CN104370980B - A kind of oligosaccharide compound and its pharmaceutical composition for suppressing endogenous factors X enzymatic activitys - Google Patents

Abstract

The present invention discloses a kind of pure oligosaccharide compound of chemical constitution for suppressing intrinsic coagulation factor X enzymes, its preparation method, the pharmaceutical composition containing the oligosaccharide compound and its application in preventing and/or treating thrombotic diseases.The oligosaccharide compound is by L 2,4 di-sulfate base fucoses, D glucuronic acids, the acetylamino 4 of 2 deoxidations of D 2,6 di-sulfate base galactolipins, 2,5 dehydration taloses or its sugar alcohol, the oligosaccharide with structure shown in formula (I) of osamine composition, wherein in formula (I), n is 0,1,2,3,4,5 or 6；R is optionally CH=O, CH (OH)₂、‑CH₂OH、‑CH₂NH₂、‑CH₂NHR ' or CH₂N(R’)₂, wherein R ' is substituted or unsubstituted C1 C6 straight or branched alkyls, substituted or unsubstituted C7 C12 aryl.

Description

A kind of oligosaccharide compound and its medicine group for suppressing endogenous factors X enzymatic activitys Compound

Technical field

The invention belongs to pharmaceutical technology field, and in particular to one kind is by L-2,4- di-sulfate bases fucose, D-Glucose Aldehydic acid, D-2- deoxidation -2- acetylaminohydroxyphenylarsonic acid 4,6- di-sulfates base-galactolipin, 2,5- dehydration taloses or its sugar alcohol, osamine group Into the pure oligosaccharide compound of chemical constitution, the preparation method of the oligosaccharide compound, containing the oligosaccharide compound Pharmaceutical composition and thrombin inhibitor that they are relied on as endogenous factors X enzyme inhibitors and/or heparin DPN I Application in preventing and/or treating thrombotic diseases.

Background technology

Cardiovascular and cerebrovascular disease seriously endangers the health of the mankind, and pathologic thrombus formation is the Etiological of cardiovascular and cerebrovascular disease One of.Antithrombotic reagent including anticoagulant is widely used in the clinical treatment of angiocardiopathy.Classical anticoagulant Thing, such as Coumarins and heparin class medicine, there is the defects of hemorrhagic tendency and pharmacokinetics aspect.In the late two decades, select The fibrin ferment of selecting property is applied to clinic successively with Xa inhibitor, but is not yet made a breakthrough in terms of hemorrhagic tendency is reduced. At present, selective intrinsic coagulation pathway inhibitor research turn into lower bleeding tendency anticoagulant research Main way it One, Hageman factor a, XIa and IXa inhibitor is preclinical and clinical experimental study deploys successively.Endogenous Xase is endogenous Last position and speed limit enzyme activity site on coagulation pathway, its selective depressant have important potential clinical value.

Glycosaminoglycan (the Fucosylated of the fucose substitution of existing studies have shown that natural origin Glycosaminoglycan, FG) there is potent anticoagulating active, and its anticoagulant mechanism is related to endogenous Xase activity suppressions (Thromb.Haemost.,2008,100:420–428；J.Biol.Chem.,1996,271:23973-23984).However, day The prototype FG in right source is also present extensively and the pharmacological action of contradiction, including induced platelet aggregation, produces hemorrhagic tendency and swash XII living etc. (Thromb.Haemost., 1988,59:432-434；Thromb.Haemost.,1997,65:369–373； Thromb.Haemost.,2010,103:994-1004).The FG of appropriate depolymerization can reduce platelet activation activity (Thromb.Haemost.,1991,65:369-373).The Chinese B of granted patent CN 101724086 and CN 101735336 B The method for preparing Depolymerized fucose-containing glycosaminoglycan is disclosed, it uses peroxidative depolymerization method to prepare FG depolymerization products, and the hemorrhagic tendency of products therefrom shows Writing reduces.In addition, patent of invention discloses the deacylation deaminizating depolymerization method that the A of CN 103214591 report FG, this method uses It is partially deacetylated that N- acetyl group -2- amino contained by FG -2-DGal base (D-GalNAc) occurs for hydrazinolysis method, after And make FG that deaminizating depolymerization occur by nitrous acid treatment, be derived from containing end 2,5- dehydrating tower sieve glycosyls (anTal) or The FG depolymerization products of its reductive derivative；, should and patent of invention discloses β-elimination depolymerization that CN103145868 A report FG D-Glucose aldehydic acid (D-GlcUA) carboxyl of method first contained by partial esterification FG, then handled and obtained by anhydrous alkali solvent It is undersaturated Δ to obtain non reducing end^4,5The FG depolymerization products of-hexuronic acid base (Δ UA).

In the eighties in last century so far 30 years, the either prototype FG of natural origin, or pass through different depolymerization skills The depolymerization product that art obtains, it is the mixture for the series compound that chemical constitution has differences, rather than is tied with identical chemistry The neat compounds of structure.Although these mixtures can be used for the treatment and/or prevention of thrombotic diseases, but be limited to its mixture Property, its quality controllability is relatively limited, pharmacological effect poor activity caused by the chemical constitution difference of different component contained by mixture Misgivings in terms of different and side effect and security are difficult to avoid that, for example, a certain proportion of big point contained in these mixtures Son amount component there may be XII Activation Activities and platelet activation activity.

Because FG chemical constitutions are complicated, especially because FG is height sulfating product and the sulfuric acid of its contained construction unit Change form is complicated and changeable, and preparing and purifying the pure FG classes compound of chemical constitution has very high technical difficulty, therefore, so far Untill, it there is no the research report that the pure FG classes compound of chemical constitution is related.Just because of the pure change of no chemical constitution Compound reports that the precise structure of FG class compounds, which still leaves a question open, asks, for example, the existence form of side chain fucose (L-Fuc) (may be Monosaccharide side-chains or disaccharides side chain) and link position (may be D-GlcUA C3 positions, D-GalNAc C4 or C6 positions etc.)；With this Correlation, the basic chemical structure that FG classes compound suppresses needed for endogenous Xase do not obtain in-depth study yet and sufficiently recognized Know.

The present invention is it has surprisingly been found that be mainly α-L-2,4- di-sulfates base-fucose (L-2,4- using side chain Disulfate-fucosyl- α 1-, Fuc2S4S) FG be initiation material, using selective splitting D-GalNAc- β 1-4-D- Raw material FG described in the deacylation deaminizating depolymerization depolymerization of GlcUA glycosidic bonds, it is then comprehensive using exclusion chromatography, ion exchange color Spectrometry and/or ultrafiltration purifying products therefrom, the present invention obtain a series of FG class chemical combination that chemical constitutions are single, pure first Thing.

Due to the preparation and discovery of the pure FG class compounds of sequence of chemical structure, the present invention can not only be to gained FG classes The precise structure of compound is accurately parsed, and can suppress endogenous factors X enzymes (Intrinsic to FG classes compound Xase, Tenase) the required basic chemical structure of activity and FG classes compound it is related to platelet activation and factor XI, plasma thromboplastin antecedent I activation Chemical constitution be compared analysis.

On this basis, the present invention is surprised finds that the FG class compounds containing two to seven " trisaccharide construction unit " have The antithrombin activity that potent Xase and/or heparin DPN I (HCII) is relied on, swashs without XII Activation Activities and blood platelet It is activity.Thus, these compounds are respectively provided with significant anti-freezing antithrombotic acitivity, and in the absence of small with XII Activation Activities and blood The related safety concerns of plate activation.

Obviously, compared with the mixture of known Depolymerized fucose-containing glycosaminoglycan class compound, the pure compound of chemical constitution has more The potential using value of the related cardiovascular and cerebrovascular disease of important treatment thrombosis.The reason is that because active component is The pure compound of chemical constitution, active component and pharmaceutical composition quality control level, the predictability of pharmacotoxicological effect show Write and improve.Due to being inevitably present the component of a certain proportion of higher molecular weight in Depolymerized fucose-containing glycosaminoglycan class mixture, therefore still It there may be the XII doubts related to platelet activation；And for neat compounds component, the doubt can obtain effectively Eliminate.

The content of the invention

Taken off it is an object of the invention to provide one kind by L-2,4- di-sulfate bases fucose, D-Glucose aldehydic acid, D-2- Oxygen -2- acetylaminohydroxyphenylarsonic acid 4,6- di-sulfates base-galactolipin, 2,5- dehydration taloses or its sugar alcohol, the chemical constitution of osamine composition Pure oligosaccharide compound, the preparation method of the oligosaccharide compound, the pharmaceutical composition containing the oligosaccharide compound And they are preventing and/or controlled as endogenous factors X enzyme inhibitors and/or heparin DPN the I thrombin inhibitor relied on Treat the application in thrombotic diseases.

In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme：

The present invention obtains the rock of the thrombin-inhibiting activity relied on endogenous Xase inhibitory activity and/or HCII first The sterling oligosaccharide compound of algae saccharification glycosaminoglycan, therefore, present invention firstly provides a kind of pure oligosaccharides of chemical constitution Class compound and its pharmaceutically acceptable salt, the oligosaccharide compound is by L-2,4- di-sulfate bases fucose, D- grapes Uronic acid, D-2- deoxidation -2- acetylaminohydroxyphenylarsonic acid 4,6- di-sulfates base-galactolipin, 2,5- dehydration taloses or its sugar alcohol, osamine Composition, shown in its chemical constitution such as formula (I)：

In formula (I),

N is 0,1,2,3,4,5 or 6；

R is optionally CH=O ,-CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR ' or-CHNH (R ')₂, wherein R ' is substitution Or unsubstituted C1-C6 straight or branched alkyls, substituted or unsubstituted C7-C12 aryl.

Due to there is no sterling fucosylated glycosaminoglycan compound to report so far, oligosaccharide compound of the present invention Prominent features be that it is " sterling compound chemically ".Usually, sterling compound of the present invention refers to, using analysis Type high productivity computing (HPGPC) is analyzed, such as the serial chromatograph of Agilent high performance liquid chromatography (HPLC) and gel chromatographic columnses, Detected, calculated according to area normalization method, the purity of its single compound is not less than using the Composition distribution (RI) of versatility 95%；And the chemical constitution of single compound can obtain¹H、 ¹³The confirmation of C and 2D NMR spectras detection.

Oligosaccharide compound of the present invention contains sulfate group, thus can with pharmaceutically acceptable cation into Salt.Usually, pharmaceutically acceptable salt of the present invention is alkali metal salt, alkali salt or organic ammonium salt.Preferably The pharmaceutically acceptable salt of oligosaccharide compound of the present invention is sodium salt, sylvite or calcium salt.The present invention also provides a kind of excellent The oligosaccharide compound and its pharmaceutically acceptable salt of choosing, it is chemical constitution such as formula (II) institute of the oligosaccharide compound Show：

In formula (II), n 0,1,2,3,4,5 or 6.

In chemical nature, formula (II) compound is unreduced end 2 in compound shown in formula (I), and 5- is dehydrated talose The hydrate of the end group aldehyde radical of base.

Preferred the compounds of this invention is n=2,3 or 4 formula (II) compound.

The present invention also provides another preferable oligosaccharide compound and its pharmaceutically acceptable salt, is the oligosaccharide kind Shown in the chemical constitution of compound such as formula (III)：

In formula (III), n 0,1,2,3,4,5 or 6.

In chemical nature, compound shown in formula (III) is the end 2 in compound shown in formula (I), 5- dehydrating tower sieve glycosyls Aldehyde radical reduzate.

Preferred the compounds of this invention also includes n=2,3 or 4 formula (III) compound.

Oligosaccharide compound and its pharmaceutically acceptable salt of the present invention have significant pharmacological activity, and its is main It is characterised by, the oligosaccharide compound has the blood coagulation that endogenous Xase (Intrinsic tenase) and/or HCII is relied on Enzyme (Thrombin, factor IIa) inhibitory activity.Usually, detected using vitro enzyme live system, compound of the present invention suppresses Xase IC₅₀Value can be between about 5~200ng/ml；In the presence of HCII, suppress IIa IC₅₀Value can be in about 200~2000ng/ Between ml.

There is potent suppression intrinsic coagulation to live for oligosaccharide compound and its pharmaceutically acceptable salt of the present invention Property, in terms of people's Quality Control plasma A PTT times of doubling, the μ of drug concentration about 2~30 of required oligosaccharide compound of the present invention g/ml.In terms of people's Quality Control blood plasma PT times of doubling, oligosaccharide compound of the present invention is right in about 2000 μ g/ml concentration ranges Extrinsic coagulation does not make significant difference.Present invention research be also found, caused coagulation process is activated for intrinsic coagulation system, this Invent the oligosaccharide compound and mainly realize its anti-freezing antithrombotic acitivity by suppressing endogenous Xase；And weaker HCII according to Bad thrombin activity is then advantageous to remove established thrombin activity in pathologic thrombus forming process.

The research of the present invention also confirms, in anti-freezing drug effect concentration range, oligosaccharide compound and its medicine of the present invention Acceptable salt not factor of influence XII activation on；In higher concentration range (about 2000 μ g/ml), it is to people Platelet aggregation also has no significant effect.

The present invention also provides the preparation method of oligosaccharide compound and its pharmaceutically acceptable salt of the present invention, institute Stating preparation method includes step shown in following route：

In the route, its step (1) is that the mixture 1 of fucosylated glycosaminoglycan class compound is dissolved in into anhydrous hydrazine Or in hydrazine hydrate solution, without or 0.5%~5% hydrazine sulfate in the presence of, react, be allowed at a temperature of room temperature or 60~120 DEG C The mixture 2 of partially deacetylated acquisition fucosylated glycosaminoglycan class compound occurs.

The mixture 1 of the fucosylated glycosaminoglycan class compound, typically using natural products, x is usually equal in formula The natural number of value about 40~100；R₁、R₂、R₃And R₄For H or-SO₃ ^-.The mixture 1 and trisaccharide construction unit in bracket In, preferably R₁=R₂=R₃=R₄=-SO₃ ^-Construction unit in entire infrastructure unit proportion be not less than about 60%.

In the mixture 2 of the fucosylated glycosaminoglycan class compound, R₁、R₂、R₃And R₄And x is the same as fucosylated sugar The definition of the mixture 1 of the poly- saccharide compound of amine, R₅For H or-COCH₃, and with molar percent, H is in whole R₅Institute in group The ratio accounted for is in the range of about 15%~60%.

Step (1) products therefrom fucosylated glycosaminoglycan mixture 2 (hereinafter referred to as product 2) can be by reaction solution The high concentration ethanol aqueous solution of middle addition ethanol in proper amount or salt (such as NaCl or sodium acetate) saturation makes product 2 be precipitated from reaction solution Out collect.

Because the desamination reaction of nitrous acid treatment shown in step (2) is quick and thorough, therefore, second is taken off shown in step (1) The degree of deacetylation of acyl group reaction products therefrom 2 is to determine being averaged for product 3 (mixture of serial depolymerization product compound) The key factor of the degree of polymerization.And the degree of deacetylation of product 2 can by the reaction condition such as reaction temperatures of rate-determining steps (1) and Reaction time and realize.Usually, it is to obtain oligosaccharide compound of the present invention, the average degree of deacetylation of product 2 should be In the range of about 15%~60%.The average degree of deacetylation of preferable product 2 is in the range of about 30%~50%.

In theory, the FG classes compound that GalNAc4S6S be present containing Fuc2S4S side chains and main chain can pass through the present invention The technology path prepares oligosaccharide compound of the present invention, still, due to the influence to subsequent purification process and to end Products collection efficiency and the influence for preparing cost, present invention starting raw material used is typically using Fuc2S4S in whole Fuc contained by FG Shared mol ratio is not less than about 60% FG class compounds.Thus, the natural FGization according to the present inventor to different sea cucumber sources The comparative studies of compound structure, the mixture 1 of currently preferred fucosylated glycosaminoglycan class compound is from sea cucumber Stichopus monotuberculatus, Stichopus variegates Semper (Stichopus variegatus (Sempen)), Apostichopus The fucosylated glycosaminoglycan extract of acquisition is extracted in japonicas (imitative stichopus japonicus) body walls and/or internal organ.

In the circuit, its step (2) is that fucosylated glycosaminoglycan mixture 2 is dissolved in into water, adds HNO₂Solution The pH value of reaction solution is adjusted to make mixture 2 that deaminizating depolymerization reaction occur to 1~4.5, obtain fucosylated glycosaminoglycan class The mixture 3 of compound.From the point of view of end-product separation and purification treatment, the fucosylated glycosaminoglycan class compound is mixed Compound 3, m are usually 0~20 natural number；R₁、R₂、R₃And R₄Mixture 1 with fucosylated glycosaminoglycan class compound is determined Justice, and the mixture 3 of fucosylated glycosaminoglycan class compound can exist with its hydrate forms.

Step (2) reaction is general quick and thorough, and therefore, the reaction typically can be low temperature (such as ice bath is 0 DEG C) Or carry out at room temperature, the reaction time is also can be controlled within 30min.The fucosylated glycosaminoglycan mixture 3 is (hereinafter referred to as Product 3) reaction solution to adjust pH be 7~8, use molecular cut off to be dialysed for 500~1000Da bag filters or ultrafiltration, also can be After neutralization reaction solution, by adding ethanol or the high concentration ethanol of salt (such as NaCl or sodium acetate) saturation into reaction solution The aqueous solution makes product 3 be precipitated out and collect from reaction solution；Suitable gel column can also be passed through after neutralization reaction solution After (such as Sephadex G25 gel columns) desalination, by be dried under reduced pressure or freeze-drying obtain products therefrom 3.Product 3 is in water General (its reducing end is that 2,5- is dehydrated talose glycol-based) in the form of hydrates is present in solution, is done using conventional drying method It is dry, such as freeze-drying dries, its products therefrom also exists in the form of hydrates.Dried using heating hypobaric drying method, after And it is that solvent dissolving detects its NMR spectra then its visible reducing end is 2,5- dehydrating towers in the form of aldose by deuterated dimethyl sulfoxide Sieve glycosyl compound.

In the circuit, its step (3) is will using gel chromatography chromatography and/or ion-exchange chromatography and/or ultrafiltration The mixture 3 of fucosylated glycosaminoglycan class compound is isolated and purified as oligosaccharide compound 4.Wherein, in oligosaccharide compound 4, n is 0~6 natural number.

As it was previously stated, the mixture 1 of starting fucosylated glycosaminoglycan class compound to purification step (3) processing procedure and Yield has a great influence.Using Stichopus monotuberculatus, Stichopus variegates Semper sources FG when being initial compounds, during by suitable gel chromatography, be typically readily available purifying oligosaccharide compound 4 and Can have higher efficiency of pcr product, contained a small amount of same polymeric degree and substituted vitriol ester group type difference compound can pass through Ion-exchange treatment removes.When using the FG in Apostichopus japonicas sources as initial compounds, it can pass through first Suitable gel chromatography obtains the depolymerization product of same polymeric degree, is then removed and substituted by suitable ion exchange chromatography The different types of impurity compound of sulfate group, comparatively, its ion exchange chromatography process are more complicated, and efficiency of pcr product Decrease.For the FG in the complicated and diversified other sea cucumber sources of side chain fucose sulfate type, because depolymerization is produced Thing it is complicated various, despite the presence of theoretic feasibility, the process for actually obtaining oligosaccharide compound of the present invention is general It is excessively complicated, therefore it is generally not used for preparing the oligosaccharide compound of the present invention.

Gel rubber material used in the gel chromatography of the step (3) selects according to the molecular weight ranges for the target product for intending purifying Take, generally comprise but be not limited to P6 gels (separating ranges 1000-6000Da), P10 gels (separating ranges 1500-20000Da), P2 gels (separating ranges 100-1800Da), Sephadex G50 gels (separating ranges 1500-30000Da) and meet molecule Measure the gel rubber material of separating ranges requirement or into capo etc..Loading elution action can be determined according to applied sample amount used in column material The parameters such as amount, column volume, post bed height.Usually, in order to obtain preferable separating degree, post bed height is no less than 1 meter.Eluent Can be 0.01~0.5M inorganic salts or organic salt, such as NaCl, ammonium hydrogen carbonate.Molecular cut off is less than 700Da bag filter Or the gel rubber material of milipore filter and molecular weight separating ranges less than 700Da can be in target product preliminary purification and/or desalination.

When using above-mentioned gel column separating purification target compound or carrying out desalination, each fraction of fraction collector reception It can be detected using Phenol sulfuric acid procedure, sulfuric acid carbazole method or cysteine phynol method, draw elution curve, it is bent further according to outflow Chemical composition identical fraction contained by line merging.

In the circuit, its step (4) is that compound 4 is changed into compound 5 by reduction or reductive amination reaction, Namely formula (I) compound.

Usually, use sodium borohydride or sodium cyanoborohydride can be by alkaline aqueous solution (typically using NaOH for reducing agent Adjust pH about 8~10) in compound 4 reducing end under neutral 2,5- dehydration talose (anTal) be reduced into sugar alcohol (anTal-ol)；

, can be by the end of compound 4 in the presence of ammonium salt such as ammonium hydrogen carbonate, organic amine and reducing agent such as sodium cyanoborohydride AnTal reductive aminations, i.e. ammonium salt or organic amine and anTal aldehyde radicals reaction generation schiff bases (Schiff base), the latter can be with It is reduced agent and is reduced into secondary amine.To those skilled in the art, reacted using reductive amination, R can be readily available₅ For H, substituted or unsubstituted C1-C6 straight or branched alkyls, the compound 5 of substituted or unsubstituted C7-C12 aryl.

Oligosaccharide compound 4 and oligosaccharide compound 5 are in the range of the oligosaccharide compound that the present invention defines.Step (3) and (4) after products therefrom preferably uses suitable gel column (such as Sephadex G10 or P2) desalination, obtained by freeze-drying Gained oligosaccharide compound 4 and 5.

End-product obtained by the inventive method can also be prepared into mono-salt form by base exchange method, including alkali metal, Alkali salt or organic ammonium salt, its preferable mono-salt form are sodium salt, sylvite or calcium salt etc..Product of the present invention into The oligosaccharide compound can be exchanged into Hydrogen by salt process using ion-exchange, then using corresponding alkali neutralize To corresponding salt；Also desired mono-salt form can be converted it into by cation exchange column.The pre- place of ion exchange resin column Reason, sample loading and elution can be carried out according to a conventional method.

As it was previously stated, the anticoagulating active that oligosaccharide compound of the present invention is potent, the activation of its people's Quality Control blood plasma that doubles Drug concentration needed for partial thromboplastin time (APTT) in the range of about 2~30 μ g/mL shows that endogenous can be significantly inhibited Coagulation process caused by coagulation pathway activation.In about 2000 μ g/mL concentration range, these compounds do not influence hostage typically The prothrombin time (PT) of blood plasma is controlled, prompts it may not influence the related physiological haemostasis work(of exogenous cruor pathway activation Energy.Oligosaccharide compound of the present invention has relatively weak influence to the thrombin time (TT) of people's Quality Control blood plasma, in theory, its There is certain deactivation to established fibrin ferment in thrombosis.

Heparin class anticoagulation (such as heparin, low molecular weight heparin, the sulphur of oligosaccharide compound and Clinical practice of the present invention Up to liver last of the ten Heavenly stems sodium) anticoagulant active mechanism it is different, oligosaccharide compound of the present invention does not have significant antithrombase The factor Xa and factor IIa inhibitory activity, its intrinsic coagulation pathway inhibitory activity that (ATIII, AT) is relied on depend on it Direct Xase inhibitory activity.

In vitro enzyme biopsy examining system, oligosaccharide compound of the present invention can have potent suppression endogenous Xase to live Property.Surprised discovery of the invention, (contain the composition monose of 9 or more, including dehydrating tower sieve containing no less than three trisaccharide construction units Sugar or its reductive derivative) compound of the present invention can have potent endogenous Xase inhibitory activity, its suppression in Source property Xase IC₅₀Value can be about in the range of 5~200ng/mL；Containing two trisaccharide construction units (containing 6 composition monose, including Dehydration talose and its reductive derivative) the endogenous Xase inhibitory activity of oligosaccharide compound significantly reduce.

Oligosaccharide compound of the present invention typically also has the IIa inhibitory activity that certain HCII is relied on.It is endogenous with it Property Xase inhibitory activity it is different, (contain the composition monose of 6 or more, including dehydrating tower containing no less than two trisaccharide construction units Sieve sugar and its reductive derivative) compound of the present invention can have HCII rely on IIa inhibitory activity (IC₅₀About 200~ 1500ng/mL).For the present invention containing the composition monose of 9 or more (including dehydration talose or its reductive derivative) For compound, the IIa inhibitory activity that its endogenous Xase inhibitory activity is relied on much stronger than its HCII is (with IC₅₀Value meter, Xase suppressions System activity is strong about 10~25 times).The endogenous Xase inhibitory activity of the compounds of this invention of six sugared structures be then weaker than its HCII according to Bad IIa inhibitory activity, however, six sugar compounds also have certain anticoagulating active, it is clear that those skilled in the art can To understand, now, its anticoagulating active mechanism should be similar to dermatan sulfate.

Correlative study of the present invention is shown, in about 2000ng/mL concentration ranges, oligosaccharide compound of the present invention is to solidifying Other clotting factor or co-factor in blood approach, for example, Xa, XIa, XIIa, III-VIIa compound, Xa-Va compounds, ATIII etc. does not make significant difference.Particular, it is important that it is different from prototype FG class natural products, under effective anticoagulating active concentration Oligosaccharide compound of the present invention has no significant effect to XII and biologically active pdgf.

Therefore, oligosaccharide compound of the present invention is that a kind of endogenous factors Xase with good selectivity suppresses The IIa inhibitor that agent and/or HCII are relied on.Existing research data shows that intrinsic coagulation pathway forms close with pathologic thrombus Cut pass rather than physiological haemostasis institute are necessary, and selective intrinsic coagulation pathway inhibitor can be used for suppressing pathologic thrombus shape Into, and its hemorrhagic tendency can obtain effectively it is relatively low.Factor X enzymes are last zymetology sites in intrinsic coagulation pathway, It is the speed limit site of intrinsic coagulation process, therefore, oligosaccharide compound of the present invention has important anti-freezing antithrombotic Application value.

Therefore, the present invention also provides a kind of medicine group containing the oligosaccharide compound and its pharmaceutically acceptable salt Compound, described pharmaceutical composition contain the oligosaccharide compound or its pharmaceutically acceptable salt of effective anti-freezing dosage with And pharmaceutically acceptable excipient.

In view of the physicochemical property of oligosaccharide compound of the present invention, pharmaceutical composition of the present invention is preferably prepared to stomach Parenteral form of administration, such as aqueous solution for injection or prepared before use are into the lyophilized formulations of aqueous solution for injection.

Oligosaccharide compound of the present invention has good water solubility, is easy to obtained aqueous solution；Due to active component Molecular weight is relatively low, can remove pathogenic microorganism by aqueous solution ultrafiltration (for example with molecular cut off about 10kD milipore filter) And heat source substance；In the preparation process of its aqueous solution and/or lyophilized formulations, selectable pharmaceutical excipient may include to adjust solution Osmotic pressure and/or the inorganic salts of pH value such as sodium chloride, buffer salt such as phosphate etc., preferably without cosolvent and/or surface-active Agent.For the freeze drying powder injection of prepared before use parenteral solution, except regulation osmotic pressure and the pharmaceutically acceptable nothing of pH value Outside machine salt and/or buffer salt, it is also an option that contributing to the pharmaceutically acceptable excipient of preparations shaping using mannose etc..

Although oral administration biaavailability is relatively limited, relative to natural prototype FG classes compound and there is certain molecule For the mixture for measuring the low molecule amount FG class compounds of distribution, the gastrointestinal administration of oligosaccharide compound of the present invention is still With certain drug activity.Therefore pharmaceutical composition of the present invention can also be prepared into stomach and intestine well known to those skilled in the art Canal drug administration formulation, such as tablet, capsule etc..It is of the present invention for specified disease, such as the treatment of DVT Preparation can also be prepared into special parenteral dosage forms, such as spray through respiratory tract administration etc..

Oligosaccharide compound and its pharmaceutically acceptable salt of the present invention have potent anti-freezing antithrombotic acitivity, It can be used for the prevention and treatment of thrombotic diseases, such as thrombotic angiocardiopathy, thrombotic cerebrovascular disease, pulmonary vein Thrombus, PeV thrombus, DVT, peripheral arterial thrombus etc., therefore, the present invention also provides the oligosaccharide compound And its pharmaceutically acceptable salt and the pharmaceutical composition containing the oligosaccharide compound and its pharmaceutically acceptable salt are being made Application in the medicine of standby preventing and treating thrombotic diseases, the thrombotic diseases are venous thronbosis or Arterial thrombosis or lacked Courageous and upright heart disease or ischemic cerebrovascular disease.

Oligosaccharide compound of the present invention is the fucosylated glycosaminoglycan class compound of sterling compound form.By In the complicated of fucosylated glycosaminoglycan class compound, the prototype for seeing the natural origin of report so far is fucosylated Depolymerization product obtained by glycosaminoglycan and different depolymerization methods is a series of mixture of the approximate compound of chemical constitutions. The suitable starting material selection of integrated use of the present invention, the Depolymerization Technique for the glycosidic bond selectivity that the present invention establishes, end group reduction are spread out Biochemical treatment and suitable separating and purifying technology obtain a series of fucosylated glycosaminoglycan class of sterling compound forms Compound.On this basis, the pharmacological activity comparative studies of first passage sterling compound of the present invention, has been inquired into fucosylated Glycosaminoglycan compound chemical structure and its endogenous Xase inhibitory activity, other clotting factor or co-factor activities influence, The correlation that biologically active pdgf influences, find three trisaccharide construction units and two trisaccharide construction units for the oligosaccharides in surprise Compound suppresses the importance of endogenous Xase and HCII inhibitory activity.

Brief description of the drawings

Fig. 1 is FG depolymerization products (A) and compound a and compound b (B) liquid chromatogram；

Fig. 2 is compound a and compound b¹H(A)/¹³C (B) NMR spectra；

Fig. 3 is compound b's¹H-¹H COSY spectrograms (A) and¹H-¹³C hsqc spectrums figure (B)；

Fig. 4 is compound a (A) and compound b (B) Q-TOF mass spectrograms；

Fig. 5 is compound c, d, e, f's¹H NMR spectras；

Fig. 6 is compound c, d, e, f's¹³C NMR spectras；

Fig. 7 is compound g, i, k's¹H NMR spectras；

The suppression Xase activity that Fig. 8 is compound a~f；

Fig. 9 is influences of the compound a~f to factor XI, plasma thromboplastin antecedent I activity；

Figure 10 is influences of the compound a~f to human blood platelets Activation Activity.

Embodiment

Below in conjunction with accompanying drawing, with the specific embodiment of the present invention come the present invention is described in further detail, but it is described Embodiment does not limit present disclosure and protection domain.

【Embodiment 1】

The preparation of compound a and compound b：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base- (β 1,3) -2,5- dehydration talitols (trisaccharide)；The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3) sulphation galactosyl-(β 1,4) of-D-N- acetyl group -2- deoxidations -2- amino -4,6- two-[sulphation rock algaes of L-2,4- bis- Glycosyl-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3) -2,5- dehydrations talitol (six sugar)

1.1 material

The fucosylated glycosaminoglycan (FG, sodium salt) in Stichopus Variegatus Semper body walls source, by document Prepared by method (Marine Drugs, 2013,11,399-417) extraction purification, weight average molecular weight about 70kDa.Hydrazine hydrate, sulfuric acid The reagents such as hydrazine, sodium chloride, natrium nitrosum, the concentrated sulfuric acid, sodium borohydride, absolute ethyl alcohol, sodium hydroxide are ommercially available AR.

1.2 method

It is prepared by the partially deacetylated FG of step 1.：The FG raw material 2.0g in Stichopus Variegatus sources are weighed, are put In 250ml round bottom reaction bulbs, hydrazine sulfate 500mg is added, then addition hydrazine hydrate 50mL, under nitrogen atmosphere, 250rpm rotating speeds Lower stirring, heating response 24h at 90 DEG C.After reaction terminates, into reaction solution plus 200ml ethanol makes resulting solution determining alcohol about 80% (v/v) must be precipitated, and supernatant is removed in centrifugation.Gained precipitation with 50ml it is water-soluble after, adding 200ml absolute ethyl alcohols, (resulting solution is pure and strong Spend about 80%, v/v), repeat alcohol precipitation 4 times, then precipitation is dissolved with water, is the 3500Da bag filters (U.S. with molecular cut off Union Carbide Corp's product) to dialyse, dialysis trapped fluid is dried under reduced pressure at 55 DEG C, obtains deacetylated intermediate product sample about 1.9g, yield are about 95%.¹The deacetylated rate of H NMR detection products therefroms is 51%.

It is prepared by step 2. deaminizating depolymerization product：Partially deacetylated intermediate product 1.5g obtained by step 1 is taken to justify in 250ml In the reaction bulb of bottom, water 30mL is added to dissolve (final concentration 50mg/mL)；At ambient temperature, 5.5M is added, the nitrous acid that pH is 4 is molten Liquid 60mL, under room temperature condition after stirring reaction 20min, 1M sodium hydroxide solution is added to adjust the terminating reaction of pH value of solution about 9, and add 0.25M sodium borohydride solutions 30mL, 50 DEG C are heated lower stirring reaction 2h to reduce the terminal aldehyde groups of depolymerization product.Reaction terminating Afterwards, reaction solution is cooled to room temperature, and 0.5M sulfuric acid adjusts reaction solution, to remove excessive sodium borohydride, then to use 0.5M again to pH3 Sodium hydroxide adjust solution gained reaction solution be saturating with molecular cut off 1000Da bag filters (Union Carbide Corp) to pH about 7 Analysis, collect dialysis trapped fluid and be freeze-dried, obtain depolymerization product 1.2g, yield about 80%.

Step 3. compound a and compound b's isolates and purifies：Step 2 gained depolymerization product 1000mg is taken with 20mL 0.2M NaCl dissolves (final concentration 50mg/mL), 0.22 μm of membrane filtration；P6 (Bio-Rad) gel column that upper 0.2M NaCl have been balanced (Φ 2cm, l 140cm), the elution of 0.2M NaCl solutions, flow velocity 8.6mL/h, 14min/ pipe are received, and elution is collected by every part of 2ml Fraction, it is combined sulfuric acid carbazole method and HPGPC detection methods monitoring (Superdex Peptide 10/300GL (10mm × 300mm)). 61~68 and 75~81 fractions are merged according to HPGPC testing results respectively, freezed after merging again respectively with P6 gel columns by above-mentioned Operation is purified and detected, and is purified more than 3 times repeatedly, until collect sample is detected as simple spike with HPGPC, then will be obtained Sterling Sephadex G10 or P2 gel column (60cm × 1cm) desalination, after freezing, compound a about 60mg, compound are obtained respectively B about 120mg.

It is attached：(1) desalination of oligosaccharides：It will treat that desalination oligosaccharides dried frozen aquatic products is dissolved in 2~12mL ultra-pure waters, 0.22 μm of membrane filtration The good Sephadex G10 gel columns (60cm × 1cm) of upper ultrapure water balance afterwards.Flow velocity about 22.2mL/h, 5min/ pipes receive, and put down About 1.85mL/ is managed, and sulfuric acid carbazole method detects each pipe sugared content, 0.1mol/LAgNO₃Detect each fraction whether saliferous (NaCl). Not saliferous and each fraction containing sugar are merged, freezed.

(2) P6 elutes sulfuric acid carbazole method colour developing monitoring method P6 gel column elutions every part of 20 μ L of sampling of fraction of fraction, pure water 0.5mL is diluted to, 0.0125mol/L sodium tetraborate-concentrated sulfuric acid solution 2.5mL is added in ice-water bath, is shaken up, 85 DEG C of water-baths 20min, room temperature is cooled to, each pipe plus 0.1% carbazole solution 0.1mL, shakes up, boiling water bath 15min, be cooled to room temperature, be divided Optical density (OD values) at photometric detection 520nm.

1.3 compound purity and chemical structure analysis

(1) purity analysis method and result

It is prepared by sample solution：Step 2 gained depolymerization product, step 3 gained compound a and compound b samples 1 are weighed respectively ~2mg, adds ultra-pure water to dissolve, and crosses 0.22 μm of filter membrane, takes filtrate to carry out HPGPC detections.

HPGPC analysis conditions：Agilent technologies 1200series high performance liquid chromatographs, Superdex Peptide 10/300GL (10mm × 300mm) post, 30 DEG C, mobile phase 0.2MNaCl, flow velocity 0.4mL/min of temperature, detection Device is differential refraction detector (G1362A), and 0~60min collection of illustrative plates is recorded after sample feeding.

As a result：HPGPC detection displays, the HPGPC collection of illustrative plates of step 2 gained depolymerization product show multiple different retention times Eluting peak, show its be series compound mixture；Compound a and compound b HPGPC collection of illustrative plates show single guarantor The eluting peak (accompanying drawing 1) of time is stayed, it is sterling compound to prompt them, and area normalization method calculates, compound a and compound b Purity be respectively about 98.5% and about 96.3%.

(2) chemical structure analysis and result

Analysis method：Mass Spectrometer Method uses micrOTOF-QII ESI-MS (Bruker-Daltonik, Germany) mass spectrum Instrument, Mass Spectrometry Conditions are capillary voltage 2500V, sprayer voltage 0.6bar, dry gas stream speed 4.0L/min, dry temperature degree+ 180 DEG C, m/z scanning ranges 50-3000.Data analysis uses the (Bruker- of Bruker Compass Data-Analysis 4.0 Daltonik, Germany) software progress.¹H/¹³C and 2D NMR detections use Bruker DRX 800MHz NMRs, compose A width of 16 025.6Hz, acquisition time 2.0447s, pulse width 9.5s, relaxation time 1s, scanning times are 32 times.Sample is dense Spend for 10-15g/L, freezed repeatedly three times with heavy water before detection.

Compound a and compound b's¹H/¹³CNMR collection of illustrative plates is shown in attached Fig. 1 and 2, and signals assignment is shown in Table 1.Compound b 2D NMR spectra is shown in accompanying drawing 3.NMR structural analyses show that compound a and b are the pure compound of structure, wherein, compound a Structure is that the sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3) -2,5- is dehydrated talitol (L-Fuc2S4S-(α1,3)-D-GlcUA-(β1,3)-anTal-ol)；Compound b is the sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-sulphation the galactosyls of (β 1,3)-D-N- acetyl group -2- deoxidation -2- amino -4,6- two-(β 1,4)-[sulphation fucosido-(α 1,3)-of L-2,4- bis-]-D-Glucose aldehydic acid base-(β 1,3) -2,5- dehydration talitols (L-Fuc2S4S-(α1,3)-D-GlcUA-(β1,3)-D-GalNAc4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D- GlcUA-(β1,3)-anTal-ol)。

Compound a and compound b Q-TOF Mass Spectrometer Method collection of illustrative plates are shown in accompanying drawing 4.The Q-TOF collection of illustrative plates of compound a is shown [M-Na]^-(m/z 892.90)、[M-2Na]^2-(m/z 434.96)、[M-3Na]^3-Signal peaks such as (m/z 282.32), compound b Q-TOF collection of illustrative plates then show [M-2Na]^2-(m/z 912.40)、[M-3Na]^3-(m/z 600.61)、[M-4Na]^4-(m/z 444.71)、[M-5Na]^5-Signal peaks such as (m/z 351.17), further prove compound a and compound b chemical constitution.

The compound a of table 1. and compound b's¹H/¹³H NMR signals belong to and coupling constant (ppm, Hz)

Note：In table, F, U, T in compound a represent fucosido, glucuronic acid base and dehydration talitol respectively Base；In compound b, F, U, T represent the fucosido positioned at reducing end, glucuronic acid base and dehydration talitol base respectively, F ', U ', A represent fucosido, glucuronic acid base and the acetylamino galactosamine base positioned at non-reducing end respectively.

【Embodiment 2】

Compound c~f preparation：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)- { sulphation galactosyl-(β 1,4) of D-N- acetyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucoses of L-2,4- bis- Base-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3)-}_n- 2,5- is dehydrated talitol.In its compound c, n=2；Compound d In, n=3；In compound e, n=4；In compound f, n=5.

2.1 material

With embodiment 1.

2.2 method

It is prepared by the partially deacetylated FG of step 1.：FG (sodium salt) 2.0g in Stichopus Variegatus sources is weighed, Handled with deacetylation described in embodiment 1 and post-processing approach, but the hydrazinolysis reaction time is about 18h.Pass through the reaction Deacetylated intermediate product sample about 1.93g is obtained, yield is about 96.5%.¹H NMR detect the deacetylated of products therefrom Rate is 40%.

It is prepared by step 2. deaminizating depolymerization product：Partially deacetylated intermediate product 1.50g obtained by step 1 is taken in 250ml In round bottom reactor, water 30mL is added to dissolve；Under the conditions of ice bath (0 DEG C), 5.5M is added, pH is 1.5 nitrous acid solution 60mL, Under ice bath after stirring reaction 30min, 1M sodium hydroxide solution is added to adjust the terminating reaction of pH value of solution about 9.Add then in reaction solution Enter 0.25M sodium borohydride solutions 40mL, the lower stirring reaction 2h of 60 DEG C of heating (terminal aldehyde groups of reduction depolymerization product).Reaction terminates Afterwards, reaction solution is cooled to room temperature, 0.5M hydrochloric acid adjusts reaction solution then to use 0.5M to pH about 3 (removing excessive sodium borohydride) Sodium hydroxide adjusts back solution to pH about 7.Reaction solution is dialysed with molecular cut off 1000Da bag filters (American Association carbonization) Afterwards, dialysis trapped fluid is freezed, obtains depolymerization product 1.25g, yield about 83%.

Isolating and purifying for step 3. compound c~f takes step 2 gained depolymerization product 1000mg, 0.2MNaCl 20mL to dissolve (final concentration 50mg/mL), 0.22 μm of membrane filtration；P10 (Bio-Rad) gel column (the Φ 2cm, l that upper 0.2M NaCl have been balanced 200cm), 0.2M NaCl solutions are eluted, and flow velocity 27.6mL/h, 5min/ pipe receive, and elution fraction is collected by every part of about 2.3ml, It is combined sulfuric acid carbazole method and HPGPC detection methods monitoring (Superdex Peptide 10/300GL (10mm × 300mm)).According to HPGPC testing results merge 76~83,86~93,96~103 and 105~112 fractions respectively, freeze after merging and use respectively again P10 gel columns are purified and detected by aforesaid operations, are purified more than 3 times repeatedly, until collect sample is detected as list with HPGPC One peak, method desalination in the step 3 of embodiment 1 is pressed using P2 gel columns (60cm × 1cm), freeze-drying, obtains compound c about respectively 73mg, compound d about 140mg, compound e about 185mg, compound f about 108mg.

2.3 compound purities and chemical structure analysis

(1) purity analysis method and result

Method：Detected with HPGPC analysis methods described in embodiment 1.

As a result：HPGPC detection displays, compound c~f HPGPC collection of illustrative plates show the eluting peak of single retention time, It is sterling compound to prompt them, and area normalization method calculates, and compound c~f purity is above 95%.

(2) nmr chemical structural analysis and result

¹H/¹³C and 2D NMR testing conditions are the same as described in embodiment 1.NMR structural analyses show that compound c~f is structure Pure compound, its chemical constitution are respectively：

Compound c：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₂- 2,5- dehydration talitol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β 1, 3)-{-D-GalNAc 4S6S-(β1,4)-[L-Fuc2S4S-α1,3)-]D-GlcUA-(β1,3)-}₂-anTal-ol；

Compound d：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₃- 2,5- dehydration talitol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β 1, 3)-{-D-GalNAc 4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-}₃-anTal-ol；

Compound e：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₄- 2,5- dehydration talitol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β 1, 3)-{-D-GalNAc 4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-}₄-anTal-ol；

Compound f：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₅- 2,5- are dehydrated talitol, i.e.,

L-Fuc2S4S-(α1,3)-D-GlcUA-(β1,3)-{-D-GalNAc 4S6S-(β1,4)-[L-Fuc2S4S-(α 1,3)-]D-GlcUA-(β1,3)-}₅-anTal-ol；

Compound c~f's¹H/¹³C NMR spectras are shown in accompanying drawing 5 and 6, its structural formula of compound and¹H/¹³C NMR signals belong to It is shown in Table 2.

Compound c~the f of table 2 chemical constitution and¹H/¹³C NMR signals belong to (δ, ppm)

Note：In table, F, F ', F ", U, U ', U ", A, A ', A " and T represent saccharide residue shown in structural formula respectively.

【Embodiment 3】

Compound g~m preparation：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)- { sulphation galactosyl-(β 1,4) of D-N- acetyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucoses of L-2,4- bis- Base-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3)-}_n- 2,5- is dehydrated talose glycol.In its compound g, n=0；Chemical combination In thing h, n=1；In compound i, n=2；In compound j, n=3；In compound k, n=4；In compound l, n=5, chemical combination In thing m, n=6.

3.1 material

The fucosylated glycosaminoglycan (FG, sodium salt) in Stichopus monotuberculatus body walls source, by document Prepared by method (Marine Drugs, 2013,11,399-417) extraction purification, weight average molecular weight about 68kDa.Other reagents and material Material is the same as embodiment 1.

3.2 method

It is prepared by the partially deacetylated FG of step 1.：Take the FG raw materials in Stichopus monotuberculatus sources 2.0g, it is slightly changed with deacetylation described in embodiment 1 and post-processing approach processing.Wherein, the hydrazinolysis reaction temperature Degree is about 75 DEG C, the reaction time is about 24h.Deacetylated intermediate product sample about 1.87g is obtained by the reaction, yield is about For 93.5%.¹The deacetylated rate of H NMR detection products therefroms is 35%.

It is prepared by step 2. deaminizating depolymerization product：Partially deacetylated intermediate product 1.50g obtained by step 1 is taken in 200ml In round bottom reactor, water 30mL is added to dissolve；Under the conditions of ice bath (0 DEG C), add 5.5M, pH be 3 nitrous acid solution 60mL, ice After the lower stirring reaction 30min of bath, 1M sodium hydroxide solution is added to adjust the terminating reaction of pH value of solution about 7.Cold bath cools down reaction solution To room temperature, after gained reaction solution is dialysed with molecular cut off 1000Da bag filters (American Association carbonization), by trapped fluid of dialysing It is lyophilized, obtain depolymerization product 1.32g, yield about 88%.

Isolating and purifying for step 3. compound g~m takes step 2 gained depolymerization product 1000mg, 0.2MNaCl 20mL to dissolve (final concentration 50mg/mL), 0.22 μm of membrane filtration；P10 (Bio-Rad) gel column (the Φ 2cm, l that upper 0.2M NaCl have been balanced 200cm), 0.2M NaCl solutions are eluted, and flow velocity 30mL/h, 5min/ pipe receive, and elution fraction, connection are collected by every part of about 2.5ml With sulfuric acid carbazole method and HPGPC detection methods monitoring (Superdex Peptide 10/300GL (10mm × 300mm)).According to HPGPC testing results merge 65~69,71~78,81~89,94~100,105~111,115~122 and 130~138 respectively Fraction, freezed after merging and purified and detected by aforesaid operations with P10 gel columns respectively again, purified more than 3 times repeatedly, until Collect sample and be detected as simple spike with HPGPC, the step of embodiment 1 is pressed using Sephadex G10 or P2 gel column (60cm × 1cm) Method desalination in rapid 3, freeze-drying, compound g about 36mg, compound h about 62mg, compound i about 115mg, chemical combination is obtained respectively Thing j about 126mg, compound k about 155mg, compound l about 118mg, compound m about 54mg.

3.3 compound purities and chemical structure analysis

(1) purity analysis method and result：Detected with HPGPC analysis methods described in embodiment 1.As a result show, compound g ~m HPGPC collection of illustrative plates shows the eluting peak of single retention time, and it is sterling compound to prompt them, area normalization method Calculate, compound g~m purity is above about 96%.

(2) nmr chemical structural analysis and result

¹H/¹³C and 2D NMR testing conditions are the same as described in embodiment 1.NMR structural analyses show that compound g~m is structure Pure compound, its chemical constitution are respectively：

Compound g：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3) -2,5- dehydrations Talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β 1,3)-anTal-diol；

Compound h：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-D-N- acetyl Sulphation galactosyl-(β 1,4)-[sulphation fucosido-(α 1,3)-of L-2,4- bis-] of base -2- deoxidation -2- amino -4,6- two D-Glucose aldehydic acid base-(β 1,3) -2,5- dehydration talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β 1,3)-D- GalNAc 4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-anTal-diol；

Compound i：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₂- 2,5- are dehydrated talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β1,3)-{-D-GalNAc4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-}₂-anTal-diol；

Compound j：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₃- 2,5- are dehydrated talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β1,3)-{-D-GalNAc4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-}₃-anTal-diol；

Compound k：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₄- 2,5- are dehydrated talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β1,3)-{-D-GalNAc4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-}₄-anTal-diol；

Compound l：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -] D-Glucose aldehydic acid base-(β 1,3)-₅- 2,5- are dehydrated talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β1,3)-{-D-GalNAc4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]D-GlcUA-(β1,3)-}₅-anTal-diol；

Compound m：The sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-{-D-N- second Sulphation galactosyl-(β 1,4) of acyl group -2- deoxidation -2- amino -4,6- two-[the sulphation fucosidos of L-2,4- bis--(α 1, 3) -]-D-Glucose aldehydic acid base-(β 1,3)-₆- 2,5- are dehydrated talose glycol, i.e. L-Fuc2S4S- (α 1,3)-D-GlcUA- (β1,3)-{-D-GalNAc 4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]-D-GlcUA-(β1,3)-}₆-anTal- diol。

Wherein, compound g, compound i and compound k¹H NMR are shown in accompanying drawing 7.Compound g, compound h, compound j, Compound l's¹H/¹³C NMR signals ownership is shown in Table 3.

Table 3. compound g, h, j, l chemical constitution and¹H/¹³C NMR signals belong to (δ, ppm)

Note：In table, F, F ', U, U ', A and T represent the saccharide residue shown in structural formula respectively.

【Embodiment 4】

By the FG prepare compounds b in Apostichopus japonicas sources

4.1 material

The fucosylated glycosaminoglycan (FG) in Apostichopus japonicas (imitative stichopus japonicus) body wall source, by document Prepared by method (Marine Drugs, 2013,11,399-417) extraction purification, weight average molecular weight about 65kDa.Other reagent materials With embodiment 1.

4.2 method

It is prepared by the partially deacetylated FG of step 1.：The FG 2.0g in Apostichopus japonicas sources are weighed, with real The processing of the methods described of example 1 is applied, obtains deacetylated intermediate product sample about 1.84g, yield is about 92%.¹H NMR detect institute The deacetylated rate for obtaining product is 53%.

It is prepared by step 2. deaminizating depolymerization product：Partially deacetylated intermediate product 1.5g obtained by step 1 is taken to justify in 250ml In the reactor of bottom, deaminizating depolymerization is carried out with the methods described of embodiment 1, obtains depolymerization product 1.17g, yield about 78%.

Step 3. compound b's isolates and purifies：Step 2 gained depolymerization product 1000mg, 0.2M NaCl20mL is taken to dissolve (final concentration 50mg/mL), 0.22 μm of membrane filtration；P6 (Bio-Rad) gel column (the Φ 2cm, l that upper 0.2M NaCl have been balanced 140cm), 0.2M NaCl solutions are eluted, flow velocity 8.6mL/h, and elution fraction is collected by every part of 2ml, combination sulfuric acid carbazole method and HPGPC detection methods monitor (Superdex Peptide10/300GL (10mm × 300mm)).Distinguished according to HPGPC testing results Merge 62~68 fractions, freezed after merging and purified and detected by aforesaid operations with P6 gel columns respectively again, purified 3 times repeatedly More than, until collect sample is detected as simple spike with HPGPC, by method desalination in the step 3 of embodiment 1, purify to obtain six sugar mixing Thing 130mg.The saccharic composition of gained six is through DEAE (Macro-Prep DEAE, Bio-rad) and anion-exchange resin column (Boston Poly SAX, Boston Analytics, Inc.) purify repeatedly, Sephadex G10 gel column desalinations, obtain sterling compound group Divide (compound b) about 90mg.

4.3 compound purities and chemical structure analysis

(1) with embodiment 1, testing result is shown, pure obtained by step 3 for purity analysis method and result HPGPC analysis conditions The HPGPC collection of illustrative plates of product compound component shows the eluting peak of single retention time, and retention time and the gained chemical combination of embodiment 1 Thing b is identical, and area normalization method calculates, and its purity is respectively about 96.3%.

(2) chemical structure analysis and result NMR testing results are shown, above 4.2 step (3) the sterling compound Chemical constitution is identical with the chemical constitution of the gained compound of embodiment 1.

【Embodiment 5】

Compound n, compound o preparation：Compound n is the sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose Aldehydic acid base-(β 1,3)-{ the sulphation galactosyls of-D-N- acetyl group -2- deoxidation -2- amino -4,6- two-(β 1,4)-[L-2,4- Two sulphation fucosidos-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3)-}₂- 1- deoxidation -1- amino -2,5- dehydrating tower sieve Sugar；Compound o be the sulphation fucosidos of L-2,4- bis--(α 1,3)-D-Glucose aldehydic acid base-(β 1,3)-- D-N- acetyl group- Sulphation galactosyl-(β 1,4) of 2- deoxidation -2- amino -4,6- two-[sulphation fucosido-(α 1,3)-of L-2,4- bis-] D- Glucuronic acid base-(β 1,3)-}₂- N- (4- leptodactylines) -1- deoxidation -1- amino -2,5- is dehydrated talose.

5.1 material

Compound i, the gained sterling compound i of embodiment 3.The reagents such as Ammonium bicarbonate food grade, sodium cyanoborohydride are commercially available point Analyse pure.

5.2 method

(1) 100mg compounds i is dissolved in 5mL 0.2mM phosphate buffers (pH 9.0), added in stirring at room temperature Ammonium hydrogen carbonate 10mg and sodium cyanoborohydride 30mg, reaction 12h and 24h is separately added into ammonium hydrogen carbonate 10mg again, after reacting 36h, Add 95% ethanol 15mL, centrifuging to precipitate, and after gained precipitation is washed twice with 95% ethanol 15mL, answered with 35mL 0.1%NaCl Insoluble matter is removed in molten gained precipitation, centrifugation, and it is 1000Da bag filters (American Association carbonization) that gained supernatant, which is placed in molecular cut off, In, deionized water dialysis 24h, target compound n about 76mg are obtained after gained dialysis trapped fluid freeze-drying.

(2) 100mg compounds i is dissolved in 5mL 0.2mM phosphate buffers (pH 8.0), be separately added into stirring 80mg tyrasamines and 30mg sodium cyanoborohydrides, react about 72h in 35 DEG C of waters bath with thermostatic control.After completion of the reaction, 95% ethanol 15mL is added, Centrifuging to precipitate, and after gained precipitation is washed twice with 95% ethanol 15mL, redissolve gained with 35mL 0.1%NaCl and precipitate, centrifugation Insoluble matter is removed, gained supernatant is placed in molecular cut off as in 1000Da bag filters (American Association carbonization), deionized water is dialysed 24h, target compound o about 85mg are obtained after gained dialysis trapped fluid freeze-drying.

5.3 result

¹H NMR detections data show that compound n is L-2, the sulphation fucosidos of 4- bis--(α 1,3)-D-Glucose aldehyde Acidic group-(β 1,3)-{ sulphation galactosyl-(β 1,4) of-D-N- acetyl group -2- deoxidation -2- amino -4,6- two-[L-2,4- bis- Sulphation fucosido-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3)-}₂- 1- deoxidation -1- amino -2,5- is dehydrated talose. Its¹H NMR signals and compound i are basically identical, only with compound i contained by end -2,5- dehydration talose glycol terminal hydrogen Signal is about δ 5.06ppm, and the end group hydrogen signal of end -1- deoxidations -1- amino -2,5- dehydrating tower sieve glycosyls goes out contained by compound n Present about δ 2.57ppm and 2.83ppm (pass through¹H-¹H COSY belong to), and the integration of its signal peak and non-reducing end L-Fuc it The signal integration ratio about 1 of H1 signals (δ 5.56ppm):1.

¹H NMR detections data show that compound n is L-2, the sulphation fucosidos of 4- bis--(α 1,3)-D-Glucose aldehyde Acidic group-(β 1,3)-{ sulphation galactosyl-(β 1,4) of-D-N- acetyl group -2- deoxidation -2- amino -4,6- two-[L-2,4- bis- Sulphation fucosido-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3)-}₂- N- (4- leptodactylines) -1- deoxidation -1- ammonia Base -2,5- is dehydrated talose.In addition to end N substitutes dehydrating tower sieve Sugar signal, compound o's¹H NMR signals and compound i bases This is consistent.Its¹H NMR show the end group of end-N- (4- leptodactylines) -1- deoxidation -1- amino -2,5- dehydrating tower sieve glycosyls For hydrogen at about δ 2.91 and 3.22ppm, the phenyl ring hydrogen signal of its 4- leptodactyline respectively appears in about δ 7.26ppm (H ' 2 6) and about 6.88ppm (H ' 3 and H ' 5) place and H ', and the H1 signals (δ of its phenyl ring hydrogen signal integration and non-reducing end L-Fuc Signal integration ratio about 4 5.56ppm):1.

【Embodiment 6】

Anticoagulant active detects

6.1 materials and instrument

(1) sample：Compound a~f, compound i~k, compound n.Embodiment 1~3 and embodiment 5 prepare gained.

(2) reagent：People's Quality Control blood plasma, activated partial clotting time (APTT) kit, prothrombin time (PT) reagent Box, thrombin time (TT) kit are Teco Medical (moral) product；Low molecular weight heparin (Enoxaparin Sodium, LMWH), Sanofi Aventis companies (method) product；The FG in Stichopus monotuberculatus sources, with embodiment 1 Source.

(3) instrument：MC-4000 coagulo meters.

6.2 method

By sample and reference substance LMWH 0.02M, pH7.4 Tris-HCl buffers and/or to be diluted to series dense Degree, the influence of sample and reference substance solution to people's Quality Control plasma A PTT, PT, TT time is detected according to kit specification method.

6.3 result

Testing result is shown in Table 4.Testing result shows that prototype FG, compound e~f and compound j~k are respectively provided with potent Intrinsic coagulation inhibitory activity, drug concentration that it doubled needed for people's standard plasma APTT times in about 1.8~5.6 μ g/ml, its Activity is better than LMWH；The activity of compound c~d, compound i and compound n prolonged human Quality Control plasma A PTT times are close or omit Less than LMWH；There is compound b relatively weak APTT to extend activity, and compound a is in about 128 μ g/ml experimental concentration model APTT time values are had substantially no effect in enclosing.In the range of about 128 μ g/ml experimental concentration, the compound is to the equal nothings of PT and TT Significantly affect, show that it does not influence exogenous cruor pathway and common coagulation pathway influenceed weaker.

4. compound as of table~f, compound i~k, compound n anticoagulant active

Note：^aThe drug concentration (μ g/mL) to double needed for APTT, PT, TT；^bRequired drug concentration>128μg/ml

As control, in the range of identical experimental concentration, LMWH also has a significant impact to APTT and TT, on PT influence compared with It is weak；Prototype natural products FG can significantly affect APTT, and PT is not made significant difference, TT is had a significant effect.

【Embodiment 7】

Clotting factor and the detection of coagulation cofactor activity influence

7.1 materials and instrument

(1) sample：With embodiment 6.

(2) reagent：Plasma thromboplastin antecedent a (100IU/ branch) is ASSAYPRO Products；XIa chromogenic substrates S-2366 (25mg/ branch) is CHROMOGENIX Products；Hageman factor a (100IU/ branch), IXa (100IU/ branch), FX (100IU/ Branch), fibrin ferment (100IU/ branch), fibrin ferment chromogenic substrate CS-01 (38) (25mg/ branch), XIIa chromogenic substrates CS-31 (02) (25mg/ branch), IXa chromogenic substrates CS-51 (09) (25mg/ branch), VIIa chromogenic substrates SXa-11 (25mg/ branch), heparin it is auxiliary because Sub- II (HCII) (100 μ g/ branch), HeparinAnti-IIa kits, Factor IX detection kit are HYPHEN BioMed companies (method) product；Factor IX (f.VIII) (250IU/ branch), Bayer Healthcare LLC companies (U.S.) production Product.Dermatan sulfate (DS) is Sigma companies (U.S.) product；Chondroitin polysulfate (OSCS), Chinese pharmaceutical biological product calibrating There is provided；Low molecular weight heparin and FG, with the source of embodiment 6.DFG, low molecule amount FG mixtures, the methods described of embodiment 1 it Step 2 gained depolymerization product.

(3) instrument：MC-4000 coagulo meters, TICO Gmbh companies (moral) product；ELIASA, Bio Tek companies (U.S.) production Product；Chronolog-700 types platelet aggregation instrument (U.S.).

7.2 method

(1) endogenous factors X enzymes (Xase) inhibitory activity detects：The given the test agent and the μ of reference substance solution 30 of series concentration L mixed with 30 μ L f.VIII after according to f.VIII detection kit specification methods be added sequentially f.IXa reagents, FX reagents, F.Xa substrates (SXa-11) detect OD afterwards₄₀₅/min.According to literature method (Blood, 2006,107:3876-3882, similarly hereinafter) meter Calculate the IC that each sample suppresses f.Xase₅₀Value.

(2) antithrombase (IIa) Activity determination that HCII is relied on：The given the test agent of series concentration is separately added into 96 orifice plates And reference substance solution, 30 μ L HCII (1 μM), f.IIa (20IU/mL) and CS-01 (38) (2.5mg/mL) are then added sequentially, 37 DEG C of incubation 2min, detect OD₄₀₅/ min simultaneously calculates the IC that each sample suppresses fibrin ferment₅₀Value.

(3) antithrombase (IIa) Activity determination that AT is relied on：Be separately added into 96 orifice plates series concentration given the test agent and Reference substance solution, 30 μ L AT (0.25IU/mL), 30 μ Lf.IIa (24IU/mL) are added sequentially according to kit specification method And CS-01 (38) (1.25mM), detect OD₄₀₅/ min simultaneously calculates the IC that each sample suppresses IIa₅₀Value.

(4) the anti-factor Xa activity detection that AT is relied on：Given the test agent and the control of series concentration are separately added into 96 orifice plates Product solution, it is added sequentially 30 μ L AT (0.25IU/mL), 30 μ L Xa (24IU/mL) according to kit specification method and adds lustre to Substrate CS-11 (65) (1.25mM), detect OD₄₀₅/ min simultaneously calculates the IC that each sample suppresses IIa₅₀Value.

(5) anti-XIIa, XIa, IXa, VIIa Activity determination that AT is relied on：The tested of series concentration is separately added into 96 orifice plates Sample and reference substance solution, according to kit specification method be added sequentially 30 μ L AT (0.25IU/mL), 30 μ L XIIa or XIa or IXa or FX (24IU/mL) and corresponding chromogenic substrate (CS-31 (02) or S-2366 or CS-51 (09) or SXa- 111.25mM), detect OD₄₀₅/ min simultaneously calculates influence of each sample to XIIa, XIa, IXa, VIIa activity.

7.3 result

(1) endogenous Xase inhibitory activity：Compound a~f, i~k, n is shown in Table 5 (compound a~f suppression endogenous Xase activity see also accompanying drawing 8).Compound e~f, k and the IC for suppressing Xase as the FG and dFG compareed₅₀Value close to (about 8.9~ 13ng/mL)；Compound c~d, i~j also have more strongly active (IC₅₀It is worth about 49~103ng/ml), and compound a and b then live Property is weaker.Accordingly, for the pure Depolymerized fucose-containing glycosaminoglycan class compound of chemical constitution, the basic structure of Xase activity is effectively suppressed at least Need containing three or more trisaccharide construction units.LMWH also has Xase inhibitory activity, and DS activity is weaker.

(2) AT and HCII IIa inhibitory activity is relied on：Compound c~f, i~k, n and as control FG, LMWH, DS is respectively provided with the IIa inhibitory activity that certain HCII is relied on, and compound b activity is weaker；In the presence of HCII, compound a is not high IIa activity is not made significant difference in the range of about 5000ng/ml experimental concentration.

The influence of 5. compound as of table~f, compound i~k, compound n to blood coagulation (auxiliary) factor active

Remarks：^aSuppress the required drug concentration (ng/mL) of 50% endogenous Xase activity；^bSuppress 50% in the presence of HCII Drug concentration (ng/mL) needed for IIa activity；^cSuppress the required drug concentration (ng/mL) of 50%IIa activity in the presence of AT；^dAT is deposited In the required drug concentration (ng/mL) of lower suppression 50%Xa activity；^eDrug concentration>3000ng/mL；^fDrug concentration>3000ng/ mL

(3) in the presence of the anticoagulin activity AT for relying on AT, no more than about in 5000ng/ml concentration range, by Compound a~f, i~k, n is tried to the activity of the clotting factor such as prothrombin a, Xa, XIIa, XIa, IXa, VIIa without significantly Influence or only minor way (influences of such as compound f to IIa activity), and in the range of similar experimental concentration, LMWH is then Can potent suppression IIa, xa activity, also have to XIIa, IXa compared with strong inhibitory activity, can also influence XIa and VIIa activity.In addition, AT is present, and prototype FG also has a significant impact to IIa activity.

Obviously, in tested the compounds of this invention, compound c~f, i~k, n are respectively provided with potent Xase inhibitory activity；By In these compounds rely on HCII anti-IIa activity it is relatively weak, and in the presence of AT to IIa, Xa, XIIa, XIa, IXa, The activity of the clotting factor such as VIIa has no or only minor way, and therefore, these compounds are that have preferably to endogenous Xase The inhibitor of selectivity, this is clearly distinguishable from LMWH more extensive pharmacological activity.In addition, compound b have it is relatively weak HCII rely on anti-IIa activity, this should to its APTT extend activity it is related.

【Embodiment 8】

XII and platelet activation Activity determination

8.1 materials and instrument

(1) sample：Compound a~f.Embodiment 1~3 prepares gained.

(2) reagent：People's Quality Control blood plasma is Sigma companies (U.S.) product；Chondroitin polysulfate (OSCS), Chinese medicine biology Product calibrating is provided；Kallikrein (KK) chromogenic substrate CS-31 (02) (25mg/ branch).Low molecular weight heparin (LMWH), FG And dFG sources are the same as embodiment 7.

(3) instrument：ELIASA, Bio Tek companies (U.S.) product；Chronolog-700 types platelet aggregation instrument (U.S.).

8.2 method

(1) factor XI, plasma thromboplastin antecedent I Activation Activities detect：The sample and the μ of reference substance solution 30 of series concentration are separately added into 96 orifice plates L, people's Quality Control blood plasma that 30 μ L dilute 4 times with TS buffer solutions (0.02M, pH7.4Tris-HCl, 0.15M NaCl) is then added, 37 DEG C of incubation 2min, add 30 μ L CS-31 (02) (6mM, chromogenic substrate), detect OD₄₀₅/min。

(2) platelet activation Activity determination：Collection healthy volunteer's anticoagulation prepares platelet rich plasma (PRP) and anaemia Platelet-poor plasma (PPP), using Chronolog-700 type platelet aggregation instruments, prepared with turbidimetry detection physiological saline solution The blood platelet induced aggregation activity of sample solution described in series concentration.

8.3 result

(1) to the influence of XII activation：As shown in Figure 9, FG has similar OSCS XII Activation Activities, and dFG XII swashs It is activity to significantly reduce, but under higher concentration (>16 μ g/mL) remain to activate XII；And compound a~f in the range of experimental concentration Obvious XII Activation Activities are then not present.

(2) to the influence of human blood platelets activation：As shown in Figure 10,30 and 120 μ g/mL FG can significantly induce people's blood small Plate assembles (72.8% ± 4.7%, 68.3% ± 7.9% and 68.3% ± 6.2%, p<0.001vsCon.)；And 30,120 μ g/ Obvious blood platelet induced aggregation activity is not present in mL compound as~f, and (platelet aggregation rate is below 10%, with Con. groups Compared to without significant difference).

Obviously, compared with prototype FG, compound a~f be substantially not present the blood platelet induced aggregation related to prototype FG and XII Activation Activities, compared with Low Molecular Weight Compound dFG, these compounds do not influence XII activity.Therefore, as anticoagulating active Composition, these compounds show the obvious security advantages relative to prototype FG and Low Molecular Weight Compound dFG.

【Embodiment 9】

It is prepared by freeze-dried products

9.1 material

Compound j, according to the methods described of embodiment 3, using the FG in Stichopus Variegatus sources as starting material system It is standby to obtain.

9.2 prescription

9.3 preparation technology

Technical process：The compound j (500g) and NaCl (50g) of 10 times of recipe quantities are weighed, adds water for injection 10L to dissolve, After stirring and dissolving is complete, Mi Libo (MILLIPORE) ultrafiltration apparatus and molecular cut off is used to remove heat for 10kD milipore filters bag Source.Under gnotobasis, after 0.2 μm of membrane filtration is degerming, the filling cillin bottle in capacity 2mL of resulting solution, fills every bottle of 0.5mL Process monitoring loading amount is filled, half tamponade, puts in freeze drying box, is freezed by the freeze-drying curve of setting, tamponade, outlet, roll lid, It is qualified through examining, obtain finished product.

Freeze-drying process：By sample inlet, drop dividing plate temperature keeps 1h to -25 DEG C, is cooled to -45 DEG C, keeps 3h；Cold-trap drops To -50 DEG C, start to be evacuated to 40Pa.Start to distil：1h is at the uniform velocity warming up to -30 DEG C, keeps 2h；2h is at the uniform velocity warming up to -20 DEG C, 6h is kept, vacuum keeps 40~30Pa.It is dried again：2h is warming up to -5 DEG C, keeps 2h, and vacuum keeps 30~20Pa；0.5h 10 DEG C are warming up to, keeps 3h, vacuum keeps 30~20Pa；0.5h is warming up to 40 DEG C, keeps 4h, vacuum is evacuated to minimum.

Claims (17)

1. a kind of oligosaccharide compound and its pharmaceutically acceptable salt, it is characterised in that：The oligosaccharide compound by L-2, 4- di-sulfate bases fucose, D-Glucose aldehydic acid, D-2- deoxidation -2- acetylaminohydroxyphenylarsonic acid 4,6- di-sulfates base-galactolipin, 2, 5- is dehydrated talose or its sugar alcohol, osamine composition, shown in its chemical constitution such as formula (I),

In formula (I),

N is 0,1,2,3,4,5 or 6；

R is optionally-CH=O ,-CH (OH)₂、-CH₂OH、-CH₂NH₂、-CH₂NHR ' or-CH₂N(R’)₂, wherein R ' is unsubstituted C1-C6 straight or branched alkyls, unsubstituted C7-C12 aryl.

2. oligosaccharide compound as claimed in claim 1 and its pharmaceutically acceptable salt, it is characterised in that the oligosaccharide kind Compound is sterling compound chemically, refers to analyze using analytic type high productivity computing, shows poor inspection using versatility Device detection is surveyed, is calculated according to area normalization method, the purity of its single compound is not less than 95%；And the chemistry knot of single compound Structure can obtain¹H、¹³The confirmation of C and 2D NMR spectras detection.

3. oligosaccharide compound and its pharmaceutically acceptable salt as described in any one of claim 1~2, its feature exist In described pharmaceutically acceptable salt is alkali metal salt or alkali salt or organic ammonium salt.

4. oligosaccharide compound as claimed in claim 3 and its pharmaceutically acceptable salt, it is characterised in that described pharmacy Upper acceptable salt is its sodium salt, sylvite or calcium salt.

5. oligosaccharide compound and its pharmaceutically acceptable salt as described in any one of claim 1~2, its feature exist In shown in the chemical constitution such as formula (II) of the oligosaccharide compound：

In formula (II), n 0,1,2,3,4,5 or 6.

6. oligosaccharide compound as claimed in claim 5 and its pharmaceutically acceptable salt, it is characterised in that the formula (II) In shown oligosaccharide compound, n 2,3 or 4.

7. oligosaccharide compound and its pharmaceutically acceptable salt as described in any one of claim 1~2, its feature exist In shown in the chemical constitution such as formula (III) of the oligosaccharide compound：

In formula (III), n 0,1,2,3,4,5 or 6.

8. oligosaccharide compound as claimed in claim 7 and its pharmaceutically acceptable salt, it is characterised in that the formula (III) in oligosaccharide compound shown in, n 2,3 or 4.

9. oligosaccharide compound and its pharmaceutically acceptable salt as described in any one of claim 1~2, its feature exist In the oligosaccharide compound has the thrombin-inhibiting activity that endogenous X enzymes and/or heparin DPN I are relied on.

10. the preparation method of the oligosaccharide compound and its pharmaceutically acceptable salt described in any one of claim 1~9, Characterized in that, the preparation method includes the step shown in following route：

In the route,

Its step (1) is that the mixture 1 of fucosylated glycosaminoglycan class compound is dissolved in anhydrous hydrazine or hydrazine hydrate solution, Without or 0.5%~5% hydrazine sulfate in the presence of, reacted at a temperature of room temperature or 60~120 DEG C, be allowed to occur partially deacetylated Obtain the mixture 2 of fucosylated glycosaminoglycan class compound；Wherein, the mixture 1 of fucosylated glycosaminoglycan class compound In, x is the natural number that average is 40~100；R₁、R₂、R₃And R₄For H or-SO₃ ^-, and in bracket in trisaccharide construction unit, R₁=R₂ =R₃=R₄=-SO₃ ^-Construction unit in entire infrastructure unit proportion be equal to or higher than 60%；Fucosylated osamine gathers In the mixture 2 of saccharide compound, R₁、R₂、R₃And R₄And mixtures 1 of the x with fucosylated glycosaminoglycan class compound is determined Justice, R₅For H or-COCH₃, and with molar percent, H is in whole R₅Scope of the shared ratio 15% to 60% in group It is interior；

Its step (2) is that the mixture 2 of fucosylated glycosaminoglycan compound is dissolved in into water, adds HNO₂Solution adjusts reaction molten The pH value of liquid makes mixture 2 that deaminizating depolymerization reaction occur, obtains the mixed of fucosylated glycosaminoglycan class compound to 1~4.5 Compound 3；Wherein, in the mixture 3 of fucosylated glycosaminoglycan class compound, m is 0~20 natural number；R₁、R₂、R₃And R₄Together The definition of the mixture 1 of fucosylated glycosaminoglycan class compound, and the mixture 3 of fucosylated glycosaminoglycan class compound with Its hydrate forms is present；

Its step (3) is to use gel chromatography chromatography and/or ion-exchange chromatography and/or ultrafiltration by fucosylated glycosaminoglycan The mixture 3 of class compound is isolated and purified as oligosaccharide compound 4；Wherein, in oligosaccharide compound 4, n is 0~6 natural number；It is few Sugar compounds 4 exist with its hydrate forms；

Its step (4) is that oligosaccharide compound 4 is changed into compound 5, i.e. formula (I) chemical combination by reduction or reductive amination reaction Thing.

11. the preparation method of oligosaccharide compound as claimed in claim 10 and its pharmaceutically acceptable salt, its feature exists In the mixture 1 of, fucosylated glycosaminoglycan class compound be from sea cucumber Stichopus monotuberculatus, The rock of acquisition is extracted in Stichopus variegates Semper, Apostichopus japonicas body walls and/or internal organ Algae saccharification glycosaminoglycan extract.

12. containing effective anti-freezing dosage claim 1~8 any one described in oligosaccharide compound or its can pharmaceutically connect The salt and the pharmaceutical composition of pharmaceutically acceptable excipient received.

13. pharmaceutical composition as claimed in claim 12, it is characterised in that the formulation of described pharmaceutical composition is water for injection The freeze drying powder injection of solution or prepared before use into aqueous solution for injection.

14. oligosaccharide compound and its pharmaceutically acceptable salt are preparing treatment and/or prevention as described in 1~9 any one Application in the medicine of thrombotic diseases, it is characterised in that described oligosaccharide compound and its pharmaceutically acceptable salt be Endogenous factors X enzyme inhibitors.

15. oligosaccharide compound and its pharmaceutically acceptable salt are preparing treatment and/or prevention as described in 1~9 any one Application in the medicine of thrombotic diseases, it is characterised in that described oligosaccharide compound and its pharmaceutically acceptable salt be The thrombin inhibitor that heparin DPN I is relied on.

16. any one of claim 1~9 oligosaccharide compound and its pharmaceutically acceptable salt prepare treatment and/ Or the application in the medicine of prevention thrombotic diseases, it is characterised in that the thrombotic diseases are venous thronbosis, arterial blood Bolt is formed and/or ischemic angiocardiopathy and cerebrovascular disease.

17. the pharmaceutical composition described in any one of claim 12~13 is preparing treatment and/or is preventing thrombotic diseases Application in medicine, it is characterised in that the thrombotic diseases are venous thronbosis, Arterial thrombosis and/or ischemic Cardiovascular and cerebrovascular disease.