CN108285498A

CN108285498A - A kind of oligosaccharide compound and preparation method thereof and purposes inhibiting intrinsic coagulation factor X multienzyme complexes

Info

Publication number: CN108285498A
Application number: CN201710018307.8A
Authority: CN
Inventors: 赵金华; 李振国; 高娜; 吴明; 吴明一; 陈艳明; 赵龙岩; 务勇圣; 李姿; 肖创; 郑顺亮; 南志远; 周剑波; 许建萍; 周路坦; 郭亚芳; 秦红波; 刘吉开
Original assignee: Mudanjiang Youbo Pharmaceutical Co Ltd; Jiuzhitang Co Ltd
Current assignee: Mudanjiang Youbo Pharmaceutical Co Ltd; Jiuzhitang Co Ltd
Priority date: 2017-01-10
Filing date: 2017-01-10
Publication date: 2018-07-17
Anticipated expiration: 2037-01-10
Also published as: CN108285498B

Abstract

The mixture and its pharmaceutically acceptable salt of the present invention relates to a kind of purifying oligosaccharide compound or its homologous compound with antithrombotic acitivity and the preparation method of the mixture of the oligosaccharide compound or its homologous compound, the pharmaceutical composition of mixture containing the oligosaccharide compound or its homologous compound and they as endogenous factors X enzymes (Xase) inhibitor prevent and/or the medicine preparation for the treatment of thrombotic diseases in purposes.

Description

It is a kind of inhibit intrinsic coagulation factor X multienzyme complexes oligosaccharide compound and its preparation Method and purposes

Technical field

The invention belongs to pharmaceutical technology fields, and in particular to it is a kind of with the purifying oligosaccharide compound of antithrombotic acitivity or its The mixture and its pharmaceutically acceptable salt of homologous compound, preparation method and use.

Background technology

Thrombotic diseases including Ischemic Stroke, coronary heart disease, venous thromboembolism etc. are most important mankind's lethals The cause of disease.The antithrombotic reagents such as fibrinolytic, anti-freezing, antiplatelet are the basic means of clinical medicine prevention and treatment thrombotic diseases, But there is general character defect in existing antithrombotic reagent：Hemorrhagic tendency and severe haemorrhage are dangerous.Reduce hemorrhagic tendency and bleeding danger Danger is the core objective of novel antithrombotic reagent research and development.In recent years the study found that intrinsic coagulation pathway and pathologic thrombus Formed it is closely related, and may not be hemostatic function institute it is necessary, therefore, intrinsic coagulation factor inhibitors have become low bleeding and incline The emphasis direction studied to antithrombotic reagent.Wherein, the preclinical and clinical test of Hageman factor a, XIa and IXa inhibitor Research is unfolded successively.Endogenous factors X multienzyme complexes (Xase) are the enzyme activity of the last position and speed limit on intrinsic coagulation pathway Site, selective depressant have important potential clinical value.

Fucosylated glycosaminoglycan (Fucosylated Glycosaminoglycan, FG) is to be detected in spine skin so far to move A kind of glycosaminoglycan with special chemical structure and pharmacological activity in object, has similar chondroitin sulfate main chain, and deposit In the glycosaminoglycan that fucosido (Fucosyl, Fuc) side chain replaces similar to object (Yoshida et.al, Tetrahedron Lett,1992,33:4959-62；et.al,J Biol Chem,1996,271:23973-84).Studies have shown that day Right FG has a potent anticoagulating active, and the main (Thromb related to endogenous Xase activity is inhibited of its anticoagulant mechanism Haemost,2008,100:420-8；J.Biol.Chem.,1996,271:23973-84).However, there is also extensive by natural FG And contradictory pharmacological action, including induced platelet aggregation and cause recycle platelet count reduce, activation XII and lead to low blood Pressure etc. (Thromb Haemost, 1988,59:432-4；Thromb Haemost,2010,103:994-1004).It is administered systemically Under the application value of FG be thus limited.The FG of appropriate depolymerization can reduce induced platelet aggregation activity (Thromb Haemost, 1991,65:369-73), to improve the selectivity that it inhibits endogenous factors Xase.

The chemical depolymerization to natural FG and the chemistry and pharmacological characteristics of depolymerization product carry out system to the present inventor before this Research.For example, 101724086 B of Chinese invention patent CN disclose the peroxidative depolymerization method of FG, gained depolymerization product can press down Thrombosis processed and hemorrhagic tendency significantly reduce, but this method products therefrom is difficult to further detach because of its end structure complexity Purifying.

It has been found that conventional method usually also contains molecular weight distribution from the natural FG that the extraction of echinoderm body wall obtains With glucan similar in natural FG, fucosan (Fucan) and/or containing the polysaccharide compound of aminohexose.These mix in Polysaccharide constituents in natural FG are difficult to clean by the even ion-exchange chromatography removal of gel chromatography, ultrafiltration.The present invention The comparative studies that people carries out is shown, using technical method described in document above, the natural FG of extraction gained out of echinoderm body Usual also other polysaccharide constituents containing mass ratio about 10%~20% in extract.Since these polysaccharide constituents can quilt Peroxidative depolymerization, therefore the oligosaccharides composition of the peroxidative depolymerization product of natural FG is considerably complicated.

103214591 A of Chinese invention patent CN disclose the deacylation deamination depolymerization of FG, and this method alternative is split D- acetylamino galactosamines (GalNAc)-(β 1 → 4)-D-Glucose aldehydic acid (D-GlcA) glycosidic bond is solved, acquisition contains reproducibility End is the depolymerization product of 2,5- dehydrating tower sieve glycosyls (anTal), and the oligosaccharides homologue in gained depolymerization product is by 3m Monosaccharide residue composition (m is natural number, is herewith defined hereinafter).Deacylation deamination depolymerization is unable to depolymerization and mixes the Portugal in natural FG Glycan and fucosan, after deacylation deamination disaggregation processing, these polysaccharide impurity for being unable to depolymerization can be easily by solidifying Glue chromatography or ultrafiltration remove.The deacylation deamination depolymerization product of natural FG has more regular structure feature, contained oligosaccharides It can further be isolated and purified (CN 104370980A).Homology obtained by the natural FG deacylations deamination depolymerization of studies have shown that In oligosaccharides compounds, nine sugared (nonasaccharide, NSac) are that have the potent active minimal structure segments of inhibition Xase (Proc Natl Acad Sci USA.2015；112(27):8284-9).

The authorized application for a patent for invention CN of the present inventor 201310099800 discloses β-elimination depolymerization of natural FG Method, this method are alkali process FG carboxylates and selective splitting D-GalNAc- (β 1 → 4)-D-GlcA glucosides in nonaqueous solvents Key is undersaturated Δ thus to obtain non-reducing end^4,5The depolymerization product of hexuronic acid base (Δ UA), the depolymerization product are one The mixture of series of oligosaccharides compound.Similarly, it since β-elimination depolymerization has good glycosidic bond selectivity, cannot solve Gather the other types of polysaccharide compound contained in natural FG extracts, therefore is conducive to remove the non-FG classes in FG extracts Polysaccharide impurity component.

But not with depolymerization product obtained by deacylation deamination depolymerization described in 103214591 A of application for a patent for invention CN Together, although the non-reducing end of β described in CN 201310099800-null method depolymerization product is relatively regular, the group at reproducibility end At relative complex：The reducing end glycosyl had both included "-D-GalNAc ", also included the " -4-D- of L-Fuc- (α 1 → 3) substitutions GlcA”.Due to reduction end structure it is relative complex, from the depolymerization product separation obtain purifying oligosaccharides there are larger technology hardly possible Degree, therefore be typically chosen directly using existing depolymerization product as a mixture.

Pharmaceutical field technical staff is readily appreciated that, the chemical constitution for purifying oligosaccharides is pure, quality control level higher, thus There can be higher application value.For the depolymerization product of natural FG, the regular degree of end structure can be notable It influences to prepare the technical feasibility for purifying oligosaccharide compound.

Theoretically, β-elimination depolymerization alternative cracks "-D-GalNAc- (β 1 → 4)-D-GlcA- " glycosidic bond, institute The reducing end under neutral for obtaining depolymerization product should be "-D-GalNAc " residue, and the oligosaccharides homologue in gained depolymerization product is general It should be made of 3m monosaccharide residue.The β of natural FG disclosed in application for a patent for invention CN 201310099800-elimination depolymerization production There are the oligosaccharide compound that a certain amount of reducing end is "-[L-Fuc- (α 1 → 3)]-D-GlcA- " in object, show in its item It, should be there is also certain side reaction, in particular, the reducing end of its depolymerization product during the beta-elimination reaction carried out under part Glycosyl receive a degree of destruction.

By the further research of the beta-elimination reaction condition to natural FG, the inventors discovered that will be natural using reducing agent The glycosyl of the reducing end under neutral of FG is reduced into sugar alcohol base, and carboxylic esterification product can be described in patent ZL 201310099800 Occur beta-elimination reaction in alkaline nonaqueous solvents, but it with "-D-GlcA- " residue is still to contain in its depolymerization product a certain amount of The oligosaccharide compound of originality end is analyzed according to the HPGPC of depolymerization product and is calculated with area normalization method, is with-D-GlcA The oligosaccharide compound of reducing end under neutral can account for about the 10%~30% of oligosaccharide compound total amount, result and the end under the same terms Hold the depolymerization product of unreduced natural FG approximate.The glycosyl of the reducing end under neutral of natural FG is restored saccharogenesis by its studies have shown that Alcohol has no effect on the progress of the beta-elimination reaction of FG carboxylates, but can not reduce the destruction of the reducing end glycosyl of depolymerization product.

Directly add in the alkaline nonaqueous solvents that application for a patent for invention CN 201310099800 carries out described in beta-elimination reaction Enter reducing agent (such as sodium borohydride), as a result, it has been found that the beta-elimination reaction of natural FG carboxylates can be normally carried out.It is surprising that The reducing end under neutral of its products therefrom substantially on be acetylamino galactosamine alcohol radical (- 3-D-GalNAc-ol)；And with " -3-D- GlcA (- ol) " residues, which are the content of the oligosaccharide compound of reducing end under neutral, then to be significantly reduced, in terms of HPGPC area normalization methods It calculates, content can be below about 5%, and even lower than HPGPC detections limit, as a result, the homology oligosaccharides in its gained depolymerization product More regular terminal chemical structure feature can be had by closing object：The homology oligosaccharide compound is made of 3m monosaccharide residue； The glycosyl of its non-reducing end is " L-Fuc- (α 1-3)-Δ UA-1- " and reproducibility end glycosyl is " -3-D-GalNAc-ol ".

Using in the alkaline non-aqueous organic solvent in the presence of reducing agent beta-elimination reaction and chromatographic separation technology, this hair It is novel " purifying oligosaccharide compound " that a person of good sense isolates and purifies out a series of chemical constitutions from the β of FG-elimination depolymerization product for the first time. There are common chemical structure characteristics to be for the purifying oligosaccharide compound：The purifying oligosaccharides is made of 3m monosaccharide residue, Its non-reducing end structure is " L-Fuc- (α 1-3)-Δ UA-1- ", reproducibility end glycosyl is " -3-D-GalNAc-ol ".

The present inventor has found that the oligosaccharides containing 3m monosaccharide residue can pass through " the peeling of reducing end after further research Reaction " loses a monosaccharide residue, thus generates the oligosaccharides of " containing (3m-1) a monosaccharide residue ", and the reducing end of these oligosaccharides is “-D-GlcA”.By the further investigation of the beta-elimination reaction condition to FG carboxylates, the present inventor is also surprised to be found：

A small amount of highly basic (such as NaOH) aqueous solution is added in non-aqueous alkaline reaction solution described above, it is described to contain 3m Monosaccharide residue and " peeling reaction " is easily occurred with the FG oligosaccharides that " -3-D-GalNAc " is reducing end under neutral and loses end "-D- GalNAc " glycosyls.It is surprising that the reducing end under neutral generated by " peeling is reacted " is the oligosaccharides of "-D-GlcA " Object (it contains (3m-1) a monosaccharide residue) is closed but " unexpectedly " to be difficult to happen further " peeling reaction ".Therefore, change Into alkali process condition of the FG carboxyl esters in nonaqueous solvents, D-GalNAc- (β 1 → 4)-D-GlcA can be cracked in β-null method After glycosidic bond, further depolymerization product is set to lose "-the 3-D-GalNAc " of end by " peeling react " of reducing end under neutral Glycosyl, it is possible thereby to obtain the oligosaccharides homologue with novel and regular chemical structure characteristic.

HPGPC chromatography and NMR structural analyses are shown, in anhydrous organic solvent, highly basic processing FG carboxylates are allowed to Occur " β-elimination depolymerization ", a small amount of strong alkali aqueous solution is then added into reaction solution makes β-elimination depolymerization product further occur " peeling reaction ", homology oligosaccharide compound contained by depolymerization product can have fairly regular chemical constitution, i.e.,：The homology Oligosaccharide compound is made of (3m-1) a monosaccharide residue；The glycosyl of the non-reducing end of the homology oligosaccharide compound is " L- Fuc- (α 1-3)-Δ UA-1- ", and its reducing end glycosyl is then C3 that there are " -4-D-GlcA " that L-Fuc replaces.

Oligosaccharides homologue of the natural FG carboxylates through " β-elimination " and " peeling reaction " processing gained has more regular Chemical structure characteristic, therefore be easy to be separated further purifying and obtain a series of purifying oligosaccharide compounds.The serial purification The structural features of oligosaccharide compound are：The oligosaccharide compound contains (3m-1) a monosaccharide residue；Its non-reducing end is equal For " L-Fuc- (α 1-3)-Δ UA-1- "；Its reducing end is " -4- [Fuc- (α 1-3)]-D-GlcA ".

In summary as it can be seen that the present invention can be obtained respectively by further improving β-conditions to release of natural FG carboxylates Obtain the more regular FG depolymerization products of compound structure (especially reducing end glycosyl structure)：First, be reducing end be "-D- The depolymerization product of GalNAc-ol ", second, being the depolymerization product that reducing end is "-D-GlcA ".Since the end of depolymerization product is tied Structure is more regular, and present invention firstly discloses the purifying oligosaccharides from natural FG obtained is detached from these depolymerization products Close object.By the structural modification of the specified chemical group to these purifying oligosaccharides, the present invention further discloses different series FG oligosaccharide compounds derivative.

In addition, by FG oligosaccharide compounds anticoagulating active, auxiliary to the inhibitory activity and its heparin of endogenous factors Xase Antithrombase (i.e. factor Xa IIa) active structure activity study that factor II (HC-II) is relied on, the present inventor also send out It is existing：

It is potent for reducing end is "-D-GalNAc-ol ", the FG oligosaccharides homologues containing 3m monosaccharide residue It is nine sugared (NSac) to inhibit the simple structure segment of intrinsic coagulation factor Xase；It is "-D-GlcA " for reducing end, contains (3m- 1) for the FG oligosaccharides homologues of a monosaccharide residue, the simple structure segment of potent inhibition intrinsic coagulation factor Xase is Eight sugared (octasaccharide, OSac)；All there is also the anticoagulations that the HC-II of varying strength is relied on for purifying oligosaccharide compound Enzymatic activity and external anticoagulating active, and the pharmacology with the arteriovenous thrombosis in Inhibition test animal pathological model is lived Property.

It is coagulated since the oligosaccharide derivative obtained by purifying oligosaccharide compound of the present invention and its structural modification all has inhibition Blood factor activity and significant anti-freezing and antithrombotic acitivity, therefore, these oligosaccharide compounds, which have, potentially to be prevented and/or controls Treat the application value of thrombotic diseases.

Generally, present invention firstly discloses obtained by " β-elimination depolymerization " or " β-elimination depolymerization+end peeling reaction " The technical method of chemical constitution (especially reducing end glycosyl structure) more regular natural FG depolymerization products is obtained, and passes through institute State the FG oligosaccharides homologues of the homogeneity structure of method acquisition.The present invention also makes public for the first time with non-reducing end unsaturation hexose The FG oligosaccharide compounds of the purifying of aldehydic acid based structures, the derivative of its structural modification and their mixture.Due to the widow Sugar compounds have anti-freezing and antithrombotic acitivity, the invention also discloses the oligosaccharide compound and its mixture prevent and/ Or the purposes in the medicine preparation for the treatment of thrombotic diseases.

Invention content

There is anti-freezing and the purifying oligosaccharide compound of antithrombotic acitivity, its preparation side the purpose of the present invention is to provide a kind of Method, the pharmaceutical composition containing the purifying oligosaccharide compound or oligosaccharide mixture and its pharmaceutically acceptable salt, and The oligosaccharide compound, oligosaccharide mixture and its pharmaceutical composition are in preventing and/or treating the medicine preparation of thrombotic diseases Purposes.

Present invention firstly provides one kind having antithrombotic acitivity, especially has the work for inhibiting intrinsic coagulation factor Xase Property, oligosaccharide compound and its pharmaceutically acceptable salt, the oligosaccharide compound have formula (I) shown in general formula structure：

In formula (I),

R₁、R₂、R₃、R₄、R₅Optionally mutually independent-H or-SO₃H；

R₆Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

R₇Optionally-H ,-SO₃H, C2-C5 acyl groups；

R₈Optionally group shown in formula (II), formula (III) or formula (IV)：

In its formula (II), formula (III) and formula (IV),

R₁、R₂、R₃、R₄、R₅、R₆And R₇It is defined above；

R₉And R₁₀Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

R₁₁Optionally-NHR₁₂Or-OR₁₃, wherein R₁₂And R₁₃Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

N is optionally 0 or 1~8 natural number.

Oligosaccharide compound of the present invention with general formula structure shown in formula (I) refers to " purifying oligosaccharide compound ".One As for, the purity of " purifying oligosaccharide compound " is not less than 95%.For example, using analytic type high productivity computing Method (HPGPC) is analyzed, for example, showing poor inspection using Agilent high performance liquid chromatograph and gel chromatographic columns, and using universal It surveys device (RID) to detect, be calculated according to area normalization method, the purity of the purifying oligosaccharide compound is generally not less than 95%.

In the oligosaccharide compound of formula of the present invention (I) structure, a kind of preferred oligosaccharide compound is R₈For formula (II) The compound of shown group, that is, the oligosaccharide compound has general formula structure shown in formula (V)：

In formula (V),

R₁、R₂、R₃、R₄、R₅Optionally mutually independent-H or-SO₃H；

R₆Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

R₇Optionally-H ,-SO₃H, C2-C5 acyl groups；

R₉Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；And

N is optionally 0 or 1~8 natural number.

In the compound of more preferably formula (V) structure, R₁=-H；R₂=R₃=R₄=R₅=-SO₃H；

In another more preferably compound of formula (V) structure, R₁=R₃=R₄=R₅=-SO₃H；R₂=-H.

In the oligosaccharide compound of formula of the present invention (I) structure, another preferred oligosaccharide compound is its R₈For formula (III) compound of group shown in, i.e., the described oligosaccharide compound have general formula structure shown in formula (VI)：

In formula (VI),

R₁、R₂、R₃、R₄、R₅Optionally mutually independent-H or-SO₃H；

R₆Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

R₇Optionally-H ,-SO₃H, C2-C5 acyl groups；

R₁₀Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；And

N is optionally 1~8 natural number.

Similarly, in the compound of more preferably formula (VI) structure, R₁=-H；R₂=R₃=R₄=R₅=-SO₃H；

In another more preferably compound of formula (VI) structure, R₁=R₃=R₄=R₅=-SO₃H；R₂=-H.

In the oligosaccharide compound of formula of the present invention (I) structure, another preferred oligosaccharide compound is its R₈For formula (VII) compound of group shown in, i.e., the described oligosaccharide compound have general formula structure shown in formula (VII)：

In formula (VII),

R₁、R₂、R₃、R₄、R₅Optionally mutually independent-H or-SO₃H；

R₆Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

R₇Optionally-H ,-SO₃H, C2-C5 acyl groups；

R₁₁Optionally-NHR₁₂Or-OR₁₃, wherein R₁₂And R₁₃Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；And

N is optionally 0 or 1~8 natural number.

Similarly, in the compound of more preferably formula (VII) structure, R₁=-H；R₂=R₃=R₄=R₅=- SO₃H；

In another more preferably compound of formula (VII) structure, R₁=R₃=R₄=R₅=-SO₃H；R₂=-H.

It should be readily apparent to one skilled in the art that the technical difficulty for isolating and purifying oligosaccharide compound of the present invention can be with oligosaccharides The increase of the degree of polymerization and increase.Therefore, the oligosaccharides chemical combination of the structure shown in the above-mentioned formula (I), (V), (VI) or (VII) of the present invention In object, preferred oligosaccharide compound is that n is optionally 1,2,3 or 4.

There are sulfate group substituent group and/or free carboxies in purifying oligosaccharide compound of the present invention, therefore can be with Pharmaceutically acceptable inorganic and/or organic ion is combined into salt.In general, oligosaccharide compound of the present invention is pharmaceutically Acceptable salt may optionally be alkali metal salt, alkali salt or organic ammonium salt.

The pharmaceutically acceptable salt of preferred oligosaccharide compound of the present invention is sodium salt, sylvite or calcium salt.

It should be readily apparent to one skilled in the art that by the purifying oligosaccharide compound of the present invention of homologue form, for example, will Compound shown in the homologue or (VII) of compound shown in the homologue of compound or formula (VI) shown in formula (V) described above Homologue mix, you can obtain with specific structure type homology oligosaccharide compound mixture.Particularly, according to The preparation method of this specification the compounds of this invention described hereinafter, the present invention can also pass through specific technical method Learn the FG oligosaccharide mixtures of structure (especially reducing end glycosyl structure type) more regular homologue form.

For this purpose, the present invention also provides one kind having antithrombotic acitivity, especially has and inhibit intrinsic coagulation factor X enzymes Active oligosaccharide mixture and its pharmaceutically acceptable salt, the oligosaccharide mixture is by above-mentioned formula (I) oligosaccharide compound Homologue forms；Also, the R of formula (I) oligosaccharide compound in the composition oligosaccharide mixture₈Be all group shown in formula (II), Or it is all group shown in formula (III) or is all group shown in formula (IV).Specifically, with molar ratio computing, R₈For formula (II) Formula (I) the structure oligosaccharide compound of shown group ratio shared in the mixture is not less than 95%；Or R₈For formula (III) formula (I) oligosaccharide compound of group shown in ratio shared in the mixture is not less than 95%；Or R₈For formula (IV) formula (I) oligosaccharide compound of group shown in ratio shared in the mixture is not less than 95%.

In oligosaccharide mixture of the present invention, a kind of preferred oligosaccharide mixture is that have general formula shown in formula above (V) The mixture of the homology oligosaccharide compound of structure.A kind of oligosaccharide mixture more preferably of the present invention is knot shown in formula (V) The mixture of the homology oligosaccharide compound of structure, and its R₁=-H；R₂=R₃=R₄=R₅=-SO₃H；It is another more preferably In the mixture of the homology oligosaccharide compound of structure shown in formula (V), R₁=R₃=R₄=R₅=-SO₃H；R₂=-H.

In oligosaccharide mixture of the present invention, another preferred oligosaccharide mixture is then with general formula shown in formula (VI) The mixture of the homology oligosaccharide compound of structure.Similarly, the homology oligosaccharides chemical combination of the structure shown in more preferably formula (VI) In the mixture of object, R₁=-H；R₂=R₃=R₄=R₅=-SO₃H；And another more preferably structure shown in formula (VI) In the mixture of homology oligosaccharide compound, R₁=R₃=R₄=R₅=-SO₃H；R₂=-H.

In oligosaccharide mixture of the present invention, another preferred oligosaccharide mixture is then with logical shown in formula (VII) The mixture of the homology oligosaccharide compound of formula structure.Similarly, a kind of homology oligosaccharides of more preferably structure shown in formula (VII) In the mixture of compound, R₁=-H；R₂=R₃=R₄=R₅=-SO₃H；And knot shown in another more preferably formula (VII) In the mixture of the homology oligosaccharide compound of structure, R₁=R₃=R₄=R₅=-SO₃H；R₂=-H.

For the oligosaccharide compound and oligosaccharide mixture of the present invention described above, the present invention furthermore provides describedization Close the preparation method of object and its mixture.

First, the oligosaccharide compound and its pharmaceutically acceptable salt of structure shown in a kind of formula (I) of present invention offer Preparation method.The preparation method be with the fucosylated glycosaminoglycan in echinoderm source (FG) be start material, appoint Select depolymerization with the following method：

Be esterified FG carboxyls, and make in anhydrous organic solvent in the presence of highly basic and reducing agent FG carboxyl esters occur " β-disappears Except reaction " and depolymerization, and-D- acetylamino galactosamine the bases (- D-GalNAc) of the reducing end of depolymerization product are reduced agent also Original is at sugar alcohol (- D-GalNAc-ol), thus to obtain the mixture of homology oligosaccharide compound；Or

FG carboxyls are esterified, and make FG that " beta-elimination reaction " and depolymerization occur in anhydrous organic solvent in the presence of highly basic, Then make depolymerization product that the-D-GalNAc that end group " peeling is reacted " loses reducing end occur by adding alkaline aqueous solution, obtain Reducing end is-mixture of the homology oligosaccharide compound of D-Glucose aldehydic acid base (- D-GlcA).

By the homology oligosaccharides obtained by " β-elimination depolymerization and the end group reduction " or " β-elimination depolymerization and peeling are reacted " The mixture for closing object obtains required purifying oligosaccharide compound by isolating and purifying and optionally carrying out structural modification.

Particularly, the preparation method of oligosaccharide compound of the present invention be it is a kind of prepare with structure shown in formula (I) and its R₈For the method for the oligosaccharide compound of group shown in formula above (II).It is so-called " to there is structure and its R shown in formula (I)₈For formula (II) The oligosaccharide compound of shown group " actually is equivalent to the oligosaccharide compound of structure shown in formula defined above (V).

The oligosaccharide compound preparation method of structure shown in the formula (V) is " β-elimination depolymerization+end group reduction " method, the side Method is to make the FG of carboxylic esterification that " beta-elimination reaction " occur and crack in anhydrous organic solvent in the presence of highly basic and reducing agent Its " D-GalNAc- (β 1 → 4)-GlcA " glycosidic bond, and the reducing end-D-GalNAc of its depolymerization product be reduced agent be reduced into- D-GalNAc-ol is then pure by detaching thus to obtain the mixture of end structure regular homology oligosaccharide compound relatively Change and structural modification optionally is carried out to obtain required purifying oligosaccharide compound to specified substituent.Its specific steps includes：

(a) natural FG is changed into quaternary ammonium form, and in organic solvent, it will be on the D-GlcA in the FG quaternary ammonium salts Carboxyl be completely or partially converted into carboxylate；

(b) in the organic solvent there are reducing agent, made with the FG quaternary ammonium salts of highly basic processing step (a) carboxylic esterification Generation β-elimination depolymerization, and-the D-GalNAc of the reducing end of depolymerization product is made to be reduced into-D-GalNAc-ol, thus to obtain The mixture of end structure regular homology oligosaccharide compound relatively；

(c) mixture of homology oligosaccharide compound obtained by step (b) is changed into alkali metal salt, and in aqueous solution In, the carboxylate of basic hydrolysis homology oligosaccharide compound obtains the mixture of the homology oligosaccharide compound containing free carboxy；

(d) the homology oligosaccharide compound contained by oligosaccharide mixture obtained by chromatography purification procedures (c) is used；

(e) further structural modification optionally is carried out to the purifying oligosaccharide compound obtained by step (d).

Step (a)~(e) is as shown in route map 1：

Route map 1

In route map 1：

Natural FG 1 are the natural FG in echinoderm source, are a series of mixing of homology polysaccharide compounds Object；

FG ammonium salt 2 are the FG of quaternary ammonium form；

FG ester ammonium salt 3 are the products that the carboxyl in FG on D-GlcA is partly or entirely esterified, Exist with quaternary ammonium form；

DFG ester ammonium salt 4 are the depolymerization products that FG carboxylates occur that beta-elimination reaction generates, also Former end glycosyl-D-GalNAc can be present in the reduction of the reducing agent in reaction solution and generate reduced dFG ester ammonium salt 5；

Reduced dFG 6 are with existing for alkali metal salt 5 carboxylate hydrolysate；Depolymerization product 4,5 and 6 It is the mixture of homology oligosaccharide compound；

Purified oligosaccharide 7 are that the purifying oligosaccharides obtained is detached from depolymerization product 6；And it purifies few Sugar 8 is then the purifying oligosaccharides of 7 gained for optionally carrying out substituent structure modification.

In chemical constitution described in route map 1, R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₉It is defined with formula above (I) with n；X is equal It is worth the natural number in about 40~80 ranges；Y is the natural number in about 0~15 range；- COOX is carboxylate and/or carboxylic acid Quaternary ammonium salt.

In route map 1, by the chemical constitution of shown natural FG as it can be seen that it has the backbone structure similar to chondroitin sulfate, And there are the L-Fuc side chain substituents of Sulfation.In general, natural FG be appreciated that serve as reasons " trisaccharide structural unit "- 4)-[L-FucS- (α 1-3)]-D-GlcA- (β 1-3)-D-GalNAcS- (β 1- } sequently connected polysaccharide compound (formula In, FucS and GalNAcS indicate the Fuc of Sulfation and the GalNAc of Sulfation respectively).In general, natural FG is usual Contain about 40~80 trisaccharide structural units of mean value (in short, the mean value of x is in about 40~80 ranges).

The existence form of natural FG salt depends on the process route of its extraction purification.In general, the FG is with alkali metal Or the form of alkali salt (such as its sodium salt, sylvite or calcium salt etc.) exists.To realize in the organic solvent in subsequent step Chemical reaction, the step (a) converts natural FG to quaternary ammonium form (the 2 of route map 1).

The quaternary ammonium salt conversion of natural FG optionally can use technical method well known in the art to carry out.For example, the season Quaternary salt deposit method can be used in ammonium salt conversion, and the method was added into the aqueous solution of the alkali or alkaline earth metal salt of FG The organic ammonium salt compounds of amount, the FG quaternary ammonium salts of water-insoluble, which are consequently formed, to be easily settled out from the aqueous solution Come；In addition, ion exchange resin can also be used is exchanged into H-type FG by the alkali metal salt of FG or alkali salt, alkali is then used Property organic ammonium neutralize the H-type FG and also can get FG quaternary ammonium salts.

The step (a2) as shown in route map 1 is completely or partially to turn the carboxyl in FG quaternary ammonium salts (2) on D-GlcA residues Turn to carboxylate (3).The purpose of the carboxyl esterification reaction of FG is to make it easy to that beta-elimination reaction occurs.Existing for carboxy form The more difficult generation β-elimination depolymerization reactions of GlcA, and its carboxylate then due to the electronic effect of ester group make it easy to occur β-elimination it is anti- It answers.

In general, the carboxyl esterification reaction of the GlcA in the FG is in organic solvent, such as dimethylformamide (DMF) or the in the mixed solvent of DMF and lower alcohol, ketone and/or ether, make the halogenated of GlcA carboxyls in the FG and stoichiometry Hydrocarbon reaction can be readily available the required FG carboxylates of different degree of esterification.The degree of esterification of the FG carboxylates refers to ester Change the ratio of the molal quantity of the carboxylate generated after reaction and the molal quantity of the free carboxy before esterification；The halogenated hydrocarbons It may optionally be and be not limited to：C1-C6 linear chain or branched chains, saturation or unsaturation, substituted or unsubstituted aliphatic hydrocarbyl；Substitution or not Substituted C7-C12 aromatic hydrocarbyls etc..The applicant discloses FG in another application for a patent for invention CN 201110318704.X The preparation method of carboxyl ester derivative, the full text of the application for a patent for invention is attached in this specification by this as a reference.

Step (b) shown in route map 1 is to make FG carboxylates that β-elimination depolymerization (b1) occur to obtain depolymerization product 4, and depolymerization Reducing end-the D-GalNAc of product 4 can be reduced agent and be reduced into-D-GalNAc-ol (b2) and obtain depolymerization product 5.

As it was noted above, CN 201310099800 discloses β-elimination depolymerization of natural FG, the method can obtain Non-reducing end is the depolymerization product of undersaturated Δ UA, but the structure type of the reducing end glycosyl of its depolymerization product is relative complex： The reducing end glycosyl had both included "-D-GalNAc ", also included "-the D-GlcA " of L-Fuc substitutions.Due to reducing end structure type Complexity, from the depolymerization product separation obtain purifying oligosaccharides it is more difficult, therefore be typically chosen directly use form of mixtures Existing depolymerization product.Theoretically, β-elimination depolymerization alternative cracks " D-GalNAc- (β 1 → 4)-D-GlcA " glucosides The reducing end of key, gained depolymerization product is "-D-GalNAc " residue.Using β described in CN 201310099800-elimination depolymerization There are the oligosaccharide compound that a certain amount of reducing end is "-D-GlcA " in the depolymerization product of the natural FG prepared, show in its institute State under reaction condition that there is also certain side reactions, in particular, the glycosyl of the reducing end of a certain amount of depolymerization product receives brokenly It is bad.The full text of patent application CN 201310099800 is attached in this specification by this as a reference.

It should be readily apparent to one skilled in the art that the chemical constitution of purifying oligosaccharides is pure, quality control level higher, thus can With higher application value.By the further research of the beta-elimination reaction condition to natural FG, the present inventor has found in surprise：

The glycosyl of the reducing end of natural FG is reduced by sugar alcohol, carboxylic esterification product using reducing agent (such as sodium borohydride) Beta-elimination reaction can occur in alkaline nonaqueous solvents, but be also with-D-GlcA according to the HPGPC atlas analysis of depolymerization product The oligosaccharide compound of originality end can account for about 10%~30% (area normalization method calculating) of oligosaccharide compound total amount, result It is approximate with the unreduced depolymerization product of natural FG in the end under the same terms.This result shows that, end reduction have no effect on FG The progress of the beta-elimination reaction of carboxylate.

Further studies have shown that is directly added into reducing agent (such as sodium borohydride), natural FG into the alkaline nonaqueous solvents The beta-elimination reaction of carboxylate can be also normally carried out.Unexpectedly, the reducing end under neutral of products therefrom substantially on be- 3-D-GalNAc-ol, and reducing end be -3-D-GlcA-ol oligosaccharide compound content it is little (content can be below about 5%, very To less than HPGPC detections limit).This result shows that, the reduction of the end of depolymerization product is it is possible to prevente effectively from reduction under alkaline condition Glycosyl is held to destroy, it is possible thereby to realize that the reduction end structure of depolymerization product is relatively regular.

Those skilled in that art are readily appreciated that, since there are reducing agent (such as sodium borohydride), β-elimination solution in reaction solution Consor at the terminal saccharide of depolymerization product can be reduced into sugar alcohol rapidly.On the one hand, reducing end glycosyl is reduced into sugar alcohol Have no effect on the generation that beta-elimination reaction further occurs for hexuronic acid ester；On the other hand, the terminal saccharide quilt in depolymerization product Destruction and degradation it is possible to prevente effectively from the reducing end glycosyl under alkaline condition are reduced into after sugar alcohol.Therefore, exist in reducing agent Lower β-elimination the depolymerization for carrying out FG carboxylates can obtain the more regular depolymerization product of chemical constitution.

" chemical constitution is more regular " refers to：(1) homology oligosaccharide compound contained in depolymerization product is by 3m Monosaccharide residue forms；(2) the non-reducing end glycosyl of the homology oligosaccharide compound be " L-Fuc- (α 1-3)-Δ UA-1- " and Its reproducibility end glycosyl is " -3-D-GalNAc-ol ".

It is anti-that the β-elimination depolymerization is carried out under the conditions of the technical characteristic of the step (b) is existing for reducing agent as a result, It answers, the beta-elimination reaction condition is to handle FG carboxylates using highly basic in nonaqueous solvents.Due to step (a) the FG carboxylic acids The reaction solution of esterification is nonaqueous solvents, and therefore, after the completion of the carboxylic esterification reaction, reaction solution is further without carrying out Processing and be directly used in the β-elimination depolymerization reaction carried out described in step (b).

Step (b) reducing agent is the compound that reducing end glycosyl can be reduced into sugar alcohol by those, such as hydroboration Sodium；The dosage of reducing agent is related to the production quantity of depolymerization product.It will be understood by those skilled in the art that ensure depolymerization product Yield and structural homogeneity should generally use the excessive reductant in Chemical Measurement in the reaction.On the other hand, step (b) The highly basic generally may optionally be rudimentary sodium alkoxide, diazabicylo etc..

Step (c) shown in route map 1 is that homology oligosaccharide mixture 5 obtained by the β of FG-elimination depolymerization is changed into alkali metal Salt, method are optionally to be realized using inorganic salts (such as sodium chloride) aqueous solution for adding saturation into reaction solution；Its institute Basic hydrolysis homology oligosaccharide compound carboxylate is stated, inorganic base aqueous solution (such as NaOH or KOH of 0.05M~1M) generally can be selected Processing can be realized, it is possible thereby to obtain the homology oligosaccharide mixture 6 containing free carboxy；

Step (d) shown in route map 1 is a series of oligosaccharide compound 7 for isolating and purifying oligosaccharide mixture and obtaining purifying. In general, it refers to being handed over using gel chromatography and/or ion that step (d) the use chromatography, which isolates and purifies oligosaccharide compound, The purifying that chromatography carries out oligosaccharide compound is changed, the gel chromatography and/or ion exchange chromatography are people in the art Method known to member.In addition, the gel chromatography and/or ion exchange chromatography can also optionally be combined ultrafiltration, salt The technical methods such as analysis method are to improve the efficiency isolated and purified.

Step (e) shown in route map 1 is optionally to carry out further structure to the oligosaccharide compound 7 obtained by step (d) to repair Decorations, thus to obtain oligosaccharide compound 8.The compound 8 is R₈For the oligosaccharides chemical combination of formula (I) structure of group shown in formula (II) Object is equal to oligosaccharide compound shown in formula above (V).Wherein：

Oligosaccharide compound 7 is subjected to quaternary ammonium salt conversion, it then can be in organic solvent using this field convenient technical process In with halohydrocarbons reaction can be readily available R₆For C1-C6 aliphatic hydrocarbyls or formula (V) oligosaccharide compound of C7-C12 aryl.

Using 103214591 A of CN and pertinent literature (Proc Natl Acad Sci USA.2015；112(27): Hydrazinolysis method described in 8284-9) can then remove the acetyl group in oligosaccharide compound 7 on D-GalNAc, obtain deacetylation Oligosaccharide compound, this i.e. R₇For formula (V) oligosaccharide compound of-H.The deacetylation oligosaccharide compound and acid anhydrides or Et₃N.SO₃ Reaction can obtain that N- is acylated again or the oligosaccharide compound of sulphation, i.e. R again₇For C2-C5 acyl groups or-SO₃It is few shown in the formula (V) of H Sugar compounds.

In addition, for the alcoholic extract hydroxyl group of the positions C1 on 7 reducing end-D-GalNAc-ol of oligosaccharide compound, it can be optionally in acidity Under the conditions of reacted with alcohol compound generate terminal alkylations product.It should be readily apparent to one skilled in the art that passing through the alkyl R can be obtained by changing reaction₉For formula (V) compound of substituted or unsubstituted C1-C6 aliphatic hydrocarbyls or C7-C12 aryl.

Obviously, above structure method of modifying is applied in combination then to can get shown in the formula (V) for the various structures that the present invention defines Oligosaccharide compound.

For preparation method shown in route map 1, a kind of preferred embodiment is：

Its step (a) FG quaternary ammonium salts are N, N- dimethyl-N -s [2- [2- [4 (1,1,3,3- tetramethyl butyls) benzene oxygen Base] ethyoxyl] ethyl] benzene ammonium carbamate, i.e. benzyl rope ammonium salt (Benzethonium)；The organic solvent is dimethylformamide (DMF) or DMF- alcohol mixtures；The carboxylate is its benzyl ester；It refers to mixture that all or part, which is converted into carboxylate, 3 carboxyl esterification degree is in the range of about 30% to about 100%；

Step (b) organic solvent is DMF or DMF- alcohol mixtures；The reducing agent is sodium borohydride；It is described strong Alkali is sodium ethoxide.

In step (c), it is described by quaternary ammonium mixture be changed into alkali metal salt be directed in reaction solution be added saturation chlorination Thus gained oligosaccharides homologue 5 is transformed into sodium-salt form by sodium water solution；Basic hydrolysis in the aqueous solution refers to a concentration of The carboxylate of homology oligosaccharide compound is hydrolyzed in the NaOH aqueous solutions of 0.05M~1M；

Step (d) chromatography includes but not limited to gel chromatography chromatography and/or ion-exchange chromatography；

Step (e) the further structural modification includes but not limited to：D-Glucose aldehydic acid base in oligosaccharide compound (GlcA) and the carboxyl esterification on unsaturated hexuronic acid base (Δ UA)；D- acetylamino galactosamines base (GalNAc) is deacetylated And optionally acylation or again sulphation again；The alkylation of reducing end sugar alcohol (D-GalNAc-ol).

Difference may be present in the Sulfation form of the natural FG in known different plant species source.Wherein, the sides FG of report are seen The Sulfation form of chain L-Fuc includes its 2,4- di-sulfate (L-Fuc_2S4S), 3,4- di-sulfates (L-Fuc_3S4S), 3- sulphur Acid esters (L-Fuc_3S) and 4- sulfuric esters (L-Fuc_4S) and without sulfate group substitution；See the D-GalNAc in the main chain of report Sulfation form includes then its 4,6- di-sulfate (D-GalNAc_4S6S), 4- sulfuric esters (D-GalNAc_4S), 6- sulfuric esters (D- GalNAc_6S) and without sulfate group substitution.Also, the L-FucS of different Sulfation forms can be existed simultaneously in some natural FG And/or D-GalNAcS, and the Sulfation form of L-FucS and/or D-GalNAcS contained by other natural FG is then relatively regular With it is single (referring to：Pomin VH.Mar Drugs.2014,12,232-54).

By the preparation method of the formula (V) oligosaccharide compound as it can be seen that its described step does not influence sulfate group on glycosyl Stability, therefore the sulfate form of gained oligosaccharide compound depend on natural FG sulfate form.Clearly for sulfuric acid For the relatively regular natural FG of change form, the type of the oligosaccharide compound in β-elimination depolymerization product is relatively fewer, identical The chemical constitution of the oligosaccharide compound of the degree of polymerization is identical, it is possible thereby to easily prepare purifying oligosaccharides chemical combination of the present invention Object.

For example, from Stichopus variegatus (Stichopus variegatus (Sempen)), Bohadschia argus (Bohadschia argus Jaeger) and In the echinoderms body wall such as Stichopus monotuberculatus in the natural FG of extraction purification, side chain fucosido master To be L-Fuc_2S4S, and the aminohexose in its main chain is mainly D-GalNAc_4S6S.Therefore, these natural FG are suitable for preparation Oligosaccharide compound and its pharmaceutically acceptable salt shown in formula (VIII), the compound are actually a kind of R₁=R₃=R₄= R₅=-SO₃H, and R₂Formula (V) oligosaccharide compound of=- H.

In formula (VIII), R₆、R₇、R₉As hereinbefore defined.

From Holothuria scabra (Holothuria scabra), Holothuria fuscopunctata (Roscoea intermedia), In the echinoderms body wall such as Stichopus horrens (rough stichopus japonicus) and Pearsonotheia graeffei (leather Pi Shi ginsengs) In the natural FG of extraction, side chain fucosido is mainly L-Fuc_3S4S, and the aminohexose in its main chain is mainly D- GalNAc_4S6S.Therefore, these natural FG are suitable for preparing oligosaccharide compound and its pharmaceutically acceptable shown in formula (IX) Salt, the compound are actually a kind of R₁=-H and R₂=R₃=R₄=R₅=-SO₃Formula (V) oligosaccharide compound of H.

In formula (IX), R₆、R₇、R₉As hereinbefore defined.

The preparation method of another oligosaccharide compound of the present invention is prepared with structure and its R shown in formula (I)₈For The method of the oligosaccharide compound of group shown in formula (III) or formula (IV) above.It is so-called " to there is structure and its R shown in formula (I)₈For The oligosaccharide compound of group shown in formula (III) or formula (IV) above " actually is equivalent to formula defined above (VI) or formula (VII) Shown in oligosaccharide compound.The preparation method is " β-elimination depolymerization+peeling reaction " method：In the organic solvent without reducing agent In make the natural FG of carboxylic esterification that β-elimination depolymerization occur, then so that FG depolymerization products is lost reducing end D- by " peeling react " GalNAc residues, thus to obtain the mixture for the homology oligosaccharide compound that reducing end is-D-GlcA.The specific steps of the method Including：

(a) natural FG is changed into quaternary ammonium form, then in organic solvent, by the carboxylic in FG quaternary ammonium salts on D-GlcA Base is completely or partially converted into carboxylate；

(b) in anhydrous organic solvent, the FG carboxylates is handled with highly basic and are allowed to that β-elimination depolymerization occurs, are then led to It crosses and a small amount of strong alkali aqueous solution is added depolymerization product is made " peeling reaction " further to occur to lose reducing end-D-GalNAc residues, Thus to obtain the mixture that reducing end is-D-GlcA homology oligosaccharide compounds；

(c) oligosaccharide mixture obtained by step (b) is changed into its alkali metal salt, then basic hydrolysis homology is few in aqueous solution The carboxylate of sugar compounds obtains the mixture of the homology oligosaccharide compound containing free carboxy；

(d) oligosaccharide compound in chromatography purification procedures (c) oligosaccharide mixture；

The product of the processing of step (a)~(e) described in this method is as shown in roadmap 2：

Route map 2

In route map 2：

Natural FG 1, FG ammonium salt 2, FG ester ammonium salt 3 and dFG ester Ammonium salt 4 are the same as described in route map 1；

DFG ester ammonium salt 5, which are depolymerization products 4, to be lost end-D-GalNAc through " peeling reaction " and is formed Reducing end be-D-GlcA depolymerization product.DFG 6 is with existing for alkali metal salt 5 carboxylate hydrolysate.Solution Poly- product 4,5 and 6 is the mixture of homology oligosaccharide compound；

Purified oligosaccharide 7 are that the purifying oligosaccharides obtained is detached from depolymerization product 6；And it purifies few Sugar 8 and 9 optionally carries out the purifying oligosaccharides of the gained of substituent structure modification for 7.

In chemical constitution described in route map 2, R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₁₀、R₁₁It is defined with formula above (I) with n；x、 Y and-COOX go the same way the definition in circuit 1.

Its quaternary ammonium salt of natural FG shown in the step (a) of route map 2 convert and the carboxyl esterification of FG with " route above Fig. 1 " the methods are identical.

Step (b) shown in route map 2 is to make FG carboxylates that β-elimination depolymerization (b1) generation depolymerization product 4 occur (wherein to contain A small amount of product 5), depolymerization product 5 is then obtained by the reducing end-D-GalNAc residues (b2) of " peeling is reacted " removal 4.

As it was noted above, both there is reducing end in β-elimination depolymerization product of natural FG disclosed in CN 201310099800 For the oligosaccharide compound of-D-GalNAc, there is also the oligosaccharide compounds that a certain amount of reducing end is "-D-GlcA- ", and reason can It can be related with β-destruction of reducing end glycosyl of elimination depolymerization product.Since reduction end structure is relative complex, from the depolymerization product In isolate and purify oligosaccharides difficulty it is higher.

Known to a person skilled in the art, " peeling reaction (peeling reaction) " under basic conditions can make one A little polysaccharide compounds (such as cellulose) lose monosaccharide residue (Whistler RL, the et al.Alkaline of reducing end one by one Degradation of Polysaccharides.Advances in Carbohydrate Chemistry.1958,13: 289–329).In fact, it has been found by the present inventors that depolymerization under the conditions of natural FG is described in CN 201310099800, gained production There are a certain amount of by-product containing unsaturated micromolecular compound in object, the reducing end reacted with the peeling for seeing report is sugared The destruction product of base is approximate.Beta-elimination reaction in view of natural FG disclosed in CN 201310099800 be under basic conditions into Capable, it can therefore be concluded that the generation that the reducing end in depolymerization product is the oligosaccharide compound of "-D-GlcA- " may be with depolymerization " the peeling reaction " of product is related.

It will be understood by those skilled in the art that adding reducing agent into the reaction solution of β-elimination depolymerization makes the end of depolymerization product End is reduced into the generation that sugar alcohol is conducive to avoid " peeling reaction "；On the other hand, β-elimination solution of FG is utilized under controlled condition A series of poly- " peeling reaction ", it is also possible to obtain FG oligosaccharides with "-D-GlcA " reducing end.Accordingly, by FG carboxylic acids The further research of the beta-elimination reaction condition of ester, the present inventor have found in surprise：

(1) a small amount of highly basic (such as NaOH) aqueous solution is added in alkaline non-aqueous solution described above, reducing end is "-D- The FG oligosaccharides of GalNAc " easily occurs " peeling reaction " and loses the reducing end glycosyl；It is surprising that reducing end under neutral It is but difficult to happen similar " peeling reaction " for the FG oligosaccharide compounds of "-D-GlcA ".

(2) HPGPC chromatography and NMR structural analyses are shown, in anhydrous organic solvent, highly basic processing FG carboxylates make Generation " β-elimination depolymerization ", a small amount of strong alkali aqueous solution is then added into reaction solution makes β-elimination depolymerization product further occur " peeling reaction ", contained by depolymerization product "Homology oligosaccharide compound" there can be fairly regular chemical constitution, i.e.,：It is described same It is that oligosaccharide compound is made of (3m-1) a monosaccharide residue；The glycosyl of the non-reducing end of the homology oligosaccharide compound is " L-Fuc- (α 1-3)-Δ UA-1- ", and its reducing end glycosyl is then C3 that there are " -4-D-GlcA " that L-FucS replaces.

(3) natural FG carboxylates are through further point of oligosaccharides homologue warp obtained by β-elimination depolymerization and peeling reaction treatment A series of " purifying oligosaccharide compounds " are can get from purifying, these purifying oligosaccharide compounds there can be common chemical constitution special Sign：The oligosaccharide compound contains (3m-1) a monosaccharide residue；Its non-reducing end is " L-Fuc- (α 1-3)-Δ UA-1- "；Its Reducing end is " -4- [L-Fuc- (α 1-3)]-D-GlcA ".

It is that FG carboxylates are handled using highly basic in the nonaqueous solvents without reducing agent first as a result, in the step (b) It is allowed to that β-elimination depolymerization (b1 of route map 2) acquisition depolymerization product 4 (can wherein contain a small amount of oligosaccharides 5) occurs.Similarly, due to The reaction dissolvent of FG carboxyl esterifications is nonaqueous solvents, and reaction solution is directly used in without being further processed carries out step (b) institute The β stated-elimination depolymerization reaction.In general, step (b) highly basic may optionally be rudimentary sodium alkoxide, diazabicylo etc..

Depolymerization product 4 is also further completely transformed into depolymerization product by step (b) shown in route map 2 by " peeling is reacted " 5 (b2 of route map 2), method are that a small amount of strong alkali aqueous solution is added into the beta-elimination reaction liquid.In general, described strong Aqueous alkali may optionally be NaOH, KOH of 0.25M~2M or the Ca (OH) of saturation₂Aqueous solution；" a small amount of " strong alkali aqueous solution It refer to the strong alkali aqueous solution that addition is equivalent to reaction solution total volume about 1/5~1/10.

Step (c) shown in route map 2 is that depolymerization product 5 is changed into alkali metal salt and hydrolyzes its carboxylate, technical side Method is the same as 1 the method for route map；

Similarly, shown in step (d) as shown in roadmap 2, the mixture of oligosaccharide compound 6 can get after isolating and purifying A series of oligosaccharide compound 7 of purifying.In general, the purification process refers to using gel chromatography and/or ion exchange Chromatography, and the technical methods such as ultrafiltration, salting out method can be optionally combined to improve the efficiency isolated and purified.

Equally, step (e) is optionally to carry out further structural modification to oligosaccharide compound 7 obtained by step (d), is thus obtained Obtain R₈To purify oligosaccharide compound 8 or 9.Wherein：

The oligosaccharide compound 8 actually is equivalent to formula described above (VI) oligosaccharide compound, and compound 9 is actually etc. It is same as formula described above (VII) oligosaccharide compound.

Oligosaccharide compound 7 is subjected to quaternary ammonium salt conversion, it then can be in organic solvent using this field convenient technical process In with halohydrocarbons reaction can obtain R₆For substituted or unsubstituted C1-C6 alkyl or the oligosaccharide compound of C7-C12 aryl；

Acetyl group in oligosaccharide compound 7 on D-GalNAc can be removed using hydrazinolysis method, obtain deacetylated oligosaccharides Compound.The deacetylated oligosaccharide compound again can be with acid anhydrides or Et₃N.SO₃Reaction obtains that N- is acylated again or sulphation again Oligosaccharide compound, i.e. R₇For C2-C5 acyl groups or-SO₃The formula (VI) or formula (VII) oligosaccharide compound of H.

7 reducing end-D-GlcA of oligosaccharide compound can optionally be reacted with alcohol compound in acid condition generates end Alkylate, thus to obtain R₁₀Formula (VI) chemical combination of optionally substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl Object.

The positions the C1 aldehyde radical of-D-GlcA of 7 reducing end of oligosaccharide compound can in the presence of organic amine by reducing end under neutral also Former amination, reaction are that organic amine reacts generation schiff bases with the positions the C1 of terminal saccharide aldehyde radical, and the latter is in the presence of a reducing agent It is reduced into secondary amine, it is possible thereby to obtain R₁₁For-NHR₁₂Formula (VII) compound (9).

The positions the C1 aldehyde radical of-D-GlcA of 7 reducing end of oligosaccharide compound can optionally use reducing agent such as sodium borohydride reduction at- D-GlcA-ol sugar alcohols, moreover, its-D-GlcA-ol further optionally can also react life with alcohol compound in acid condition At terminal alkylations product, it is possible thereby to obtain R₁₁For-OR₁₃, R₁₃Optionally-H, substituted or unsubstituted C1-C6 alkyl or Formula (VII) compound (9) of C7-C12 aryl；

Obviously, above structure method of modifying, which is applied in combination, can obtain the formula (VI) for the various specific structures that the present invention defines Or oligosaccharide compound shown in formula (VII).

For route preparation method shown in Fig. 2, a kind of preferred embodiment is：

Its step (a) FG quaternary ammonium salts are benzethonium chloride ammonium salt；The organic solvent mixes for DMF or DMF- ethyl alcohol Object；The carboxylate is its benzyl ester, and it refers to the carboxyl esterification degree of compound 3 about that all or part, which is converted into carboxylate, In the range of 30% to about 100%；

Step (b) organic solvent is DMF or DMF- alcohol mixtures, and the highly basic is sodium ethoxide；

Step (c) it is described by quaternary ammonium mixture be changed into alkali metal salt be directed in reaction solution be added saturated sodium-chloride water Thus gained oligosaccharides homologue is transformed into sodium-salt form by solution；Basic hydrolysis in the aqueous solution refers in a concentration of 0.05M The carboxylate of homology oligosaccharide compound is hydrolyzed in the NaOH aqueous solutions of~1M；

Step (e) the further structural modification includes but not limited to：In oligosaccharide compound on D-GlcA and Δ UA Carboxyl esterification；D-GalNAc is deacetylated and optionally acylated again or sulphation again；The alkane of the positions the C1 hemiacetal of reducing end-D-GlcA Base, reduction, reductive amination or reductive alkylation.

Similarly, in the preparation method of oligosaccharide compound described in route map 2, the sulfate form of gained oligosaccharide compound Also depend on the sulfate form of natural FG.

Therefore, Stichopus variegatus, Stichopus horrens and Stichopus The natural FG in the echinoderms such as monotuberculatus source is suitable for oligosaccharide compound shown in preparation formula (X) and formula (XI) And its pharmaceutically acceptable salt, the compound are actually a kind of R₁=R₃=R₄=R₅=-SO₃H, and R₂The formula of=- H (VI) and formula (VII) oligosaccharide compound.

In formula (X) and formula (XI), R₆、R₇、R₁₀And R₁₁As hereinbefore defined.

And Holothuria scabra, Holothuria fuscopunctata and Pearsonotheia The natural FG in the echinoderms such as graeffei source is then suitable for oligosaccharide compound shown in formula (XII) and formula (XIII), The compound is actually a kind of R₁=-H and R₂=R₃=R₄=R₅=-SO₃ ^-Formula (VI) and formula (VII) oligosaccharides chemical combination Object.

In formula (XII) and formula (XIII), R₆、R₇、R₁₀And R₁₁As hereinbefore defined.

Obviously, the beta-elimination reaction that natural FG is carried out under technical conditions of the present invention can be also used for preparing chemical constitution The mixture of more regular FG class oligosaccharide compounds.

For this purpose, the present invention further provides the systems of a kind of oligosaccharide mixture of the present invention and its pharmaceutically acceptable salt Preparation Method.Wherein：(1) the homologue group of the oligosaccharide compound of structure shown in the formula (I) that the mixture is defined by this specification At, and the R in the homology oligosaccharide compound of the formula (I) structure₈It is all formula (II), is all formula (III) or is all formula (IV) Shown group.Specifically, with molar ratio computing, R₈It is all formula (II), is all formula (III) or is all the formula of group shown in formula (IV) (I) oligosaccharide compound ratio shared in the mixture is not less than 95%.(2) preparation method is reaction with natural FG Starting material optionally makes FG carboxylates that " β-elimination depolymerization " and end group " reduction reaction " acquisition occur in the presence of highly basic and reducing agent The mixture of homology oligosaccharide compound；Or make FG carboxylates that " β-elimination depolymerization " and end group " peeling occur in the presence of highly basic Reaction " obtains the mixture of homology oligosaccharide compound.Then, it is repaiied by post-processing and optionally carrying out further substituent structure Decorations obtain the oligosaccharide mixture of required molecular weight distribution.

As it was noted above, non-reducing end can be obtained according to the processing of step (a)~(c) shown in route map 1 natural products FG For L-FucS- (α 1-3)-Δ UA-1-, the homology oligosaccharide mixture that reducing end is-D-GalNAcS.Similarly, in institute of the present invention In the preparation method for stating oligosaccharide mixture, one of which method is：Make FG carboxylates that " β-occur in the presence of highly basic and reducing agent Elimination depolymerization " and end group " reduction reaction " obtain the mixture of homology oligosaccharide compound；Its gained oligosaccharide mixture contains same Being oligosaccharide compound has general formula structure shown in formula defined above (I), and its R₈For group shown in formula defined above (II). The specific steps of the method include：

(a) natural FG is changed into quaternary ammonium form, and in organic solvent will be in gained FG on hexuronic acid residue Carboxyl is completely or partially converted into carboxylate；

(b) in organic solvent, in the presence of reducing agent and highly basic, FG carboxylates is made to be allowed to that β-elimination depolymerization and end group occurs Reduction reaction, thus to obtain the mixture for the homology oligosaccharide compound that reducing end is-D-GalNAc-ol；

(c) oligosaccharide mixture obtained by step (b) is changed into alkali metal salt, and basic hydrolysis homology oligosaccharides in aqueous solution The carboxylate of object is closed, the mixture of the homology oligosaccharide compound containing free carboxy is obtained and carries out post-processing appropriate；

(d) further substituent structure modification optionally is carried out to the change oligosaccharide mixture obtained by step (d).

In a preferred embodiment：

Its step (a) quaternary ammonium salt is benzyl rope ammonium salt；The organic solvent is DMF or DMF- alcohol mixtures；It is described Carboxylate is benzyl ester；" being completely or partially converted into carboxylate " refer in FG carboxyl esterification degree about 30% to about In the range of 100%.

Its step (b) organic solvent is DMF or DMF- alcohol mixtures；The reducing agent is sodium borohydride；It is described Highly basic is sodium ethoxide.

Its step (c) is described to be changed into alkali metal salt by quaternary ammonium mixture and is directed to that saturated sodium-chloride is added in reaction solution Gained oligosaccharides homologue is changed into sodium-salt form by aqueous solution；Basic hydrolysis in the aqueous solution refer to a concentration of 0.05M~ The carboxylate of oligosaccharide compound is hydrolyzed in the NaOH aqueous solutions of 1M.

As it was noted above, usually also containing a certain amount of molecule in the natural FG prepared according to state of the art method Amount distribution is similar to the fucosan, glycogen and the polysaccharide containing aminohexose of FG.These polysaccharide constituents are through above-mentioned steps (a) After step (b) processing, the change of molecular weight is smaller.Therefore, in step (c) post-processing step, select ultrafiltration, thoroughly Analysis method or gel chromatography can easily remove these polysaccharide impurities.

As shown in route map 1 above, it is also possible to have through the depolymerization product obtained by β-elimination depolymerization and end reduction treatment Broader molecular weight distribution (in route oligosaccharide mixture 6 shown in FIG. 1, n values can be about 0-15 integers), therefore, in step In (c) described post-processing step, select ultrafiltration, dialysis or gel chromatography processing that can also remove higher degrees of polymerization suddenly Oligosaccharides and micromolecular compound, thus to obtain the oligosaccharide mixture of required molecular weight distribution.

Its step (d) further substituent structure, which is modified, includes but not limited to：D-GlcA in oligosaccharide compound With the carboxyl esterification on unsaturated Δ UA；D-GalNAc is deacetylated and optionally acylated again or sulphation again；Reducing end D- The positions the C1 hydroxy alkylated of GalNAc-ol.

Obviously, compared with oligosaccharide mixture described in patent of invention ZL 201310099800, R of the present invention₈It is all formula (II) chemical constitution of the homology oligosaccharide mixture of group is more regular.In the former contained oligosaccharide compound, about 10%-30%'s The reduction end structure of oligosaccharide compound be D-GlcA (or derivatives thereof), the reduction end structure of remaining oligosaccharide compound is D- GalNAc (or derivatives thereof)；And the reducing end of oligosaccharide compound contained by oligosaccharide mixture of the present invention is then D-GalNAc (or derivatives thereof), be free of or contain only micro about reducing end be D-GlcA (or derivatives thereof) oligosaccharide compound.

In addition, by mentioned earlier as it can be seen that can be obtained according to the processing of step (a)~(c) shown in route map 2 natural products FG The homology oligosaccharide mixture that non-reducing end is L-FucS- (α 1-3)-Δ UA-1-, reducing end is-D-GlcA.Therefore, in the present invention In the preparation method of the oligosaccharide mixture, another method is：Make FG carboxylates that " β-elimination solution occur in the presence of highly basic It is poly- " and end group " peeling reaction " acquisition homology oligosaccharide compound mixture；The homology oligosaccharides that its gained oligosaccharide mixture contains Compound has general formula structure shown in the formula (I) that description of the invention defines, and its R₈For formula defined above (III) or formula (IV) group shown in.The specific steps of the method include：

(a) natural FG is changed into quaternary ammonium form, and in organic solvent by the carboxylic on gained FG hexuronic acid residues Base is completely or partially converted into carboxylate.

(b) it in anhydrous organic solvent, using highly basic processing FG carboxylates is allowed to that β-elimination depolymerization occurs, then by adding Entering a small amount of strong alkali aqueous solution makes FG depolymerization products that " peeling reaction " further occur and loses the-D-GalNAc residues of reducing end, Thus to obtain the mixture for the homology oligosaccharide compound that reducing end is-D-GlcA.

(c) oligosaccharide mixture obtained by step (b) is changed into alkali metal salt, and in aqueous solution, the mixing of basic hydrolysis oligosaccharides Carboxylate in object obtains the mixture of the homology oligosaccharide compound containing free carboxy and carries out post-processing appropriate.

(d) further substituent structure modification optionally is carried out to the oligosaccharide mixture obtained by step (c).

In a preferred embodiment：

Its step (a) quaternary ammonium salt benzyl rope ammonium salt；The organic solvent DMF or DMF- alcohol mixtures；The carboxylic acid Ester is benzyl ester；The carboxyl esterification degree of the FG carboxylates is in the range of about 30% to about 100%.

Its step (b) organic solvent is DMF or DMF- alcohol mixtures；The highly basic is sodium ethoxide；It is described a small amount of Strong alkali aqueous solution refers to the NaOH aqueous solutions for the 1M~2M for being equivalent to about 1/5 to 1/10 volume of reaction solution total volume；

Step (c) method that quaternary ammonium mixture is changed into alkali metal salt is directed to that saturation chlorine is added in reaction solution Change sodium water solution, gained oligosaccharides homologue is thus changed into sodium-salt form；Basic hydrolysis in the aqueous solution refers in concentration To hydrolyze the carboxylate of homology oligosaccharide compound in the NaOH aqueous solutions of 0.05M~1M.Similarly, the post-processing can appoint Choosing removes the macromolecular polysaccharide class impurity of non-depolymerization using gel chromatography, ultrafiltration and/or dialysis and to remove the degree of polymerization higher Oligosaccharide compound and small molecular weight impurity, thus to obtain the oligosaccharide mixture of required molecular weight ranges.

Its step (d) further structural modification includes but not limited to：D-GlcA in oligosaccharide compound and Δ UA On carboxyl esterification；D-GalNAc is deacetylated and optionally acylated again or sulphation again；The positions the C1 hemiacetal of reducing end-D-GlcA Alkylation, reduction, reductive amination or reductive alkylation.

Similarly, compared with oligosaccharide mixture described in patent of invention ZL 201310099800, R of the present invention₈It is all formula (III) or the chemical constitution of group oligosaccharide mixture shown in formula (IV) is more regular.In the former contained oligosaccharide compound, about The reduction end structure of the oligosaccharide compound of 10%-30% be D-GlcA (or derivatives thereof), the reducing end of remaining oligosaccharide compound Structure be D-Gal-NAc (or derivatives thereof)；And the reducing end of oligosaccharide compound contained by oligosaccharide mixture of the present invention is then equal For D-GlcA (or derivatives thereof), be free of or contain only micro about reducing end be D-GalNAc (or derivatives thereof) oligosaccharides chemical combination Object.

Obviously, using the echinoderms such as S.variegatus, S.horrens and S.monotuberculatus source Natural FG is starting material, and formula described above (VIII), formula (X) can be prepared according to oligosaccharide mixture preparation method of the present invention With the mixture of homology oligosaccharide compound shown in formula (XI).Using H.scabra, H.fuscopunctata and The natural FG in the echinoderms such as P.graeffei source is starting material, then may be used according to oligosaccharide mixture preparation method of the present invention To prepare the mixture of homology oligosaccharide compound shown in formula described above (IX), formula (XII) and formula (XIII).

Available data is shown, containing L-Fuc in one kind_2S4SIt is same obtained by the deacylation deamination depolymerization of the natural FG of side chain substituents It is in oligosaccharides compounds, nine sugared (nonasaccharide, NSac) are that have the potent active minimums of inhibiting factor Xase Structure fragment (Zhao LY et al., PNAS, 2015,112:8284-8289.).The present inventor is to purifying of the present invention widow Inhibition endogenous factors Xase (people and the factor Xase in the experimental animal source) activity of sugar carries out structure activity study, as a result sends out It is existing：

(1) the selective activity for inhibiting endogenous factors Xase of oligosaccharide compound of the present invention.Usually, It is detected using vitro enzyme live system, the IC of oligosaccharide compound inhibiting factor Xase of the present invention₅₀Value can be in about 5~200ng/ml In the range of.The activity of the selective depression endogenous factors Xase, which refers to these oligosaccharide compounds, significantly inhibits Xase at it Concentration under, in the presence of with or without antithrombase (AT), to the coagulation factors such as prothrombin a, Xa, XIIa and blood platelet Activity do not make significant difference, but may be present some strength heparin cofactor II (HC-II) rely on IIa inhibitory activity.

(2) for be Δ UA containing 3m monosaccharide groups and non-reducing end, the series of oligosaccharides that reducing end is-D-GalNAc-ol For closing object, the potent active minimal structure segments of inhibition Xase are also nine sugared (nonasaccharide, NSac).Its result table Bright, the glycosyl structure variation of non-reducing end and reducing end is smaller to its inhibiting factor Xase activity influences.

(3) for containing (3m-1) a monosaccharide residue and reducing end is the series of oligosaccharides compound of-D-GlcA (- ol), The potent active minimal structure segments of inhibiting factor Xase are then eight sugared (octasaccharide, OSac), and result prompts, on It may be its potent active necessary structure of inhibiting factor Xase to state the reducing end-D-GalNAc-ol in NSac.

(4) generally, the degree of polymerization not less than OSac oligosaccharide compound of the present invention all have potent endogenous because Sub- Xase inhibitory activity (its IC₅₀Value is below about 100ng/ml)；The higher oligosaccharides activity of the degree of polymerization slightly enhances.

(5) in oligosaccharide compound of the present invention, six sugar or pentasaccharides at higher concentrations also may be used to endogenous Xase With with certain inhibitory activity, although its activity intensity is relatively weak；In addition, six sugar and pentasaccharides also have some strength Heparin cofactor II (HC-II) rely on IIa inhibitory activity.

(6) in oligosaccharide compound of the present invention, substituent structure modifies the physics and chemistry that can significantly affect oligosaccharide compound Matter, such as water-soluble and Determination of oil-water partition coefficient, and inhibit the influence of coagulation factor activity and anti-freezing antithrombotic acitivity general to it It is smaller.

(7) oligosaccharide compound of the present invention can have the significant anticoagulant active for inhibiting intrinsic coagulation pathway, with The activated partial thromboplastin time (APTT) of prolonged human Quality Control blood plasma is counted, and the drug concentration to double needed for APTT is generally about Within the scope of 2~18 μ g/mL.And oligosaccharide compound exogenous blood coagulation of the present invention does not make significant difference.

(8) in the pathological model of experimental animal, arteriovenous blood that oligosaccharide compound of the present invention can significantly inhibit Bolt is formed.For example, the studies have shown that of the present inventor in kinds of experiments animal model, in terms of thrombus weight, is subcutaneously injected (sc) Or the compounds of this invention (such as nine sugar or eight sugar) of intravenous injection (sc) about 2mg/kg~20mg/kg dosage is to experimental (as follows Caused by vena cava ligation) inhibiting rate of venous thronbosis is up to 70%~100%, it is described and under equivalent antithrombotic dosage Influence of the oligosaccharide compound to bleeding time and amount of bleeding can be substantially less than the low molecular weight heparin class drug of Clinical practice.

(9) there is oligosaccharide mixture of the present invention the inhibition endogenous factors Xase for being similar to purifying oligosaccharide compound to live Property and anti-freezing antithrombotic acitivity.

In summary as it can be seen that oligosaccharide compound of the present invention and its mixture all have significant anti-freezing antithrombotic work Property, and when the oligosaccharides degree of polymerization is not less than eight sugar, oligosaccharide compound and its mixture of the present invention are with good choosing The endogenous factors Xase inhibitor of selecting property.Existing research data shows that intrinsic coagulation pathway forms close with pathologic thrombus Cut phase is closed, and may not be that physiological haemostasis institute is necessary, and selective intrinsic coagulation pathway inhibitor can inhibit pathologic blood Bolt is formed, and hemorrhagic tendency can be reduced effectively.Since factor Xase is last position and the speed limit of intrinsic coagulation pathway Enzyme activity site, the anti-freezing antithrombotic reagent that endogenous factors Xase has become lower bleeding tendency research and develop drug target of interest.

There is significant anti-freezing antithrombotic acitivity, these oligosaccharides and widow in view of oligosaccharides of the present invention and oligosaccharide mixture Sugared mixture should have the clinical value of prevention and/treatment thrombus disease.The present invention further provides containing thus State the pharmaceutical composition of oligosaccharides or oligosaccharide mixture.

First, the present invention provides a kind of pharmaceutical composition with antithrombotic acitivity, and described pharmaceutical composition contains effectively The oligosaccharide compound of the present invention or their pharmaceutically acceptable salt of antithrombotic dosage and pharmaceutically acceptable Excipient.Wherein, the oligosaccharide compound refer to there is present specification to define formula (I) shown in structure compound.

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 be at the lyophilized preparation of aqueous solution for injection, can also be through The spray of the respiratory tract administration or transdermal patch of percutaneous dosing, paste or gelling agent etc..

Oligosaccharide compound of the present invention may generally be of good water solubility, be easy to obtained aqueous solution；Due to activity The molecular weight of ingredient is relatively low, can remove pathogenic microorganism and pyrogenic substances by ultrafiltration；Its aqueous solution and/or lyophilized preparation can The pharmaceutical excipient of selection may include adjusting solution osmotic pressure and/or the inorganic salts such as sodium chloride, buffer salt of pH value, preferably be free of Cosolvent and/or surfactant.For the freeze drying powder injection of prepared before use injection, except the inorganic salts and/or Outside buffer salt, it is also an option that contributing to the pharmaceutically acceptable excipient of preparations shaping using mannose etc..

In general, the oral administration biaavailability of oligosaccharide compound is relatively limited, but oligosaccharide compound of the present invention The gastrointestinal administration of (especially substituent structure modification gained oligosaccharides) can still have certain drug activity.Therefore institute of the present invention Gastrointestinal administration dosage form, such as tablet, capsule well known to those skilled in the art etc. can be prepared by stating pharmaceutical composition also.

It will be understood by those skilled in the art that for the pharmaceutical composition of particular formulations form, the oligosaccharides chemical combination Effective antithrombotic dosage of object and its pharmaceutically acceptable salt and its dosage form, administration route and the weight of patient and physiology shape The factors such as state are related.In general, in the unit preparation form of pharmaceutical composition of the present invention, the oligosaccharides active constituent Content about in the range of 5mg~100mg；In the unit preparation form of preferred pharmaceutical composition, the oligosaccharides activity The content of ingredient can be in the range of about 20mg~80mg.

Similarly, the present invention also provides a kind of pharmaceutical composition with antithrombotic acitivity, described pharmaceutical composition contains The oligosaccharide mixture of the present invention or its pharmaceutically acceptable salt of effective antithrombotic dosage and pharmaceutically acceptable Excipient.

For the pharmaceutical composition containing oligosaccharide mixture of the present invention, the relevant preparation formulation of administration route and Dosage selection is approximate with the above-mentioned pharmaceutical composition containing oligosaccharide compound.Such as：Its preferred administration route is parenteral gives Medicine, especially subcutaneous administrations or intravenous injection administration；Its preferred dosage form is aqueous solution for injection or freeze-dried powder Agent；In the unit preparation form of preferred pharmaceutical composition, the content of the oligosaccharides active constituent can be about 20~100mg's In range.

Oligosaccharide compound, oligosaccharide mixture and its pharmaceutically acceptable salt of the present invention have the potent anti-blood of anti-freezing Thrombus activity can be used for the prevention and treatment of thrombotic diseases, such as thrombotic angiocardiopathy, the thrombotic cerebrovascular Disease, pulmonary vein thrombus, PeV thrombus, deep vein thrombosis, peripheral arterial thrombus etc., for this purpose, the present invention also provides described Oligosaccharide compound and/oligosaccharide mixture and its pharmaceutically acceptable salt are preparing treatment and/or are preventing the medicine of thrombotic diseases Application in object, the thrombotic diseases include but not limited to venous thronbosis, Arterial thrombosis and/or ischemic heart and brain Vascular diseases.

Similarly, the present invention also provides contain the oligosaccharide compound and/oligosaccharide mixture and its pharmaceutically acceptable Application of the pharmaceutical composition of salt in preparing treatment and/or preventing the drug of thrombotic diseases, the thrombotic diseases include But it is not limited to venous thronbosis, Arterial thrombosis and/or ischemic angiocardiopathy and cerebrovascular disease.

Abbreviation：

Description of the drawings

The HPGPC of Fig. 1 compounds A1~A4 schemes

Fig. 2 compounds A1's¹H NMR spectras and ownership

Fig. 3 compounds A2's¹³C NMR spectras and ownership

Fig. 4 compound A-13s¹³C-¹H hsqc spectrums figure and ownership

The Q-TOF MS spectrograms and ownership of Fig. 5 compounds A1

Fig. 6 compound B-11s¹H NMR spectras and ownership

Fig. 7 compounds B2's¹³C NMR spectras and ownership

Fig. 8 compounds B3's¹³C-¹H hsqc spectrums figure and ownership

The Q-TOF MS spectrograms of Fig. 9 compounds B2 and ownership

The HPGPC collection of illustrative plates of Figure 10 compound B-26s-B8

Figure 11 mixture Cs 1¹³C NMR spectras and ownership

The HPLC collection of illustrative plates of Figure 12 mixture Ds 1

Figure 13 mixture Ds 1¹³C NMR spectras and ownership

The influence that Figure 14 .A2 and D1 forms mouse inferior vena cava thrombosis

Influences of Figure 15 .A2 and D1 to mouse amount of bleeding

Specific implementation mode

Following embodiment is the detailed description to the content of present invention, but is not limit the scope of the invention.

【Embodiment 1】

The preparation of compound A1, A2, A3, A4 and A5：Bis- sulfated fucose bases of L-2,4--(α 1 → 3)-L-4- deoxidations- Threo- hex- 4- alkene pyrans uronic acids base-(α 1 → 3)-{ two sulfuric acid galactolipins of-D-N- acetyl group -2- deoxidation -2- amino -4,6- Base-(β 1 → 4)-[bis- sulfated fucose bases of L-2,4--(α 1 → 3)]-D-Glucose aldehydic acid base-(β 1 → 3) }_(n+1)- D-N- acetyl Two sulphation galactitol (n=0,1,2,3 and 4 of base -2- deoxidation -2- amino -4,6-；Six sugar, nine sugar, ten disaccharides, ten pentasaccharides and 18 sugar)

1.1 material

The natural FG (sodium salt) in the sources SvFG, Stichopus Variegatus.It presses literature method (Zhao LY et al.,PNAS,2015,112:It 8284-8289) prepares, purity 98% (HPGPC, area normalization method), weight average molecular weight (Mw) About 70kDa.

The agents useful for same such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride and ethyl alcohol are the commercially available pure examination of analysis Agent.

Sephadex G10,medium(50-100μm),GE Healthcare；Bio-Gel P-6/P-2 gel,fine (45-90μm),Bio-Rad；Bio-Gel P-10 gel,medium(90-180μm),Bio-Rad；HPLC chromatogram instrument, Agilent 1200/1260 serial chromatograph.

1.2 method

(1) the quaternary ammonium salt conversion of SvFG：2.0g SvFG are taken to be dissolved in 30mL deionized waters；Separately 5.0g benzethonium chlorides is taken to be dissolved in 80mL deionized waters.Under stirring, white precipitate is obtained with benzethonium chloride solution titration SvFG solution.Gained precipitation 55mL deionizations Water washing three times, vacuum drying obtain SvFG quaternary ammonium salts 5.34g.

(2) carboxyl esterification of SvFG：SvFG quaternary ammonium salts obtained by step (1) are placed in round-bottomed flask, add 26mL DMF molten Xie Hou is added 0.769mL benzyl chlorides, is reacted for 24 hours in being stirred at 35 DEG C；Standing makes reaction solution be down to room temperature (25 DEG C).Sampling inspection Survey product¹H NMR and the carboxyl esterification degree for calculating FG are about 41%；

(3) β in the presence of reducing agent-elimination depolymerization：The 8.9mL newly prepared is added into step (2) described reaction solution 0.08M sodium ethoxides-ethanol solution (NaBH containing 0.4M₄), it is stirred to react 30min.

(4) the sodium salt conversion of depolymerization product and carboxyester hydrolysis：35mL saturations are added into step (3) described reaction solution NaCl solution and 284mL absolute ethyl alcohols, 4000rpm × 10min centrifuge to obtain precipitation.Gained is precipitated and dissolved in 90mL water, is added After being stirred to react 30min at room temperature, 6M HCl neutralization reactions liquid (pH~7.0) is added dropwise in 1.5mL 6M NaOH solutions.Reaction solution With 0.45 μm of membrane filtration, gained filtered fluid is with the desalination of G10 gel filtration chromatographies and is lyophilized, and obtains depolymerization product dSvFG (depolymerized SvFG) total 1.059g (yield 53%).

(5) compound A1~A5 is isolated and purified：It takes 1g dSvFG to be dissolved with 10mL 0.2M NaCl, is splined on Bio-Gel P-10 gel columns (L 200cm), it is eluted with 0.2M NaCl solutions, flow velocity 10mL/h, 2.5mL/ pipe collects elution stream Part.Cysteine-sulfuric acid process monitoring elution fraction simultaneously draws elution curve, merges the identical elution fraction of component.HPGPC methods (TSK gel G2000SW XL,Column) detection purity.Non- purification of samples is solidifying with Bio-Gel P-10 Rubber column gel column continues to purify.Purifying oligosaccharides with Sephadex G-10 or Bio-Gel P-2 gel columns desalination and is lyophilized.

(6) Spectrum Analysis：¹H-/¹³C- and 2D-NMR detections use Bruker DRX 800MHz Nuclear Magnetic Resonance, spectrum width 16025.6Hz, acquisition time 2.0447s, pulse width 9.5s, relaxation time 1s are scanned 32 times.Sample concentration is (10~15) G/L is lyophilized three times before detection with heavy water repeatedly；ESI-Q-TOF MS using micrOTOF-QII ESI-MS (Bruker, Germany) spectrometer analysis.Mass Spectrometry Conditions are capillary voltage 2500V, sprayer voltage 0.6bar, dry gas stream speed 4.0L/ Min, dry+180 DEG C of temperature degree, m/z scanning ranges 50~3000.Data use Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany) softwares are analyzed.

1.3 result

(1) compound A1 35mg, A2 45mg, A3 55mg, A4 35mg, A5 20mg, HPGPC are obtained according to the method Method detects its purity about 99%.The HPGPC figures of oligosaccharide compound A1~A5 are as shown in Fig. 1.

(2) compound A1~A5 structure elucidations：Oligosaccharide compound A1's¹H NMR spectras and ownership are as shown in Fig. 2；Change Close object A2's¹³C NMR spectras and ownership are as shown in Fig. 3；Compound A-13¹³C-¹H hsqc spectrums figure and ownership are as shown in Figure 4； The Q-TOF MS spectrograms and ownership of compound A1 is as shown in Fig. 5；Compound A1~A2's¹H/¹³C NMR signals ownership is shown in respectively Table 1 and 2.

According to¹H-/¹³C- and 2D-NMR and Q-TOF MS analyses, the chemical constitution of compound A1~A5 is L-Fuc_2S4S-(α 1,3)-ΔUA-(α1,3)-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-GlcA-(β1,3)}_n+1-D- GalNAc_4S6S- ol, wherein n=1,2,3,4 and 5, that is to say, that compound A1-A5 is six sugar, nine sugar, ten disaccharides, ten respectively Pentasaccharides and 18 sugar, structural formula are：

In its A1, n=1；In A2, n=2；In A3, n=3；In A4, n=4；In A5, n=5.

1. compound A1's of table¹H/¹³C NMR signals belong to and coupling constant (ppm, Hz)

Note：RA indicates reducing end D-GalNAc-ol；DU and dF indicates Δ UA and the L-Fuc for being connected to Δ UA.

2. compound A2's of table¹H/¹³C NMR signals belong to (ppm, Hz)

Note：RA, rU and rF indicate D-GlcA and the L-Fuc sugar of the GalNAc-ol of reducing end, nearly reducing end respectively in table Base；DU, dA and dF indicate Δ UA, the D-GalNAc connected with Δ UA and the L-Fuc for connecting Δ UA respectively.

【Embodiment 2】The preparation of compound B-11, B2, B3, B4 and B5：Bis- sulfated fucose bases of L-3,4--(α 1,3)-L-4- Deoxidation-threo- hex- 4- alkene pyrans uronic acids base-(α 1,3)-{ two sulfuric acid of D-N- acetyl group -2- deoxidation -2- amino -4,6- half Lactose base-(β 1,4)-[L-3,4- bis- sulphation fucosidos-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3) }_n- D-N- second Two sulphation galactolipins of acyl group -2- deoxidation -2- amino -4,6--[L-3,4- bis- sulphation fucosidos-(α 1,3) -]-D- grapes Alditol acid alcohol (n=0,1,2,3 and 4, pentasaccharides, eight sugar, 11 sugar, ten tetroses and 17 sugar).

2.1 material

The natural FG (sodium salt) in HsFG, Holothuria fuscopunctata (Roscoea intermedia) source；It presses literature method (Zhao LY et al.,PNAS,2015,112:8284-8289), purity 98% (HPGPC methods), weight average molecular weight (Mw) is about 50kDa。

The agents useful for same such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium borohydride, sodium chloride and ethyl alcohol are commercially available Analytical reagents.

Sephadex G10/G25, medium (50-100 μm), GE Healthcare；Bio-Gel P-12 gel,fine (45-90μm),Bio-Rad；Bio-Gel P-10 gel, medium (90-180 μm), Bio-Rad；1200/1260 series HPLC Chromatograph, Agilent.

2.2 method

(1) the quaternary ammonium salt conversion of HsFG：3.5g HsFG are taken, 1.2 (1) the methods processing with embodiment 1 obtains HsFG Quaternary ammonium salt 10.3g.

(2) carboxyl esterification of HsFG：1.2 (2) the methods processing HsFG quaternary ammonium salts with embodiment 1 obtain HsFG carboxylic acids Ester, sample detection¹H NMR, the carboxyl esterification degree for calculating products therefrom are about 44%；

(3) β of HsFG-elimination depolymerization is reacted with end peeling：It is added into reaction solution obtained by step (2) and newly to prepare The alcohol sodium alcohol solution of 16.7mL 0.08M, after being stirred to react 30min at room temperature, be added 2.5mL 2M NaOH solutions and It is stirred to react 30min at 60 DEG C.

(4) the sodium salt conversion, carboxylate hydrolysis of depolymerization product are restored with end：It is suitable into step (3) described reaction solution Secondary addition 67mL is saturated NaCl solution, 536mL absolute ethyl alcohols, 4000rpm × 10min centrifugations；Gained is precipitated and dissolved in 120mL water In, add 6M NaOH solution 1.0mL, is stirred to react 30min at room temperature；NaBH is added₄To final concentration about 0.1M, stir at room temperature 30min is reacted, 6M HCl, which are then added dropwise, makes reaction solution neutralize (pH~7.0).Reaction solution is with 0.45 μm of membrane filtration, filtered solution warp 30kDa ultrafiltration membrane packet ultrafiltration；The desalination of G25 gel filtration chromatographies is used after ultrafiltrate concentration and is lyophilized, thus to obtain depolymerization product DHsFG 1.53g (yield 43.7%).

(5) compound B-11~B5 is isolated and purified：Step (4) the 1g dHsFG are dissolved in 10mL 0.2M NaCl, loading In Bio-Gel P-10 gel columns (L 200cm), it is eluted with 0.2M NaCl solutions, flow velocity 15mL/h, 2.5mL/ pipe is received Collection elution fraction.Ultraviolet spectrophotometry (λ_max234nm) monitor.HPGPC (TSK G2000SW columns) detects the sample of elution fraction Product purity and composition.Non- purified components continuation is purified with Bio-Gel P-10 gel columns, until the HPGPC collection of illustrative plates of product is in Existing single eluting peak.Purified components after Sephadex G-10 or Bio-Gel P-2 gel column desalinations to be lyophilized.

(6) Spectrum Analysis：With 1.2 (6) the methods of embodiment 1, using Bruker DRX 800MHz Nuclear Magnetic Resonance Detection¹H-/¹³C- and 2D-NMR, using microTOF-QII ESI-MS (Bruker, Germany) spectrometer analysis ESI-Q- TOF MS.Detection data uses Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany) Software is analyzed.

2.3 result

(1) compound B-11 54mg, B2 177mg, B3 154mg, B4 86mg, B5 57mg are obtained according to the method, It is equal that HPGPC methods detect its purity>99%.

(2) compound B-11~B5 structure elucidations：Oligosaccharide compound B1's¹H NMR spectras are as shown in Fig. 6；Compound B2 's¹³C NMR spectras and ownership are as shown in Fig. 7；Compound B3's¹³C-¹H hsqc spectrums figure and ownership are as shown in Fig. 8；Chemical combination The Q-TOF MS spectrograms and ownership of object B2 is as shown in Fig. 9；Compound B-11~B2's¹H/¹³C NMR signals ownership is shown in Table 3 respectively With 4.

In conjunction with¹H-/¹³C-/2D-NMR and Q-TOF MS analyses, the chemical constitution of compound B-11~B5 is L-Fuc_3S4S-(α 1,3)-L-ΔUA-(α1,3)-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_3S4S-(α1,3)]-D-GlcA-(β1,3)}_n-D- GalNAc_4S6S-(β1,4)-[L-Fuc_3S4S(α 1,3)]-D-GlcA-ol (wherein n=0,1,2,3 and 4).That is, chemical combination Object B1~B5 is pentasaccharides, eight sugar, 11 sugar, ten tetroses and 17 sugar respectively.Its chemical structural formula is：

In its B1, n=0；In B2, n=1；In B3, n=2；In B4, n=3；In B5, n=4.

3. compound B-11 of table¹H/¹³C NMR signals belong to and coupling constant (ppm, Hz)

Note：RU, dU are respectively the D-GlcA-ol of reducing end and the Δ UA of non-reducing end in table；RF, dF are respectively to be connected to The L-Fuc glycosyls of reducing end D-GlcA-ol and Δ UA.A is D-GalNAc.

4. compound B2's of table¹H-/¹³C-NMR signals assignments (ppm, Hz)

Note：RA indicates the GalNAc of nearly reducing end in table, in table rU, dU be respectively reducing end D-GlcA-ol and it is non-also The Δ UA at former end；RF, dF are respectively the L-Fuc glycosyls for being connected to reducing end D-GlcA-ol and Δ UA.

【Embodiment 3】The preparation of compound B-26, B7, B8：Bis- sulfated fucose bases of L-3,4--(α 1,3)-L-4- deoxidations- Threo- hex- 4- alkene pyrans uronic acids base-(α 1,3)-{ two sulfuric acid galactolipins of D-N- acetyl group -2- deoxidation -2- amino -4,6- Base-(β 1,4)-[L-3,4- bis- sulphation fucosidos-(α 1,3) -] D-Glucose aldehydic acid base-(β 1,3) }_n- D-N- acetyl group- The Portugals-D- 2- deoxidation -2- amino -4,6- two sulphation galactolipin-(β 1,4)-[L-3,4- bis- sulphation fucosidos-(α 1,3) -] Grape uronic acid (n=0,1 and 2).

3.1 material：

The FG sodium salts in HsFG, Holothuria fuscopunctata (Roscoea intermedia) source, source with embodiment 2 2.1 It is described.

3.2 method：

(1) the quaternary ammonium salt conversion of HsFG：With 2.2 (1) the methods of embodiment 2, HsFG is prepared with 3.5g HsFG Quaternary ammonium salt 9.55g.

(2) carboxyl esterification of HsFG：2.2 (2) the methods with embodiment 2 prepare carboxyl esterification HsFG,¹H It is about 44% that NMR, which detects its carboxyl esterification degree,；

(3) β of HsFG-elimination depolymerization：Sodium ethoxide-second of the 0.08M newly prepared is added into reaction solution obtained by step (2) Alcoholic solution 16.0mL, is stirred to react 30min at room temperature.

(4) the sodium salt conversion of depolymerization product is hydrolyzed with carboxylate：Saturation chlorination is added into reaction solution obtained by step (3) Sodium solution 67mL and enter absolute ethyl alcohol 536mL, 4000rpm × 10min centrifugations；After gained precipitation dissolving water (125mL), add 1.05mL 6M NaOH solutions after being stirred to react 30min at room temperature, are added dropwise 6M HCl and are allowed to neutralize (pH~7.0).Reaction solution with 0.45 μm of membrane filtration, the filtered solution ultrafiltration membrane packet ultrafiltration through 30kDa again, ultrafiltrate is through the desalination of G25 gel filtration chromatographies and is lyophilized, Obtain depolymerization product dHsFG ' 1.623g (yield 46.4%).

(5) compound B-26~B8 is isolated and purified：Take 1g depolymerization products dHsFG ' with loading after 10mL 0.2M NaCl dissolvings In Bio-Gel P-10 gel columns (L 200cm), it is eluted with 0.2M NaCl solutions, flow velocity 15mL/h, 2.5mL/ pipe is received Collection elution fraction.Ultraviolet spectrophotometry (λ max 234nm) monitors and merges the identical elution fraction of component.HPGPC(TSK G2000 SW columns) detection chromatography samples purity and composition.Non- purification of samples continues pure with Bio-Gel P-10 gel filtration chromatographies Change.Oligosaccharides is purified to be lyophilized after Sephadex G-10 or Bio-Gel P-2 gel column desalinations.

(6) Spectrum Analysis：With 1.2 (6) the methods of embodiment 1, examined using Bruker DRX 800MHz nuclear magnetic resonance spectrometers It surveys¹H-/¹³C- and 2D-NMR, microTOF-QII ESI-MS (Bruker, Germany) spectrometer analysis Q-TOF MS.Detection Data are divided using Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany) software Analysis.

3.3 result

(1) 3.2 step process are pressed, compound B-26 47mg, B7 55mg, B8 35mg, HPGPC methods is obtained and detects it Purity is equal>99% (area normalization method calculating).

(2) compound B-26~B8 structure elucidations：The HPGPC collection of illustrative plates of compound B-26~B8 is as shown in Fig. 10.In conjunction with¹H-/¹³C-/2D-NMR and Q-TOF MS analyses, compound B-26, B7 and B8 are L-Fuc_3S4S-(α1,3)-L-ΔUA-(α1,3)-{D- GalNAc_4S6S-(β1,4)-[L-Fuc_3S4S-(α1,3)]-D-GlcA-(β1,4)}_n-D-GalNAc_4S6S-(β1,4)-[L- Fuc_3S4S(α 1,3)]-D-GlcA (wherein n=0,1 and 2, i.e. pentasaccharides, eight sugar and 11 sugar).Its chemical structural formula is：

In its B6, n=0；In B7, n=1；In B8, n=2.

【Embodiment 4】The preparation of compound A6, A7 and A8：L-Fuc_2S4S-(α1,3)-[6-Me-ΔUA-(α1,3)]-{D- Gal-NAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]-D-6-Me-GlcA-(β1,4)}₂-D-GalNAc_4S6S-ol、L- Fuc_2S4S-(α1,3)-L-ΔUA-(α1,3)-{D-GalNS_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]-D-GlcA-(β1, 3)}₂-D-GalNS_4S6S- ol and L-Fuc_2S4S-(α1,3)-[6-Me-ΔUA-(α1,3)]-{D-GalNAc_4S6S-(β1,4)-[L- Fuc_2S4S-(α1,3)-]-D-6-Methyl-GlcA-(β1,3)}₂- D-1-Me-GalNAc_4S6S-ol

4.1 material

Compound A2, preparation method and chemical constitution are as described in Example 1.

Hydrazine sulfate, hydrazine hydrate, Et₃N.SO₃([Et₃N-SO₃H]Cl,N,N-diethyl-N-sulfoethanammonium Chloride) it is ommercially available AR.

4.2 methods and result

(1) preparation of compound A6：Compound 10mg A2 are taken to be dissolved in 0.5mL water, through Dowex 50X8 hydrogen form cations Exchange resin column is converted to H⁺Type, eluent are lyophilized after being neutralized with tetrabutylammonium hydroxide, obtain A2 4-butyl ammoniums 19mg.By institute It obtains A2 4-butyl ammoniums and is dissolved in 1mL dimethyl sulfoxide (DMSO)s (DMSO), 15 μ L2M trimethyl silicanes diazomethanes (TMSD) are added, at room temperature After reacting 60min, 15 μ L acetic acid are added and remove remaining TMSD, absolute ethyl alcohol 4mL, 4000rpm × 30min centrifugation is added at 4 DEG C, Precipitation plus 1mL are water-soluble, through Dowex/r50w × 8 50-100 (Na⁺Type) exchanger resin is converted into sodium form.Products therefrom with Simultaneously A6 8.35mg are lyophilized to obtain in Sephadex G-10 columns desalinations.1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR confirms that the structure of compound A6 (the carbomethoxy signal on Δ UA is located at 3.70ppm) is L-Fuc_2S4S-(α1,3)- [6-Methyl-ΔUA-(α1,3)]-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]-D-6-Methyl-GlcA- (β1,4)}₂-D-GalNAc_4S6S- ol, structural formula are：

(2) preparation of compound A7：Compound A2 10mg are taken, 2.5mg hydrazine sulfates and 0.25mL hydrazine hydrates, nitrogen is added Under protection, 15h is stirred to react at 105 DEG C；0.5mL 16%NaCl solution and 3mL absolute ethyl alcohols are added into reaction solution, 4000rpm × 20min centrifuge, gained precipitation with 10mL it is water-soluble after with 500-1000Da bag filters dialyse, trapped fluid is lyophilized de- Acetylate 8mg.Confirm deacetylated product (at 2.0ppm with 1.2 (6) the methods detection NMR spectra of embodiment 1 Ac methyl signals disappear, GalNH₂2 H signals present in 3.0ppm) chemical constitution be：

A2 deacetylated products are dissolved in 1mL water, 36mg Na are added₂CO₃And 55 DEG C are heated to, after starting reaction 0,5 and 10h be added Et₃N.SO₃15mg is stirred to react 15h at 55 DEG C, 1mL16%NaCl is then added into reaction solution Solution and 8mL absolute ethyl alcohols, 4000rpm × 20min centrifugations；It is water-soluble with 10mL to collect precipitation, then with the saturating of 500-1000Da Bag dialysis is analysed, dialysis trapped fluid is freeze-dried to obtain N- sulfating product A7 6.8mg.

1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR confirms that the chemical constitution of compound A7 is L-Fuc_2S4S-(α1,3)-L-ΔUA-(α1,3)-{D-GalNS_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]-D-GlcA-(β 1,3)}₂-D-Gal-NS_4S6S- ol, structural formula are：

(3) preparation of compound A-28：Take 20mg compounds A2 with Dowex 50X8 hydrogen type cation exchange resins for catalysis 5mL methanol solutions are added in agent, heated overnight at reflux under nitrogen protection, are filtered to remove resin, and filtrate rotates to remove solvent, obtains The positions the C1 hydroxy alkylated product of A2, the product are converted to H through Dowex 50X8 cation exchange resin columns⁺Type, eluent are used Tetrabutylammonium hydroxide is lyophilized after neutralizing, and obtains the 4-butyl ammonium 37mg of the positions the C1 hydroxy alkylated product of A2.Products therefrom with 2mL DMSO dissolving, adds 30 μ L 2M TMSD, reacts at room temperature and 30 μ L acetic acid are added after 60min remove remaining TMSD, and 4 2mL 16%NaCl solution and 8mL absolute ethyl alcohols are added at DEG C, 4000rpm × 30min centrifugations, gained precipitation is water-soluble with 2mL, warp Dowex/r50w×8 50-100(Na⁺Type) exchange resin column is converted into sodium form.Products therefrom is with Sephadex G-10 column desalinations And A8 13.5mg are lyophilized to obtain.

1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR confirms the compound A-28 (first of Δ UA methyl esters Base signal is located at 3.70ppm, and reducing end methyl signals are located at 3.23ppm) structure be L-Fuc_2S4S-(α1,3)-[6- Methyl-ΔUA-(α1,3)]-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)-]-D-6-Methyl-GlcA-(β 1,3)}₂-D-1-Methyl-Gal-NAc_4S6S- ol, structural formula are：

Similarly, the linear chain or branched chain alkane or the corresponding alcohol of alkene that C2-C6 also may be selected, according to the side of the present embodiment Method prepares corresponding hydroxy alkylated product A8 '

【Embodiment 5】The preparation of compound B9, B10 and B11：L-Fuc_2S4S-(α1,3)-L-ΔUA-(α1,3)-D-Gal- NAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-GlcA-(β1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α 1,3)]-D-β1-Bn-GlcA、L-Fuc_2S4S-(α1,3)-L-6-Me-ΔUA-(α1,3)-D-GalNAc_4S6S-(β1,4)-[L- Fuc_2S4S-(α1,3)]-D-6-Me-ΔUA-GlcA-(β1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D- β 1-Bnz-6-Me-GlcA and L-Fuc_2S4S-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S- (α1,3)]-D-GlcA-(β1,3)}₂-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-1-deoxy-1- amino-GlcA-ol-N-4-benzoic ethyl ester

5.1 material

It is that raw material is sugared (B3 ') according to preparation 11 described in embodiment 2 using SvFG, it is sugared according to preparing eight described in embodiment 3 (B7’)。

4-aminobenzoic acid ethyl ester, tetrabutylammonium hydroxide, dichloromethane, pyridine, acetic anhydride, benzyl alcohol, boron trifluoride Ether etc. is ommercially available AR.

5.2 methods and result

(1) preparation of compound B9 takes 40mg compounds B7 ' to be dissolved in 4mL water, is handed over through Dowex 50X8 hydrogen form cations It changes resin column and is converted to H⁺Type, eluent are lyophilized after being neutralized with tetrabutylammonium hydroxide, obtain B7 ' 4-butyl ammoniums 80mg.Take institute B7 ' 4-butyl ammoniums are obtained, 8mL pyridines and 8mL acetic anhydrides is added, 30min is stirred to react at 100 DEG C, is blown at room temperature with nitrogen It is dry, 4mL dichloromethane is dissolved the residue in, 128 μ L benzyl alcohols are added, boron trifluoride ether (BF is added dropwise at 0 DEG C₃OEt₂)20μ L is heated to reflux 36h, and water is added to terminate reaction, and oscillation takes CH after standing₂Cl₂Layer simultaneously volatilizes CH₂Cl₂；Add at room temperature into residue Enter 4mL 0.02M sodium methoxides-methanol solution, is stirred to react 10min deacetylates；After volatilizing methanol, through Dowex 50X8 hydrogen Type cation exchange resin column is converted to H⁺Type, eluent are neutralized with sodium hydroxide, are isolated and purified through Bio-gel P6, merging contains Sugar-like product, concentration is with Sephadex G-10 columns desalinations and is lyophilized, and obtains compound B9 24mg.

1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR confirms compound B9 (benzyl-CH₂Signal Positioned at 4.6ppm, phenyl ring signal is located at 7.3ppm) structure be L-Fuc_2S4S-(α1,3)-L-ΔUA-(α1,3)-D- GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-GlcA-(β1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S- (α 1,3)]-D-1-Benzyl-GlcA-ol, structural formula is：

(2) preparation of compound B-11 0：Take compound B9 10mg to be dissolved in 0.5mL water, through 8 Hydrogen sun of Dowex 50X from Sub-exchange resin column is converted to H⁺Type, eluent are lyophilized after being neutralized with tetrabutylammonium hydroxide, obtain B9 4-butyl ammoniums 19mg.It will Gained B9 4-butyl ammoniums are dissolved in 1mL dimethyl sulfoxide (DMSO)s (DMSO), and 15 μ L 2M trimethyl silicanes diazomethanes (TMSD), room is added After warm lower reaction 60min, 15 μ L acetic acid are added and remove remaining TMSD, add absolute ethyl alcohol 4mL, 4000rpm × 30min at 4 DEG C Centrifugation, precipitation plus 1mL are water-soluble, through Dowex/r50w × 8 50-100 (Na⁺Type) exchanger resin is converted into sodium form.Products therefrom with Bio-Gel P-6 purifying, with Sephadex G-10 columns desalinations and is lyophilized to obtain B10 8.35mg.

1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR confirms (the carboxyl ester methyl of compound B-11 0 Signal is located at about 3.7ppm, benzyl-CH₂Signal is located at 4.6ppm, and phenyl ring signal is located at 7.3ppm) structure be L-Fuc_2S4S-(α 1,3)-L-6-Methyl-ΔUA-(α1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-6-Methyl-Δ UA-GlcA-(β1,3)-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-β-1-Benzyl-6-Methyl- GlcA, structural formula are：

(3) preparation of compound B-11 1 takes that 4-aminobenzoic acid ethyl ester 330mg is dissolved in 80 μ L glacial acetic acid and the mixing of methanol is molten (1 in liquid:9) sodium cyanoborohydride 70mg dissolving, is separately added, compound B3 ' 20mg are dissolved in 2mL water, and the mixed solution, 60 DEG C 4h is reacted in water bath with thermostatic control, 2mL chloroforms is added to extract, and water phase is purified through Bio-gel P10 column chromatographies, through Sephadex G-10 columns Desalination is simultaneously lyophilized up to the about 14mg of compound B-11 1.

1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR confirmations compound B-11 1 (ethoxycarbonyl- CH₃With-CH₂Signal is located at 1.3ppm and 4.3ppm, and phenyl ring signal is divided into two groups, is located at 6.78ppm and 7.68ppm) Structure be L-Fuc_2S4S-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D- GlcA-(β1,3)}₂-D-GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-1-deoxy-1-amino-GlcA-ol- N-4-benzoic ethyl ester, structural formula are：

Similarly, benzyl alcohol is changed into corresponding C8-C12 aromatic hydrocarbons alcohol (such as to methylbenzyl alcohol, to amyl benzyl alcohol), According to the preparation method of the present embodiment B9, the series derivates B9 ' with corresponding C8-C12 aryls is obtained；4- aminobenzoics Acetoacetic ester changes 4- amino-(C8-C12) aromatic hydrocarbons formic acid esters (such as 4- amino-benzoic acids propyl ester, 4- amino-benzoic acids pentyl ester) into, According to the preparation method of the present embodiment B11, the series derivates B11 ' with corresponding C8-C12 aryls is obtained.

【Embodiment 6】The preparation of oligosaccharide mixture C1

6.1 material

SvFG, source is the same as described in embodiment 1.

The agents useful for same such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium borohydride, sodium chloride and ethyl alcohol are commercially available Analytical reagents.Sephadex G-50 (medium, 50-100 μm), GE Healthcare products.

6.2 method

(1) the quaternary ammonium salt conversion of SvFG：SvFG 70g are taken to be dissolved in 1L water；Separately benzethonium chloride 175g is taken to be dissolved in 2.8L water.It stirs Lower benzethonium chloride solution titration SvFG solution is mixed, after the completion of titration, centrifugation, vacuum is dry after precipitation is washed with deionized three times It is dry to obtain SvFG quaternary ammonium salts 210g.

(2) carboxyl esterification of SvFG：SvFG quaternary ammonium salts obtained by step (1) are dissolved in 1.020L DMF, add 29mL benzyl chlorides, After being stirred to react for 24 hours at 35 DEG C, standing makes reaction solution be down to room temperature (25 DEG C), sample detection¹H NMR spectras and the carboxylic for calculating FG Base degree of esterification is about 46%；

(3) β of the SvFG in the presence of reducing agent-elimination depolymerization：333mL is added into step (2) described reaction solution to contain 0.4M NaBH₄The 0.08M newly prepared alcohol sodium alcohol solution, be stirred to react 30min at room temperature.

(4) it post-processes：1.333L saturated nacl aqueous solutions are added into reaction solution obtained by step (3) and 10.7L is anhydrous Ethyl alcohol, 4000rpm × 10min centrifugations, gained precipitation are dissolved in 5L water and 40mL 6M NaOH solutions are added, react at room temperature After 30min, 6M HCl neutralization reactions liquid (pH~7.0) is added dropwise.Products therefrom is sequentially through 0.1m²10kDa and 3kDa ultrafiltration (Millipore) ultrafiltration of film packet obtains oligosaccharide mixture C1 35g to remove macromolecular and small molecular weight impurity.

(5) Spectrum Analysis：1.2 (6) the methods of embodiment 1 detect¹H-/¹³C-/2D NMR spectras.

6.3 result

(1) oligosaccharide mixture C1 35g, yield 50% are obtained according to the method；

(2) HPGPC analysis shows that, containing six sugar, nine sugar, ten disaccharides, ten pentasaccharides, 18 sugared and 21 sugar are respectively in C1 14.2%, 23.1%, 24.1%, 16.0%, 8.9%, 5.1%.

(3) oligosaccharide mixture C1¹³C NMR spectras and ownership are as shown in Fig. 11.Oligosaccharide mixture C1's¹In H NMR, Visible three groups of stronger signal peaks within the scope of 5.4~5.7ppm, wherein the signal peak positioned at 5.77ppm is to be located at oligosaccharide mixture 4 H signals of the non-reducing end Δ UA of C1.Signal at 5.6ppm and 5.43ppm is L- in nearly reducing end sugar chain respectively Fuc_2S4SEnd group hydrogen signal and be connected to the L-Fuc of non-reducing end Δ UA_2S4SEnd group hydrogen signal.

Through the signal analysis to reducing end under neutral, especially-D-GalNAc_4S6SThe positions the C1 (- CH of-ol and-D-GlcA-ol₂) Carbon signal analysis, reduction end structure be-D-GalNAc_4S6SThe content of the oligosaccharide compound of-ol is more than 95%.

In conjunction with¹³C-NMR and 2D-NMR analyses, it is L-Fuc that C1, which is by structure,_2S4S-(α1,3)-L-ΔUA-(α1,3)-{D- GalNAc_4S6S-(β1,4)-[L-Fuc_2S4S-(α1,3)]-D-GlcA-(β1,3)}_n-D-GalNAc_4S6S- ol's (n is natural number) Homology oligosaccharide compound forms, the sum of compounds content that wherein n is 1~7 about 95%.

【Embodiment 7】The preparation of oligosaccharide mixture D1 and D2

7.1 material

HsFG, source are as described in Example 2.

It is tried used in benzethonium chloride, benzyl chloride, DMF, DMSO, TMSD, sodium hydroxide, sodium borohydride, sodium chloride and ethyl alcohol etc. Agent is ommercially available AR.Molecular cut off is the ultrafiltration membrane packet (0.5m of 30kDa, 10kDa, 3kDa²), Merk Millipore。

7.2 method

(1) the quaternary ammonium salt conversion of HsFG：330g HsFG are taken to add 4.9L water dissolutions；Separately 825g benzethonium chlorides is taken to add 13.2L water Dissolving, acquired solution are added under stiring in HsFG solution, and 4000rpm centrifuges 10min, and precipitation washs three with 9L deionized waters Time, it is dried in vacuo to obtain HsFG quaternary ammonium salts 804g.

(2) carboxyl esterification of HsFG：HsFG quaternary ammonium salts obtained by step (1) are placed in 30L reaction kettles, add 3.9L DMF molten Solution.At 35 DEG C, 97mL benzyl chlorides are added, after being stirred to react for 24 hours, standing makes reaction solution be down to room temperature (25 DEG C).Sample detection¹H NMR and the carboxyl esterification degree for calculating product are about 46%；

(3) β of HsFG-elimination depolymerization is reacted with end peeling：1.3L is added into step (2) described reaction solution newly to prepare 0.08M sodium ethoxides-ethanol solution, after being stirred to react 30min at room temperature, into reaction solution be added 2.5mL 2M NaOH solutions And it is stirred to react 90min at 60 DEG C.

(4) it post-processes：5.36L saturated nacl aqueous solutions and the anhydrous second of 57L are added into step (3) described reaction solution Alcohol, 4000rpm × 10min centrifugations, gained are precipitated and dissolved in 14.5L water, the NaOH solution of 122mL 6M are added, stirs at room temperature Reaction 30min is mixed, 54.3g NaBH are then added₄, continue to be stirred to react 30min at room temperature；6M HCl neutralization reaction liquid is added dropwise (pH~7.0).Gained reaction solution uses 0.5m successively with 0.45 μm of membrane filtration, filtered fluid²30kDa (taking permeate), 10kDa (taking permeate) and (Mi Libo products) ultrafiltration of 3kDa (taking trapped fluid) ultrafiltration membranes packet are simultaneously lyophilized, thus to obtain oligosaccharides Mixture D 1 (98.7g).[note：After testing, obtained by the ultrafiltration of 30kDa ultrafiltration membranes in trapped fluid contained non-depolymerization macromolecular components In heteroglycan containing fucosan and containing aminohexose]

(5) D1 carboxyl esters：It takes oligosaccharide mixture D1 20g to be dissolved in 300mL water, is added with stirring 800mL 6.25% benzethonium chloride solution, 4000rpm × 10min is centrifuged after standing, vacuum after precipitation washs three times with 300mL deionized waters It is dry, obtain D1 quaternary ammonium salts 58g.Gained D1 quaternary ammonium salts are dissolved in 5.8L DMSO, 87mL 2M TMSD are added, stir at room temperature 87mL acetic acid is added after middle reaction 60min to remove remaining TMSD；Be added sequentially in stirring 5.9L saturated nacl aqueous solutions and 95% ethyl alcohol of 63L, 4000rpm × 30min centrifugations, gained is precipitated and dissolved in 2L deionized waters, through 3kDa ultrafiltration membranes Bao Chao Desalination is filtered, D2 (16.3g) is lyophilized to obtain in trapped fluid.

(6) Spectrum Analysis：1.2 (6) the methods with embodiment 1 detect¹H-/¹³C- and 2D-NMR.

7.3 result

(1) oligosaccharide mixture D1 yield and chemical composition analysis

Oligosaccharide mixture D1 98.7g are obtained according to the method, and yield is about 30%；

HPGPC analyzes other containing pentasaccharides, eight sugar, 11 sugar, ten tetroses, 17 sugar, 20 sugars in (attached drawing 12) display D1 It is 4.3%, 17.1%, 18.0%, 16.3%, 14.1%, 11.1%.The sugared total content in pentasaccharides~29 is about 96.0%.

Oligosaccharide mixture D1's¹³C-NMR spectrograms are as shown in Fig. 13.D1's¹There is stronger letter in H-NMR, at 5.685ppm Number, this is 4 hydrogen of Δ UA.Three groups of stronger signal peaks (5.283,5.201 and 5.030ppm) are presented in 5.0~5.6ppm, Respectively it is connected to the L-Fuc of D-GlcA, Δ UA and D-GlcA-ol_3S4Sα-end group hydrogen signal.By to reducing end glycosyl The hydrocarbon signal of end group is analyzed, and the oligosaccharide compound content that reducing end glycosyl is-D-GlcA-ol in D1 is more than 95%.

In conjunction with¹³C- and 2D-NMR analyses are as it can be seen that it is L-Fuc that mixture D 1, which is by structure,_3S4S-(α1,3)-L-ΔU-(α1, 3)-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_3S4S-(α1,3)]-D-GlcA-(β1,3)}_n-D-GalNAc_4S6S-(β1,4)- [L-Fuc_3S4S(α 1,3)]-D-GlcA-ol (n is natural number) homology oligosaccharide compound mixture.

(2) oligosaccharide mixture D2 yield and chemical composition analysis

Oligosaccharide mixture D2 16.3g are obtained according to the method, and yield is about 80%；

HPGPC analysis shows that wherein contain pentasaccharides, eight sugar, 11 sugar, ten tetroses, 17 sugar, 20 sugared respectively 3.34%, 16.71%, 17.03%, 17.25%, 13.78%, 12.25%；

With D1's¹H NMR spectras are compared, D2's¹First (ester) base letter for being connected to hexuronic acid (alcohol) is increased in H NMR newly Number, it is located at 3.7ppm and 3.2ppm.In conjunction with¹H-/¹³C- and 2D-NMR analyses are as it can be seen that it is L- that mixture D 2, which is by structure, Fuc_3S4S-(α1,3)-L-6-Me-ΔUA-(α1,3)-{D-GalNAc_4S6S-(β1,4)-[L-Fuc_3S4S-(α1,3)]-D-6-Me- GlcA-(β1,4)}_n-D-GalNAc_4S6S-(α1,3)-[L-Fuc_3S4S(α 1,3)]-D-6-Me-GlcA-ol (n is natural number) The mixture of homology oligosaccharide compound.Wherein, the sum of compounds content of n=1~9 is about 96%.

【Embodiment 8】Anti-freezing and the analysis of coagulation factor inhibitory activity

8.1 material

Sample：Oligosaccharide compound A1~A8, B1~B11, oligosaccharide mixture C1, D1, D2, by Examples 1 to 7 the method It prepares.

Reference substance：Enoxaparin sodium injection (3500~5500Da of LMWH, Mw, Sanofi-Aventis product)；

Reagent：Blood coagulation Quality Control blood plasma (047B-D024A), activated partial thromboplastin time (APTT), prothrombin time (PT) assay kit is German TECO GmbH Products；Factor IX detection kit, heparin cofactor II (HCII), anti-factor Xa detection kits, the fibrin ferment (factor of AT is relied on anti-factor IIa detection kit, AT dependences IIa), thrombin substrate CS01 (38), KK substrates CS31 (02) are HYPHEN BioMed companies (France) product；The factor VIII (FVIII), Bayer Healthcare LLC company (Germany) product；ADP, Chronolog company (U.S.) product；Chinese holly Rafter acid sodium, chloraldurate, physiological saline, are commercial reagent.

Instrument：XS105 electronic balances, FE20pH meters, METTLER TOLEDO products；HH-4 thermostat water baths, Gongyi City Give magnificent product；VOR76X-6 vortex oscillators, its woods Bell's product of Hainan；Spectrafuge-24D907386 centrifuges, Labnet products；MC-4000 coagulo meters, TICO GmbH companies (Germany) product；808 enzyme marks of Microplate Reder ELx Instrument, Bio-Tek Products；Chronolog-700 type platelet aggregation instruments, Chrono-log companies (U.S.) product.

8.2 method

(1) sample solution is prepared：Oligosaccharide compound A1~A8, B1~B11 and oligosaccharide mixture C1, D1, D2 are with Tris- HCl buffer solutions are simultaneously diluted to required serial solubility.

(2) anticoagulating active detects：After 90 μ L hostages control blood plasma addition sample or 10 μ L reference substance solutions are mixed, according to APTT With PT kit specification the methods, the clotting time (APTT and PT) is detected with MC-4000 coagulo meters.

(3) coagulation factor inhibitory activity is analyzed：

Xase inhibitory activity is analyzed：Be combined Factor IX and Factor IX detection kit, reference reagent box specification and Literature method is detected.Specifically, in 96 orifice plates, each hole be separately added into 30 μ L by test sample solution, reference substance solution or After Tris-HCl buffer solutions (negative control), 30 μ l FVIII (2IU/ml) are added sequentially, (60nM FIXa, contain 30 μ l R2 FIIa、PC/PS、Ca²⁺), vibration plate mixing, 37 DEG C of incubation 2min；Then 30 μ L R1 are added, and (50nM FX press down containing direct fibrin ferment Preparation), vibration plate mixing, 37 DEG C of incubation 1min；Add 30 μ L R3 (FXa chromogenic substrate SXa-11, about 8.4mM), microplate reader inspection Survey light absorption value (OD at 405nm₄₀₅), continuously detect 7.5min by interval of 30s.According to OD₄₀₅Changing value calculate Xase activity with And given the test agent inhibits the IC of Xase₅₀Value.

The Xa inhibitory activity analysis that AT is relied on：It is detected using heparin Anti-FXa kits.30 μ L samples are added in 96 orifice plates After product, reference substance solution or Tris-HCl buffer solutions (negative control), 30 μ L 1IU/mL AT solution, mixing and at 37 DEG C is added Lower incubation 1min, adds 30 μ L, 8 μ g/mL FXa solution, and mixing is simultaneously incubated 1min at 37 DEG C, is eventually adding 30 μ of preheating L 1.25mM Xa chromogenic substrate SXa-11, microplate reader detect OD₄₀₅。

The IIa inhibitory activity analysis that AT is relied on：It is detected using heparin Anti-FIIa kits.30 μ L are added in 96 orifice plates After sample, reference substance solution or Tris-HCl buffer solutions (negative control), 30 μ L 1IU/mL AT solution are added, vibration plate mixing exists It is incubated 2min at 37 DEG C；30 μ L 24IU/mL FIIa solution are added, after vibration plate mixing is incubated 2min at 37 DEG C, preheating is added 30 μ L 1.25mM FIIa specificity chromogenic substrate CS-01 (38), vibration plate mixing, microplate reader detect OD₄₀₅And calculate each sample Inhibit the IC of IIa₅₀Value.

The IIa inhibitory activity analysis that HC-II is relied on：It is (cloudy that 30 μ L samples, reference substance solution or Tris-HCl buffer solutions is added Property control) after, add 30 μ L, 1 μM of HCII solution, 2min be incubated at 37 DEG C；30 μ L 20NIH/mL FIIa are added, at 37 DEG C It is incubated 1min；30 μ L 4.5mM FIIa chromogenic substrates CS-01 (38) of preheating are eventually adding, microplate reader detects OD₄₀₅And it calculates Each sample inhibits the IC of FIIa₅₀Value.

Data processing：The OD for taking multiple holes to detect₄₀₅Detected value of the mean value as each concentration of test product and reference substance, with detection It is worth slope (the change rate OD of light absorption value to the linear fit of time value₄₀₅/ min) indicate coagulation factor enzymatic activity；With feminine gender The coagulation factor activity of control wells is 100%, calculates the coagulation factor activity (percentage) in the presence of given the test agent；With test sample Coagulation factor activity in the presence of product maps to given the test agent concentration, and is fitted as the following formula, calculates IC₅₀Value：

B=(IC₅₀)ⁿ/{(IC₅₀)ⁿ+[I]ⁿ}

In formula, B is the coagulation factor activity (percentage) in the presence of given the test agent, and [I] is given the test agent concentration, IC₅₀For Half-inhibition concentration (inhibits given the test agent concentration needed for 50% activity), and n is Hill coefficients.

(4) surface activation and biologically active pdgf influence：

FXII Activation Activities detect：It is separately added into 30 μ L series concentrations samples and reference substance solution in 96 orifice plates, adds 4 times of 30 μ L of people's standard plasma, 37 DEG C of incubations are diluted with 0.02M Tris-HCl (pH7.4) buffer solution of the NaCl containing 0.15M After 2min, 30 μ L 6mM kallikrein chromogenic substrates CS-31 (02) are added, microplate reader detects OD₄₀₅Value.

Platelet activation Activity determination：Acquisition healthy volunteer's anticoagulation prepares platelet rich plasma (PRP) and anaemia is small Plate blood plasma (PPP), the series concentration that Chronolog-700 types platelet aggregation instrument, turbidimetry detection physiological saline solution are prepared The blood platelet induced aggregation activity of given the test agent solution.

8.3 result

(1) anti-freezing and coagulation factor inhibitory activity：The results are shown in Table 5.Oligosaccharide compound of the present invention and its mixing Object has significant extension APTT activity, without influencing PT, TT, illustrates that it can have significant inhibition intrinsic coagulation pathway Anticoagulant active, and exogenous blood coagulation does not make significant difference.Oligosaccharide compound and its mixture of the present invention are to factor Xase There is significant inhibitory activity；In the presence of with or without antithrombase (AT), to the coagulation factors such as prothrombin a, Xa, XIIa without The IIa inhibitory activity that the heparin cofactor II (HC-II) for significantly affecting, but some strength may be present is relied on.

The linear chain or branched chain alkane or the corresponding alcohol of alkene of selection C2-C6 prepares corresponding hydroxyl according to embodiment 4 Alkylate A8 '.It is tool to the progress of these series derivates activity the study found that they have the activity similar with A8 Have and extend APTT activity (drug concentration in multiplication APTT clotting times is 7.0-10 μ g/mL), without influencing PT, TT；To the factor Xase has significant selective inhibitory activity (IC₅₀, 50-100ng/mL), to the coagulation factors such as prothrombin a, Xa, XIIa without It significantly affects, the IIa inhibitory activity that the heparin cofactor II (HC-II) with some strength is relied on.

According to the preparation method of the present embodiment B9, the series derivates B9 ' with corresponding C8-C12 aryls is obtained, according to The preparation method of the present embodiment B11 obtains the series derivates B11 ' with corresponding C8-C12 aryls.They be respectively provided with The drug concentration of activity similar with B11 B9, multiplication APTT clotting times is 6.0-9 μ g/mL, without influencing PT, TT；To the factor Xase has significant selective inhibitory activity (IC₅₀, 40-110ng/mL), the heparin cofactor II (HC-II) with some strength The IIa inhibitory activity of dependence.

The anti-freezing of 5. oligosaccharide compound of table and oligosaccharide mixture and coagulation factor inhibitory activity

Note：#,>128μg/mL；##,>5000ng/mL

(2) surface activation is influenced with biologically active pdgf：

XII Activation Activities are analyzed：In not higher than 100 μ g/ml concentration ranges, whole oligosaccharide compounds and oligosaccharide mixture Without significant XII Activation Activities；

Platelet activation activity analysis：In not higher than 50 μ g/ml concentration ranges, whole oligosaccharide compounds and oligosaccharides mixing Object is without significant platelet activation activity；

【Embodiment 9】Antithrombotic acitivity is influenced with bleeding

9.1 material

A2 preparation as described in Example 1, D1 prepare it is as described in Example 7.

Reference substance：Low molecular weight heparin (LMWH), Sanofi-Aventis companies (France) product, lot number 4SH69.

Reagent：Chloraldurate (chloral hydrate), Sinopharm Chemical Reagent Co., Ltd.；Physiological saline, Kun Mingnan Boundary pharmaceutical Co. Ltd.

Experimental animal：SD rats, 250~350g weight, male, Hunan SJA Laboratory Animal Co. , Ltd carry For licensing number SCXK (Hunan) 2011-0003；New zealand rabbit is provided by Kunming Medical University, SCXK (Yunnan) 2011-0004, For making rabbit brain powder leachate.

9.2 method

9.2.1 anti-venous thronbosis experiment

Grouping and administration：Rat is randomly divided into 8 groups, every group 8.The dosage of experiment packet and each group animal is (1) Physiological saline (NS) control group；(2) LMWH 4.0mg/kg groups；(3) A2 2.5mg/kg groups；(4) A2 groups 5.0mg/kg；(5)A2 10mg/kg groups；(6) D1 2.5mg/kg groups；(7) D1 5.0mg/kg groups；(8) D1 10mg/kg groups.Each group rat is through back skin Lower injection (sc.) administration, administered volume is 1mL/kg.Modeling experiment is carried out after 1h is administered.

It is prepared by rabbit brain powder leachate：After new zealand rabbit is put to death, rabbit brain is taken out immediately, by literature method (Thromb Haemost,2010,103(5):Rabbit brain powder leachate 994-1004) is prepared, is stored for future use at -20 DEG C.

Rabbit brain powder leachate induction inferior vena cava thrombosis is formed：10% chloral hydrate anesthesia (300mg/ of rats by intraperitoneal injection Kg), stomach wall is longitudinally cut off along ventrimeson, removes internal organ, inferior caval vein and its branch are detached, under the left renal vein of inferior caval vein Edge passes through ligature, ligatures the inferior caval vein branch under left renal vein.2% rabbit brain powder leachate (1mL/ of vena femoralis injection Kg), after 20 seconds, left renal vein lower edge ligature is ligatured.Postoperative that internal organ are put back to abdominal cavity, hospital gauze (physiological saline infiltration) covers It covers, after 20min, blood vessel is closed with hemostasis clamp at 2cm under ligature, longitudinally split blood vessel, removal of thromboses measures thrombus length And wet weight of thrombus is weighed, 50 DEG C of dryings weigh dry weight afterwards for 24 hours.

Data processing and statistics：It is arranged using SPSS softwares, analysis data, measurement data is with mean ± standard deviation (x ± s) It indicates.The data test of normality of difference group is examined using One-Sample K-S, and homogeneity test of variance is examined using Levene, If data fit normal distribution, variance is neat, then judges its conspicuousness using One-Way ANOVA, on the contrary then use Two- Independent-Samples Test judge its conspicuousness.

9.2.2 hemorrhagic tendency detects

Grouping and administration：Mouse is randomly divided into 10 groups, every group 8.The dosage of experiment packet and each group animal is, (1) physiological saline (NS) control group；(2) LMWH 4.0mg/kg groups；(2) LMWH 20mg/kg groups；(3)LMWH 100 mg/kg Group；(4) A2 5mg/kg groups；(5) A2 25mg/kg groups；(6) A2 125mg/kg groups；(7) D1 5mg/kg groups；(8)D1 25mg/ Kg groups；(10) D1 125mg/kg groups.Each group rat is administered through dorsal sc injection (sc.), and administered volume is 10mL/kg.

Test method：After each experimental group subcutaneous administration 60min, mouse is placed in mouse fixing device, tail method is cut and cuts off tail Rat-tail is immersed and is filled in the beakers of 40mL pure water (37 DEG C) by sharp 5mm, since cutting rat-tail outflow the 1st and bleeding timing, And keep stirring, when 60min, remove after beaker places 60min, UV spectrophotometer measuring solution absorbance (OD₅₄₀)。

Healthy mice whole blood separately is taken, is added in 40mL pure water with different volumes Mouse whole blood, is stood after stirring evenly 60min, with method detection solution absorbance (OD₅₄₀) and draw volume-absorbance curve as the standard curve for calculating amount of bleeding. The amount of bleeding of each experimental mice is calculated with standard curve.

9.3 result

(1) antithrombotic acitivity：As shown in Fig. 14, the results showed that, A2 and D1 under test dose all have significant anti- Thrombus activity, at 5mg/kg~10mg/kg, thrombosis inhibiting rate can reach 70% or more.

(2) hemorrhagic tendency influences：As shown in Fig. 15, equivalent antithrombotic dosage equimultiple high dose administration under, A2 and The amount of bleeding of D1 administration groups is substantially less than LMWH administration groups.

【Embodiment 10】It is prepared by A3 medicinal composition freezing-dried powder injections

10.1 materials

Compound A-13,12 sugar compounds of purifying prepared according to 1 the method for embodiment.

NaCl, commercially available, pharmaceutical grade；Sterile water for injection；Pyrex control injection bottle, Millipore in 2mL 2 ultrafiltration systems of Pellicon (Merk Millipore), 35 EL freeze driers of VirTis Ultra.

10.2 prescriptions

10.3 preparation processes

(1) process route：The A3 (40g) and NaCl (8g) for weighing 2 times of recipe quantities add water for injection 1.0L to dissolve, stirring After dissolving completely, Millipore ultrafiltration apparatus is used to remove pyrogen removal with molecular cut off for 10kDa ultrafiltration membrane packets.Gnotobasis Under, after 0.22 μm of membrane filtration degerming, by the filling cillin bottle in capacity 2mL of acquired solution, every bottle of 0.5mL, pouring process monitoring Loading amount, half tamponade in the drying box for setting pilot scale type freeze drier (U.S. VirTis), are frozen by the freeze-drying process of setting Dry, tamponade, outlet rolls lid, examines.

(2) freeze-drying process：

Precooling：By sample inlet, drop partition board temperature keeps 1h to -25 DEG C, is cooled to -45 DEG C, keeps 3h；Cold-trap is down to -50 DEG C, start to be evacuated to 40Pa.

Distillation：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, keeps 6h, and vacuum keeps 40~ 30Pa。

It is dry：2h is warming up to -5 DEG C, and 2h, vacuum is kept to keep 30~20Pa；0.5h is warming up to 10 DEG C, keeps 3h, vacuum Keep 30~20Pa；0.5h is warming up to 40 DEG C, keeps 4h, vacuum is evacuated to minimum.

10.4 results

A3 lyophilized preparations certified products 1960, product qualified rate about 98% are prepared by the preparation process.On inspection, Its lyophilized cake appearance is regular；Sterile, heat source and particulate matter check qualified；Water content detection result shows that its water content is less than About 3%, loading amount testing result shows its loading amount in the range of planning the 95~115% of loading amount.

【Embodiment 11】It is prepared by D1 medicinal composition freezing-dried powder injections

11.1 materials

Oligosaccharide mixture D1 is prepared according to 7 the method for embodiment.

NaCl, commercially available, pharmaceutical grade；Sterile water for injection；Pyrex control injection bottle, Millipore in 2mL 2 ultrafiltration systems of Pellicon (Merk Millipore)；Freeze dryer (LYO-20m²), the limited public affairs of Shanghai Dong Fulong science and technology shares Department.

11.2 prescriptions

11.3 preparation processes

(1) technical process：The D1 (1000g) and NaCl (180g) for weighing 20 times of recipe quantities add water for injection 20L to dissolve, After stirring and dissolving is complete, Millipore ultrafiltration apparatus is used to remove pyrogen removal with molecular cut off for 10kDa ultrafiltration membrane packets.It is sterile Under environment, after 0.22 μm of membrane filtration degerming, by the filling cillin bottle in capacity 2mL of acquired solution, every bottle of 0.5mL, pouring process Loading amount is monitored, type of production freeze drier (LYO-20m is set in half tamponade², Shanghai Dong Fulong) drying box in, by setting jelly Dry process is lyophilized, tamponade, outlet, rolls lid, qualified through examining, and obtains finished product.

(2) freeze-drying process

Precooling：Sample inlet, drop partition board temperature keep 1h to -25 DEG C；- 45 DEG C are cooled to, 3h is kept；Cold-trap is down to -50 DEG C, start to be evacuated to 40Pa.

11.4 results：

D1 lyophilized preparations certified products 17,600, product qualified rate about 88% are prepared by the preparation process.

Appearance/character：This product is white loose block.

Loading quantity inspection：Gravimetric method inspection meets regulation.

Sterility test：Take this product appropriate, check in accordance with the law (《Chinese Pharmacopoeia》Version four in 2015 is 1101).Inspection result table Bright, this batch sample meets injection quality requirement.

Pyrogen test：This product is taken, the solution of the 3.5mg containing D1 in every 1mL is made, in accordance with the law (《Chinese Pharmacopoeia》Version four in 2015 Portion 1142) it checks, the results showed that, this batch sample meets the quality requirement of injection pyrogen test.

Claims

1. a kind of oligosaccharide compound or its pharmaceutically acceptable salt, which is characterized in that the oligosaccharide compound has antithrombotic Activity, and with general formula structure shown in formula (I)：

In formula,

R₁、R₂、R₃、R₄、R₅Optionally mutually independent-H or-SO₃H；

R₆Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 aryl；

R₇Optionally-H ,-SO₃H, C2-C5 acyl groups；

R₈Optionally group shown in formula (II), formula (III) or formula (IV)：

In its formula (II), (III) and formula (IV),

R₁、R₂、R₃、R₄、R₅、R₆And R₇It is defined above；

R₁₁Optionally-NHR₁₂、-OR₁₃, wherein R₁₂And R₁₃Optionally-H, substituted or unsubstituted C1-C6 alkyl or C7-C12 virtues Base；And

N is optionally 0 or 1~8 natural number.

2. oligosaccharide compound as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that work as R₈For formula (II) institute When showing group, the oligosaccharide compound has general formula structure shown in formula (V)：

Wherein, R₁=-H, R₂=R₃=R₄=R₅=-SO₃ ^-；Or R₁=R₃=R₄=R₅=-SO₃ ^-；R₂=-H.

3. oligosaccharide compound as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that work as R₈For formula (III) institute When showing group, the oligosaccharide compound has general formula structure shown in formula (VI)：

Wherein R₁=-H, R₂=R₃=R₄=R₅=-SO₃ ^-；Or R₁=R₃=R₄=R₅=-SO₃ ^-、R₂=-H.

4. oligosaccharide compound as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that work as R₈For formula (IV) institute When showing group, the oligosaccharide compound has general formula structure shown in formula (VII)：

Wherein：R₁=-H, R₂=R₃=R₄=R₅=-SO₃ ^-；Or R₁=R₃=R₄=R₅=-SO₃ ^-、R₂=-H.

5. such as Claims 1 to 4 any one of them oligosaccharide compound or its pharmaceutically acceptable salt, which is characterized in that institute It states in its formula (I), (V), (VI), general formula structure shown in (VII), n is optionally 1,2,3 or 4.

6. oligosaccharide compound as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that it is described pharmaceutically Acceptable salt is optionally alkali metal salt, alkali salt or organic ammonium salt.

7. oligosaccharide compound as claimed in claim 6 or its pharmaceutically acceptable salt, which is characterized in that it is described pharmaceutically Acceptable salt is optionally sodium salt, sylvite or calcium salt.

8. a kind of oligosaccharide compound described in claim 1 forms and oligosaccharide mixture or its pharmacy with antithrombotic acitivity Upper acceptable salt, it is characterised in that：The oligosaccharide mixture by oligosaccharide compound described in claim 1 homologue group At；And in the oligosaccharide compound described in the formula (I) for forming the oligosaccharide mixture, R₈It is all formula (II), is all formula (III) or it is all group shown in formula (IV), and with molar ratio computing, R₈It is all formula (II), is all formula (III) or is all formula (IV) formula (I) oligosaccharide compound of group shown in ratio shared in the mixture is not less than 95%.

9. the preparation method of a kind of oligosaccharide compound described in claim 1 or its pharmaceutically acceptable salt, it is characterised in that： After the carboxylate for preparing natural fucosylated glycosaminoglycan, then in organic solvent, optionally in the presence of highly basic and reducing agent Make the mixture that " beta-elimination reaction " occurs for fucosylated glycosaminoglycan and end group " reduction reaction " obtains homology oligosaccharide compound； Or make fucosylated glycosaminoglycan that " beta-elimination reaction " and end group " peeling reaction " acquisition homology widow occur in the presence of highly basic The mixture of sugar compounds；Then few to obtain required purifying by isolating and purifying and optionally carrying out substituent structure modification Sugar compounds.

10. preparation method as claimed in claim 9, it is characterised in that：The oligosaccharide compound has defined in claim 1 General formula structure shown in formula (I), and its R₈For group shown in formula (II) defined in claim 1；The method includes：

(a) fucosylated glycosaminoglycan is changed into quaternary ammonium form, then in organic solvent, by the fucosylated osamine of gained Carboxyl in glycan quaternary ammonium salt on hexuronic acid residue is completely or partially converted into carboxylate；

(b) in the anhydrous organic solvent there are reducing agent, with highly basic processing step (a) the fucosylated glycosaminoglycan carboxylic acid Ester is allowed to that β-elimination depolymerization and end group reduction reaction occurs, thus to obtain the homology that reducing end is D- acetylamino galactosamine alcohol The mixture of oligosaccharide compound；

(c) homology oligosaccharide mixture obtained by step (b) is changed into alkali metal salt, then its carboxylic acid of basic hydrolysis in aqueous solution Ester obtains the mixture of the homology oligosaccharide compound containing free carboxy；

(d) oligosaccharide mixture obtained by chromatography separating step (c) is used, thus to obtain the oligosaccharide compound of purifying；

(e) substituent structure modification optionally is carried out to purifying oligosaccharide compound obtained by step (d).

11. preparation method as claimed in claim 10, which is characterized in that in the preparation process：

In its step (a), the fucosylated glycosaminoglycan quaternary ammonium salt be benzyl rope ammonium salt (N, N- dimethyl-N-[2- [2- [4 (1, 1,3,3- tetramethyl butyls) phenoxy group] ethyoxyl] ethyl] benzene ammonium carbamate)；The organic solvent is dimethylformamide (DMF) Or DMF- alcohol mixtures；The carboxylate is benzyl ester, and it refers to fucosylated osamine that all or part, which is converted into carboxylate, Carboxyl esterification degree in glycan is in the range of about 30% to about 100%；

Step (b) organic solvent is dimethylformamide (DMF) or DMF- alcohol mixtures；The reducing agent is hydroboration Sodium；The highly basic is sodium ethoxide；

Step (c) it is described by oligosaccharide mixture be changed into alkali metal salt be directed in reaction solution be added saturated sodium-chloride water solution will Oligosaccharide mixture is transformed into sodium-salt form；The basic hydrolysis refers to being hydrolyzed together in the NaOH aqueous solutions of a concentration of 0.25M~1M It is the carboxylate of oligosaccharide compound；

Step (d) chromatography includes but not limited to gel chromatography chromatography and/or ion-exchange chromatography.

Step (e) the further substituent structure, which is modified, includes but not limited to：D-Glucose aldehydic acid in oligosaccharide compound Carboxyl esterification on base and unsaturated hexuronic acid base；D- acetylamino galactosamine bases are deacetylated and optionally acylated again or sulphur again Acidification；The positions the C1 hydroxy alkylated of reducing end-D-GalNAc-ol.

12. preparation method as claimed in claim 11, it is characterised in that：The oligosaccharide compound has defined in claim 1 Formula (I) shown in general formula structure, and its R₈For group shown in formula (III) or formula (IV) defined in claim 1；The method Specific steps include：

(a) fucosylated glycosaminoglycan is changed into quaternary ammonium form, and in organic solvent, the fucosylated osamine of gained is gathered The carboxyl on hexuronic acid residue in sugared quaternary ammonium salt is completely or partially converted into carboxylate；

(b) it in anhydrous organic solvent, with highly basic processing step (a) the fucosylated glycosaminoglycan carboxylate is allowed to that β-occurs Depolymerization is eliminated, then makes depolymerization product that " peeling is reacted " occur to lose reducing end glycosyl-by the way that a small amount of strong alkali aqueous solution is added D-GalNAc, thus to obtain the mixture for the homology oligosaccharide compound that reducing end glycosyl is-D-GlcA；

(c) and by homology oligosaccharide mixture obtained by step (b) it is changed into alkali metal salt, and basic hydrolysis homology oligosaccharides in aqueous solution The carboxylate of compound obtains the mixture of the homology oligosaccharide compound containing free carboxy；

(e) further substituent structure modification optionally is carried out to the purifying oligosaccharide compound obtained by step (d).

13. preparation method as claimed in claim 12, which is characterized in that in the preparation process：

In step (a), the quaternary ammonium salt is benzyl rope ammonium salt；The organic solvent is DMF or DMF- alcohol mixtures；The carboxylic acid Ester is benzyl ester；" being completely or partially converted into carboxylate " refers to the carboxyl esterification degree of fucosylated glycosaminoglycan about In the range of 30% to about 100%；

In step (b), the organic solvent is DMF or DMF- alcohol mixtures；The highly basic is sodium ethoxide；A small amount of highly basic Aqueous solution refers to the NaOH aqueous solutions for the 1M~2M for being equivalent to about 1/5 to 1/10 volume of reaction solution total volume；

In step (c), it is described by oligosaccharide mixture be changed into alkali metal salt be directed in reaction solution be added saturated sodium-chloride it is water-soluble Gained oligosaccharide mixture is changed into sodium-salt form by liquid；The basic hydrolysis refers to the NaOH aqueous solutions in a concentration of 0.05M~1M The carboxylate of middle hydrolysis oligosaccharide compound；

In step (d), the chromatography includes but not limited to gel chromatography chromatography and/or ion-exchange chromatography；

In step (e), the further substituent structure modification includes but not limited to：D-GlcA in oligosaccharide compound and Δ Carboxyl esterification on UA；D-GalNAc is deacetylated and optionally acylated again or sulphation again；It contracts the positions C1 of reducing end-D-GlcA half The alkylation of aldehyde, restores aminated or reductive alkylation at reduction.

14. a kind of pharmaceutical composition with antithrombotic acitivity, which is characterized in that containing a effective amount of active ingredient of antithrombotic and Pharmaceutically acceptable excipient, the active ingredient is oligosaccharide compound described in claim 1 or its is pharmaceutically acceptable Salt or oligosaccharide mixture according to any one of claims 8 or its pharmaceutically acceptable salt.

15. pharmaceutical composition as claimed in claim 14, which is characterized in that the preparation formulation of described pharmaceutical composition is injection With aqueous solution or freeze-dried powder injection, and the active constituent containing 20mg~100mg in its unit preparation form.

16. oligosaccharides described in oligosaccharide compound and its pharmaceutically acceptable salt or claim 8 described in claim 1 mixes The application of object and its pharmaceutically acceptable salt in preparing treatment and/or preventing the drug of thrombotic diseases, the thrombotic Disease is venous thronbosis, Arterial thrombosis and/or ischemic angiocardiopathy and cerebrovascular disease.