector), and further detecting the heptasaccharide component with Dionex IonPac^TMPurifying AS11-HC semi-preparative column with elution condition of eluent H within 60min₂The volume ratio of O (pH 3.5) -2M NaCl (pH 3.5) was increased from 25% to 85% in a gradient, and the resulting oligosaccharide was desalted by Sephadex G25 gel column and lyophilized.

(6) And (3) spectrum analysis:¹H/¹³c-and 2D-NMR detection adopts a Bruker DRX 800MHz nuclear magnetic resonance instrument, the spectrum width is 16025.6Hz, the acquisition time is 2.0447s, the pulse width is 9.5s, the relaxation time is 1s, and scanning is carried out for 32 times. The sample concentration is 10g/L, and the sample is dried repeatedly with heavy water for three times before detection; ESI-Q-TOF MS was analyzed using a microOTOF-QII ESI-MS (Bruker, Germany) mass spectrometer. The mass spectrum conditions are that the capillary voltage is 2500V, the atomizer voltage is 0.6bar, the drying air flow rate is 4.0L/min, the drying air temperature is +180 ℃, and the m/z scanning range is 50-3000. Data were analyzed using Bruker Compass Data-analysis4.0(Bruker-Daltonics, Germany) software.

Results

(1) The procedure described gave 150 mg of compound.

(2) Structural analysis of compound 1:¹the H NMR spectrum and the attribution are shown in figure 1;¹³the C NMR spectrum and the attribution are shown in figure 2;¹H-¹³the C HSQC spectrogram and attribution are shown in figure 3; the Q-TOF MS spectrogram and attribution are shown in figure 4;¹H/¹³the C NMR signals are assigned in Table 1.

(3) According to¹H-/¹³C-and 2D-NMR and Q-TOF MS analysis, the chemical structure of the compound 1 is as follows:

D-Gal_4S6S-(α1,2)-L-Fuc_3S-(α1,3)-L-Δ^4,5GlcUA-(α1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]-D-GlcUA-(β1,3)-D-GalNAc_4S6S-ol having the formula:

TABLE 1 preparation of Compound 1¹H/¹³C NMR Signal assignment and coupling constants (ppm, Hz)

Note: glycosyl A is D-alpha-4, 6-disulfated galactosyl (G); glycosyl B is L-alpha-3-fucosyl sulfate (dF); glycosyl C is L-alpha-4-deoxy-threo-hex-4-enal (dU); glycosyl D is D-alpha-2-deoxy-2-acetamido-4, 6-disulfated galactosyl (A); glycosyl E is D-beta-glucuronyl (U); glycosyl F is L-alpha-2, 4-disulfated fucosyl (F); glycosyl G is D-alpha-2-deoxy-2-acetamido-4, 6-dithio-galactitol (rA). () The labels are as in Table 1 and FIGS. 1-3.

Example 2

material

TaFG, a natural FG (sodium salt) from Thelenota ananas, is as in example 1.

Reagents used for hydrazine hydrate, hydrazine sulfate, sodium chloride, sodium nitrite, concentrated sulfuric acid, ethanol and the like are all commercial analytical pure reagents.

The materials and apparatus required for oligosaccharide isolation were the same as in example 1.

Method

(1) Preparation of partially deacetylated TaFG: 10g TaFG was placed in a 500mL round bottom reaction flask, 2.5g hydrazine sulfate was added, followed by 250mL hydrazine hydrate, N₂Protecting, heating and stirring at 90 ℃ to react for 24 h. Adding absolute ethyl alcohol into the reaction solution after the reaction is finished until the final concentration of the system ethyl alcohol is 80% (v/v), precipitating, and separatingThe heart is deprived of the supernatant. The resulting precipitate was taken up in 250mL of H₂After dissolving, 1000mL of absolute ethanol (the pure concentration of the obtained solution is 80%, v/v) is added, and the alcohol precipitation is repeated for 4 times. Adding water into the precipitate for redissolving, dialyzing by a dialysis bag (product of United states Union carbonization) with the molecular weight cutoff of 3500Da, and freeze-drying the retentate to obtain about 8g of an intermediate product sample of deacetylated cellulose, wherein the yield is about 80%.

(2) Deaminizing and depolymerizing: dissolving the TaFG partially deacetylated intermediate obtained in step (1) in 160mLH₂And adding 320mL of 5.5M nitrous acid solution with the pH value of 4 into O at room temperature, stirring for reacting for 20min, and adding 1M NaOH to adjust the pH value of the solution to about 9 to terminate the reaction. Dialyzing the obtained reaction solution by using a dialysis bag with the molecular weight cutoff of 1000Da, collecting the cutoff solution, and freeze-drying to obtain about 6g of depolymerization product with the yield of about 75%.

(3) Separation and purification of heptasaccharide and spectral analysis: the same as in example 1.

Results

(1) Compound 230 mg was obtained as described.

(2) The chemical structure of the compound 2 is determined by NMR spectrum and Q-TOF MS analysis as follows: D-Gal_4S6S-(α1,2)-L-Fuc_3S-(α1,3)-D-GlcUA-(β1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]The spectrum of-D-GlcUA- (beta 1,3) -D-anTal, 2 is similar to that of compound 1, and the new signal peak appeared at about 5.0-5.1 ppm comes from the end group and 4-position hydrogen of characteristic reducing end 2, 5-anhydrotalose (an-Tal). The structural formula is as follows:

wherein the sugar group C is D-. beta. -glucuronic acid group, the sugar group G is4, 6-disulfuric acid-2, 5-anhydrotalose, and the sugar group A, B, D, E, F is as defined in example 1.

Example 3

3.2 methods

230 mg of the compound obtained in example 2 was dissolved in 1mL of 0.2mM phosphateTo a buffer solution (pH 8), 25mg of tyramine and 10mg of sodium cyanoborohydride in excess were added with stirring, and the mixture was reacted in a constant-temperature water bath at 35 ℃ for 72 hours. After the reaction is finished, adding 5mL of absolute ethyl alcohol, and centrifuging to obtain a precipitate; the precipitate is repeatedly washed twice with absolute ethyl alcohol and redissolved in H₂In O, insoluble matter was removed by centrifugation, and the supernatant was freeze-dried to obtain 20mg of the terminal reductively aminated derivative.

3.3 results

The product yield is about 66 percent based on the charge; the structure of the product is judged by an NMR spectrum, and the reduction end of the compound 2 is aminated by the reduction casein according to the integral ratio of the aromatic proton hydrogen signal (7.0 ppm) to the L-Fuc end group hydrogen (5.3 ppm).

Example 4

4.1 materials

Compound 2, prepared as in example 2. 1 phenyl-3-methyl-5 pyrazolone (PMP), biochemical reagent, purity 99%.

4.2 methods

30mg of the oligosaccharide obtained in example 2 was dissolved in H₂Adding 1.5mL of 0.5M PMP methanol solution and 1mL of 0.6M NaOH solution into O (50mg/mL), stirring at 50 ℃ for reaction for 90min, adjusting the pH to be neutral after the reaction is finished, desalting the reaction solution by a Bio-Gel P2 Gel chromatographic column, combining oligosaccharide parts, and freeze-drying to obtain a derivative product of 25 mg.

4.3 results

The structure of the derivative is judged by an NMR spectrum, and the reductive alkylation of the compound 2 is determined according to the integral ratio of an aromatic proton hydrogen signal (-7.0 ppm) to an L-Fuc terminal hydrogen signal (-5.3 ppm) in PMP.

Example 5

Preparation of aqueous injection solution of oligosaccharide compound

Material

Oligosaccharide Compound 1 prepared by the method of example 1

Sterile water for injection; 2mL of a medium borosilicate glass tube injection bottle; millipore Pellicon 2 ultrafiltration system (Merk Millipore)

The ingredients of the prescription are shown in Table 2.

TABLE 2 formulation ratio of oligosaccharide compound for injection aqueous solution

Name of raw and auxiliary materials	Dosage of
		1	100g
Water for injection	1000mL
		Are co-produced into	1000 pieces

Preparation process

Weighing the oligosaccharide compound 1 with the prescription amount, adding water for injection to the full amount, stirring and dissolving completely, and removing pyrogen by using an ultrafiltration membrane package with the molecular weight cutoff of 10kDa by using a Millipore ultrafiltration device. And (3) filtering and sterilizing the solution by using a 0.22-micron membrane in a sterile environment, filling the solution into penicillin bottles with the capacity of 2mL, wherein each penicillin bottle is 1mL, monitoring the filling amount in the filling process, inspecting the product to be qualified, and packaging the product to obtain a finished product.

Example 6

Preparation of freeze-dried powder injection of oligosaccharide compound

Material

Oligosaccharide Compound 1 prepared by the method of example 1

Sterile water for injection; 2mL of a medium borosilicate glass tube injection bottle; millipore Pellicon 2 ultrafiltration system (Merk Millipore), VirTis Ultra 35EL freeze dryer.

The ingredients of the prescription are shown in Table 3.

TABLE 3 lyophilized powder for injection compounding ratio of oligosaccharide compound

Name of raw and auxiliary materials	Dosage of
		1	100g
Water for injection	500mL
		Are co-produced into	1000 pieces

Preparation process

Weighing the oligosaccharide compound 1 with the prescription amount, adding water for injection to the full amount, stirring and dissolving completely, and removing pyrogen by using an ultrafiltration membrane package with the molecular weight cutoff of 10kDa by using a Millipore ultrafiltration device. And (2) in an aseptic environment, filtering and sterilizing by using a 0.22-micron membrane, filling the obtained solution into penicillin bottles with the capacity of 2mL, wherein each penicillin bottle is 0.5mL, monitoring the filling amount in the filling process, performing half tamponade, performing freeze drying according to the set freeze drying process in a drying box of a pilot scale freeze dryer (VirTis USA), performing tamponade, discharging, capping and inspecting.

And (3) freeze-drying: pre-cooling: putting the sample into a box, cooling the partition plate to-25 ℃, keeping for 1h, cooling to-45 ℃, and keeping for 3 h; the cold trap is cooled to-50 ℃, and vacuum pumping is started to 40 Pa. Sublimation: heating to-30 deg.C at constant speed for 1h, and maintaining for 2 h; raising the temperature to-20 ℃ at a constant speed for 2h, keeping the temperature for 6h, and keeping the pressure in vacuum at 40-30 Pa. And (3) drying: heating to-5 ℃ for 2h, keeping for 2h, and keeping vacuum at 30-20 Pa; heating to 10 ℃ within 0.5h, keeping for 3h, and keeping vacuum at 30-20 Pa; heating to 40 deg.C for 0.5h, maintaining for 4h, and vacuum pumping to minimum.

Test example 1

This test example mainly includes the anticoagulant and blood coagulation factor inhibitory activity analysis of the oligosaccharide compound (compound 1) provided in example 1.

Material

Sample preparation: heptasaccharide compound 1

Comparison products: enoxaparin sodium injection (LMWH, Mw 3500-5500 Da, Xenofiat-Anvant product);

reagent: human blood coagulation quality control plasma, Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) determination kits which are all products of the Germany TECO GmbH company; factor VIII detection kit, AT-dependent anti-factor IIa detection kit, AT-dependent anti-factor Xa detection kit, thrombin (factor IIa), thrombin substrate CS-01(38) are all HYPHEN BioMed (France) products; factor VIII (FVIII), product of Bayer Healthcare LLC (Germany).

The instrument comprises the following steps: MC-4000 coagulometer, product of the company TECO GmbH, Germany; microplate reader ELx 808 Microplate reader, product of BioTek corporation, usa; vortex oscillator, product of SCIENTIFIC INDUSTRIES, usa; XS105 electronic balance, FE20 pH meter, METTLER TOLEDO, USA; HH-4 constant temperature water bath, consolidation of the product in market; VOR76X-6 vortex oscillator, linbel product, hainan; Spectrafuge-24D907386 centrifuge, Labnet product.

Method

(1) Preparing a sample solution: heptasaccharide compound 1 and control were dissolved in Tris-HCl buffer and diluted to the desired serial solubility.

(2) And (3) anticoagulant activity detection: after 5. mu.L of sample or control solution was added to 45. mu.L of human plasma for quality control and mixed uniformly, the clotting times (APTT, PT and TT) were measured with MC-4000 hemagglutination apparatus according to the method described in the kit instructions for APTT, PT and TT.

(3) Coagulation factor inhibitory activity assay:

analysis of Xase inhibitory Activity: the detection is carried out by combining factor VIII and a factor VIII detection kit according to kit instructions and literature methods. Specifically, 30. mu.L of heptasaccharide 1 or Tris-HCl buffer (negative buffer) was added to a 96-well plate at a series of concentrationsSexual control) 30. mu. L R2(Activation Reagent,60nM FIXa, containing FIIa, PC/PS, Ca were added² ⁺) 30 microliter FVIII solution (2IU/mL) is placed in an enzyme-linked immunosorbent assay device, the plates are uniformly mixed and incubated for 2min at 37 ℃; taking out 96-well plate, adding 30 μ L R1(50nM FX containing direct thrombin inhibitor), placing in enzyme labeling instrument, vibrating plate, mixing, and incubating at 37 deg.C for 1 min; the 96-well plate was removed, 30. mu. L R3(FXa chromogenic substrate Sxa-11, about 8.4mM) was added thereto, the resulting mixture was placed in a microplate reader, and the absorbance (OD) at 405nm was measured_405nm) The detection was continued for 5min at intervals of 30 s. According to OD₄₀₅Calculation of Xase Activity and IC for Compound 1 to inhibit Xase₅₀The value is obtained.

AT-dependent Xa inhibitory activity assay: and detecting by adopting a heparin Anti-FXa kit. Specifically, after 30 μ L of heptasaccharide compound 1 or Tris-HCl buffer solution (negative control) with serial concentrations is respectively added into a 96-well plate, 30 μ L R1(1IU/mL AT) is added, the mixture is placed in a microplate reader, the plate is shaken and mixed uniformly, and the mixture is incubated for 1min AT 37 ℃; adding 30 mu L R2(8 mu g/mL F Xa solution), placing in a microplate reader, vibrating the plates, mixing uniformly, and accurately incubating at 37 ℃ for 1 min; finally, 30. mu.L of preheated R3(1.25mM FXa specific chromogenic substrate Sxa-11) was added, and the absorbance (OD) at 405nm was measured with a microplate reader_405nm) Continuously detecting for 5min at intervals of 30 s; and calculating IC for inhibition of Xa by Compound 1₅₀The value is obtained.

AT-dependent IIa inhibition activity assay: and detecting by adopting a heparin Anti-FIIa kit. Adding 30 μ L heptasaccharide compound 1 or Tris-HCl buffer solution (negative control) with serial concentration into each well of 96-well plate, adding 30 μ L R1(0.25IU/mL AT), vibrating, mixing, and incubating AT 37 deg.C for 1 min; taking out the 96-well plate, adding 30 mu L R2(24IU/mL F IIa solution), vibrating the plate, mixing uniformly, and incubating accurately at 37 ℃ for 1 min; finally, 30. mu.L of preheated R3(1.25mM FIIa-specific chromogenic substrate CS-01(38)) was added, and the absorbance (OD) at 405nm was measured with a microplate reader_405nm) Continuously detecting for 5min at intervals of 30s, and calculating IC of IIa inhibition by Compound 1₅₀The value is obtained.

Data processing: OD of taking multiple hole detection₄₀₅The mean value was used as the measurement value of the test article and the control article at each concentration, and the slope of the linear fit of the measurement value to the time value (the change rate OD of the absorbance value)₄₀₅Min) representsThe enzymatic activity of coagulation factors; calculating the coagulation factor activity (percentage) in the presence of the test sample with the coagulation factor activity of the negative control well as 100%; the clotting factor activity in the presence of the test sample was plotted against the test sample concentration and fitted as follows to calculate the IC₅₀The value: b ═ IC (IC)₅₀)ⁿ/{(IC₅₀)ⁿ+[I]ⁿWherein B is the activity (percentage) of the blood coagulation factor in the presence of a test sample, [ I ]]Is the concentration of the test sample, IC₅₀Is the median inhibitory concentration (the concentration of test sample required to inhibit 50% of activity) and n is the Hill coefficient.

Figure 5 shows the doubling of APTT prolongation activity results. FIG. 6 shows the inhibitory activity of the compound prepared in example 1 of the present application against endogenous factor Xase.

Results

The research on anticoagulant activity shows that the heptasaccharide compound 1 obtained in the example 1 of the application has remarkable APTT prolonging activity, the concentration required by APTT multiplication is 90.45 mu g/mL, PT and TT are not influenced, and the heptasaccharide compound can have remarkable anticoagulant activity for inhibiting an intrinsic coagulation pathway and has no remarkable influence on extrinsic coagulation.

The research on the inhibitory activity of the blood coagulation factor shows that the compound 1 has obvious inhibitory activity (IC) on the factor Xase₅₀397.9 ± 57.5 nM); in the presence of Antithrombin (AT), no significant effect was observed on coagulation factors IIa, Xa.

Using the same assay as described above, the compound prepared in example 3 also showed potent inhibitory activity against factor Xase, the IC thereof₅₀The value was 420.5. + -. 60.3 nM; in the presence of antithrombin (AT-III), no significant effect was observed on coagulation factors IIa, Xa.

Test example 2

This test example was conducted mainly to test the antithrombotic activity and hemorrhagic effect of the oligosaccharide compound (compound 1) provided in example 1.

Material

Sample preparation: heptasaccharide compound 1

reagent: chloral hydrate (chloral hydrate), national pharmaceutical group chemical agents ltd; physiological saline, south Kunming pharmaceutical Co.

Experimental animals: SD rat, 250-350 g weight, male, provided by Schlekschada laboratory animals Co., Ltd, Hunan, license number SCXK (Xiang) 2016-; kunming mouse, 18-22 g weight, male, provided by Schleickzeda laboratory animals Co., Ltd, Hunan, license number SCXK (Xiang) 2016-; new Zealand rabbits are supplied by Kunming medical university, SCXK (Dian) 2011-.

Method

Anti-venous thrombosis formation experiment

Grouping and administration: the rats were randomly divided into 5 groups of 5 rats each. Experimental groups and the dose administered to each group of animals were: (1) saline (NS) control group; (2) LMWH 3.6mg/kg group; (3) heptasaccharide compounds 14.8 mg/kg group; (4) heptasaccharide compounds 18.0 mg/kg group; (5) heptasaccharide compounds 113.6 mg/kg group. The rats in each group were administered by dorsal subcutaneous injection (sc.) in a volume of 1 mL/kg. The molding experiment was performed 1h after the administration.

Preparing a rabbit brain powder leaching solution: after the New Zealand rabbits are sacrificed, the rabbit brains are immediately taken out, and rabbit brain powder leachate is prepared according to the literature method (Thromb Haemost,2010,103(5): 994-.

The rabbit brain powder extract induces the formation of the inferior vena cava thrombus: rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300mg/kg), the abdominal wall was cut longitudinally along the midline of the abdomen, the viscera was removed, the inferior vena cava and its branches were separated, the inferior vena cava of the left renal vein of the inferior vena cava was threaded through a ligature at the inferior border of the left renal vein, and the inferior vena cava branches below the left renal vein were ligated. The femoral vein is injected with 2% rabbit brain powder leachate (1mL/kg), and after 20 seconds, the lower margin ligature of the left renal vein is ligated. After operation, the viscera was returned to the abdominal cavity, covered with medical gauze (infiltrated with normal saline), 20min later, the blood vessel was closed with hemostatic forceps 2cm below the ligature, the blood vessel was dissected longitudinally, the thrombus was taken out, the length of the thrombus was measured and the wet weight of the thrombus was weighed, and the dry weight was weighed after drying at 50 ℃ for 24 h.

Data processing and statistics: the data were collated and analyzed using SPSS software, and the data were expressed as means. + -. standard deviation (x. + -.s). And (3) performing One-Sample K-S Test on the data normality tests of different groups, performing Leven Test on the homogeneity of variance Test, judging the significance of the data by using One-Way ANOVA if the data conforms to normal distribution and the variance is uniform, and judging the significance of the data by using Two-Independent-Samples Test if the data does not conform to normal distribution and the variance is uniform.

Bleeding tendency detection

Grouping and administration: the mice were randomly divided into 3 groups of 4 mice each. Experimental groups and the dose administered to each group of animals were: (1) saline (NS) control group; (2) LMWH 36mg/kg group; (3) heptasaccharide compounds 180 mg/kg group. Each group of mice was administered via dorsal subcutaneous injection (sc.) in a volume of 10 mL/kg.

The test method comprises the following steps: placing the mice in a mouse fixer after subcutaneous administration for 60min, cutting tail tip by 5mm, soaking the rat tail in a beaker filled with 40mL of purified water (37 deg.C), timing from the 1 st drop of blood flowing out from the cut rat tail, stirring, standing for 60min, and detecting solution absorbance (OD) with ultraviolet spectrophotometer₅₄₀)。

Adding mouse whole blood of different volumes into 40mL of purified water, stirring, standing for 60min, and detecting absorbance (OD) of the solution by the same method₅₄₀) And a volume-absorbance curve was plotted as a standard curve for calculating the amount of bleeding. The amount of bleeding was calculated from the standard curve for each experimental group of mice.

Results

(1) Antithrombotic activity: as shown in figure 7, the results show that the compound 1 under the experimental dosage has obvious antithrombotic activity, and the thrombosis inhibition rate can reach more than 80% under 4.8-13.6 mg/kg.

(2) Bleeding effects: as shown in figure 8, the amount of oligosaccharide compound 1 that bleeds was significantly lower than the LMWH group given an equal fold high dose of the equivalent antithrombotic agent amount.

In summary, it can be seen that: the compound prepared by the application has strong inhibition activity on iXase, and has remarkable antithrombotic activity with low bleeding tendency.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

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-alpha-4, 6-disulfated galactosyl;

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

glycosyl C is D-beta-glucuronyl or L-alpha-4-deoxy-threo-hex-4-enouronyl;

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

glycosyl E is D-beta-glucuronyl;

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

wherein, when C is L-alpha-4-deoxy-threo-hex-4-enouronyl, R is₁Is composed of

R₂is-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-beta-glucuronyl, R₁Is composed of

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-alpha-4, 6-disulfated galactosyl;

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

glycosyl C is L-alpha-4-deoxy-threo-hex-4-enal;

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

glycosyl E is D-beta-glucuronyl;

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

glycosyl G is D-alpha-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 is₂when-CH ═ O is used, the terminalThe aldehyde group forms a hemiacetal six-membered sugar ring structure with the hydroxyl group at position 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-alpha-4, 6-disulfated galactosyl;

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

glycosyl C is D-beta-glucuronyl;

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

glycosyl E is D-beta-glucuronyl;

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

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

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.

5. Oligosaccharide compound according to claim 2 and pharmaceutically acceptable salts thereof, characterized in thatIn the structural formula shown in the formula (II): r₂is-CH ═ O or-CH (OH)₂。

6. The oligosaccharide compound as claimed in claim 2, wherein the structural formula of the formula (II): r₂is-CH₂OH。

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

8. The oligosaccharide compound as claimed in claim 3, wherein the structural formula of the formula (III) is: r₂is-CH ═ O or-CH (OH)₂。

9. The oligosaccharide compound as claimed in claim 3, wherein the structural formula of the formula (III) is: r₂is-CH₂OH。

10. 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 beta-elimination reaction on benzyl esterified carboxylate to break beta 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-alpha-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;

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

11. The process for producing an oligosaccharide compound according to claim 10,

the quaternary ammonium salt of the quaternary ammonium salinized fucosylated glycosaminoglycan is N, N-dimethyl-N- [2- [2- [4(1,1,3, 3-tetramethylbutyl) phenoxy ] ethoxy ] ethyl ] benzyl ammonium salt, and the carboxylic ester is benzyl ester.

12. 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 the beta 1,4 glycosidic bond of D-GlcUA to obtain a deamidated depolymerization product with a 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.

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

14. Use of an oligosaccharide compound as claimed in any of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prevention of a thrombotic disorder.

15. The use according to claim 14, wherein the thrombotic disease is venous thrombosis, arterial thrombosis, or ischemic cardiovascular and cerebrovascular disease.

16. A medicament for inhibiting the activity of an endogenous factor X enzyme, comprising an excipient and the oligosaccharide compound of any of claims 1-9, or a pharmaceutically acceptable salt thereof.

17. The agent for inhibiting the activity of an endogenous factor X enzyme according to claim 16, wherein the agent is administered parenterally.