WO2014166282A1 - 一种含末端2，5−脱水塔罗糖或其衍生物的低分子量糖胺聚糖衍生物 - Google Patents

一种含末端2，5−脱水塔罗糖或其衍生物的低分子量糖胺聚糖衍生物 Download PDF

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WO2014166282A1
WO2014166282A1 PCT/CN2013/090124 CN2013090124W WO2014166282A1 WO 2014166282 A1 WO2014166282 A1 WO 2014166282A1 CN 2013090124 W CN2013090124 W CN 2013090124W WO 2014166282 A1 WO2014166282 A1 WO 2014166282A1
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molecular weight
low molecular
glycosaminoglycan
derivative
product
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PCT/CN2013/090124
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English (en)
French (fr)
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赵金华
吴明一
高娜
李姿
赖森森
赵龙岩
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中国科学院昆明植物研究所
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Priority to US14/779,934 priority Critical patent/US10494452B2/en
Priority to CA2908959A priority patent/CA2908959C/en
Priority to JP2016506758A priority patent/JP6248179B2/ja
Priority to EP13881924.8A priority patent/EP2985298B1/en
Publication of WO2014166282A1 publication Critical patent/WO2014166282A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a low molecular weight glycosaminoglycan containing a terminal 2,5-anhydrous talose or a derivative thereof.
  • 2,5-anhydrated Talose terminal Low-molecular-weight Fucosylated Glycos - aminoglycan, aTFG 2,5-anhydrated Talose terminal Low-molecular-weight Fucosylated Glycos - aminoglycan, aTFG
  • a pharmaceutical composition comprising the aTFG and its use in the preparation of a medicament for preventing and/or treating cardiovascular and cerebrovascular diseases.
  • Cardiovascular and cerebrovascular diseases have high incidence, high disability rate, high mortality, high recurrence rate and many complications, which seriously endanger human health and quality of life.
  • Thrombosis is one of the main causes of cardiovascular and cerebrovascular diseases.
  • Antithrombotic drugs including anticoagulants, are clinical first-line drugs for the treatment of cardiovascular diseases and occupy an important position in the pharmaceutical market.
  • Anticoagulant drugs mainly include coumarins and heparin anticoagulants. These drugs have clear pharmacological effects and pharmacological mechanisms, but there are also obvious clinical application defects: Defects of coumarin anticoagulants include It inhibits the severe bleeding tendency caused by the synthesis of coagulation factors, slow onset, large individual differences, etc.
  • Heparin drugs mainly act as a common pathway for the target factors Ila and Xa (fn a , f.Xa) in the coagulation cascade.
  • the application of drugs is mainly due to the serious bleeding risk associated with the target and thrombocytopenia. Therefore, a new type of anticoagulant drug with superior pharmacological effects is required clinically.
  • the core of the development of new anticoagulant drugs is to effectively avoid bleeding tendency, and innovative drug research with low bleeding tendency has not made breakthrough progress.
  • Fusarsin-derived fucosylated glycosaminoglycan is a class of glycosaminoglycan derivatives with fucose side chain substitutions, which have glucuronic acid (GlcUA) and acetamidogalactose ( GalNAc) consists of a chondroitin-like backbone with a fucose L-Fuc side chain linked to the main chain glucuronic acid group by a ct-l,3 glycosidic bond, with both the main chain and the side chain sugar hydroxyl groups present.
  • GlcUA glucuronic acid
  • GalNAc acetamidogalactose
  • FGAG still has a wide range of contradictory pharmacological effects, including induction of platelet aggregation, bleeding tendency, and activation of XII (Thromb. Haemost, 1988, 59: 432-434; Thromb. Haemost, 1997, 65 (4): 369 -373; Thromb. Haemost., 2010, 103 : 994-1004).
  • An appropriately depolymerized oligomeric FGAG retains the anticoagulant activity of native FGAG and reduces its platelet activation activity (73 ⁇ 4ramb. Haemost., 1991, 65: 369-373).
  • the Chinese patents CN 101724086 B and CN 101735336 B disclose a process for preparing oligomeric FGAG, which is obtained by depolymerization of FGAG by hydrogen peroxide depolymerization to obtain an oligomerization product, and the bleeding tendency of the obtained product is remarkably lowered.
  • FGAG is a glycosaminoglycan derivative with a large molecular weight and a complicated structure, under the premise of reducing the side effects and retaining the pharmacological activity, the process can be effectively and depolymerized to obtain a low molecular weight derivative having a characteristic end structure. Technically, it is very difficult.
  • the present invention establishes a new FGAG depolymerization method...deacylation deamination depolymerization method, which firstly makes FGAG by deacetylation reaction
  • the partial deacetylation reaction of D-2-(N-acetyl)amino-2-deoxygalactose (D-GalNAc) is carried out to obtain D-2-amino-2-deoxygalactosyl (D-GalNH) 2 )
  • the FGAG partially deacetylated product is then subjected to a deamination depolymerization reaction by nitrous acid treatment, thereby obtaining a FGAG depolymerization product containing a terminal 2,5-anhydro talose group or a reduced derivative thereof.
  • aTFG Low-molecular-weight Fucosylated Glycosaminoglycan
  • the present invention provides the following technical solutions:
  • a low molecular weight glycosaminoglycan derivative and a pharmaceutically acceptable salt thereof wherein the monosaccharide of the low molecular weight glycosaminoglycan derivative comprises hexuronic acid, hexose hexose, deoxyhexose and 2,5 - dehydrated talose or a reduced derivative thereof; wherein, hexuronic acid is D- ⁇ -glucuronic acid, and aminohexose is 2-N-acetylamino-2-deoxy-D- ⁇ -galactose or 2 -amino-2-deoxy-D- ⁇ -galactose or - ⁇ -oxime-2-sulfate amino-2-deoxygalactose, deoxyhexose as L-ct-fucose, 2,5-anhydro tartarose
  • the reduced derivative is 2,5-anhydrous tartanol, 2,5-anhydro tartaramide, hydrazine-substituted
  • the proportion of the 5-dehydrated talose and/or its reduced derivative in the total constituent monosaccharide is not less than 3.0%.
  • the weight average molecular weight Mw of the aTFG of the present invention ranges from 2.5 kD to 20 kD;
  • the polydispersity index (Mn/Mw) of the aTFG of the present invention is between 1.0 and 1.8.
  • the aTFG of the present invention is a mixture of homologous glycosaminoglycan derivatives having the structure of formula (I),
  • n is an integer having a mean value of 3 to 21;
  • -D-GlcUA- ⁇ - is - ⁇ -D-glucuronic acid-1-yl
  • -D-GalN- ⁇ - is - ⁇ - ⁇ -2-acetamido-2-deoxygalactose-1-yl or - ⁇ -indole-2-amino-2-deoxygalactose or -PD-2-sulfate Amino-2-deoxygalactose;
  • L-Fuc- ⁇ - is a-L-fucose-1-yl
  • R' is independently of each other -OH or -OS0 3 _;
  • R 3 is -H, -S0 3 - or acetyl
  • R is -H or P-D-2-acetamido-2-deoxygalactose sulfate-1-yl
  • R 2 is -H or - ⁇ -D-glucurono-1-yl, or a group of the formula ( ⁇ ):
  • anTal is 2,5-dehydrated talose, its sugar alcohol, sugar amine or N-substituted sugar amine,
  • n 1 or 2;
  • R 2 is a formula ( ⁇ ) group in terms of molar ratio The ratio of the compound to the compound wherein R 2 is -H or - ⁇ -D-glucuron-1-yl is not less than 2:1.
  • the molecular weight of the aTFG of the present invention can be detected by high performance gel chromatography (HPGPC).
  • HPGPC high performance gel chromatography
  • the molecular weight of the selected &1?0 of the present invention is in the range of about 2,500 to 20,000 0", that is, the homolog of the homologue of the formula (I) has a mean value of about 3 to 21, preferably a molecular weight range of The average value of n of about 5,000 to 12,000 Da, BP, is about 5 to 15.
  • the polydispersity index (PDI, weight average/number average molecular weight ratio, Mw/Mn) of the aTFG of the present invention is generally between 1.0 and 1.8; the preferred aTFG has a PDI between 1.1 and 1.5.
  • the aTFG of the present invention may be a salt of a pharmaceutically acceptable alkali metal, alkaline earth metal or the like.
  • the aTFG may be an ester formed with a basic organic group.
  • the pharmaceutically acceptable salt of aTFG of the present invention is preferably a sodium salt, a potassium salt or a calcium salt of aTFG.
  • the aTFG of the present invention is a deamination depolymerization product of fucosylated Glycosaminoglycan (FGAG) of the body wall and/or visceral origin of the echinoderms, or the reductive property of the depolymerized product.
  • FGAG fucosylated Glycosaminoglycan
  • the reduced derivatization product of the terminal is to further provide a method for preparing the aTFG of the present invention by using FGAG as a raw material, and the preparation method of the aTFG comprises the following steps:
  • Step 1 The echinoderma-derived fucosylated glycosaminoglycan (FGAG) is subjected to partial deacetylation of the aminoglycol (GalNAc) contained therein to obtain a partially deacetylated product of FGAG;
  • the acetylated product can be subjected to a sulfonation reaction to sulfonate the free amino group to a sulfated amino derivative.
  • Step 2 Partially deacetylating the FGAG obtained by the nitrous acid treatment step 1, deamination reaction and depolymerization, and obtaining a low molecular weight fucosylated glycosaminoglycan having a reducing end of 2,5-anhydro talose Sugar, the resulting low molecular weight fucosylated glycosaminoglycan optionally undergoing a reducing end reduction reaction comprising reducing a 2,5-anhydro tartarose end group to a sugar alcohol, a sugar amine or an N substituted sugar amine,
  • a low molecular weight fucosylated glycosaminoglycan having a terminal 2,5-anhydroglucopyryl group or a sugar alcohol thereof, a sugar amine, or an N-substituted sugar amine is the aTFG of the present invention.
  • the method for treating the deacetylation reaction in the first step is: adding the fucosylated glycosaminoglycan derived from echinoderma to the anhydrous hydrazine or hydrazine hydrate solution, with or without In the presence of a catalyst, the reaction is carried out at a temperature of from 75 ° C to 125 ° C for 2 to 14 hours with stirring.
  • the deacetylation reaction in the first step is preferably carried out in the presence of a catalyst, and the catalyst may be selected from barium sulfate, barium hydrochloride, etc., or a small amount of a strong acid such as sulfuric acid and/or hydrochloric acid may be added as a catalyst to the reaction solvent. And/or hydrochloric acid can be reacted with a solvent hydrazine or hydrazine hydrate to form hydrazine sulfate and/or hydrazine hydrochloride, which can act as a catalytic reaction.
  • the concentration of the catalyst in the reaction solution is from 0.5% to 2.5%.
  • the reaction solution may be evaporated to dryness under reduced pressure.
  • an alcohol precipitation method may be employed, such as adding an equal volume of 80% ethanol to precipitate the obtained product. Adding an appropriate amount of saturated sodium chloride solution during the alcohol precipitation process can make the precipitation more complete.
  • the partially deacetylated product of the FGAG obtained by the reaction may be directly dried and used for the deamination depolymerization reaction of the second step, or may be selected. After removing the excess hydrazine and hydrazide derivative by iodine oxidation, it is selected to be purified by an appropriate method, dried, and used for the deamination depolymerization of the second step.
  • the FGAG partial deacetylated product obtained in the first step can be determined by nuclear magnetic resonance spectroscopy (NMR) detection technology. Specifically, the degree of deacetylation is compared between D-GalNAc and L-Fuc in the raw material compounds and products. The ratio of the area of the methyl proton-containing peak, the degree of deacetylation of the reaction product can be calculated by the integral ratio of the methyl peak.
  • NMR nuclear magnetic resonance spectroscopy
  • the degree of deacetylation of the partially deacetylated product of the FGAG obtained in step one can determine the molecular weight of the deaminated depolymerization end product aTFG.
  • the present inventors have found that when the degree of deacetylation of the FGAG partial deacetylated product obtained in the first step is about 5% to 35%, the molecular weight of the deaminated depolymerization end product aTFG can range from about 4 000 Da to 20 000. Within the Da range.
  • the specific step of the deamination depolymerization method of the nitrous acid treatment is generally selected as follows: the FGAG partial deacetylated product obtained in the first step is used in an ice bath or at room temperature. (1.5 to 4.5) Nitrous acid solution (4 to 6) mol/L After treatment for 5 min to 60 min, the pH is adjusted to 8 or more with an alkaline solution such as NaOH to terminate the reaction. Then choose to proceed:
  • Step 2 (2) of the end group reduction reaction generally after sodium hydroxide pH adjustment (8 ⁇ 9) to terminate the deamination depolymerization reaction, the concentration of sodium borohydride and / or sodium cyanoborohydride is added (0.05 ⁇ 0.5) mol/L, stirring at 50 ° C for 20 min ⁇ 60 min to convert anTal into a nTalOH. After cooling the reaction solution to room temperature, the pH is adjusted with an acid (3 to 4) to remove excess sodium borohydride and/or sodium cyanoborohydride, and then neutralized with an alkaline solution such as NaOH, followed by purification according to the procedure described in (1). The product obtained.
  • Step (3) is to reductively amination of the terminal anTal in the presence of ammonium hydrogencarbonate or an organic amine and a reducing agent, that is, an ammonium salt or an organic amine reacts with an Tal aldehyde group to form a Schiff base, the latter being a reducing agent such as a cyano group. Reduction to a secondary amine in the presence of sodium borohydride.
  • a reducing agent that is, an ammonium salt or an organic amine reacts with an Tal aldehyde group to form a Schiff base, the latter being a reducing agent such as a cyano group.
  • the aTFG product obtained in the present invention can be detected by an NMR method.
  • the compound having the terminal 2,5-anhydroglucopyran or a reduced derivative thereof may account for 60% of the total depolymerized product. ⁇ 97%.
  • the D-GalNAc deacetylation, D-GalNAc deamination and anTal carbonyl reduction and reductive amination routes of the process of the present invention are shown in the "Reaction Process Roadmap". In the route, R' and R 5 in each of the compounds (1) to (5) are as defined above.
  • the prototype natural product FGAG has a complex main side chain structure, and it is a key technical difficulty to effectively realize its selective deacetylation reaction.
  • the side chain L-Fuc is prone to acid hydrolysis, which makes the deamination reaction condition of HN0 2 deamination. Control is especially important.
  • the technical route and its main technical parameters described in the present invention can ensure the smooth progress of these reactions.
  • the final product of the aTFG reaction can be purified by a method known in the art (CN)
  • 101735336A for example, removal of impurities such as small molecule salts by dialysis or ultrafiltration, or further purification by gel chromatography or DEAE ion exchange chromatography.
  • a dialysis membrane or an ultrafiltration membrane package suitable for molecular weight cutoff may be selected according to the target molecular weight requirement, preferably a dialysis membrane with a molecular weight cutoff of 1000 Da or an ultrafiltration membrane package for dialysis removal.
  • the dialysis time is determined according to the specific treatment conditions, usually not less than 6 hours. Dialysis can also be selected to remove other macromolecular impurities as well as undepolymerized FGAG or over the molecular weight range aTFG.
  • the final aTFG product obtained by the process of the present invention can also be prepared by cation exchange to form a single salt form such as a sodium salt, a potassium salt or a calcium salt.
  • the salt forming process of the aTFG product of the present invention may be carried out by ion exchange to exchange the aTFG into a hydrogen form, and then neutralized with a corresponding base to obtain a salt corresponding to aTFG; preferably, the dynamic ion exchange salt method is directly
  • the column is exchanged for salt. Resin column pretreatment, sample loading and elution can be carried out in a conventional manner.
  • the starting material of the "Step 1" is fucosylated glycosaminoglycan (FGAG) derived from echinoderms.
  • FGAG fucosylated glycosaminoglycan
  • the FGAG has a similar chondroitin sulfate backbone, which is generally composed of glucuronic acid (D-GlcUA) and 2-(N-acetyl)amino-2-deoxygalactose (D-GalNAc).
  • the two constituent monosaccharides are sequentially linked in the form of -4)-D-GlcUA - (; ⁇ -l- and -3)-D-GalNAc- (; ⁇ -l- glycosidic linkage; FGAG also has fucoal Sugar (L-FucM chain, in general, L-Fuc side chain is linked to D-GlcUA in the main chain by ct-l, 3 glycosidic bond; in addition, D-GalNAc and side chain sugar L- in the FGAG backbone There may be varying degrees of sulfation of the hydroxyl groups on Fuc (J. Biol. Chem., 1996, 271: 23973-23984; Mar. Drugs, 2013, 11: 399-417).
  • the FGAG used to prepare the aTFG of the present invention and a pharmaceutically acceptable salt thereof may be derived from, but not limited to, the following sea cucumbers: Sric/iopws van'egii es Semper, Scabra Jaeger) Holothuria leucospilota Brandt, Holothuria edulis Lesson, Bohad randt, Chinese sea cucumber (Holothuria sinica Liao) Molpadioides Semper) ⁇ Pearsonothuria graejfei Semper) and black ginseng (Ho/o /iwn' ⁇ 3 ⁇ 4 nobilis Selenka).
  • sea cucumbers Sric/iopws van'egii es Semper, Scabra Jaeger
  • Holothuria leucospilota Brandt Holothuria edulis Lesson
  • Bohad randt Chinese sea cucumber (Holothuria sinica Liao) Molpadioides Semper)
  • the method of the present invention is not limited by the particular sea cucumber variety.
  • aTFG has potent anticoagulant activity, which multiplies human-controlled plasma-activated partial clotting activity.
  • concentration of the drug required for the enzyme time (APTT) is not higher than 12 g/mL.
  • the aTFG has potent inhibitory activity against the endogenous Factor X enzyme with an EC 5Q of about 5 to 50 ng/mL. Since factor X enzyme is the last enzyme point in the endogenous coagulation pathway and is the rate-limiting site of the coagulation process induced by various factors, its inhibitor can have significant antithrombotic activity and has an effect on physiological hemostasis. Smaller (Blood, 2010, 116(22), 4390-4391; Blood, 2009, 114, 3092-3100).
  • aTFG of the present invention and its pharmaceutically acceptable salt have an exact anticoagulant activity and thus have a clear potential medicinal value.
  • aTFG has good water solubility and is therefore easy to prepare into a solution preparation or a lyophilized preparation.
  • the polysaccharide component its oral bioavailability is limited, and therefore its pharmaceutical composition is preferably prepared into a parenteral dosage form, and preparation of the preparation can be carried out according to a technical method well known in the art.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-described low molecular weight glycosaminoglycan derivative and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, comprising an effective anticoagulant dose
  • said pharmaceutical composition may be an aqueous solution for injection or a lyophilized powder for injection.
  • the aTFG of the invention has potent anticoagulant activity and can therefore be used for the prevention and treatment of different degrees of thrombotic diseases, such as thrombotic cardiovascular diseases, thrombotic cerebrovascular diseases, pulmonary vein thrombosis, peripheral venous thrombosis, Deep vein thrombosis, peripheral arterial thrombosis, etc.
  • thrombotic diseases such as thrombotic cardiovascular diseases, thrombotic cerebrovascular diseases, pulmonary vein thrombosis, peripheral venous thrombosis, Deep vein thrombosis, peripheral arterial thrombosis, etc.
  • the present invention also provides the use of the low molecular weight glycosaminoglycan derivative (aTFG), a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising aTFG or a salt thereof for the preparation of a medicament for preventing and treating a thrombotic disease
  • aTFG low molecular weight glycosaminoglycan derivative
  • a pharmaceutical composition comprising aTFG or a salt thereof for the preparation of a medicament for preventing and treating a thrombotic disease
  • the thrombotic disease is venous thrombosis or arterial thrombosis or ischemic heart disease or ischemic cerebrovascular disease.
  • the FGAG depolymerization method reported here mainly consists of a hydrogen peroxide depolymerization method which lacks glycosidic bond selectivity and is complicated in process control.
  • the invention establishes a new FGAG depolymerization method-deacylation deamination depolymerization method, which firstly causes D-2-(N-acetyl)amino-2- contained in FGAG by deacetylation reaction.
  • Deacetylated galactose undergoes partial deacetylation to obtain a FGAG partially deacetylated product containing D-2-amino-2-deoxygalactosyl (D-GalNH 2 ), which is then treated with nitrous acid.
  • a deamination depolymerization reaction occurs, thereby obtaining a FGAG depolymerization product containing a terminal 2,5-anhydro talose group or a reduced derivative thereof.
  • the present invention discloses for the first time a method for obtaining a complex structure FGAG deamination depolymerization having a characteristic terminal structure derivative, and for the first time discloses a FGAG depolymerization product containing a terminal 2,5-anhydro talose group or a reduced derivative thereof.
  • FGAG deacylation deamination depolymerization has glycosidic linkage Selectively, selective cleavage of D-GalNH 2 Cpi-) glycosidic linkages without cleavage of L-FucCcd-) and D-GlcUACpi-) glycosidic linkages, thus resulting in better structural homogeneity of the depolymerized product;
  • the FGAG deacyl deamination depolymerization reaction product may have a characteristic 2,5-anhydrated Talose (anTal) or its reduced derivative terminal, and the presence of a characteristic terminal facilitates the depolymerization product chemistry.
  • Figure 1 shows the 1H NMR spectrum of FGAG and its deacetylated products dAFG-1 and dAFG-2;
  • Figure 2 is a 13 C NMR spectrum of FGAG and its deacetylated products dAFG-1 and dAFG-2;
  • Figure 3 is a 1H NMR spectrum of FGAG and its deacylation deamination depolymerization products aTFG-a, aTFG-b and aTFG-c;
  • Figure 4 shows FGAG and its deacyl deamination depolymerization products aTFG-a, aTFG-b and 13 C NMR spectrum of aTFG-c;
  • Figure 5 is a superposition of COGA (A), NOESY (B), TOCSY (C) spectra of FGAG and its deacyl deamination depolymerization products aTFG-a, aTFG-b (partial) Figure
  • Figure 6 is a 1H COSY spectrum comparison of FGAG (A) and its deacylated deamination depolymerization product aTFG-b (B);
  • Figure 7 is an anti-endogenous factor X enzyme activity of aTFG;
  • FGAG The fucosylated glycosaminoglycan derived from the body of Thelenota ananas, prepared by literature method (Marine Drugs, 2013 11, 399-417), has a molecular weight of 69,930 Da.
  • Reagents such as hydrazine hydrate, barium sulfate, guanidine hydrochloride, hydrochloric acid, sodium chloride, absolute ethanol, iodic acid, hydroiodic acid, and sodium hydroxide are commercially available analytical reagents.
  • Deacetylation reaction Accurately weigh 60 mg of FGAG raw material, put it in the reaction tube, add 14.5 mg of catalyst barium sulfate or guanidine hydrochloride 12.2 mg or 2.304 mol/L hydrochloric acid 0.1 mL or without catalyst, then add hydrazine hydrate or no Otter 1.45 mL, stir under a nitrogen atmosphere at 250 rpm, and heat at 75 to 105 °C for 2 to 14 h.
  • the reaction solution is precipitated in 80% ethanol, centrifuged to obtain a precipitate, and then vacuum-dried under reduced pressure to obtain a sample of deacetylated intermediate product, which can be directly used for nitrous acid depolymerization, or further processed to obtain a relatively pure intermediate.
  • the treatment method is as follows: The dried sample is cooled in an ice bath, and a 0.25 mol/L iodic acid solution is added dropwise; the dropping is continued until the precipitate is insoluble and a black suspension is obtained.
  • DD degree of deacetylation
  • the molecular weight of the product was determined by high performance liquid phase exclusion (HPGPC).
  • HPGPC high performance liquid phase exclusion
  • G1362A refractive index detector
  • Preparation of standard and reference solutions Take a reference series of dextran of known molecular weight, accurately weighed, dissolve and dilute with 0.1 ml/L sodium chloride solution to make a 10 mg/mL solution as a narrow calibration solution.
  • a FGAG reference substance with a known molecular weight was accurately weighed and dissolved and diluted with a 0.1 mol/L sodium chloride solution to prepare a 10 mg/mL solution as a broad calibration solution.
  • 25 L of each sample, standard and reference solution were injected into the liquid chromatograph to record the chromatogram. The data is processed using GPC-specific software.
  • Figures 1 and 2 show the 1H, 13 C-NMR spectra of two different degrees of deacetylation samples dAFG-1 and dAFG-2 prepared according to the method of the present example.
  • the degrees of deacetylation calculated from the NMR spectra were 48% and 88%, respectively.
  • the spectral data also indicated that the basic structure of the D-GalNAc was not significantly changed except that part of D-GalNAc was deacetylated to form D-GalNH 2 before and after deacetylation.
  • composition monosaccharide (D-GlcUA): (D-GalNAc + D-GalNH 2 ): (L-Fuc) has a molar ratio of about 1: (1 ⁇ 0.3) ): (1 ⁇ 0.3), this result further suggests that the basic structure of the polysaccharide remains stable except for partial deacetylation of D-GalNAc.
  • dAFG that is, a partially deacetylated product of FGAG, prepared according to the method described in Example 1; reagents such as sodium nitrite, concentrated sulfuric acid, sodium borohydride, sodium carbonate, sodium hydroxide, and anhydrous ethanol are commercially available analytical reagents. .
  • Preparation of the product Accurately weigh about 20 mg of deacetylated intermediate product in the reactor, add 1 mL of water to dissolve, add 5.5 mol/L nitrous acid solution (pH 4) 2 mL in ice bath or room temperature, and dissolve After 2 to 30 min of polymerization, the reaction was terminated by adding 1 mol/L sodium carbonate solution to adjust pH 8 to 9 to depolymerize nitrous acid with 1 mL of 0.1 mol/L sodium hydroxide solution containing 0.25 mol/L sodium borohydride. The aldehyde group of the product is reduced, heated at 50 °C for 40 min, cooled to room temperature after the reaction, and removed by 0.5 mol/L sulfuric acid. Excess sodium borohydride was finally neutralized with 0.5 mol/L sodium hydroxide solution and dialyzed against a 1000 Da dialysis bag. The dialysate in the dialysis bag was collected and lyophilized.
  • Product detection BUCKER DRX-500 NMR spectrometer for depolymerization samples; gel exclusion chromatography to determine the molecular weight of the depolymerized sample; Conductivity method to determine the molar ratio of depolymerized sample sulfuric acid carboxylic acid.
  • the yield of the deaminated depolymerization end product is greater than 90%, and the sample purity is greater than 95%.
  • the NMR nuclear magnetic spectra of the three products aTFG-a, aTFG-b and aTFG-c prepared according to this example are shown in Figures 3 to 6.
  • the nuclear magnetic resonance spectra of the raw material FGAG and the intermediate product dAFG were compared according to 1H NMR, 13 C NMR and 1H homonuclear correlation spectra COSY, TOCSY, NOESY and 1H- 13 C heteronuclear correlation spectra HQSC, HMBC, for different degree of deacetylation ( 15%, 48%, 88%)
  • the spectral signals of the nitrous acid depolymerization products aTFG-a, aTFG-b and aTFG-c were assigned, and the NMR signal data are shown in Table 3.
  • the 1H NMR signal of the deaminated depolymerization product is closer to the prototype FGAG than the deacetylated product.
  • the imitation bundle 1 J ginseng Apostichopus japonicus, the red-bellied sea cucumber Holothuria edulis, the Brazilian ginseng Ludwigothurea grisea, the seaweed sea cucumber Holothuria leucospilota, the black milk sea cucumber Holothhuria nobilis, are all commercially available dry body walls.
  • AJG, HEG, LGG, HLG and HNG were respectively taken, and the deaminated depolymerization product aTFG was prepared according to the methods described in Examples 1 and 2, and was referred to as aAJG, aHEG, aLGG, aHLG and B aHNG, respectively.
  • the yields of AJG, HEG, LGG, HLG and HNG from the dried body wall of sea cucumber, red ginseng, Brazilian ginseng, jade ginseng and black ginseng were about 1.4%, 0.9% and 0.8%, respectively. %, its weight average molecular weight is between about 50 kD and 80 kD.
  • the basic characteristics of AJG, HEG, LGG, HLG and HNG as FGAG compounds were determined by 1H NMR spectroscopy: the terminal groups and related characteristic proton signals on a-L-Fuc ⁇ -D-GalNAc and ⁇ -D-GlcUA were clearly defined.
  • the yield of aAJG (8.0 kD), aHEG (10.5 kD), aLGG (7.3 kD), HLG (10.2 kD) and aHNG (8.7 kD) from AJG, HEG, LGG, HLG and HNG is about 40% ⁇ 70. Within the range of %. Determined by 1 H NMR spectrum Understand the anTal related characteristic signals formed by the aggregation.
  • Terminal reductive amination The aTFG O. lg obtained in Example 2 was dissolved in 3.5 mL of 0.2 mM phosphate buffer (pH 8.0), and an excess of 80 mg of tyramine and 30 mg of sodium cyanoborohydride were respectively added thereto, 35 The reaction was carried out in a constant temperature water bath for about 72 h. After the reaction was completed, 10 mL of 95% ethanol was added, and the precipitate was obtained by centrifugation.
  • the obtained precipitate was washed twice with 30 mL of 95% ethanol, and then the precipitate was reconstituted with 35 mL of 0.1% NaCl, centrifuged to remove insoluble matter, and the supernatant was dialyzed at 1 KD. In the bag, deionized water was dialyzed for 24 h, and lyophilized to obtain dLFG-2A 82 mg.
  • HPGPC detects molecular weight and distribution
  • Conductivity method detects -OS0 3 7-COO-molar ratio
  • Elson-Morgon method detects acetylgalactose (D-GalNAc) content
  • carbazole method detects glucose aldehyde Acid (D-GlcUA) content
  • iHNMR methyl peak integrated area Calculated D-GalNAc/L-Fuc molar ratio (as in Example 1).
  • NMR spectra were detected by the Bruker AVANCE AV 500 superconducting nuclear magnetic resonance apparatus (500 MHz).
  • the product yield was about 72% based on the feed; the product component test results showed that D-GalNAc: D-GlcUA: L-Fuc: -OS0 3 _ was about 1.00:0.98: 1.10:3.60, Mw was about 9,969, and PDI was about 1.32. .
  • the deacetylated sample dAFG-1 was derived from the ginseng prepared according to Example 1, and the degree of deacetylation was 35%. 5.2 method
  • aTFG samples ATFG samples of different molecular weights prepared according to the methods described in Examples 2 and 3; the physical and chemical properties of these samples are shown in Table 4.
  • Coagulation-controlled plasma, activated partial thromboplastin time (APTT) assay kit, CaC are produced by TECO GmbH, Germany; other reagents are commercially available analytical grades.
  • APTT activated partial thromboplastin time
  • aTFG-6 y 5.3358x + 37.032 0.9978 6.14
  • aTFG-7 y 4.5807x + 38.73 0.9925 6.78
  • the results in Table 5 show that the deamination depolymerization product aTFG of FGAG can significantly prolong human plasma APTT, and the drug concentration of doubling APTT is below 12 ⁇ ⁇ / ⁇ 1, indicating that these derivatives can effectively inhibit endogenous coagulation. . Comparing the molecular weight of these derivatives with the corresponding drug concentration of doubling APTT, it was found that the greater the molecular weight, the stronger the anticoagulant activity, and the molecular weight is one of the main factors affecting its anticoagulant activity. According to this regular result, from the viewpoint of retaining the FGAG hematological activity, the molecular weight of the preferred aTFG of the present invention is not less than 5,000 Da in terms of weight average molecular weight.
  • aTFG sample Prepared according to the method of Example 2, molecular weight 8777 Da.
  • Reagents and instruments Factor vm (f.vm), 200 ⁇ /, Shanghai Lai Shi Blood Products Co., Ltd.; f.vm test trial U box, trial U including Reogew iv Rl: Human Factor X; R2: Activation Reagent, human Factor IXa, containing human thrombin, calcium and synthetic phospholipids; R3: SXa-11, Chomogenic substrate, specific for Factor Xa; R4: Tris-BSA Buffer; HYPHEN BioMed (France) product. Bio Tek-
  • Inhibition of endogenous factor X enzyme (anti-f.Xase) activity assay A detection method established using the f.VIII assay kit in combination with the f.VIII reagent. After serial concentration of the test solution or blank control solution (Tris-BSA buffer) 30 ⁇ and 2.0 IU/ml factor VIII (30 ⁇ ), add the reagents R 2 (30 ⁇ 1), Ri (30 ⁇ ). ), after incubation at 37 ° C for 2 min, add R 3 (30 ⁇ ), 37 . C was precisely incubated for 2 min, the reaction was stopped with 20% acetic acid (30 ⁇ M) and OD 4Q5 nm was detected.
  • test solution or blank control solution Tris-BSA buffer
  • the ⁇ was calculated from the blank control (R 4 ), and the EC 5 of each sample inhibiting f.Xase was calculated according to the formula provided in the literature (Sheehan JP & Walke EK, Blood, 2006, 107: 3876-3882). value.
  • the aTFG prepared by reacting the crude sea cucumber-derived FGAG according to Examples 1 and 2 had a weight average molecular weight of 9476 Da.
  • Freeze-drying process The sample is placed in the box, the temperature of the separator is lowered to -40 °C for 3 hours; the cold trap is lowered to -50 °C, and the vacuum is started to 300 bar. Start sublimation: 1 h at a constant temperature to -30 °C for 2 h; 2 h at a constant temperature to -20 °C, hold for 8 h, vacuum to maintain 200 ⁇ 300 bar; then dry: 2 h to -5 V, Hold for 2 h, keep the vacuum at 150 ⁇ 200 bar; 0.5 h to 10 °C for 2 h, keep the vacuum at 80 ⁇ 100 bar; 0.5 h to 40 °C for 4 h, vacuum to the lowest.

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Abstract

一种含有2,5−脱水塔罗糖、其糖醇、糖胺或N取代的糖胺单糖组分的低分子量岩藻糖化的糖胺聚糖(aTFG)，其制备方法，含所述aTFG的药用组合物及其在预防和/或治疗血栓性疾病中的应用。所述aTFG具有强效抗凝血活性，其作用于内源性凝血因子X酶，抑制血栓形成，因此可用于预防和或治疗心脑血管疾病药物。

Description

一种含末端 2,5-脱;^罗糖或其衍生物的低分子量糖胺聚糖衍生物
【技术领域】
本发明属于医药技术领域， 具体涉及一种含末端 2，5-脱水塔罗糖或其衍生物的低分子量 糖胺聚糖翁于生物 (2,5-anhydrated Talose terminal Low-molecular-weight Fucosylated Glycos- aminoglycan, aTFG), 其制备方法， 含所述 aTFG的药用组合物及其在制备预防和 /或治疗心脑 血管疾病的药物中的应用。
【背景技术】
心脑血管疾病的发病率高、 致残率高、 死亡率高、 复发率高和并发症多， 严重危害人类 的健康和生活质量。 血栓形成是心脑血管疾病的主要病因之一， 包括抗凝药物在内的抗血栓 药物是心血管疾病治疗的临床一线用药， 在医药市场上占有重要地位。 抗凝血药物主要包括 香豆素类和肝素类抗凝药物等， 这些药物具有明确的药效以及药理学作用机制， 但也存在明 显的临床应用缺陷： 香豆素类抗凝药物的缺陷包括其抑制系列凝血因子合成所致的严重出血 倾向、 起效慢、 个体差异大等； 肝素类药物主要作用靶点因子 Ila和 Xa (f.na，f.Xa)位于凝血 瀑布的共同途径，此类药物的应用缺陷主要是与靶点相关的严重出血危险和血小板减少症等。 因此， 临床需要具有优势药理药效作用特点的新型抗凝药物。 新型抗凝血药研发的核心是有 效避免出血倾向， 而低出血倾向的创新药物研究尚未取得突破性进展。
棘皮动物来源的岩藻糖化糖胺聚糖 （Fucosylated Glycosaminoglycan, FGAG)是一类具有 岩藻糖侧链取代的糖胺聚糖类衍生物，其具有葡萄糖醛酸 (GlcUA)和乙酰氨基半乳糖 (GalNAc) 构成的类似硫酸软骨素的主链， 又具有以 ct-l，3 糖苷键连接于主链葡萄糖醛酸基的岩藻糖 L-Fuc)侧链， 主链及侧链糖羟基均存在不同程度的硫酸酯化 CJ Biol. Chem. , 1996, 271 : 23973 -23984; Mar. Drugs, 2013, 11 : 399-417) 天然来源的 FGAG具有很强的抗凝血活性 (7 romb. Haemost , 2008, 100: 420-428; J. Biol. Chem., 1996， 271 : 23973-23984)
然而， 天然 FGAG仍存在广泛而矛盾的药理作用， 包括诱导血小板聚集、 产生出血倾向 和激活 XII等 (Thromb. Haemost, 1988， 59: 432-434； Thromb. Haemost , 1997, 65 (4): 369-373； Thromb. Haemost., 2010, 103 : 994-1004)。 适当解聚的低聚 FGAG可保留天然 FGAG的抗凝活 性而降低其血小板激活活性（7¾ramb. Haemost., 1991 , 65 : 369-373)。 中国授权专利 CN 101724086 B和 CN 101735336 B公开了制备低聚 FGAG的方法， 采用过氧化氢解聚法解聚 FGAG得到了低聚产物， 所得产物的出血倾向显著降低。 由于 FGAG为分子量较大、 结构复杂的糖胺聚糖衍生物， 在降低其副作用而保留药理学 活性的前提下， 实现工艺有效可控的解聚以获得具有特征末端结构的低分子量衍生物， 技术 上有很大难度。 鉴于过氧化氢解聚法缺乏糖苷键选择性并且过程控制较复杂， 本发明建立了 一种新的 FGAG解聚方法…脱酰脱氨基解聚法， 该方法首先通过肼解脱乙酰基反应使 FGAG 所含的 D-2-(N-乙酰基)氨基 -2-脱氧半乳糖 （D-GalNAc)发生部分脱乙酰化反应， 获得含 D-2- 氨基 -2-脱氧半乳糖基 (D-GalNH2)的 FGAG部分脱乙酰化产物， 继而采用亚硝酸处理使之发生 脱氨基解聚反应， 由此获得含有末端 2，5-脱水塔罗糖基或其还原衍生物的 FGAG解聚产物。 现有技术中尚无 FGAG脱酰脱氨基解聚法的报道， 也没有具有末端 2，5-脱水塔罗糖或其还原 衍生物的低分子量岩藻糖化糖胺聚糖的报道。
【发明内容】
针对上述现有技术中存在的问题， 本发明的目的在于提供一种低分子量糖胺聚糖衍生物 及其药学上可接受的盐， 所述低分子量糖胺聚糖衍生物是具末端 2，5-脱水塔罗糖或其还原衍 生物的低分子量岩藻糖化糖胺聚糖 (2，5-anhydrated Talose or its reduced derivative terminal Low-molecular-weight Fucosylated Glycosaminoglycan , aTFG), 其制备方法， 含所述 aTFG的 药用组合物及其在制备预防和 /或治疗心脑血管疾病的药物中的应用。
为了实现本发明的上述目的， 本发明提供了如下的技术方案：
一种低分子量糖胺聚糖衍生物及其药学上可接受的盐， 所述低分子量糖胺聚糖衍生物的 组成单糖包括己糖醛酸、 氨基己糖、 脱氧己糖及 2，5-脱水塔罗糖或其还原衍生物； 其中， 己 糖醛酸为 D-β-葡萄糖醛酸， 氨基己糖为 2-N-乙酰基氨基 -2-脱氧 -D-β-半乳糖或 2-氨基 -2-脱氧 -D-β-半乳糖或 -β-ϋ-2-硫酸氨基 -2-脱氧半乳糖， 脱氧己糖为 L-ct-岩藻糖， 2，5-脱水塔罗糖还原 衍生物为 2，5-脱水塔罗糖醇、 2，5-脱水塔罗糖氨、 Ν-取代的 2，5-脱水塔罗糖氨；
以摩尔比计， 所述 aTFG的组成单糖含量比例范围是， 己糖醛酸：氨基己糖：脱氧己糖= 1 : (1±0.35) : (1±0.3)； 以摩尔比计， 2，5-脱水塔罗糖和 /或其还原衍生物在全部组成单糖中所占 比例不低于 3.0 %。
本发明所述 aTFG的重均分子量 Mw范围为 2.5 kD〜 20 kD;
本发明所述 aTFG的多分散指数 （Mn/Mw) 介于 1.0至 1.8之间。
本发明所述 aTFG是具有式（I)结构的同系糖胺聚糖衍生物的混合物，
Figure imgf000005_0001
(I)
式 (I) 中：
n是均值为 3〜 21的整数；
-D-GlcUA-βΙ-, 为 -β-D-葡萄糖醛酸 -1-基；
-D-GalN-βΙ- , 为 -β-ϋ-2-乙酰氨基 -2-脱氧半乳糖 -1-基或 -β-ϋ-2-氨基 -2-脱氧半乳糖或 -P-D-2-硫酸氨基 -2-脱氧半乳糖；
L-Fuc-αΙ-, 为 a-L-岩藻糖 -1-基；
R'相互独立地为 -OH或 -OS03_;
R3为 -H、 -S03—或乙酰基；
R 为 -H或 P-D-2-乙酰氨基 -2-脱氧半乳糖硫酸酯 -1-基；
R2为 -H或 -β-D-葡萄糖醛酸 -1-基， 或式（Π)所示基团：
-D-GlcUA-β Ι -
Figure imgf000005_0002
式中， -D-GlcUA-βΙ-, L-Fuc-al-, R'同上文定义；
anTal为 2，5-脱水塔罗糖、 其糖醇、 糖胺或 N-取代糖胺，
m为 1或 2;
R4任选为 =0、 -0、 -NH2、 -NHR5， 其中 R5为 C1-C6直链或支链烷基， C7-C12芳基； 并且， 所述式（I)结构同系糖胺聚糖衍生物的混合物中， 以摩尔比计， R2为式（Π) 基团 的化合物与 R2为 -H或 -β-D-葡萄糖醛酸 -1-基的化合物的比例不低于 2 : 1。
本发明之 aTFG的分子量可采用高效凝胶色谱法（HPGPC)检测。 以重均分子量计， 本发 明选择的&1?0的分子量范围为约2，500〜20，000 0&， 即， 式（I)所示同系物的 n的均值约 3〜 21， 优选分子量范围为约 5，000〜12，000 Da， BP , 式 所示同系物的 n的均值约 5〜 15。
本发明之 aTFG的多分散指数（PDI,重均 /数均分子量之比， Mw/Mn)一般介于 1.0至 1.8 之间； 优选的 aTFG的 PDI介于 1.1至 1.5之间。
本发明之 aTFG可以是其药学上可接受的碱金属、 碱土金属等的盐， 类似地， 所述 aTFG 也可以是使其与碱性有机基团形成的酯。
本发明所述 aTFG的药学上可接受的盐优选为 aTFG的钠盐、 钾盐或钙盐。
本发明所述 aTFG 是棘皮动物门海参纲动物体壁和 /或内脏来源的岩藻糖化糖胺聚糖 (Fucosylated Glycosaminoglycan, FGAG) 的脱氨基解聚产物， 或者是所述解聚产物的还原性 末端的还原衍生化产物， 为此， 本发明又一目的是进一步提供一种以 FGAG为原料制备本发 明 aTFG的方法， 所述 aTFG的制备方法包括如下步骤：
步骤一、 肼处理棘皮动物来源的岩藻糖化糖胺聚糖（FGAG) , 使之所含的氨基己糖 (GalNAc)发生部分脱乙酰化反应， 获得 FGAG 的部分脱乙酰化产物； 获得的脱乙酰化产物 可进行磺化反应使游离氨基磺化为硫酸化氨基衍生物。
步骤二、 亚硝酸处理步骤一所得 FGAG的部分脱乙酰化产物， 使之发生脱氨基反应并解 聚， 获得还原性末端为 2，5-脱水塔罗糖基的低分子量岩藻糖化糖胺聚糖， 所得低分子量岩藻 糖化糖胺聚糖任选进行还原性末端的还原反应， 包括将 2，5-脱水塔罗糖端基还原成糖醇、 糖 胺或 N取代的糖胺， 所述具有末端 2，5-脱水塔罗糖基或其糖醇、 糖胺、 N取代糖胺的低分子 量岩藻糖化糖胺聚糖即为本发明所述的 aTFG。
本发明所述 aTFG制备方法中， 其步骤一所述肼处理脱乙酰化反应的方法是： 将棘皮动 物来源的岩藻糖化糖胺聚糖加入无水肼或水合肼溶液中， 在有或无催化剂存在下， 搅拌中于 75°C-125°C的温度下反应 2-14 h。
步骤一所述脱乙酰化反应优选在催化剂存在下进行， 所述催化剂可选择采用硫酸肼、 盐 酸肼等，亦可向反应溶剂中加入少量硫酸和 /或盐酸等强酸作为催化剂，所加入的硫酸和 /或盐 酸可以与溶剂肼或水合肼反应生成硫酸肼和 /或盐酸肼， 后者可起到催化反应的作用。 在本发 明优选的实施方案中， 所述反应溶液中的催化剂的浓度为 0.5%〜 2.5%。
步骤一所述脱乙酰化反应结束后， 反应溶液可减压蒸干， 亦可选择采用醇沉法， 如加入 等体积 80%乙醇， 以沉淀所得产物。醇沉过程中加适量饱和氯化钠溶液， 可使沉淀更为完全。 反应所得 FGAG部分脱乙酰化产物可选择直接干燥并用于步骤二的脱氨基解聚反应， 或者选 择采用碘酸氧化反应除去过量的肼和酰肼衍生物之后， 选择采用适当方法纯化后， 再干燥并 用于步骤二的脱氨基解聚反应。
步骤一所得 FGAG部分脱乙酰化产物可以采用核磁波谱 (NMR)检测技术进行确定脱乙酰 化程度， 具体地说， 脱乙酰化程度检测是比较原料化合物和产物中的 D-GalNAc和 L-Fuc所 含甲基质子峰的面积的比例， 通过所述甲基峰的积分比可计算反应产物的脱乙酰化程度。
由于步骤二的脱氨基解聚是快速和可化学计量的反应， 因此步骤一所得 FGAG部分脱乙 酰化产物的脱乙酰化程度可决定脱氨基解聚终产物 aTFG的分子量。 本发明人研究发现， 当 步骤一所得 FGAG部分脱乙酰化产物的脱乙酰化程度约为 5% -35%时， 其脱氨基解聚终产物 aTFG的分子量范围可在约 4 000 Da - 20 000 Da范围内。 本发明所述 aTFG制备方法的步骤二中， 所述亚硝酸处理的脱氨基解聚法的具体步骤一 般选择为：在冰浴或室温条件下，将步骤一所得 FGAG部分脱乙酰化产物用 pH (1.5〜 4.5) 的 亚硝酸溶液 （4〜 6) mol/L处理 5min〜 60 min后，用碱性溶液如 NaOH等调 pH 8或以上以终 止反应。 然后任选进行：
(1) 向反应溶液中加入 3-5倍体积的乙醇，静置，离心得沉淀，超滤或层析纯化所得产物；
(2) 采用硼氢化钠或氰基硼氢化钠，将反应产物的还原性末端 2，5-脱水塔罗糖 (anTal)还原 成糖醇 (anTalOH), 然后按 (1)所述步骤纯化所得产物；
(3) 通过还原氨基化反应将反应产物的还原性末端 2，5-脱水塔罗糖还原成糖胺或 N-取代 的糖胺， 然后按 (1)所述步骤纯化所得产物。
步骤二之 (2)所述端基还原反应，一般是在氢氧化钠调 pH (8〜 9) 终止脱氨基解聚反应后， 加入硼氢化钠和 /或氰基硼氢化钠浓度达 （0.05 〜 0.5) mol/L, 50°C下搅拌 20min〜 60 min使 anTal充分反应转化为 anTalOH。将反应液冷却至室温后，用酸调节 pH(3〜 4)除去过量的硼氢 化钠和 /或氰基硼氢化钠， 再碱性溶液如 NaOH中和， 然后按 (1)所述步骤纯化所得产物。
步骤二之 (3)是在碳酸氢铵或有机胺及还原剂存在下将末端 anTal还原氨基化， 即铵盐或 有机胺与 anTal醛基反应生成席夫碱， 后者在还原剂如氰基硼氢化钠存在下被还原成次级胺。
本发明所得 aTFG产物的可通过 NMR法检测。 一般地， 根据产物 NMR检测谱图计算， 以摩尔比计， 本发明所述 aTFG产物中， 具有末端 2，5-脱水塔罗糖或其还原衍生物的化合物 可占全部解聚产物的 60%〜 97%。 本发明所述方法之 D-GalNAc脱乙酰基、 D-GalNAc脱氨基及 anTal羰基还原与还原氨基 化反应路线如 "反应过程路线图"所示。 该路线中， 各化合物 (1) 〜（5)中的 R'、 R5均同前文 定义。对于本领域技术人员而言， 采用还原氨基化反应， 当 为15、 C1-C6直链或支链烷基、 C7-C12芳基时， 路线图中的化合物 (5)所示的反应终产物均可容易地获得。
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000008_0003
(反应过程路线）
原型天然产物 FGAG具有复杂的主侧链结构， 有效实现其选择性的脱乙酰反应是一项关 键的技术难点， 而侧链 L-Fuc容易发生酸水解， 使得 HN02脱氨基解聚反应条件的控制尤为 重要。 本发明所述的技术路线及其主要技术参数可以保证这些反应的顺利进行。 本发明所述方法中， 所述 aTFG 反应终产物可以通过本技术领域已知方法纯化 (CN
101735336A), 例如通过透析法或超滤法去除小分子盐等杂质， 或通过凝胶层析或 DEAE离 子交换层析进一步纯化等。
本发明方法中， 所述透析去杂处理过程中， 可根据目标分子量大小的要求选择适宜截留 分子量的透析膜或超滤膜包， 优选截留分子量为 1000 Da透析膜或超滤膜包透析去除小分子 物质， 透析时间需根据特定处理条件确定， 通常不少于 6小时。 透析法亦可选择用于去除其 它大分子杂质以及未解聚的 FGAG或超过分子量范围 aTFG。
本发明方法所得 aTFG终产物还可以通过阳离子交换以制备成单盐形式， 如钠盐、 钾盐 或钙盐等。 本发明所述之 aTFG产物的成盐过程可以采用离子交换法将所述 aTFG交换成氢 型， 然后采用相应的碱进行中和得到 aTFG对应的盐； 亦可优选动态离子交换成盐法直接在 柱上交换成盐。 树脂柱预处理、 样品上样与洗脱均可按常规方法进行。 本发明所述 aTFG制备方法中， 其 "步骤一"所述起始原料为棘皮动物来源的岩藻糖化 糖胺聚糖 （fucosylated glycosaminoglycan, FGAG)。 所述 FGAG具有类似的硫酸软骨素主链， 一般来说， 该主链由葡萄糖醛酸 (D-GlcUA)和 2-(N-乙酰基)氨基 -2-脱氧半乳糖 (D-GalNAc)构 成，两种组成单糖分别以 -4)- D-GlcUA - (； β-l- 和 -3)-D-GalNAc- (； β-l- 的糖苷键形式顺次连接； FGAG 还具有岩藻糖 (L-FucM则链， 一般地， L-Fuc 侧链以 ct-l，3 糖苷键连接于主链上的 D-GlcUA; 此外， FGAG主链中的 D-GalNAc及侧链糖 L-Fuc上的羟基均可能存在不同程度 的硫酸酯化 (J. Biol. Chem. , 1996， 271 : 23973 -23984; Mar. Drugs, 2013, 11 : 399—417)。
用于制备本发明所述 aTFG及其药学上可接受的盐的 FGAG可来源于可选自但不局限于 以下海参品禾中： 花束 lj参 (Sric/iopws van'egii es Semper)、
Figure imgf000009_0001
scabra Jaeger) 玉足 海参 {Holothuria leucospilota Brandt)、 红腹海参 {Holothuria edulis Lesson)、 蛇目白尼参 (Bohad randt) 中华海参 (Holothuria sinica Liao)
Figure imgf000009_0002
molpadioides Semper) 格皮氏海参 {Pearsonothuria graejfei Semper)禾口黑乳参 (Ho/o /iwn'<¾ nobilis Selenka)。 对于产于世界各地的其 它品种海参， 其来源的符合上述结构特征的 FGAG均可以采用本发明所述方法脱氨基解聚获 得所需终产物， 因此， 本发明方法不受特定海参品种的限制。
本发明人的研究显示， 海参品种及其组织来源不同或提取方法的差异可导致 FGAG的单 糖组成比例、 侧链存在形式以及多糖硫酸化程度等方面的不同。 本领域技术人员容易理解， 由于这些差异不涉及 FGAG存在的乙酰氨基的结构特征， 因此不影响本发明所述脱酰脱氨基 解聚方法的实施和应用。 本发明研究发现， 所述 aTFG具有强效的抗凝活性， 其倍增人质控血浆活化部分凝血活 酶时间 (APTT)所需的药物浓度不高于 12 g/mL。 本发研究还确认， 所述 aTFG具有强效的抑 制内源性因子 X酶的活性， 其 EC5Q约为 5〜50 ng/mL。 由于因子 X酶是内源性凝血途径中的 最后一个酶学位点， 是多种因素激发的凝血过程的限速位点， 因此其抑制剂可具有显著的抗 血栓活性而对生理性止血的影响较小 (Blood, 2010, 116(22), 4390- 4391 ; Blood, 2009, 114, 3092-3100)。
本发明所述 aTFG及其药学上可接受的盐具有确切的抗凝血活性， 因此具有明确的潜在 药用价值。 aTFG具有良好的水溶性， 因此易于制备成溶液型制剂或冻干制品。作为多糖类成 分， 其口服生物利用有限， 因此其药物组合物优选制备成胃肠外给药剂型， 其制剂制备可以 按照本领域内熟知的技术方法进行。
因此， 本发明还提供了含有有效抗凝血剂量的上述的低分子量糖胺聚糖衍生物及其药学 上可接受的盐， 以及药用赋形剂的药物组合物， 所述的药物组合物的剂型可以是注射用水溶 液或注射用冻干粉针剂。
本发明所述 aTFG具有强效的抗凝血活性， 因此可以用于不同程度的血栓性疾病的预防 和治疗， 例如血栓形成性心血管疾病、 血栓性脑血管病， 肺静脉血栓、 周围静脉血栓、 深静 脉血栓、 周围性动脉血栓等。 因此， 本发明还提供所述低分子量糖胺聚糖衍生物 (aTFG)、 其 药学上可接受的盐以及含有 aTFG或其盐的药物组合物在制备防治血栓性疾病的药物中的应 用， 所述血栓性疾病为静脉血栓形成或动脉血栓形成或缺血性心脏病或缺血性脑血管病。 与现有技术相比， 本发明所具备的优益性在于：
目前， 见于报道的 FGAG解聚方法主要由过氧化氢解聚法， 该解聚方法缺乏糖苷键选择 性并且过程控制复杂。 本发明建立了一种新的 FGAG解聚方法--脱酰脱氨基解聚法， 该方法 首先通过肼解脱乙酰基反应使 FGAG所含的 D-2-(N-乙酰基)氨基 -2-脱氧半乳糖 （D-GalNAc) 发生部分脱乙酰化反应，获得含 D-2-氨基 -2-脱氧半乳糖基 (D-GalNH2)的 FGAG部分脱乙酰化 产物， 继而采用亚硝酸处理使之发生脱氨基解聚反应， 由此获得含有末端 2，5-脱水塔罗糖基 或其还原衍生物的 FGAG解聚产物。本领域技术人员容易理解，鉴于 FGAG的复杂化学结构， 特别大量的岩藻糖 (Fuc)侧链取代基的存在， 使得选择性脱除 FGAG所含 GalNAc上的氨基存 在明显的技术困难； 而 Fuc侧链在酸性条件下容易裂解， 因此， 部分脱乙酰化 FGAG的酸性 条件下脱氨基反应同样存在技术挑战。 本发明首次公开了获得具有特征末端结构衍生物的复 杂结构 FGAG脱氨基解聚方法， 并且首次公开了含有末端 2，5-脱水塔罗糖基或其还原衍生物 的 FGAG解聚产物。 本发明方法及其所得产物的优势特征在于， (1) FGAG脱酰脱氨基解聚反应具有糖苷键选 择性， 即选择性断裂 D-GalNH2Cpi-)糖苷键， 而不裂解 L-FucCcd-)及 D-GlcUACpi-)糖苷键， 因 此所得解聚产物具有更好的结构均一性； (2) FGAG脱酰脱氨基解聚反应产物可具有特征性的 2，5-脱水塔罗糖 (2，5-anhydrated Talose, anTal)或其还原衍生物末端， 特征性末端的存在有利于 解聚产物化学结构分析以及解聚产物的质量控制， 而末端还原衍生化处理可使之容易地用于 药代动力学及药理机制分析等； （3) 部分脱乙酰化的 FGAG可经 HN02处理发生可化学计量 的脱氨基解聚反应， 因此， 通过脱乙酰化程度控制， 可以较好地控制解聚产物的分子量范围， 有效提高 FGAG解聚产物的制备工艺的可控性。
【附图说明】
图 1为 FGAG及其脱乙酰化产物 dAFG-1和 dAFG-2的 1H NMR谱；
图 2为 FGAG及其脱乙酰化产物 dAFG-1和 dAFG-2的 13C NMR谱；
图 3为 FGAG及其脱酰脱氨基解聚产物 aTFG-a、 aTFG-b和 aTFG-c的 1H NMR谱； 图 4为 FGAG及其脱酰脱氨基解聚产物 aTFG-a、 aTFG-b和 aTFG-c的 13C NMR谱； 图 5为 FGAG及其脱酰脱氨基解聚产物 aTFG-a、 aTFG-b的 COSY (A), NOESY (B), TOCSY (C) 谱图叠加 (局部)图；
图 6为 FGAG (A)及其脱酰脱氨基解聚产物 aTFG-b (B)的 1H COSY谱图比较； 图 7为 aTFG的抗内源性因子 X酶活性；
【具体实施方式】
以下具体实施例是对本发明内容的详细说明， 所述实施方案不限制本发明的权利范围。
【实施例 1】 FGAG脱乙酰化产物 (dAFG)的制备 1.1 材料
FGAG : Thelenota ananas体壁来源的岩藻糖化糖胺聚糖，按文献方法 (Marine Drugs, 2013 11, 399-417) 提取纯化制备得到， 分子量 69930 Da。 水合肼、 硫酸肼、 盐酸肼、 盐酸、 氯化 钠、 无水乙醇、 碘酸、 氢碘酸、 氢氧化钠等试剂均为市售分析纯试剂。
1.2方法
脱乙酰化反应： 准确称量 FGAG原料 60 mg， 置于反应管， 加入 14.5 mg催化剂硫酸肼或 盐酸肼 12.2 mg或 2.304 mol/L盐酸 0.1 mL或不加催化剂条件下， 然后加入水合肼或无水肼 1.45 mL， 氮气氛围下， 250 rpm转速下搅拌， 75〜 105 °C下加热反应 2〜 14 h。 反应结束后， 反应液在经 80%乙醇沉淀， 离心得沉淀， 然后减压真空干燥， 得到脱乙酰化中间产物样品， 该样品可直接用于亚硝酸解聚， 或进一步处理得到较纯的中间体， 其中处理方法为： 蒸干样 品置冰浴冷却， 逐滴加入 0.25 mol/L碘酸溶液； 继续滴加， 直至沉淀不溶解呈黑色悬液。 滴 加约 5 ml 45%氢碘酸， 再加入 3 mol/L氢氧化钠， 沉淀溶解， 继续滴加， 直至溶液变为澄清 透明或淡黄色溶液。 将该溶液调 pH至中性， 用截留分子量为 1000 Da透析袋透析， 冻干。
脱乙酰化程度的测定：精确称取上述脱乙酰化产物约 5 mg溶于约 600 mL重水 (含 TSP内 标)中， 使用 BUCKER DRX-500 核磁共振波谱仪对样品进行检测。 脱乙酰化程度 (degree of deacetylation, DD)通过 1H NMR图谱中两种甲基质子峰 (；乙酰氨基半乳糖乙酰氨基上的甲基和 侧链硫酸岩藻糖甲基)积分面积比值来计算。
产物分子量的测定：采用高效液相排阻法 (HPGPC)确定产物的分子量。 Agilent technologies 1200 series 高效液相色谱仪， Shodex Ohpak SB-804 HQ (7.8 mmx300 mm)柱， 温度 35 °C， 检 测器为示差折光检测器 (G1362A)。精密称取样品适量， 加 0.1 mol/L氯化钠溶液溶解， 并稀释 至 10 mL量瓶中， 摇匀， 过 40 μηι滤膜， 取滤液作为供试溶液。
标准品及对照品溶液制备： 取已知分子量的右旋糖酐系列对照品， 精密称定， 用 0.1 ml/L 氯化钠溶液分别溶解并稀释制成 10 mg/mL的溶液， 作为窄标的校正溶液。 精密称取已知分 子量的 FGAG对照品， 用 0.1 mol/L氯化钠溶液分别溶解并稀释制成 10 mg/mL的溶液， 作为 宽标校正溶液。 分别将样品、 标准品、 对照品溶液各 25 L, 注入液相色谱仪， 记录色谱图。 数据采用 GPC专用软件处理。
化学组成检测： 单糖组成乙酰氨基半乳糖、 葡萄糖醛酸和岩藻糖分别采用 Elson-Morgon 法、间羟联苯法和半胱氨酸苯酚法进行测定 (张惟杰，糖复合物生化研究技术 （第二版)，浙江： 浙江大学出版社， 1999),硫酸羧酸摩尔比采用电导法测定 (张惟杰，糖复合物生化技术研究 (第 二版)， 浙江： 浙江大学出版社， 1999， 409-410)。
1.3 结果
反应条件变化对脱乙酰化产物 （deacelyted FGAG，dAFG) 脱乙酰化程度的影响见表 1。结 果显示， 加入催化剂可以加快反应进程， 产物脱乙酰化程度更高； 控制反应时间、 反应温度 和肼的质量浓度可以获得不同脱乙酰化程度的产物。
表 1. 不同条件下脱乙酰化反应的实验结果
乙酰胺基上 岩藻糖侧链上 脱乙酰化程度 影响因素
甲基的积分 甲基的积分 （DD)
反应时间 2 h 1 0.92 0.065 6 h 1 1.03 0.167
10 h 1 1.23 0.301
14 h 1 1.31 0.342
24 h 1 1.79 0.519
36 h 1 2.79 0.691
48 h 1 3.98 0.784
催化剂种类 无催化剂 1 0.98 0.123
硫酸肼 1 1.25 0.310
盐酸肼 1 1.28 0.330
盐酸 1 1.24 0.306
反应温度 60 °C 1 0.88 0.010
75 °C 1 1.00 0.134
90 °C 1 1.26 0.319
105 °C 1 2.08 0.582
肼浓度 32 % 1 0.94 0.084
64% 1 1.26 0.315
100% 1 3.04 0.704
图 1和图 2给出了按照本实施例方法制备的 2个不同脱乙酰程度样品 dAFG-1和 dAFG-2 的 1H、 13C-NMR谱图。 根据 NMR谱图计算它们的脱乙酰化程度分别是 48%和 88%。 谱图数 据还提示， 脱乙酰化前后， 除了部分 D-GalNAc被脱去乙酰氨基生成 D-GalNH2外， 其基本结 构未见显著变化。对脱乙酰化前后样品的单糖组成分析，其组成单糖 （D-GlcUA)： (D-GalNAc + D-GalNH2)： (L-Fuc) 的摩尔比均约为 1： (1±0.3)： (1±0.3)， 这个结果进一步提示， D-GalNAc 发生部分脱乙酰化反应外， 多糖的基本结构保持稳定。
【实施例 2】脱氨基解聚 dAFG制备 aTFG
2.1 材料
dAFG, 即 FGAG部分脱乙酰化产物， 按照实施例 1所述方法制备获得； 亚硝酸钠、 浓 硫酸、 硼氢化钠、 碳酸钠、 氢氧化钠、 无水乙醇等试剂均为市售分析纯试剂。
2.2方法
产物制备： 精确称取脱乙酰化中间产物约 20 mg于反应器中， 加 I mL水溶解， 在冰浴或 室温条件下， 加入 5.5 mol/L的亚硝酸溶液 (pH 4) 2 mL， 解聚 2〜 30 min， 反应后加 1 mol/L 的碳酸钠溶液调 pH 8〜 9终止反应，用含 0.25 mol/L硼氢化钠的 0.1 mol/L氢氧化钠溶液 1 mL 将亚硝酸解聚产物的醛基还原， 50 °C加热 40 min， 反应后冷却至室温， 0.5 mol/L的硫酸除去 过量的硼氢化钠， 最后用 0.5 mol/L 的氢氧化钠溶液中和， 1000 Da透析袋透析， 收集透析袋 内透析液， 冻干。
产物检测： BUCKER DRX-500核磁共振波谱仪对解聚样品进行检测； 凝胶排阻色谱法确 定解聚样品的分子量； 电导法测定解聚样品硫酸羧酸摩尔比。
2.3结果
脱氨基解聚终产物的收率大于 90%， 样品纯度大于 95%。
理论上， 只有游离的氨基才能被亚硝酸消去从而使糖苷键断裂， 因此， 根据亚硝酸解聚 前样品脱乙酰化程度计算游离的氨基数量， 进而理论计算解聚产物可能的分子量。 实验结果 如表 2所示， 不同分子量的原料脱乙酰化后亚硝酸解聚获得的产物的分子量与理论计算值基 本一致。 这说明， 根据脱乙酰化程度的不同可以获得理论计算分子量的解聚终产物。
表 2. 脱乙酰化程度与产物分子量关系的实验结果 起始原料分子量 脱乙酰化程度 理论产物分子量 产物实测分子量
13710 11.50% 8245 8777
13710 13.79% 6577 6751
64300 15.96% 5680 7083
64300 8.26% 10984 9702 按照本实施例制备的 3个产物 aTFG-a、 aTFG-b和 aTFG-c的 NMR核磁图谱见附图 3至 6。 比较原料 FGAG、 中间产物 dAFG的核磁波谱， 根据 1H NMR、 13C NMR及 1H同核相关 谱 COSY、TOCSY、NOESY及 1H-13C异核相关谱 HQSC、HMBC，对不同脱乙酰化程度的 （15%， 48%, 88%) 亚硝酸解聚产物 aTFG-a、 aTFG-b和 aTFG-c的谱图信号进行归属， 其 NMR信号 数据见表 3。 表 3. FGAG及其脱氨基解聚产物 aTFG-b的 1H/13C NMR信号归属
Figure imgf000014_0001
aTFG
β-GalNAc 414,
4.54 4.02 3.92 4.77 3.95 2.02 102.3 51.8 78.1 79.0 74.4 67.7 23.2 177.6 4S6S 4.24
419,
anTal4S6S 5.05 4.02 4.58 5.02 4.50 1 91.6 84.4 79.1 80.5 80.4 68.2 1 1
4.32
β-GlcUA 4.44 3.59 3.66 3.89 3.72 1 1 106.4 76.4 80.3 79.2 79.4 177.6 1 1 a-Fuc2S4S 5.66 4.45 4.13 4.85 4.89 1.33 1 99.0 77.8 70.1 83.6 68.8 18.5 1 1 a-Fuc4S 5.31 3.83 3.97 4.80 4.86 1.33 1 101.3 71.1 72.6 79.9 69.0 17.8 1 1 a-Fuc3S 5.38 4.13 4.63 4.02 4.50 1.22 1 101.2 72.8 83.5 71.3 68.8 18.2 1 1 由表 3和附图 3〜6数据可知， FGAG脱氨基解聚产物 aTFG的 1H NMR谱图与原型 FGAG 谱图基本近似， 而存在于脱乙酰化 FGAG的约 3.1〜3.2 ppm的 D-β-氨基半乳糖 CD-P-GalNH2) 的 2位氢信号消失，表明存在游离氨基的氨基己糖均已反应， 而约 5.0〜5.1 ppm处出现的新的 信号峰则来自新的还原性末端的 2，5-脱水塔罗糖 （anTal, 端基及 4位氢)。 与脱乙酰化产物相 比， 脱氨基解聚产物的 1H NMR信号更接近于原型 FGAG。
【实施例 3】不同海参来源 FGAG的亚硝酸解聚产物制备 3.1材料
仿束1 J参 Apostichopus japonicus、红腹海参 Holothuria edulis、巴西参 Ludwigothurea grisea、 玉足海参 Holothuria leucospilota, 黑乳海参 Holothuria nobilis, 均为市售干燥体壁。
3.2方法
(1)仿刺参、红腹海参、 巴西参、玉足海参、黑乳海参干燥体壁粉碎， 分别取粉碎物 300 g， 同实施例 1方法 G)所述提取其所含 FGAG, 分别称之为 AJG、 HEG、 LGG、 HLG禾 B HNG。
2)分别取 AJG、 HEG、 LGG、 HLG和 HNG约 lg， 按照实施例 1和 2所述方法制备其脱 氨基解聚产物 aTFG, 分别称之为 aAJG、 aHEG、 aLGG、 aHLG禾 B aHNG。
3.3结果
从仿刺参、红腹海参、巴西参、玉足海参、黑乳海参干燥体壁分离纯化 AJG、 HEG、 LGG、 HLG和 HNG的得率分别为约 1.4%、 0.9 %、 0.8% 禾卩 1.1%, 其重均分子量均在约 50 kD〜 80 kD之间。 通过 1H NMR谱图确定 AJG、 HEG、 LGG、 HLG和 HNG作为 FGAG类化合物的 基本特征： a-L-Fuc β-D-GalNAc以及 β-D-GlcUA上的端基及相关的特征质子信号清晰明确。
由 AJG、 HEG、 LGG、 HLG禾口 HNG制备 aAJG(8.0 kD)、 aHEG(10.5 kD)、 aLGG(7.3 kD)、 HLG(10.2kD)和 aHNG (8.7 kD) 的得率在约 40%〜70% 范围内。 通过1 H NMR谱图分别确定 了解聚形成的 anTal相关的特征信号。
【实施例 4】末端还原氨基化产物制备
4.1材料
aTFG: 实施例 1和 2制备。 酪胺、 氰基硼氢化钠等试剂均为市售分析纯。 4.2方法
(1)末端还原氨基化：实施例 2所得 aTFG O. lg溶解于 3.5mL0.2mM磷酸缓冲液 (pH8.0)中， 搅拌中分别加入过量的 80mg酪胺和 30mg氰基硼氢化钠， 35°C恒温水浴中反应约 72 h。 反 应完毕后， 加 95%乙醇 10mL， 离心得沉淀， 所得沉淀以 95%乙醇 30mL洗涤两遍后， 以 35 mL 0.1% NaCl复溶所得沉淀， 离心去不溶物， 上清液置于 1KD的透析袋中， 去离子水透析 24h, 经冷冻干燥后获得 dLFG-2A 82 mg。
(2)产物理化及波谱检测： HPGPC检测分子量及分布； 电导法检测 -OS037-COO—摩尔比； Elson-Morgon法检测乙酰氨基半乳糖 (D-GalNAc)含量， 咔唑法检测葡萄糖醛酸 (D-GlcUA)含 量， iHNMR 甲基峰积分面积计算 D-GalNAc/L-Fuc摩尔比 (；同实施例 1)。 瑞士 Bruker公司 AVANCE AV 500超导核磁共振仪 (500 MHz)检测 NMR谱图。
4.3结果
以投料计产物得率约 72%; 产物组分检测结果显示， D-GalNAc:D-GlcUA:L-Fuc:-OS03_ 为约 1.00:0.98: 1.10:3.60， Mw约 9,969， PDI约 1.32。
1HNMR(D20,5[ppm]):7.25(2',6'H);6.83(3',5'H);5.65,5.36,5.28(L-FucalH);3.38(8'H);2.82(7' H);2.02(D-GalNAc, CH3); 1.30~1.32(L-Fuc,CH 苯环氢与 L-Fuc之 HI的积分表明所得产物 还原性末端均被还原酪氨化。
【实施例 5】氨基硫酸化
5.1材料 氯磺酸、 四丁基氢氧化胺、 二甲基甲酰胺、 吡啶和碳酸钠等试剂均为市售分析纯。
脱乙酰化样品 dAFG-1为按照实施例 1制备的花刺参来源的， 脱乙酰化程度为 35%。 5.2方法
准确称取 dAFG-1 0.10g, 10 ml去离子水溶解，调 pH 7.0。 40°C水浴反应，一次性加入 0.16g Na2C03， 在 4 h 内加入 0.20g 吡啶-氯磺酸， 加完后接着反应 1 h， 反应完后静置冷却至室温， 调 pH 7.5-8.0， 超滤除盐， 冷冻干燥。 电导法测定样品的硫酸羧酸摩尔比。
5.3结果 称重计算反应产物的得率为约 87%， 电导法测定硫酸羧酸摩尔比的结果显示， 产物的硫 酸羧酸摩尔比为 4.3，与反应原型天然产物比较可知，脱乙酰化后的游离氨基基本完全硫酸化。
【实施例 6】 aTFG抗凝血活性研究 6.1 材料
aTFG样品：按实施例 2和 3所述方法制备得到的不同分子量的系列 aTFG样品；这些样 品的理化性质等信息见表 4。
试剂及仪器： 凝血质控血浆、 活化部分凝血活酶时间 (APTT)测定试剂盒、 CaC 均为德 国 TECO GmbH公司生产； 其他试剂均为市售分析纯。 MC-4000血凝仪 (德国美创公司）。
表 4. 系列分子量 aTFG样品及其理化性质 重均分子量 数均分子量
样品编号 多分散系数 硫酸羧酸摩尔比
Mw(Da) Mn(Da)
aTFG-1 24866 13514 1.84 3.60
aTFG-2 17471 9815 1.78 3.48
aTFG-3 12567 11321 1.11 3.12
aTFG-4 11332 7358 1.54 3.24
aTFG-5 10497 6439 1.63 3.42
aTFG-6 9476 6402 1.48 3.34
aTFG-7 7000 5147 1.36 3.54
aTFG-8 6600 4748 1.39 3.34
aTFG-9 5000 4166 1.20 3.42
6.2方法 45 μΐ凝血质控血浆中加入 5 L溶于 Tris-HCl缓冲液的待测样品作为检测样 本， 使用活化部分凝血时间 (APTT)试剂盒进行凝血时间测试。
6.3结果 （见表 5 )。
表 5. 不同分子量 aTFG的 APTT数据 相关系数 倍增 APTT所需的
检测样品 曲线方程
(R2) 药物浓度 （^g/mL) aTFG-1 y = 11.577X + 31.603 0.9939 3.40
aTFG-2 y = 9.0491x + 30.793 0.9880 3.55
aTFG-3 y = = 6.134x + 37.004 0.9849 4.22
aTFG-4 y = = 5.756x + 35.005 0.9971 4.85
aTFG-5 y = = 6.6247x + 37.21 0.9958 4.92
aTFG-6 y = 5.3358x + 37.032 0.9978 6.14 aTFG-7 y = 4.5807x + 38.73 0.9925 6.78
aTFG-8 y = 4.0239x + 38.378 0.9874 7.81
aTFG-9 y = 2.5692x + 37.982 0.9874 12.38
表 5中的结果显示， FGAG的脱氨基解聚产物 aTFG均可以显著延长人血浆 APTT,其倍 增 APTT的药物浓度均在 12 μ§/ηι1以下， 表明这些衍生物均能够有效抑制内源性凝血。 比较 这些衍生物的分子量和相应的倍增 APTT的药物浓度发现， 分子量越大抗凝血活性越强， 分 子量是影响其抗凝血活性的主要因素之一。 根据这一规律性结果， 从保留 FGAG血液学活性 考虑， 以重均分子量计， 本发明优选的 aTFG的分子量不低于 5,000 Da。
【实施例 7】抑制内源性因子 X酶活性 7.1 材料
aTFG样品： 为按照实施例 2的方法制备得到， 分子量 8777Da。
试剂及仪器： 因子 vm (f.vm)， 200 ιυ/支， 上海莱士血液制品有限公司产品； f.vm检测 试齐 U盒， 试齐 U包括 Reogew iv Rl : Human Factor X； R2: Activation Reagent, human Factor IXa, containing human thrombin, calcium and synthetic phospholipids; R3 : SXa-11 , Chomogenic substrate, specific for Factor Xa; R4: Tris-BSA Buffer; 为 HYPHEN BioMed (法国）产品。 Bio Tek-
ELx 808型酶标仪 （美国）。
7.2方法
抑制内源性因子 X酶 (抗 f.Xase)活性检测： 采用 f.VIII检测试剂盒结合 f.VIII试剂建立的 检测方法。 系列浓度的待测溶液或空白对照溶液 (Tris-BSA缓冲液 )30 μΐ与 2.0 IU/ml因子 VIII(30 μΐ)混合后，顺次加入试剂盒试剂 R2(30 μ1)、 Ri (30 μΐ), 37°C孵育 2 min后，加 R3 (30 μΐ), 37 。C精确孵育 2min， 以 20%乙酸 (30 μΐ)中止反应并检测 OD4Q5nm。 根据空白对照 (R4)计算 ΔΟϋ , 按文献 (Sheehan J. P. & Walke E. K.， Blood, 2006, 107:3876-3882)中提供的公式计算各样 品抑制 f.Xase的 EC5。值。
7.3结果
见附图 7。 数据显示， aTFG的 EC5。为 22 ng/mL, 具有强效抗 f.Xase活性。 由于因子 X 酶是内源性凝血途径中的最后一个酶学位点， 是多种因素激发的凝血过程的限速位点， 因此， 作用于该位点的药物对生理性凝血与止血的影响最小 ( /θί¾， 2010, 116(22), 4390-4391 ; Blood, 2009, 114, 3092-3100)。
【实施例 8】冻干制品 8.1 材料
按照实施例 1和 2对糙海参来源的 FGAG进行反应制备的 aTFG，其重均分子量 9476 Da。
8.2处方:
原辅料名称 用
aTFG-4 50 g
注射用水 500 mL
共制成 1000支
8.3 制备工艺
工艺过程： 称取处方量的 aTFG加注射用水至全量， 搅拌使溶解完全， 间歇式热压法灭 菌。 加入 0.6%的药用活性炭， 搅拌 20min; 使用布氏漏斗及 3.0 μηι微孔滤膜脱炭过滤除去 热源。测中间体含量。合格后用 0.22 μηι的微孔滤膜过滤；灌装于管制西林瓶中，每瓶 0.5 ml, 灌装过程监测装量， 半压塞， 置冷冻干燥箱内， 按设定的冻干曲线进行冻干， 压塞， 出箱， 轧盖， 经检验合格， 得成品。 冻干过程： 将样品进箱， 降隔板温至 -40 °C, 保持 3h; 冷阱降至 -50 °C, 开始抽真空至 300 bar。 开始升华： 1 h匀速升温至 -30 °C, 保持 2 h; 2 h匀速升温至 -20 °C , 保持 8 h, 真空保 持 200 ~ 300 bar; 再进行干燥： 2 h升温至 -5 V , 保持 2 h, 真空保持 150 ~ 200 bar; 0.5 h 升温至 10°C, 保持 2h, 真空保持 80~ 100 bar; 0.5h升温至 40°C, 保持 4 h, 真空抽至最 低。

Claims

权利要求
1、 一种低分子量糖胺聚糖衍生物及其药学上可接受的盐， 其特征在于：
所述低分子量糖胺聚糖衍生物的组成单糖包括己糖醛酸、 氨基己糖、 脱氧己糖及 2，5-脱 水塔罗糖或其还原衍生物； 所述己糖醛酸为 D-β-葡萄糖醛酸， 氨基己糖为 2-N-乙酰基氨基 -2- 脱氧 -D-β-半乳糖或 2-氨基 -2-脱氧 -D-β-半乳糖或 -P-D-2-硫酸氨基 -2-脱氧半乳糖， 脱氧己糖为 L-ct-岩藻糖， 2，5-脱水塔罗糖还原衍生物为 2，5-脱水塔罗糖醇或 2，5-脱水塔罗糖氨或 N-取代的 2，5-脱水塔罗糖氨；
以摩尔比计， 所述低分子量糖胺聚糖衍生物组成单糖含量比例范围是， 己糖醛酸:氨基己 糖:脱氧己糖 =1 :(1±0.35):(1±0.3)； 以摩尔比计， 2，5-脱水塔罗糖和 /或其还原衍生物在全部组成 单糖中所占比例不低于 3.0%;
所述低分子量糖胺聚糖衍生物的重均分子量 Mw范围为 2.5kD〜20kD;
所述低分子量糖胺聚糖衍生物的多分散指数介于 1.0至 1.8之间。
2、 如权利要求 1所述的低分子量糖胺聚糖衍生物及其药学上可接受的盐， 其特征在于， 所述低分子量糖胺聚糖衍生物是具有式 （I ) 结构的同系糖胺聚糖衍生物的混合物，
Figure imgf000020_0001
( I )
式 （I ) 中：
n是均值为 3〜 21的整数；
-D-GlcUA-βΙ-, 为 -β-D-葡萄糖醛酸 -1-基；
-D-GalN-βΙ- , 为 -β-ϋ-2-乙酰氨基 -2-脱氧半乳糖 -1-基或 -β-ϋ-2-氨基 -2-脱氧半乳糖或 -P-D-2-硫酸氨基 -2-脱氧半乳糖；
L-Fuc-αΙ-, 为 a-L-岩藻糖 -1-基； Ri为 -H或 β-ϋ-2-乙酰氨基 -2-脱氧半乳糖硫酸酯 -1-基；
R'相互独立地为 -OH或 -OS03_;
R3为 -H、 -S03—或乙酰基;
R2为 -H或 -β-D-葡萄糖醛酸 -1-基， 或式 （Π) 所示基团：
-D-GlcUA-Pl-
Figure imgf000021_0001
式 （Π) 中， -D-GlcUA-βΙ-, L-Fuc-αΙ-, R'同前文定义；
anTal为 2，5-脱水塔罗糖， 其糖醇、 糖胺或 N-取代糖胺；
m为 1或 2;
R4任选为 =0、 -0、 -NH2、 -NHR5， 其中 R5为 C1-C6直链或支链烷基， C7-C12芳基； 并且， 所述式 （I) 结构同系糖胺聚糖衍生物的混合物中， 以摩尔比计， R2为式 （Π) 基 团的化合物与 R2为 -H或 -β-D-葡萄糖醛酸 -1-基的化合物的比例不低于 2:1。
3、 如权利要求 1所述的低分子量糖胺聚糖衍生物及其药学上可接受的盐， 其特征在于， 所述的低分子量糖胺聚糖衍生物的重均分子量范围为 5kD〜12kD; 所述的低分子量糖胺聚糖 衍生物的多分散指数值介于 1.1至 1.5之间。
4、 如权利要求 1或 2所述的低分子量岩藻糖化糖胺聚糖衍生物及其药学上可接受的盐， 其特征在于， 所述的药学上可接受的盐是低分子量岩藻糖化糖胺聚糖衍生物的碱金属盐或碱 土金属盐或有机铵盐。
5、 如权利要求 4所述的低分子量糖胺聚糖衍生物的药学上可接受的盐， 其特征在于， 所 述的药学上可接受的盐是低分子量糖胺聚糖衍生物的钠盐或钾盐或钙盐。
6、如权利要求 1或 2或 4所述的低分子量胺聚糖衍生物及其药学上可接受的盐， 其特征 在于， 所述的低分子量糖胺聚糖衍生物是棘皮动物门海参纲动物体壁和 /或内脏来源的岩藻糖 化糖胺聚糖的脱氨基解聚产物， 或者是所述解聚产物的还原性末端的还原衍生化产物。
7、权利要求 1或 2或 4任一项所述的低分子量糖胺聚糖及其药学上可接受的盐的制备方 法， 其特征在于， 所述制备方法包括如下步骤：
步骤一： 肼处理棘皮动物来源的岩藻糖化糖胺聚糖， 使之所含的氨基己糖发生部分脱乙 酰化反应， 获得岩藻糖化糖胺聚糖的部分脱乙酰化产物；
步骤二： 亚硝酸处理 "步骤一"所得岩藻糖化糖胺聚糖的部分脱乙酰化产物， 使之发生 脱氨基反应和解聚， 获得还原性末端为 2，5-脱水塔罗糖基的低分子量岩藻糖化糖胺聚糖； 所 得低分子量岩藻糖化糖胺聚糖任选进行还原性末端的还原反应， 包括将 2，5-脱水塔罗糖端基 还原成糖醇、 糖胺或 N取代的糖胺。
8、如权利要求 7所述的低分子量糖胺聚糖及其药学上可接受的盐的制备方法， 其特征在 于， 其 "步骤一"所述棘皮动物来源的岩藻糖化糖胺聚糖是指从棘皮动物门海参纲动物体壁 和 /或内脏提取纯化的岩藻糖化糖胺聚糖天然产物； 所述岩藻糖化糖胺聚糖的单糖组成包括 D-葡萄糖醛酸、 D-N-乙酰氨基 -2-脱氧半乳糖及 L-岩藻糖； 所述的棘皮动物门海参纲动物为梅 花参 Thelenota ananas Jaeger 花朿 (J参 Stichopus variegates Semper 造海参 Holothuria scabra Jaeger 玉足海参 Holothuria leucospilota Brandt 红腹海参 Holothuria edulis Lesson 蛇目白 尼参 Bohadschia argus Jaeger 绿朿 Ij参 Stichopus chloronotus Brandt 中华海参 Holothuria sinica Liao、 海地瓜 Acaudina molpadioides Semper、 格皮氏海参 Pearsonothuria graeffei Semper禾口黑 学 L参 Holothuria nobilis Selenka。
9、如权利要求 7所述的低分子量糖胺聚糖及其药学上可接受的盐的制备方法， 其特征在 于， 其 "步骤一"所述肼处理脱乙酰化反应的方法是向棘皮动物来源的岩藻糖化糖胺聚糖加 入无水肼或水合肼溶液中，在有或无催化剂存在下，搅拌中在 75 °C -125 °C的温度下反应 2-14h。
10、 如权利要求 9所述的低分子量糖胺聚糖及其药学上可接受的盐的制备方法， 其特征 在于， 其 "步骤一"所述肼处理脱乙酰化反应是在有催化剂存在的条件下进行的， 所述催化 剂任选硫酸肼、 盐酸肼、 盐酸或硫酸， 且所述反应溶液中的催化剂的浓度为 0.5%〜2.5%。
11、 如权利要求 7所述的低分子量糖胺聚糖及其药学上可接受的盐的制备方法， 其特征 在于， 其 "步骤二"所述亚硝酸处理脱氨基解聚的方法是： 在冰浴或室温条件下， 将 "步骤 一"所得岩藻糖化糖胺聚糖部分脱乙酰化产物加入 pH 1至 pH 5亚硝酸溶液 4〜6mol/L中反应 5min〜60min后， 碱溶液调 pH8或以上终止反应； 然后任选进行：
(1) 向反应溶液中加入 3-5倍体积的乙醇，静置，离心得沉淀，超滤或层析纯化所得产物；
(2) 采用硼氢化钠或氰基硼氢化钠， 将反应产物的还原性末端 2，5-脱水塔罗糖还原成糖 醇， 然后按 (1)所述步骤纯化所得产物； (3) 通过还原氨基化反应将反应产物的还原性末端 2，5-脱水塔罗糖还原成糖胺或 N-取代 的糖胺， 然后按 (1)所述步骤纯化所得产物。
12、 药物组合物， 其含有有效抗凝血剂量的权利要求 1或 2或 4所述的低分子量糖胺聚 糖衍生物及其药学上可接受的盐， 以及药用赋形剂。
13、 如权利要求 12所述的药物组合物， 其特征在于， 所述药物组合物的剂型为注射用水 溶液或注射用冻干粉针剂。
14、 权利要求 1或 2或 4所述的低分子量糖胺聚糖衍生物及其药学上可接受的盐在制备 防治血栓性疾病的药物中的应用， 所述血栓性疾病为静脉血栓形成或动脉血栓形成或缺血性 心脏病或缺血性脑血管病。
15、权利要求 14所述的药物组合物在制备防治血栓性疾病的药物中的应用， 所述血栓性 疾病为静脉血栓形成或动脉血栓形成或缺血性心脏病或缺血性脑血管病。
PCT/CN2013/090124 2013-04-12 2013-12-20 一种含末端2，5−脱水塔罗糖或其衍生物的低分子量糖胺聚糖衍生物 WO2014166282A1 (zh)

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CN103788222B (zh) * 2014-01-08 2016-08-31 中国科学院昆明植物研究所 Fuc3S4S取代的低聚糖胺聚糖及其制备方法
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WO2018129647A1 (zh) 2017-01-10 2018-07-19 九芝堂股份有限公司 一种抑制内源性凝血因子x酶复合物的寡糖化合物及其制备方法与用途
US11202797B2 (en) 2018-07-11 2021-12-21 Zhejiang University Mixture of fucosylated chondroitin sulfate oligosaccharides and method for rapidly producing the same
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