CN103145868B - A kind of lower molecular weight osamine polysaccharid derivative and pharmaceutical composition thereof and its preparation method and application - Google Patents

Abstract

A kind of lower molecular weight fucosylated glycosaminoglycan derivative (derivate of Low molecular weight Fucosylated Glycosaminoglycan with anticoagulating active is provided, dLFG), its preparation method, pharmaceutical composition containing described dLFG or its pharmacy acceptable salt, and dLFG and pharmaceutical composition thereof are preparing the application in treatment of thrombotic disorders medicine.

Description

Low-molecular-weight glycosaminoglycan derivative, pharmaceutical composition thereof, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a Low molecular weight Fucosylated Glycosaminoglycan derivative (dLFG) with anticoagulant activity, a preparation method thereof, a pharmaceutical composition containing the dLFG or pharmaceutically acceptable salts thereof, and application of the dLFG and the pharmaceutical composition thereof in preparation of medicines for treating thrombotic diseases.

Background

Thromboembolic diseases including ischemic stroke, coronary heart disease, venous thromboembolism and other diseases are the main lethal causes of human beings. Antithrombotic therapy is the basic means for clinical prevention and treatment of thrombotic diseases, however, antithrombotic drugs including fibrinolysis, anticoagulation and antiplatelet drugs all have major and common defects: bleeding tendency and severe bleeding risk.

The classic anticoagulant drugs heparin (f.IIa/Xa inhibitor) and coumarin anticoagulant drugs (VitK antagonist) were used clinically in the third and fourth generations of the last 70 years and have been the cornerstones of drug therapy for deep vein thrombosis, cardiac stroke and post-operative anticoagulation, but the risk of bleeding and the pharmacodynamic/pharmacokinetic deficiencies associated therewith have severely limited their clinical use. Heparin and coumarin drugs can widely inhibit serine protease (blood coagulation factor) in blood coagulation waterfall, the individual difference of drug effect is large, the influence factors of the drug effect are complex, and the clinical medication needs to be continuously monitored. For decades, one of the main approaches and objectives of anticoagulant drug development is to improve the selectivity of the pharmacological action target of a new drug, improve the drug efficacy, pharmacokinetic profile, and these studies have been actively progressed: wherein the clinical application of Low Molecular Weight Heparin (LMWH) such as enoxaparin and oral anticoagulant such as rivaroxaban and dabigatran is most representative. There is still a great clinical need for new anticoagulants with low bleeding tendency.

Fucosylated glycosaminoglycan (FGAG) is a glycosaminoglycan analog derived from the body wall or viscera of echinoderm sea cucumbers: FGAG has a chondroitin sulfate-like main chain, and its main chain is composed of disaccharide structural unit [ → 4) D-GlcUA (beta 1 → 3) D-GalNAc (beta 1 →) composed of hexuronic acid and hexosamine]Sequentially connected and formed; the main chain of FGAG is substituted with a fucosyl side chain, which is believed to be a (α 1 → 3) glycoside of fucose sulfateIs linked to D-GlcUA; both FGAG backbone and side chain sugar hydroxyl groups can be sulfated (Yoshida et al, Tetrahedron Lett,1992,33: 4959-);et.al,J Biol Chem,1996,271:23973-23984)。

FGAG from natural sources has obvious anticoagulation activityet al.,Thromb Res,2001,102:167-176;et al, J Biol Chem,1996,271:23973-&Walke,Blood,2006,107:3876-3882；Buyue&Sheehan, Blood,2009,114: 3092-. In addition to anticoagulant activity, FGAG has broad pharmacological activities such as anti-inflammatory, anti-tumor, fibrinolytic, hypolipidemic activity, etc. (Tovar et al, Atheroscherisis, 1996,126: 185-.

Structural modification of FGAG is one of the ways to improve its potential utility, such as preparation of its oligomeric products, with the goal of reducing its platelet and surface activation activities while retaining its anticoagulant activity. Chinese patent publication Nos. CN101735336A and CN101724086A disclose methods for preparing oligomeric fucosylated glycosaminoglycans by depolymerizing fucosylated glycosaminoglycans in an aqueous medium by peroxide depolymerization catalyzed by a fourth-period transition metal ion to give oligomeric products which are potent inhibitors of endogenous factor X enzyme, have good anticoagulant antithrombotic activity, and have good bleeding tendencyThe reduction is remarkable, and the composition can be used for preventing and/or treating thrombotic diseases. The European patents EP 0811635A1 and EP 0408770A1 carry out hydrogenperoxide depolymerization on prototype FGAG to obtain depolymerization products with molecular weight ranges of 3,000-80,000 and 3,000-42,000 respectively, and the depolymerization products can be used for preventing and treating intimal hyperplasia of blood vessels and thrombotic diseases. These patent applications all employ peroxide depolymerization to obtain oligomeric products of FGAG. The depolymerization peroxide method has the advantages of reducing the molecular weight of polysaccharide and simultaneously maintaining the characteristic chemical structure of FGAG and the anticoagulation activity, but has the disadvantages that the depolymerization degree of the product is difficult to control, and the reaction product generally needs continuous sampling detection to confirm the time point whether the reaction is terminated. Having a non-reducing end having a^4,5Fucosylated glycosaminoglycan derivatives having unsaturated bonds have not been reported so far.

Disclosure of Invention

The invention aims to provide a Low molecular weight Fucosylated Glycosaminoglycan derivative (dLFG) with anticoagulant activity, a preparation method thereof, a pharmaceutical composition containing the dLFG or pharmaceutically acceptable salts thereof, and application of the dLFG and the pharmaceutical composition thereof in preparing medicines for treating thrombotic diseases.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

the present invention firstly provides a low molecular weight fucosylated glycosaminoglycan derivative (dLFG) and pharmaceutically acceptable salts thereof. The constituent monosaccharides of the dLFG include hexuronic acid, hexosamine, deoxyhexose, and sulfates of these monosaccharides. Wherein the hexuronic acid is D-glucuronic acid (D-GlcUA) and delta^4,5-hexuronic acid (4-deoxy-threo-hex-4-enopyrauronic acid, Δ UA), the hexosamine being D-acetylgalactosamine (2-N-acetylamino-2-deoxy-D-galactose, D-GalNAc) or an end-group reduction product thereof, the deoxyhexose being L-fucose (L-Fuc).

Said dLFG monosaccharide and-OSO in terms of mole ratio₃ ^－The composition ratio of (1) to (3.0 +/-1.0) is that the hexuronic acid, the hexosamine, the deoxyhexose and the sulfate group are =1, (1 +/-0.3) to (1 +/-0.3). Generally, in the present invention, Δ UA accounts for not less than 2.5% of hexuronic acid in terms of molar ratio.

The molecular weight of the dLFG can be detected and calibrated by adopting a high-efficiency gel permeation chromatograph and a laser low-angle light scattering instrument (HPGPC-LALLS) online; a standard curve is prepared for a standard using a calibrated molecular weight series of dLFG whose molecular weight can be routinely measured by high performance gel chromatography (HPGPC) in combination with a uv detector (UVD) and/or a difference detector (RID).

The molecular weight of the dLFG is in the range of 3kD to 20kD in terms of weight average molecular weight (Mw); in a preferred embodiment of the invention, the Mw of the dLFG ranges from about 5kD to about 12 kD.

The polydispersity index (PDI) of the dLFG is generally between 1.0 and 1.8, and the PDI refers to the ratio of the weight average molecular weight Mw to the number average molecular weight Mn of the dLFG. In a preferred embodiment of the invention, the dLFG has a PDI value between 1.1 and 1.5.

Further, the low molecular weight glycosaminoglycan derivative of the present invention is a mixture of homologous glycosaminoglycan derivatives having the structure of formula (I),

in formula (I):

n is an integer having an average value of about 2 to 20; preferred compounds of the invention are those wherein n is an integer having an average value of about 4 to 12.

-D-GlcUA- β 1-, is-D-glucuronic acid- β 1-yl-;

-D-GalNAc- β 1-, is-2-deoxy-2-N-acetylamino-D-galactos- β 1-yl-;

L-Fuc-alpha 1-is-L-fucose-alpha 1-radical-;

r independently of one another is-H or-SO₃ ^－；

R' is-OH, C1-C6 alkoxy, C7-C12 aryloxy;

R₁the structure of the non-reducing end of the homologous glycosaminoglycan derivative is a group shown in a formula (II) or (III),

in the formulae (II), (III),

Δ UA-1-, is Δ^4,5-hexuronic acid-1-yl (4-deoxy-threo-hex-4-enopyran uronic acid-1-yl);

r, R' is as defined above.

In the mixture of the homologous glycosaminoglycan derivatives of the present invention, R is expressed in terms of molar ratio₁Is of the formula (II) and R₁The ratio of the compound of formula (III) is not less than 2: 1. In a preferred embodiment of the invention, in said mixture of homologous glycosaminoglycan derivatives, the non-reducing end R₁Is a molar ratio of the structures of formula (II) to formula (III) of not less than about 4: 1.

R₂Is a group of formula (IV) or (V),

wherein R, R' is as defined above;

R₃is carbonyl, hydroxyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, C6-C12 aryl, substituted or unsubstituted five-or six-membered nitrogen-containing heterocyclic group, or-NHR₄(ii) a Wherein,

R₄is substituted or unsubstituted straight chain or branched chain C1-C6 alkyl, substituted or unsubstituted C7-C12 aryl, or substituted or unsubstituted hetero atom-containing heterocyclic aryl.

In a preferred embodiment of the invention, said R is₃is-CHO or-CH₂OH。

The dLFG is a depolymerized product obtained by beta-elimination (FGAG) of Fucosylated glycosaminoglycan (FGAG) extracted from the body wall and/or viscera of Holothuria animals in the phylum Echinodermata and a product obtained by reducing or reductively aminating the reductive end of the depolymerized product of FGAGs. The echinoderm derived FGAG generally has the following characteristics:

(1) the FGAG is obtained by extraction from the body wall or viscera of Holothuria of Echinodermata, and the preparation method thereof is well known to those skilled in the art, and can be found in literature references such as Yoshida et al, Tetrahedron Lett,1992,33: 4959-;et.al.,J Biol Chem,1996,271:23973-23984;et al.,Thromb Res,2001,102:167-176;et.al,J Biol Chem,1996,271:23973-23984;Tovaret al.,Atherosclerosis,1996,126:185-195;Kariya et al.,J Biochem,2002,132(2):335-343;Borsig et al.,J Biol Chem,2007,282(20):14984-14991。

(2) the constituent monosaccharides of FGAG include D-glucuronic acid (GlcUA), D-N-acetyl-2-amino-2-deoxygalactose (GalNAc), and L-fucose (Fuc), and sulfate group substituents may be present on these constituent monosaccharide groups;

(3) the main chain of FGAG contains [ -4-D-GlcUA-beta 1-3-D-GalNAc-beta 1- ] repeating structural unit, and Fuc glycosyl is connected to the main chain GlcUA glycosyl in a side chain form.

Generally, the FGAG has the structure of formula (VI):

in the formula VI, the reaction mixture is shown in the specification,

n is an integer having an average number of about 40 to 90;

r is independently from each other-OH or-OSO 3-;

R₅is-H or D-GlcUA-beta 1-;

R₆is-OH, -4-D-GalNAc or its sulfate.

The animals of the class Holothuroidea of the phylum Echinodermata of the present invention may include, but are not limited to:

apostichopus japonicus;

actinopyga mauritiana, radix Cynanchi Stauntonii;

actinopyga miniris, Actinopyga miliaris;

acaudina molpadioides;

bohadschia argus, Leptospira japonica;

holothuria rosea Holothuria edulis;

holothuria nobilis selenka;

holothuria leucospilota;

holothuria sinica;

sea cucumber Holothuria vagalbuda;

american ginseng Isostichopus badionotus;

brazilian ginseng Ludwigothia grisea;

stichopus chlororonotus, Stichopus japonicus selenka;

thelenota ananas;

thelenota anax;

in a preferred embodiment of the present invention, the sea cucumber animals of Echinodermata may include Apostichopus japonicus, Holothuria leucospilota, and Holothuria japonica.

It is another object of the present invention to provide a process for the preparation of the dLFG of the present invention and pharmaceutically acceptable salts thereof, which process generally comprises the steps of:

(1) using fucosylated glycosaminoglycan (FGAG) derived from echinoderm as a raw material, and completely or partially converting free carboxyl groups on GlcUA contained in the FGAG into carboxylate groups through esterification reaction, thereby obtaining FGAG carboxylate derivatives;

(2) the FGAG carboxylic ester derivative obtained in the step (1) is subjected to beta-elimination reaction of a carboxylic ester group in a non-aqueous solvent in the presence of an alkaline reagent to obtain a product containing a non-reducing terminal delta^4,5-low molecular weight glycosaminoglycan derivatives (dLFG) of hexuronic acids (Δ UA);

(3) optionally, the dLFG obtained in step (2) may be subjected to a reducing treatment of a reducing end, thereby obtaining a dLFG with a reduced end.

(4) Optionally, the dLFG obtained in step (2) and step (3) is converted into a free carboxyl group by a base hydrolysis method.

The research of the invention finds that the natural fucosylated glycosaminoglycan FGAG is relatively stable in alkaline aqueous solution, under severe reaction conditions of increased temperature, enhanced alkalinity and the like, the FGAG generates beta-elimination reaction and simultaneously can be accompanied with the destruction of the characteristic structure of the glycosylated glycosaminoglycan, including the cracking of fucose side chains, main chain sugar ring structures, sulfate groups and the like, so that the reaction product is difficult to predict, the chemical structure of the reaction product is complex and difficult to confirm, and obviously, the direct alkali treatment is difficult to obtain the FGAG depolymerized product with uniform structure.

To achieve the objective of depolymerizing FGAG by the β -elimination method, the present invention establishes β -elimination depolymerization by selective esterification of the free carboxyl group of hexuronic acid, followed by mild conditions and maintenance of the integrity of the characteristic structure of the glycosylated glycosaminoglycan. In the invention, the term "complete characteristic structure" means: except for the reduction of molecular weight and the chemical structural modification of terminal glycosyl (including reducing terminal glycosyl and non-reducing terminal glycosyl), the basic chemical structural characteristics of the depolymerized product are equal to those of un-depolymerized polysaccharide, including monosaccharide composition and proportion, the connection mode of repeating structural unit, the number and type of sulfate groups and the like.

In the invention, the preparation method of the FGAG carboxyl esterification product comprises the following steps:

(1) conversion of FGAG to quaternary ammonium salt by ion exchange, e.g. passing aqueous FGAG solution over H⁺Cation exchange resin type converts neutral FGAG salt into H⁺Form FGAG;

(2) titration/neutralization of the resulting H with a solution of quaternary ammonium base⁺Forming FGAG solution to obtain FGAG quaternary ammonium salt solution, and freeze-drying the obtained solution to obtain the FGAG quaternary ammonium salt. The quaternary ammonium bases can include, but are not limited to, tetrabutylammonium hydroxide, dodecyltrimethylammonium hydroxide, tetramethylammonium hydroxide, tetrapropylammonium hydroxide, tetraethylammonium hydroxide, benzyltrimethylammonium hydroxide, benzyltriethylammonium hydroxide, and the like.

(3) FGAG quaternary ammonium salt reacts with stoichiometric halogenated hydrocarbon in non-protonation solvent such as Dimethylformamide (DMF), and the reaction product is separated and purified to obtain FGAG carboxyl ester derivative (quaternary ammonium salt which can be directly used for further beta-elimination reaction). The hydrocarbon group in the halogenated hydrocarbon includes, but is not limited to, a C1-C6 linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic hydrocarbon group; substituted or unsubstituted C7-C12 aromatic hydrocarbon groups, and the like.

In another patent application of the present applicant, application No. 201110318704.X (publication No. CN 102329397A) describes a preparation method of FGAG carboxyl ester derivatives.

In the preparation process of the low molecular weight heparin, carboxylic ester derivatives of unfractionated heparin can generate beta-elimination reaction in alkaline aqueous solution, so that the Low Molecular Weight Heparin (LMWH) with delta UA at the tail end can be obtained. Experimental studies of the present invention have surprisingly found that under similar conditions, such as aqueous NaOH, the FGAG carboxy esterification product can be completely hydrolyzed to the prototype FGAG with little or no β -elimination. Therefore, the present invention focuses on a nonaqueous solvent system capable of performing β -elimination reaction of FGAG carboxyl ester derivative.

In the preparation method of the dLFG and the pharmaceutically acceptable salt thereof described above in the specification, the non-aqueous solvent in the step (2) is selected from ethanol, methanol, dimethylformamide, dimethyl sulfoxide, CH₂Cl₂、CHCl₃Or a mixed solvent thereof. The alkaline agent is optionally NaOH, KOH, sodium C1-C4 alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo-ring, or mixtures thereof.

One of the key technical difficulties to be solved in carrying out the β -elimination reaction of FGAG carboxyl ester derivatives with a non-aqueous solvent is that the solubility of FGAG carboxyl ester derivatives in the non-aqueous solvent is very poor, thus severely affecting the performance of the desired β -elimination reaction. The scheme of the invention for improving the solubility of the FGAG carboxyl ester derivative in the non-aqueous solvent is to further convert the FGAG carboxyl ester derivative into a quaternary ammonium salt, thereby realizing the solubility of the FGAG carboxyl ester derivative in the non-aqueous solvent which can meet the requirement of the beta-elimination reaction.

When a single lower alcohol/ketone such as ethanol is used as a solvent, the solubility of the quaternary ammonium salt of the FGAG carboxyl esterification product is relatively low, and when other non-aqueous solvents such as Dimethylformamide (DMF) are used, the reaction efficiency may be greatly limited due to the low solubility of NaOH as a catalyst. For this purpose, the invention is designed to adopt a C1-C4 sodium alkoxide as an alkaline reagent for increasing the concentration of the alkaline reagent in the non-aqueous solvent, and a mixed solvent system as a solvent system for the beta-elimination reaction, wherein the reaction system is carried out by the following steps: dissolving quaternary ammonium salt of FGAG carboxyl ester derivative in appropriate non-aqueous solvent such as DMF; dissolving NaOH or other suitable base catalyst in anhydrous lower alcohol; mixing the solution of FGAG carboxyl esterification product quaternary ammonium salt with the solution of base catalyst, thereby obtaining a clear beta-elimination reaction system.

The basic agent includes, but is not limited to, NaOH, KOH, sodium C1-C4 alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo-ring, or mixtures thereof. The preferred alkaline agent of the present invention is sodium ethoxide, and the preferred technique is to add stoichiometric amount of metallic sodium to dry anhydrous ethanol solution, thereby preparing sodium ethoxide-ethanol solution.

In the beta-elimination reaction solution, the concentration of the quaternary ammonium salt of the FGAG carboxyl ester derivative is generally 1-150 mg/ml, and the concentration of the alkali catalyst is generally 0.1-100 mmol/L. In the beta-elimination reaction solution, the beta-elimination reaction of the FGAG carboxyl esterification product can be smoothly carried out, and the reaction is completed within 0.1-8 hr at room temperature generally. After the beta-elimination reaction is finished, neutralizing the reaction solution by acid (such as hydrochloric acid) to obtain carboxyl esterified dLFG product; or, under the condition of not changing the alkalinity of the reaction solution, adding a proper amount of water into the reaction solution, and keeping the temperature for about 0.5-1 hr, the carboxylate group contained in the depolymerized dLFG derivative can be completely hydrolyzed, and at the moment, the reaction solution is neutralized by acid, and the dLFG product containing free carboxyl can be obtained.

In general, FGAG carboxyl ester preparation and its β -elimination depolymerization can be carried out using:

FGAG neutral salt is subjected to H + cation exchange resin column and quaternary ammonium base titration neutralization to obtain FGAG quaternary ammonium salt, then subjected to reaction (carboxyl esterification) at room temperature or under heating condition by using aprotic solvent and halogenated hydrocarbon to obtain FGAG carboxyl esterification product (quaternary ammonium salt), then dissolved by using non-aqueous solvent, added with strong base, and subjected to reaction (beta-elimination) at room temperature or under heating condition to finally obtain dLFG (depolymerized product of the beta-elimination method of FGAG).

Theoretically, when the esterification rate of the carboxyl groups on FGAG is about 5% -30%, the molecular weight of the resulting product can be between about 3kD and 20kD by depolymerization by the beta-elimination method described in the present invention. In the actual reaction, in view of the influence of experimental condition control on the degree of β -elimination reaction, FGAG carboxyl esterification product having a carboxyl esterification rate of about 10% to 60% is used, and dLFG product having Mw of about 3kD to 20kD can be obtained by depolymerization of β -elimination reaction.

The dLFG obtained by the beta-elimination reaction of the invention has a reducing end, and the reducing end is mainly-4-D-N-acetyl-2-amino-2-deoxygalactose (-4-D-GalNAc). In the present invention, the reducing end may optionally be end-modified by a reduction reaction, which may include, but is not limited to:

(1) the reducing end is reduced to a sugar alcohol in the presence of a reducing agent such as sodium borohydride. Taking 4-D-N-acetyl-2-amino-2-deoxygalactose as an example, the reaction process is as follows:

wherein R is-OH or-OSO₃ ^￣。

(2) The reductive end is reductively aminated in the presence of an organic amine, which reacts with the aldehyde group at the 1-position of the terminal glycosyl group to form a Schiff base, which is reduced to a secondary amine in the presence of a reducing agent. Taking the reaction of the terminal-4-D-N-acetyl-2-amino-2-deoxygalactose with ammonium bicarbonate and primary amine as an example, the reaction process is as follows:

in the formula, R₄Is H, substituted or unsubstituted straight or branched C1-C6 alkyl, substituted or unsubstitutedC7-C12 aryl, substituted or unsubstituted heterocyclic group containing a heteroatom including, but not limited to, O, N, S.

(3) Reductive alkylation of the reducing end in the presence of a reducing compound to obtain a reductively alkylated derivative. Taking the reaction of terminal-4-D-N-acetyl-2-amino-2-deoxygalactose and pyrazolone compound reducing agent as an example, the reaction process is as follows:

reduction reactions, reductive amination reactions, and reductive alkylation reactions at the reducing end of aldoses are well known to those skilled in the art. In the present invention, the reducing end of dLFG obtained by the beta-elimination method is subjected to the reduction reaction to obtain the terminal glycosyl groups represented by the formulas (IV) and (V), wherein R is₃May be a C1-C6 alkoxy group, a C1-C6 alkoxycarbonyl group, a C6-C12 aryl group, a substituted or unsubstituted five-or six-membered nitrogen-containing heterocyclic group, or a-NHR group₄(R₄Is substituted or unsubstituted straight or branched C1-C6 alkyl, substituted or unsubstituted C7-C12 aryl, substituted or unsubstituted heteroatom-containing heterocyclic aryl).

The reaction product dLFG of the present invention can be purified by methods known in the art (Chinese patent application publication No. CN101735336A), such as removing impurities such as small molecular salts by dialysis or ultrafiltration, or further purifying by gel chromatography or DEAE ion exchange chromatography.

In the dialysis impurity removal treatment process, a dialysis membrane or an ultrafiltration membrane with a proper molecular weight cut-off can be selected according to the requirement of the molecular weight of the target dLFG, and the molecular weight cut-off is preferably 1000 Da. The dialysis time is determined according to the particular treatment conditions, and is usually not less than 6 hours.

The dLFG product of the invention can also be prepared into a single salt form by cation exchange, such as alkali metal, alkaline earth metal salts, organic ammonium salts and the like. In a preferred embodiment of the invention, the mono-salt form of the dLFG is a sodium, potassium or calcium salt.

The salifying process of the dLFG product can adopt the steps of firstly exchanging a sample into a hydrogen form, and then neutralizing by using corresponding alkali to obtain a salt corresponding to the dLFG; dynamic ion exchange salt formation directly on the column to form salts is also preferred, wherein a strongly acidic cation exchange resin is optionally used. The resin column pretreatment, sample loading and elution can be carried out according to the conventional method.

The dLFG of the invention is prepared by taking echinoderm-derived FGAG as a reaction starting material through a beta-elimination reaction. As mentioned above, in the present invention, the animal from which the starting material FGAG is derived may be selected from, but not limited to, apostichopus japonicus, radial root of white sea, radial root of dark-wrinkled root of radial root of sea, Japanese pachyrhizus, white sea cucumber, red belly sea cucumber, Holothuria nobilis, Holothuria leucospilota, Chinese sea cucumber, American ginseng, bacon ginseng, green stichopus japonicus, Japanese apricot blossom ginseng, and Japanese apricot blossom ginseng. In a preferred embodiment of the present invention, the source animals include Apostichopus japonicus selenka, Holothuria leucospilota, and Holothuria japonica.

The structural characteristics of FGAG derived from Holothuria of Echinodermata of the present invention are the presence of GlcUA, GalNAc and fucose or its sulfate in a nearly equimolar ratio (about 1: 1. + -. 0.3). The difference of the sea cucumber varieties and tissue sources thereof or the difference of extraction methods can cause the difference of the monosaccharide composition proportion and the polysaccharide sulfation degree of FGAG, and the difference does not influence the basic structural characteristics of FGAG. It will be apparent to those skilled in the art that fucosylated glycosaminoglycans derived from other species of sea cucumber and conforming to the basic structural features of FGAG may be used to obtain the dLFG derivatives of the invention.

The dLFG has strong anticoagulation activity, and the drug concentration (the drug concentration for prolonging the APTT by 1 time) for doubling the Activated Partial Thromboplastin Time (APTT) of human quality control plasma is not higher than 9 mu g/mL. The present study confirms that the dLFG has significant inhibitory activity on endogenous factor X enzyme (f.Xase, Tenase) and has heparin cofactor II (HC-II) dependent antithrombin (f.IIa) activity. Xase is the last target of the intrinsic coagulation pathway in coagulation waterfalls and is the rate-limiting step in various experimental coagulation processes (Buyue & Sheehan, Blood,2009,114: 3092-; dermatan sulfate, which has HC-II dependent antithrombin activity, has been used clinically in antithrombotic therapy, and therefore dLFG has potential for clinical treatment of thromboembolic diseases.

The dLFG of the invention has no platelet activating activity; furthermore, it has been surprisingly found that the dLFG of the present invention has no activity of activating f.XII, does not affect the f.XII-kallikrein system, and does not cause a decrease in blood pressure in experimental animals, as compared to FGAG which is naturally derived therefrom.

The nonreducing end of the dLFG can contain delta UA glycosyl, and unsaturated double bonds in the delta UA enable the dLFG to have maximum ultraviolet absorption (UV, lambda max) at the position of about 232-238 nm, and the attribute has important value for establishing a qualitative and quantitative analysis method based on an ultraviolet spectrophotometry detection method, and particularly when a high performance liquid chromatography (HGPC) method is adopted for analyzing the content of the sample dLFG, a UV detector with higher sensitivity can be used for detecting, so that the method is particularly suitable for establishing technical methods related to the content analysis of the dLFG, such as sample quality control, blood concentration analysis and the like.

The dLFG has definite anticoagulant activity, so that the dLFG has an antithrombotic application value. The dLFG has good water solubility, so that the dLFG can be easily prepared into a solution type preparation or a freeze-dried product thereof. As polysaccharide components, oral bioavailability is limited, and therefore, it is preferable to prepare a parenteral dosage form, and preparation thereof can be carried out according to a method well known in the art.

It is therefore a further object of the present invention to provide a pharmaceutical composition comprising a dLFG of the present invention and a pharmaceutically acceptable excipient.

The dLFG has strong anticoagulant activity, so that the dLFG can be used for preventing and treating thrombotic diseases of different degrees, such as thrombotic cardiovascular diseases, thrombotic cerebrovascular diseases, pulmonary vein thrombosis, peripheral vein thrombosis, deep vein thrombosis, peripheral artery thrombosis and the like. Therefore, the invention can provide the application of the composition in the preparation of medicaments for treating and preventing cardiovascular diseases.

The dLFG has strong anticoagulant activity, so that the dLFG can be used for preventing and treating thrombotic diseases of different degrees, such as thrombotic cardiovascular diseases, thrombotic cerebrovascular diseases, pulmonary vein thrombosis, peripheral vein thrombosis, deep vein thrombosis, peripheral artery thrombosis and the like. Therefore, the invention can provide the application of the composition in the preparation of medicaments for treating and preventing cardiovascular diseases.

The applicant first studied and established a β -elimination depolymerization method of FGAG. The applicant finds in the experimental research process that the prototype FGAG is relatively stable in alkaline solution and not prone to β -elimination. The carboxyl esterification and the beta-elimination are optional technical methods, but because the fucosyl side chain substitution exists in FGAG, the polysaccharide structure is more complex, on one hand, the complex steric hindrance can influence the chemical reaction properties of various chemical functional groups, and on the other hand, various chemical reaction conditions can influence the stability of the side chain fucosyl glycosidic bond. It has surprisingly been found that FGAG carboxyl esterification products do not undergo the beta-elimination reaction in alkaline aqueous solution as does heparin esterification products. In fact, in alkaline aqueous solution, the ester group of FGAG carboxyl esterification product is very easy to be hydrolyzed, and almost no beta-elimination reaction occurs, which is obviously different from the properties of typical glycosaminoglycan compounds such as heparin.

The invention successfully obtains the non-reducing end with delta through the beta-elimination reaction in the non-aqueous solvent of FGAG carboxyl esterification product^4,5Depolymerized products of fucosylated glycosaminoglycans of unsaturated bonds.

By the technical method of the invention, the depolymerized fucosylated glycosaminoglycan derivative (dLFG) with the required molecular weight range can be obtained. Physical, chemical and spectral chemical structure analysis shows that except the reducing end, the product has delta^4,5Besides the unsaturated bond, the basic structure remains stable.

By using the present inventionWhen the LFG is prepared by the method, the molecular weight of a reaction product can be effectively controlled by controlling the ratio of carboxyl esterification and/or amidation; due to the non-reducing end of the reaction product having a^4,5Unsaturated bonds, and the obtained dLFG has maximum ultraviolet absorption (lambda max) at the position of about 232-240nm, and the property can be used for quantitatively detecting products, thereby being beneficial to establishing related technical methods of content analysis such as chemical reaction control, product quality analysis, blood concentration detection and the like.

Pharmacological experimental research results show that the dLFG prepared by the method has remarkable anticoagulant activity, prolongs the Activated Partial Thrombin Time (APTT) of human quality control plasma, inhibits the activity of endogenous factor X enzyme (f.Xase, Tenase) and the activity of heparin cofactor II (HC-II) dependent antithrombin (anti-f.XIIa) and is basically consistent with the activity of a peroxide depolymerization product with approximate molecular weight, and the dLFG has good potential application value. Compared with a peroxide depolymerization product, the dLFG has the advantages that firstly, the dLFG has a non-reducing end with a specific structure, has ultraviolet absorption of lambda max about 232 and 240nm, and obviously improves the quality controllability; secondly, the preparation process has good controllability.

Further research of the invention finds that the dLFG does not have platelet activation and coagulation factor XII (f.XII) activation activity under the dosage of anticoagulation drug effect, so that a series of adverse reactions caused by platelet activation and activation of f.XII-kallikrein system can be avoided; compared with the heparin medicament with equivalent antithrombotic agent amount, the low molecular weight FGAG of the invention can further reduce the bleeding tendency and has the value of treating and/or preventing thrombotic diseases.

Drawings

FIG. 1 is an HPGPC map of TAG and dLFG-1A;

FIG. 2(a) is of TAG¹H NMR spectrum;

FIG. 2(b) is a dLFG-1A¹H NMR spectrum;

FIG. 3 shows TAG and dLFG-1A¹³C NMR spectrum;

FIG. 4 shows TAG and dLFG-1A¹H-¹H COSY NMR spectrum;

FIG. 5(a) is of TAG¹H-¹H ROESY spectrum;

FIG. 5(b) is of TAG¹H-¹H TOCSY spectrogram;

FIG. 5(c) is dLFG-1A¹H-¹H ROESY spectrum;

FIG. 5(d) is dLFG-1A¹H-¹H TOCSY spectrogram;

FIG. 6 shows dLFG-1A¹H-¹³C HSQC spectrogram;

FIG. 7 shows AJG, LGG and HNG¹H NMR spectrum;

FIG. 8 is of HEG and dHEG¹H NMR spectrum;

FIG. 9 shows dLFG-2A¹H NMR spectrum;

FIG. 10 is a dose-effect relationship of dLFG-1E dependent HC-II antithrombin activity;

FIG. 11 is a dose-effect relationship of dLFG-1E inhibiting f.Xase activity;

FIG. 12 is a flow chart showing the basic steps of the beta elimination depolymerization of FGAG.

The specific implementation mode is as follows:

the following examples are given to illustrate the essence of the present invention in detail with reference to the accompanying drawings, but do not limit the scope of the present invention in any way.

[ example 1 ]

Preparation of low molecular weight fucosylated glycosaminoglycan derivative (dLFG) derived from Thelenota ananas:

1.1 materials

Thelenota ananas Jaeger, commercially available product, eviscerated desiccated body wall; benzethonium chloride, benzyl chloride, tetrabutylammonium hydroxide (TBA), N-Dimethylformamide (DMF), sodium hydroxide, sodium chloride and ethanol are commercially available analytical reagents.

1.2 methods

(1) Extracting and preparing FGAG (FGAG from Thelenota ananas, TAG) from Thelenota ananas: TAGs were prepared in a yield of about 1.5% and purity of 98% (HPGPC, area normalization), weight average molecular weight (Mw), 65,890Da by taking 300g of the dried body wall of thelenota ananas and using the reference method (Kariya et al, J Biol Chem,1990,265(9): 5081-5085).

(2) Preparation of TAG quaternary ammonium salt: putting 1.2g of TAG obtained in the step (1) into a beaker, and adding 40mL of deionized water to dissolve the TAG; and titrating with a benzethonium chloride solution of 75mg/mL while stirring, generating a white precipitate immediately, centrifuging after the titration is finished, washing the precipitate with deionized water for three times, and finally drying the precipitate in vacuum at normal temperature for 24 hours to obtain 2.68g of FGAG ammonium salt.

(3) Preparation of TAG benzyl ester: putting the TAG quaternary ammonium salt obtained in the step (2) into a round-bottom flask, adding 27mL of DMF for dissolving, adding 13.5mL of benzyl chloride, and adding N₂The reaction is stirred for 25 hours at the temperature of 35 ℃ under protection. After completion of the reaction, saturated NaCl35mL was added to the reaction mixture, 300mL of absolute ethanol was added thereto, and the mixture was centrifuged at 3500rpm for 20min to remove the supernatant. Washing the precipitate with 200mL1:9(v/v) saturated NaCl-anhydrous ethanol for three times, dissolving with 100mL deionized water, dialyzing with 3500kD dialysis bag for 24hr, concentrating the dialysate, freeze drying to obtain FGAG benzyl ester,¹the degree of esterification was determined to be 72% by H-NMR.

(4) Preparation of TAG benzyl ester tetrabutylammonium salt: the TAG benzyl ester obtained in step (3) is dissolved in water, and converted into the hydrogen form by an ion exchange method (Dowex/r50w X850-100 (H), 60X 3cm), and the sulfate and unesterified carboxyl are all converted into ammonium salts by titration with a 0.4M tetrabutylammonium hydroxide solution under the monitoring of a conductivity meter. The resulting solution was freeze-dried to obtain 1.326g of TAG benzyl ester tetrabutylammonium salt.

(5) Depolymerization by the beta-elimination method: 800mg of TAG benzyl ester tetrabutylammonium salt obtained in step (4) was dissolved in 8.0mL of DMF, 0.8mL of tri-n-butylamine was added, the mixture was reacted at 60 ℃ for 24 hours with stirring, 80mL of 1:9(v/v) saturated NaCl and anhydrous ethanol were added to the solution, and the mixture was centrifuged at 3500rmp for 15 minutes to obtain a precipitate. Adding 8mL of 0.1M NaOH into the precipitate, reacting at 30 ℃ for 40min to hydrolyze residual carboxylic ester, adjusting pH to be neutral by 0.1MHCl, adjusting pH to be neutral by 80mL of absolute ethanol, and centrifuging at 3500rpm for 15min to obtain the precipitate. 8mL of H₂The precipitate was dissolved with O, passed through a hydrogen-based ion exchange resin column (Dowex/r50w X850-100 (H), 60X 3cm), adjusted to neutral pH with 0.1M NaOH, dialyzed with deionized water for 24 hours in a 1kD dialysis bag, and freeze-dried to obtain about 310mg of β -elimination depolymerization product dLFG-1A.

(6) TAG and its beta-elimination depolymerization product dLFG-1A physicochemical property, monosaccharide composition and spectrum detection

Molecular weight: high performance gel chromatography detection (HPGPC). The detection conditions are that an Agilent technologies1200series chromatograph, Shodex Ohpak SB-804HQ gel chromatographic column, the column temperature is 35 ℃; the mobile phase is 0.1M sodium chloride, and the flow rate is 0.5 mL/min; agilent type 1100 RID and UVD were tested in combination. And drawing a standard curve by using series FGAG with calibrated molecular weight, and calculating the molecular weight and the distribution by GPC.

-OSO₃ ^-/-COO^-The molar ratio is as follows: detecting by a conductance method;

optical rotation: measuring according to the method of appendix VI E of the second part of Chinese pharmacopoeia (2010 version);

characteristic viscosity number: measured according to the method VI G in the appendix of the second part of the Chinese pharmacopoeia (2010 edition) and by an Ubbelohde viscometer.

And (3) monosaccharide composition detection: the Elson-Morgon method is used for detecting the content of acetylgalactosamine (D-GalNAc), and the carbazole method is used for detecting the content of glucuronic acid (D-GlcUA) (Zhangjie, a glycoconjugate biochemical research technology (second edition), Zhejiang: Zhejiang university Press, 1999, 19-20); content of 4,5 unsaturated glucuronic acid residue (. DELTA.UA) according to¹H4 integral ratio of acetyl galactose in H NMRIntegral calculation of (D-GalNAc) methyl group; detection of NMR spectra (detection conditions, solvent D) by AVANCE AV500 superconducting NMR spectrometer (500MHz) from Bruker, Switzerland₂O, 99.9Atom% D (Norell corporation); an internal standard, trimethylsilyl-propionic acid (TSP-d 4); temperature 300K).

Ultraviolet absorption spectroscopy (UV) detection: 0.855mg/mL dLFG, on Shimadzu UV-2450, a scan of wavelength range 190 and 400nm was performed.

1.3 results

The detection results of the physicochemical properties and monosaccharide composition of TAG and its depolymerization product dLFG-1A are shown in Table 1. The HPGPC chromatograms of TAG and dLFG-1A are shown in FIG. 1.

The detection results show that the molecular weight and the intrinsic viscosity of dLFG-1A are obviously reduced compared with that of TAG.

The monosaccharide composition detection result shows that the composition ratio of aminohexose, hexuronic acid (UA, which is the sum of GlcUA and delta UA) and deoxyhexose (Fuc) of TAG and dLFG-1A is basically kept stable. As described hereinafter¹H NMR spectrum showed that TAG contained no Δ UA, while dLFG-1A contained Δ UA in a molar ratio to GalNAc of about 0.18:1(Δ UA in a molar ratio of about 7.5% of total hexuronic acid).

TABLE 1 results of the measurement of the physicochemical parameters and monosaccharide compositions of FGAG and dLFG-1A derived from Thelenota ananas

The UV spectrophotometer scans over the wavelength range of 190nm to 400nm and the maximum UV absorption λ max236nm exists for dLFG, which coincides with the presence of Δ UA unsaturation.

FIG. 2 of the present specification shows TAG and dLFG-1A¹H NMR spectrum; FIG. 3 shows TAG and dLFG-1A¹³C NMR spectrum; FIG. 4 shows TAG and dLFG-1A¹H-¹H COSY NMR spectrum; FIG. 5(a) shows TAG¹H-¹H ROESY spectrum;FIG. 5(b) shows TAG¹H-¹H TOCSY spectrogram; FIG. 5(c) shows dLFG-1A¹H-¹H ROESY spectrum; FIG. 5(d) shows dLFG-1A¹H-¹H TOCSY spectrogram; FIG. 6 shows dLFG-1A¹H-¹³C HSQC spectrum.

Of TAG¹The attribution of the spectrum signals of the H NMR and the related spectrums thereof can refer to the Chinese patent publication No. CN102247401A filed by the applicant.

TAG¹In the HNMR spectrogram, three groups of stronger signal peaks are visible in the range of 5.2-5.7 ppm, which are the terminal hydrogen signals of different types of sulfated alpha-fucose, wherein the signal of about 5.6ppm is the terminal hydrogen signal of L-fucose-2, 4-dithionate (Fuc2S4S), and the signal of about 5.30-5.39ppm is the terminal hydrogen signals of L-fucose-3-sulfate (Fuc3S) and L-fucose (Fuc4S) -4-sulfate.

The backbone GlcUA and GalNAc terminal beta-hydrogen signals are located at about 4.4 to 4.6 ppm. About 1.0 to 1.3ppm and 1.9 to 2.0ppm are methyl proton signal peaks on Fuc methyl and GalNAc acetyl groups, respectively. The saccharide ring hydrogens at the sulfate group substitution sites are present in the range of about 4.2 to 4.8ppm, and the signal at about 3.6 to 4.6ppm is the superposition of the saccharide ring hydrogens at the non-sulfate group substitution sites.

Of dLFG-1A in comparison with TAG spectrum¹The H NMR spectrum showed new signal peaks at about 5.76 and 5.82ppm, according to which¹The H NMR correlation spectrum can attribute these signals as 4-bit H signatures of Δ UA.

Of dLFG-1A¹H-¹The HCOSY and TOCSY spectra clearly show the coupling correlation between the H4, H3, H2 and H1 proton signals of Δ UA.¹H-¹The H ROESY spectrum shows that Fuc is linked to GlcUA and Δ UA by α (1,3) glycosidic bonds. Furthermore, the same type of Fuc end hydrogen signal attached to Δ UA appeared at higher fields compared to the end hydrogen signal of Fuc attached to GlcUA (see the positions of Fuc2S4S end hydrogen and Fuc2S4S end hydrogen signal attached to Δ UA in fig. 2(a), 2 (b)).

¹³C-NIn the MR spectrum (with TMS as an external standard), the C1 peak of GlcUA and GalNAc appears at about 97-104 ppm, the C1 peak of DeltaUA appears at about 103.5ppm, the chemical shift of C4 is 106.8ppm, and the chemical shift of C5 is about 148.5 ppm.

As can be seen from the combination of the hydrogen spectrum, the carbon spectrum and the related spectrum, in dLFG-1A, GlcUA and GalNAc are connected with each other through beta (1 → 3) and beta (1 → 4) glycosidic bonds to form a glycan backbone, thereby forming a backbone disaccharide structural unit. H2, H3 chemical shifts and binding according to GlcUA¹H-¹H ROESY、¹H-¹³Fuc is linked to GlcUA by an alpha (1 → 3) glycosidic bond, as judged by C HMBC. Apparently, in dLFG-1A, the hexuronic acid at the non-reducing end is predominantly Δ UA.

TABLE 2dLFG-1A¹H/¹³C NMR Signal assignment ([ ppm)])

Wherein: GalNAc4S6S is a 4, 6-disulfate of GalNAc; fuc2S4S, -3S, -4S are Fuc-2, 4-disulfate, -3-sulfate and-4-sulfate, respectively.

[ example 2 ]

β -elimination depolymerization of TAG to prepare a range of molecular weight oligomeric fucosylated glycosaminoglycan derivatives (dLFG):

2.1 materials:

TAG derived from Thelenota ananas was prepared as described in example 1. The other reagents were the same as in example 1.

2.2 methods

(1) Preparation of TAG quaternary ammonium salt: TAG quaternary ammonium salt 5.02g was prepared as described in example 1.

(2) Preparation of TAG benzyl esters of different degrees of benzylation: and (2) adding 50mL of DMF into the TAG quaternary ammonium salt obtained in the step (1), stirring and dissolving, adding 25mL of benzyl chloride, and reacting at 35 ℃ under stirring. About 15mL of each sample was taken at different reaction times, 100mL of 1:9(v/v) saturated NaCl-absolute ethanol was added to each of the taken solutions, and the mixture was centrifuged at 3500rpm for 20min, and the resulting precipitate was washed three times with 50mL of 1:9(v/v) saturated NaCl-absolute ethanol. The precipitate was dissolved in 40mL of deionized water and dialyzed for 24h in a dialysis bag with a molecular weight cut-off of 3500 kD. Each dialyzed trapped fluid is TAG benzyl ester solution with different esterification degrees, sampling and freeze-drying are carried out,¹the degree of esterification was determined by H-NMR spectroscopy to be 9%, 21%, 28%, 45% and 56%, respectively.

(3) TAG benzyl ester tetrabutylammonium preparation: the TAG benzyl ester solution solutions with different esterification degrees obtained in the step (2) are respectively concentrated to 6mL, the solutions are converted into hydrogen form by an exchange resin method, the sulfate radical and the unesterified carboxyl radical of the solutions are all converted into tetrabutylammonium salt (until the pH value is about 7.5-8.0) by titration of a 0.4M tetrabutylammonium hydroxide solution under the monitoring of a conductivity meter, and the obtained solution is frozen and dried to obtain 1.523g, 1.518g, 1.493g, 1.490g and 1.731g of TAG benzyl ester tetrabutylammonium with different esterification degrees.

(4) Preparation of a series of molecular weight β -elimination depolymerization products, dflg: adding DMF/CH to the tetrabutylammonium TAG benzyl ester with different esterification degrees obtained in the step (3) according to the proportion of 50mg of ammonium salt₂Cl₂DMF or CH was added at a ratio of 1mL to 0.1M NaOH/EtOH1mL₂Cl₂And fresh 100mM NaOH-EtOH, reacting at 25 deg.C for 1hr under stirring, rapidly adjusting pH to neutral with 1N HCl to stop reaction, adding saturated sodium chloride 2mL, anhydrous ethanol 20mL, centrifuging at 3500rpm for 15min, and removing supernatant to obtain precipitate. 4mL of H₂Dissolving the precipitate with O, converting the product into hydrogen form by ion exchange resin method, and adjusting pH to 7-8 with 0.1M NaOH.

(5) Purification of the series of molecular weight β -elimination depolymerization products: transferring the solution obtained in step (4) into dialysis bag of 1kD, dialyzing with deionized water for 24hr, and freeze drying to obtain series of beta-eliminating depolymerization products dLFG-1B, dLFG-1C, dLFG-1D, dLFG-1E and dLFG-1F with molecular weight.

(6) detection of dLFG-1 product: the molecular weights, -OSO, of dLFG-1B, -1C, -1D, -1E and-1F were determined as described in example 1₃ ^-/-COO^-Molar ratio, optical rotation.

2.3 results

The results of the physical and chemical property tests of dLFG-1B, dLFG-1C, dLFG-1D, dLFG-1E and dLFG-1F are shown in Table 2 below. The detection result shows that the yield of dLFG series products obtained by carrying out beta-elimination depolymerization on TAG from thelenota ananas and the like is higher, the molecular weight distribution is narrower, the detection result of a conductivity method shows that the sulfate group is not obviously changed, and the characteristic viscosity is reduced along with the reduction of the molecular weight.

TABLE 2 monosaccharide composition and physicochemical Properties of dLFG of series molecular weights

[ example 3 ]

Preparation of beta-elimination depolymerization products of FGAG from different sea cucumber sources:

3.1 materials

Apostichopus japonicus, Holothuria edulis, Brazilian ginseng Ludwigothiourea grisea, Holothuria leucospilota, Holothuria nobilis, all of which are commercially available dry body walls.

3.2 methods

(1) The dried body walls of the apostichopus japonicus, the red belly sea cucumber, the bache japonicus, the holothuria leucospilota and the holothuria nobilis are crushed, 300g of crushed materials are respectively taken, FGAG contained in the crushed materials is extracted by the same method (1) as the method in the embodiment 1, and the FGAG contained in the crushed materials are respectively called AJG, HEG, LGG, HLG and HNG.

(2) About 1g of AJG, HEG, LGG, HLG and HNG were taken, respectively, and the β -elimination depolymerization products dLFG, respectively designated dAJG, dHEG, dLGG, HLG and dHNG, were prepared according to the methods (2) to (5) described in the examples. The basic steps of the beta-elimination depolymerization of FGAG are shown in FIG. 12.

3.3 results

The yields of AJG, HEG, LGG, HLG and HNG separated and purified from the dried body walls of apostichopus japonicus, red belly sea cucumber, bache, Holothuria leucospilota and Holothuria nobilis selenka in the step (1) are respectively about 1.4%, 0.9%, 0.8% and 1.1%, and the weight average molecular weights of the AJG, the HEG, the LGG, the HLG and the HNG are all between about 50kD and 80 kD. Figure 7 of the specification¹The H NMR spectrum shows the basic characteristics of AJG, LGG and HNG as FGAG compounds: the end groups and related characteristic proton signals on alpha-L-Fuc, beta-D-GalNAc and beta-D-GlcUA are clear and definite.

The yield of dAJG (8.6kD), dHEG (11.5kD), dLGG (9.3kD), HLG (10.2kD) and dHNG (9.7kD) produced from AJG, HEG, LGG, HLG and HNG in step (2) is in the range of about 70% to 90%. FIG. 8 of the present specification by HEG and dHEG¹The H NMR spectrum shows a characteristic signal related to the non-reducing end DeltaUA formed by beta-elimination depolymerization.

[ example 4 ]

Preparation of dAJG terminal reduction product:

4.1 materials

dAJG, 8.6kD, prepared as described in example 3. Sodium borohydride, a commercially available analytical grade reagent.

4.2 methods

500mg dAJG and 250mg NaBH₄Dissolving in 20ml of 0.1N NaOH solution to obtain NaBH₄The solution was added to the dAJG solution, the resulting solution was stirred at room temperature overnight, then 200mg NaBH was added₄And stirring is continued for 5 hr. Thereafter, the pH was adjusted to about 10.3 to about 2.5 by adding 1N HCl (to destroy excess sodium borohydride), the pH was adjusted back to neutral by 1N NaOH, 150ml of absolute ethanol was added, the supernatant was centrifuged off, 50ml of absolute ethanol was washed 2 times, and the precipitate was washed with 20ml of absolute ethanolDissolving ml deionized water, dialyzing the 3kD dialysis bag in the deionized water overnight, and freeze-drying the dialysis trapped fluid to obtain the end group reduced rdAJG.

4.3 results

As a result, rdAJG386.3mg was obtained. The DNS (3,5 dinitrosalicylic acid) method shows that the terminal reduction of rdAJG is almost complete.

[ example 5 ]

End reduction of dLFG-1A amination product dLFG-2A preparation:

5.1 materials

dLFG-1A: example 1 preparation. The reagents such as tyramine, sodium cyanoborohydride and the like are all commercially available analytical reagents.

5.2 methods

(1) Reductive amination of the end of dLFG-1A: 0.1g of dLFG-1A0 obtained in example 1 was dissolved in 3.5mL of 0.2mM phosphate buffer (pH8.0), and an excess of 80mg of tyramine and 30mg of sodium cyanoborohydride were added thereto while stirring, followed by reaction in a constant temperature water bath at 35 ℃ for about 72 hours. After the reaction is finished, 10mL of 95% ethanol is added, the precipitate is obtained by centrifugation, the obtained precipitate is washed twice by 30mL of 95% ethanol, the precipitate is redissolved by 35mL of 0.1% NaCl, insoluble substances are removed by centrifugation, the supernatant is placed in a dialysis bag with 1KD, deionized water is dialyzed for 24h, and dLFG-2A82mg is obtained after freeze drying.

(2) And (3) detecting the physicochemical property and the spectrum of the product: HPGPC detects molecular weight and distribution; detection of-OSO by conductance method₃ ^-/-COO^-A molar ratio; detecting the content of acetylgalactosamine (D-GalNAc) by an Elson-Morgon method, detecting the content of glucuronic acid (D-GlcUA) by a carbazole method,¹the D-GalNAc/L-Fuc molar ratio was calculated by integrating the areas of the methyl peaks of HNMR (same as in example 1). NMR spectra were measured by a Bruker AVANCE AV500 superconducting nuclear magnetic resonance spectrometer (500 MHz).

5.3 results

The yield of the dLFG-2A product is about 82 percent based on the charging amount of the dLFG-1A; the detection result of the product components shows that D-GalNAc, D-GlcUA, L-Fuc, and-OSO₃ ^－About 1.00:0.98:1.10:3.60, Mw about 8,969, and PDI about 1.42, which is essentially consistent with theoretical calculations for a degree of polymerization of about 10 for LGC-1A structural units; of dLFG-2A¹The HNMR map is shown in figure 9.¹HNMR(D₂O,[ppm]):7.25(2’,6’H);6.83(3’,5’H);5.65,5.36,5.28(L-Fucα1H);3.38(8’H);2.82(7’H);2.02(D-GalNAc,CH₃)；1.30～1.32(L-Fuc,CH₃). The integral ratio of the benzene ring hydrogens to Δ UA of H4 was about 1:0.28, indicating that the reducing ends of the resulting product were all aminated with a reducing casein.

[ example 6 ]

Preparation of reduced alkylation product dHEG-PMP:

6.1 materials

HEG: FGAG compounds derived from Holothuria erythrosticta were prepared by the same procedure as in step (1) of example 3. 1-phenyl-3-methyl-5 pyrazolone (PMP), biochemical reagent, purity 99%.

6.2 methods and results

HEG100mg, processed in the same manner as in step (3) and (4) of example 1, to give 132mg of tetrabutylammonium salt of HEG benzyl ester. After the resulting tetrabutylammonium HEG benzyl ester (50mg/mL) was dissolved in DMF and EtONa-EtOH (final concentration: 20mM) was added thereto, the mixture was stirred at 50 ℃ for 0.5hr, and then 10mL of a 0.5mol/LPMP methanol solution was added thereto, and the reaction was further stirred for 1.5 hr. 10mL of water was added thereto, the mixture was stirred and cooled to room temperature, the reaction solution was neutralized with 1N HCl, 20mL of saturated sodium chloride and 200mL of absolute ethanol were added thereto, and the supernatant was centrifuged. The resulting precipitate was dissolved in 10mL of H2O, dialyzed against a 3kD dialysis bag and lyophilized to give the product dHEG-PMP102 mg.

According to dHEG-PMP¹And (4) performing HNMR spectrum calculation, and reducing and alkylating the tail end of the obtained product.

[ example 7 ]

Anticoagulant activity of TAG-derived dLFG

7.1 materials

Sample preparation: the series of samples dLFG-1A-1F and dLFG-2A described in examples 1, 2, 5 and dLFG-1G with an Mw of about 3.5kD prepared from TAG by the β -elimination method.

Reagent: enoxaparin sodium injection (LMWH, Mw 3500-5500, sunofil-amphetate); the blood coagulation quality control plasma, the Activated Partial Thromboplastin Time (APTT) determination kit, the Thrombin Time (TT) determination kit and the prothrombin time determination kit (PT-dry powder) are all produced by the Germany company TECO GmbH; all other reagents were commercially available analytical grade.

The instrument comprises the following steps: MC-4000 coagulometer (Meichun, Germany).

5.2 methods

Dissolving the sample with deionized water to obtain a series of concentrations, and detecting the anticoagulant activity of dLFG series compounds by using an MC-4000 hemagglutination instrument according to the method of the APTT, PT and TT detection kit.

5.3 results

The results of anticoagulation experiments with dLFG-1A-1F and dLFG-2A are shown in Table 4.

The results in Table 4 show that both dLFG-1A-1F and dLFG-2A can significantly prolong human plasma APTT, the drug concentration required for doubling APTT (time value is prolonged by 2 times) is lower than 9 mu g/mL, and is stronger than that of positive control drug enoxaparin sodium (9.31 mu g/mL), and the derivatives can effectively inhibit endogenous coagulation.

The experimental result also shows that the influence of dLFG-1A-1F and dLFG-2A on PT and TT is small, the influence activity is weaker than that of the positive control medicament enoxaparin sodium, and the influence of the compounds on the exogenous coagulation process and the coagulation common pathway is small.

Comparing the molecular weight of the dLFG series derivative and the strength of the APTT prolonging activity of the dLFG series derivative, the anticoagulant activity of the dLFG series derivative is enhanced along with the increase of the molecular weight, and therefore, the molecular weight is one of important factors influencing the anticoagulant activity of the series compounds. Generally, the preferred molecular weight of dLFG of the present invention is not less than 3,000Da on a weight average molecular weight basis from the viewpoint of anticoagulant activity.

TABLE 4 series of molecular weight dLFG anticoagulant activities

^aPT value at drug concentration of 25 μ g/mL (PT value of blank control group is 14.4 + -0.2 s);

^bTT value at drug concentration of 12.5. mu.g/mL (blank group TT of 16.0. + -. 0.3 s).

[ example 8 ]

The effect of dLFG on coagulation factor activity:

8.1 materials

Samples dLFG-1A to dLFG-1F were prepared as described in example 7.

Reagent: quality control plasma (TECO GmbH, germany); enoxaparin sodium injection (LMWH, Mw 3500-5500, sunofil-amphetate); heparin (Heparin, Mw-18000, Sigma); chondroitin polysulfate (OSCS, china institute for pharmaceutical and biological products); thrombin (IIa)100NIHU/mg, thrombin detection chromogenic substrate (CS-0138)25 mg/visual, heparin cofactor II (HC-II) 100. mu.g/visual, all from HYPHEN BioMed (France); factor VIII (f.viii)200 IU/count, shanghai leshi blood products, ltd; VIII detection kit, wherein the Reagents comprise Reagents R1: Human Factor X, R2: Activation Reagent, Human Factor IXa, stabilizing Human thrombin, calcium and synthetic phospholipids, R3: Sxa-11, Chomogenic substrate, specific for Factor Xa, R4: Tris-BSA Buffer; HYPHEN BioMed (france);FXIIa-5963(CENTERCHEM,INC)。

the instrument comprises the following steps: bio Tek-ELx808 model microplate reader (USA).

8.2 methods

(1) Inhibition of endogenous factor X enzyme (f.Xase, Tenase)And (3) activity detection: and (3) a detection method established by combining the f.VIII detection kit with the f.VIII reagent. Serial concentrations of dLFG-1A-F solutions or control solvents (Tris-BSA buffer R)₄) After mixing 30. mu.L with 2.0IU/mL f.VIII (30. mu.L), the reagent kit R2 (30. mu.L) and R were added in sequence₁(30. mu.L), incubation at 37 ℃ for 2min, then adding R₃(30. mu.L), accurately incubating at 37 ℃ for 2min to detect OD_405nm. Document (Sheehan J.P.&Walk E.K., Blood,2006,107:3876-₅₀The value is obtained.

(2) HC-II dependent antithrombin (f.IIa) activity assay: adding 30 microliter of dLFG-1A-dLFG-1F solution with serial concentrations or a control solvent (Tris-HCl buffer solution) into a 96-well enzyme label plate, adding 30 microliter of 1 microliter HC-II, mixing, and incubating for 1min at 37 ℃; then adding 30. mu.L of 10U/mL IIa, incubating at 37 ℃ for 1min, adding 30. mu.L of 4.5mM chromogenic substrate CS-0138, mixing, incubating at 37 ℃ for 2min precisely, and detecting OD_405nm. Δ OD was calculated from a blank control (Tris-HCl), reference (Sheehan J.P.&Walke E.K., Blood,2006,107:3876-₅₀The value is obtained.

(3) Activated activity assay for coagulation factor f.xii: assay for XII Activity the reference (Hojima et al, Blood,1984,63: 1453-. 30 μ L of dLFG-1A-dLFG-1F solutions with serial concentrations or a control solvent (20mM Tris-HCl buffer, pH7.4) was added to a 96-well microplate, and 30 μ L of human coagulation factor XII (containing 1mM NaAc-HCl and 40mM NaCl/0.02% NaN) with a concentration of 312nM was added₃pH5.3), mixing, and incubating at 37 deg.C for 1 min; then adding 30 μ L of 620nM prekallikrein, incubating at 37 deg.C for 1min, adding 30 μ L of 6mM kallikrein chromogenic substrate, mixing, incubating at 37 deg.C for detecting OD at certain time intervals_405nmAnd calculating the change rate of the OD value.

8.3 results

The effect of dLFG-1A to dLFG-1F on coagulation factor activity is shown in Table 5.

FIGS. 10 and 11 show the dose-effect relationship and inhibition of f.Xase activity, respectively, of dLFG-1E-dependent HC-II antithrombin activity.

TABLE 5 Effect of dLFG series of compounds on coagulation factor Activity

The data in Table 5 and FIGS. 4 and 5 show that dLFG-1A-dLFG-1F prepared by the present invention has potent anti-f.Xase activity, and the Effective Concentration (EC) of 50% of the f.Xase activity is inhibited₅₀) About 0.5-5 nM. At the same time, the compound also has remarkable HC-II-dependent antithrombin activity and EC thereof₅₀About 5.5 to about 40 nM. Xase is the last target of the intrinsic coagulation pathway in the coagulation cascade, and is the limiting step in the coagulation process, and therefore may be the key mechanism for the potent anticoagulant activity of these compounds.

Recent research data show that the selective endogenous coagulation factor inhibitor can effectively avoid the bleeding tendency when generating antithrombotic activity, so the dLFG compound has potential antithrombotic application value.

On the other hand, f.xii activation can lead to severe adverse symptoms such as hypotension through activation of prekallikrein, and serious clinical events due to contamination with polysulfated chondroitin sulfate (OSCS) are of concern. The results of the present study show that, unlike OSCS, no significant f.XII activating activity is present in any of dLFG-1A to dLFG-1F of the present invention at effective anticoagulant doses.

[ example 9 ]

Aqueous injection solution of low molecular weight fucosylated glycosaminoglycan

9.1 materials

dLFG-1E, Mw7,066Da, obtained by the same method as in example 2.

9.2 prescription

Name of raw and auxiliary materials	Dosage of
		dLFG-IE	50g
Water for injection	l000mL
		Are co-produced into	1000 pieces

9.3 preparation Process

Weighing the low molecular weight fucosylated glycosaminoglycan sodium salt with the prescription amount, adding water for injection to the full amount, stirring to completely dissolve, and sterilizing by an intermittent hot pressing method. Adding 0.6% medicinal active carbon, and stirring for 20 min; the heat source was removed by decarburizing filtration using a Buchner funnel and a 3.0 μm microporous membrane. And (5) measuring the content of the intermediate. Filtering with 0.22 μm microporous membrane; filling into a tube-type penicillin bottle with 1mL per bottle, monitoring the filling amount in the filling process, inspecting, and packaging to obtain a finished product.

[ example 10 ]

Preparing a low molecular weight fucosylated glycosaminoglycan freeze-dried powder injection:

10.1 materials

dLFG-2A, Mw8,969Da, which is obtained by the same method as the example 2.

10.2 prescription

Name of raw and auxiliary materials	Dosage of
		dLFG-2A	50g
Water for injection	500mL
		Are co-produced into	1000 pieces

10.3 the preparation process comprises the following steps:

weighing the low molecular weight fucosylated glycosaminoglycan sodium salt with the prescription amount, adding water for injection to the full amount, stirring to completely dissolve, and sterilizing by an intermittent hot pressing method. Adding 0.6% medicinal active carbon, and stirring for 20 min; the heat source was removed by decarburizing filtration using a Buchner funnel and a 3.0 μm microporous membrane. And (5) measuring the content of the intermediate. Filtering with 0.22 μm microporous membrane; and (3) subpackaging into tube-type penicillin bottles, wherein each bottle is 0.5mL, half plugging, placing into a freezing and drying box, performing freeze drying according to a set freeze drying curve, plugging, taking out of the box, rolling a cover, performing visual inspection to obtain a qualified product, and packaging to obtain a finished product.

And (3) freeze-drying: putting the sample into a box, cooling the temperature of a partition plate to-40 ℃, and keeping for 3 hours; the cold trap was lowered to-50 ℃ and vacuum was started to 300 μ bar. Starting sublimation: heating to-30 deg.C at constant speed for 1h, and maintaining for 2 h; heating to-20 ℃ at constant speed for 2h, keeping for 8h, and keeping vacuum at 200-300 mu bar; and then drying: heating to-5 ℃ for 2h, keeping for 2h, and keeping the vacuum state at 150-200 mu bar; heating to 10 ℃ within 0.5h, keeping for 2h, and keeping vacuum at 80-100 μ bar; heating to 40 deg.C for 0.5h, maintaining for 4h, and vacuum pumping to minimum.

Claims (16)

1. A low molecular weight glycosaminoglycan derivative and pharmaceutically acceptable salts thereof, wherein:

the monosaccharide of the low molecular weight glycosaminoglycan derivative comprises hexuronic acid, hexosamine, deoxyhexose and sulfate of the monosaccharide, wherein the hexuronic acid is D-glucuronic acid and delta^4,5-hexuronic acid (4-deoxy-threo-hex-4-enopyrauronic acid), the hexosamine being acetylgalactosamine (2-N-acetylamino-2-deoxy-D-galactose) or its terminal reduction product, the deoxyhexose being L-fucose;

monosaccharides and-OSO in the low molecular weight glycosaminoglycan derivative in a molar ratio₃ ^－The composition proportion of (1) is (1 +/-0.3) to (3.0 +/-1.0); in terms of molar ratio,. DELTA.^4,5-the percentage of hexuronic acid to total hexuronic acid is not less than 2.5%;

the weight average molecular weight Mw of the low molecular weight glycosaminoglycan derivative ranges from 3kD to 20 kD;

the polydispersity index of the low molecular weight glycosaminoglycan derivative is between 1.0 and 1.8.

2. The low molecular weight glycosaminoglycan derivatives of claim 1, wherein the low molecular weight glycosaminoglycan derivatives are a mixture of homologous glycosaminoglycan derivatives having the structure of formula (I),

in formula (I):

n is an integer having a mean value of 2 to 20;

-D-GlcUA- β 1-, is-D-glucuronic acid- β 1-yl-;

-D-GalNAc- β 1-, is-2-deoxy-2-N-acetylamino-D-galactos- β 1-yl-;

L-Fuc-alpha 1-is-L-fucose-alpha 1-radical-;

Δ UA-1-, is Δ^4,5-hexuronic acid-1-yl (4-deoxy-threo-hex-4-enopyran uronic acid-1-yl);

r independently of one another is-OH or-OSO₃ ^－；

R' is-OH, C1-C6 alkoxy, C7-C10 aryloxy;

R₁is a group of formula (II) or (III), and in said mixture, in terms of mole ratio, R₁Is of the formula (II) and R₁In the case of compounds of the formula (III) in a ratio of not less than 2:1,

wherein R, R' is as defined above;

R₂is a group of formula (IV) or (V),

wherein R, R' is as defined above;

R₃is carbonyl, hydroxyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, C6-C12 aryl, substituted or unsubstituted five-or six-membered nitrogen-containing heterocyclic group, or-NHR₄(ii) a wherein-NHR₄R in (1)₄Is substituted or unsubstituted straight chain or branched chain C1-C6 alkyl, substituted or unsubstituted C7-C12 aryl, or substituted or unsubstituted hetero atom-containing heterocyclic aryl.

3. The low molecular weight glycosaminoglycan derivatives and the pharmaceutically acceptable salts thereof according to claim 1 or 2, wherein the weight average molecular weight of the low molecular weight glycosaminoglycan derivatives is in the range of 5kD to 12 kD; the low molecular weight glycosaminoglycan derivative has a polydispersity index value of between 1.1 and 1.5.

4. The low-molecular weight glycosaminoglycan derivative of claim 1 or 2, wherein the low-molecular weight glycosaminoglycan derivative is a β -elimination depolymerization product of fucosylated glycosaminoglycan derived from the body wall and/or the internal organs of a holothurian belonging to the phylum echinodermata, and a reduction-derivatized product of the reducing end of the depolymerization product, and a pharmaceutically acceptable salt thereof.

5. The low molecular weight glycosaminoglycan derivatives of claim 4, wherein the echinodermata holothurian species include but are not limited to:

apostichopus japonicus;

actinopyga mauritiana, radix Cynanchi Stauntonii;

actinopyga miniris, Actinopyga miliaris;

acaudina molpadioides;

bohadschia argus, Leptospira japonica;

holothuria rosea Holothuria edulis;

holothuria nobilis selenka;

holothuria leucospilota;

holothuria sinica;

sea cucumber Holothuria vagalbuda;

american ginseng Isostichopus badionotus;

brazilian ginseng Ludwigothia grisea;

stichopus chlororonotus, Stichopus japonicus selenka;

thelenota ananas;

thelenota anax (Juhua ginseng).

6. The pharmaceutically acceptable salt of low molecular weight glycosaminoglycan according to claim 1 or 2, wherein the pharmaceutically acceptable salt is an alkali metal, alkaline earth metal salt, and organic ammonium salt of the low molecular weight glycosaminoglycan derivative.

7. The pharmaceutically acceptable salt of a low molecular weight glycosaminoglycan derivative of claim 6 wherein the pharmaceutically acceptable salt is a sodium salt, a potassium salt, or a calcium salt of the low molecular weight glycosaminoglycan derivative.

8. The process for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 1, comprising the steps of:

(1) taking fucosylated glycosaminoglycan from echinoderm as a raw material, and completely or partially converting free carboxyl on contained hexuronic acid into carboxylic ester group through esterification reaction to obtain carboxylic ester;

(2) obtaining a low molecular weight glycosaminoglycan derivative by an ester group beta-elimination reaction of the carboxylate obtained in the step (1) in a non-aqueous solvent in the presence of an alkaline reagent;

(3) carrying out reduction treatment on the reducing tail end of the low molecular weight glycosaminoglycan derivative obtained in the step (2) to obtain a tail end reduced low molecular weight glycosaminoglycan derivative;

(4) converting the carboxylic ester group into a free carboxyl group by an alkaline hydrolysis method through the low molecular weight glycosaminoglycan derivative obtained in the step (2) and the step (3).

9. The method for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 8, wherein the step (1) comprises:

(i) converting the fucosylated glycosaminoglycan from an echinoderm source to a quaternary ammonium salt;

(ii) (ii) reacting the quaternary ammonium salt obtained in (i) with halogenated hydrocarbon and halogenated aromatic hydrocarbon in an aprotic solvent system to generate a derivative with completely or partially esterified carboxyl.

10. The method for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 8, wherein the non-aqueous solvent in the step (2) is selected from the group consisting of ethanol, methanol, dimethylformamide, dimethylsulfoxide, CH₂Cl₂、CHCl₃One or a mixed solvent thereof; the alkaline agent is selected from NaOH, KOH, C1-C4 sodium alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine or a mixture thereof.

11. The method for producing low molecular weight glycosaminoglycan derivatives of claim 8, wherein the terminal reduction treatment in the step (3) is a reduction of the sugar group at the reducing terminal to sugar alcohols, sugar ethers, sugar esters, aromatic and/or heterocyclic substituted derivatives.

12. The method for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 8, wherein the terminal reduction treatment in the step (3) is reductive amination reaction of a reducing terminal to form nitrogen-containing derivatives.

13. The pharmaceutical composition of low molecular weight glycosaminoglycan or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, comprising an effective anticoagulant dose of the low molecular weight glycosaminoglycan or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is in the form of an aqueous solution for injection or a lyophilized powder for injection.

15. Use of a low molecular weight glycosaminoglycan or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 for the preparation of a medicament for the treatment and/or prevention of thrombotic disorders.

16. Use of the pharmaceutical composition according to claim 13 or 14 for the preparation of a medicament for the treatment and/or prevention of thrombotic disorders.