WO2016202310A1 - 具有抗Xa因子活性的磺酸化五糖化合物 - Google Patents

具有抗Xa因子活性的磺酸化五糖化合物 Download PDF

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WO2016202310A1
WO2016202310A1 PCT/CN2016/095464 CN2016095464W WO2016202310A1 WO 2016202310 A1 WO2016202310 A1 WO 2016202310A1 CN 2016095464 W CN2016095464 W CN 2016095464W WO 2016202310 A1 WO2016202310 A1 WO 2016202310A1
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干浩
韩健
韩芙蓉
李振重
孙福亮
周喜泽
侯文锋
闫建合
姚小青
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天津红日药业股份有限公司
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    • 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    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

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  • the invention belongs to the technical field of medicine and relates to a novel compound, in particular to a novel sulfonated pentasaccharide compound having anti-factor Xa activity.
  • thrombootic diseases are diseases that seriously endanger human health. According to the location, condition and nature of thrombosis, they are mainly divided into arterial thrombosis and venous thrombosis. Arterial thrombosis begins with arterial atherosclerotic lesions and platelet activation. The serious clinical diseases are mainly acute myocardial infarction and stroke. Venous thrombosis is induced by various causes in venous blood vessels, which can lead to venous thromboembolism. The main clinical manifestations are deep vein thrombosis and pulmonary embolism.
  • anticoagulant therapy can prevent the spread and recurrence of thrombosis and further reduce the incidence and mortality of stroke, pulmonary embolism, and the like. Therefore, anticoagulant therapy has become the core and foundation of clinical prevention and treatment of thromboembolic diseases, and the development of anticoagulant drugs has always been a hot spot in the development of new drugs.
  • new anticoagulant drugs being developed or already on the market mainly include direct thrombin inhibitors, factor Xa inhibitors, factor IX inhibitors, tissue factor inhibitors, and novel vitamin K antagonists.
  • direct thrombin inhibitors and factor Xa inhibitors are the most representative new anticoagulant drugs.
  • Fondaparinux is a synthetic selective activator (Xa) inhibitor that enhances the intrinsic neutralization of factor Xa by AT III through selective binding to AT III. Neutralization of factor Xa can be achieved. Interferes with the coagulation cascade system and simultaneously inhibits thrombin formation and thrombus development. Its chemical structure is as follows (using D, E, F, G, and H to represent 5 monosaccharides from left to right):
  • An object of the present invention is to provide a sulfonated pentasaccharide compound having an activity against Xa factor (hereinafter referred to simply as Compound A).
  • the compound A of the present invention has been determined to have very good activity against Xa factor, and its anti-Xa titer is 612 IU/mg, which is about 1.5 times that of fondaparinux sodium, and has extremely high medicinal value and remarkable The therapeutic effect.
  • the compound A of the present invention has the following chemical structural formula (represented by D, E, F, G, and H, respectively, from left to Right 5 monosaccharides):
  • Another object of the present invention is to provide a process for the preparation of Compound A.
  • the synthetic route of the compound A of the present invention is as follows:
  • the preparation method of the compound A according to the invention comprises the following steps:
  • DEF1 was dissolved in an appropriate amount of acetic anhydride, added with a solution of trimethylsilyl trifluoromethanesulfonate in dichloromethane at 0 ° C, and reacted at room temperature for 3 hours; then added with ammonia water, reacted at room temperature for 4 hours; Trichloroacetonitrile and DBU, nitrogen protection, reaction at room temperature for 3 hours, to obtain product DEF;
  • DEF and GH are dissolved in an appropriate amount of anhydrous dichloromethane, and an appropriate amount of dry 4A molecular sieve is added. After stirring at room temperature for 30 minutes, an appropriate amount of dichlorosilane of trimethylsilyl trifluoromethanesulfonate is added under nitrogen atmosphere at -20 ° C. Methane solution, the reaction is carried out for 1 hour, the appropriate amount of triethylamine is added, stirred for 30 minutes, filtered, and the filtrate is concentrated to dryness. The mixture is dissolved in methylene chloride, washed sequentially with water, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness Column, get the product DEFGH;
  • DEFGH was dissolved in THF, and purified water was added thereto, and then a NaOH solution was added dropwise thereto, and the mixture was reacted for 18 hours, neutralized with hydrochloric acid, and then extracted with chloroform.
  • the organic phase was taken and concentrated to dryness under reduced pressure.
  • the obtained sample was dissolved in DMF, added with sulfur trioxide trimethylamine complex, protected with nitrogen, reacted for 18 hours, concentrated under reduced pressure, and the filtrate was applied to a Sephadex LH-20 column to remove salt, and the collected product liquid was collected.
  • the mixture was distilled under reduced pressure, and applied to a 732 sodium type strongly acidic cation exchange resin chromatography column, and the collected product liquid was concentrated to dryness under reduced pressure.
  • the obtained sample, purified water and 10% palladium carbon were placed in a hydrogenation vessel, and the mixture was hydrogenated to 1.5 MPa in the autoclave for 68 hours. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was transferred to a reaction kettle, purified water was added, and sulfur trioxide pyridine complex was added to the reaction kettle in batches for 7 hours. After the solution was clear and transparent, it was filtered and applied to a glucan gel G. -25 column desalting, The collected product liquid was distilled under reduced pressure, and applied to a 732 sodium type strong acid cation exchange resin chromatography column, and the collected product liquid was concentrated to dryness under reduced pressure, and purified to obtain Compound A.
  • the preparation method of the compound A of the present invention comprises the following steps:
  • GH1 (16.5 g, 15.8 mmol) was dissolved in 150 ml of anhydrous dichloromethane, and triethylamine (55 ml, 294.7 mmol) was added at room temperature for 3 h, and passed through a silica gel column to give product GH (11.8 g, 14.0 mmol). .
  • the collected product liquid was distilled under reduced pressure, and applied to a 732 sodium type strong acid cation exchange resin chromatography column, and the collected product liquid was concentrated to dryness under reduced pressure.
  • the obtained sample 360 ml of purified water and 10.8 g of 10% palladium carbon were placed in a hydrogenation vessel, and the mixture was hydrogenated to 1.5 MPa, and reacted at 30 ° C for 68 hours. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure.
  • the concentrated residue was transferred to a reaction kettle, and purified water was added thereto, and sulfur trioxide pyridine complex (9.5 g, 59.7 mmol) was added to the reaction kettle in portions, and reacted at 10 ° C for 7 hours until the solution was clear and transparent, and then filtered.
  • the sample was desalted to a Sephadex G-25 column, and the collected product liquid was distilled under reduced pressure, and applied to a 732 sodium type strong acid cation exchange resin chromatography column, and the collected product liquid was concentrated under reduced pressure to Dry, purified by preparative to give compound A (2.6 g, 1.5 mmol).
  • Another object of the present invention is to provide a pharmaceutical composition containing Compound A.
  • the pharmaceutically active substance in the pharmaceutical composition of the present invention is a compound of the present invention, which may be present in the formulation in an amount of from 0.01 to 99.99% by weight, the balance being a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be prepared into any pharmaceutical preparation form as needed, such as an oral preparation form, an injection form, and the like.
  • a pharmaceutically acceptable carrier can be added as needed.
  • the pharmaceutical composition of the present invention is present in the form of a unit dosage, which means a unit of a preparation, such as each tablet of a tablet, each capsule of a capsule, each bottle of an oral solution, a granule per bag, an injection. Every one of them.
  • the pharmaceutical composition of the present invention may be in any pharmaceutically acceptable dosage form, including: tablets, sugar-coated tablets, film-coated tablets, enteric coated tablets, capsules, hard capsules, soft capsules, oral liquids. Oral preparation, granules, granules, pills, powders, ointments, granules, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, pills, Patch.
  • the pharmaceutical composition of the present invention may contain conventional excipients such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent, and a moisturizing agent.
  • a binder such as a polyethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, polypropylene glycol dimethacrylate, a steaglycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, a talct, talct, talct, talct, talct, talct copolymer, graft copolymer, graft copolymer, graft copolymer, graft copolymer, graft copolymer, graft copolymer,
  • Suitable fillers include cellulose, mannitol, lactose and other similar fillers.
  • Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.
  • the present invention can prepare a solid oral composition by a usual method such as mixing, filling, tableting or the like. Repeat mixing The active substance can be distributed throughout those compositions using a large amount of filler.
  • the oral liquid preparation may be in the form of, for example, an aqueous or oily suspension, solution, emulsion, syrup or elixir, or may be a dry product which may be formulated with water or other suitable carrier before use.
  • Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats.
  • Emulsifiers such as lecithin, sorbitan monooleate or gum arabic; non-aqueous vehicles (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters of esters such as glycerol, propylene glycol or ethanol;
  • the agent for example, p-hydroxybenzyl or propylparaben or sorbic acid, and if desired, may contain conventional flavoring or coloring agents.
  • the liquid unit dosage form prepared contains the active substance of the invention and a sterile vehicle.
  • This compound can be suspended or dissolved depending on the carrier and concentration.
  • the solution is usually prepared by dissolving the active substance in a carrier, sterilizing it by filtration before filling it into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservative and buffer may also be dissolved in such a carrier.
  • the composition can be frozen after filling the vial and the water removed under vacuum.
  • the pharmaceutical composition of the present invention may optionally be added to a suitable pharmaceutically acceptable carrier when prepared as a medicament, the pharmaceutically acceptable carrier being selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite, sulfur Sodium sulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid , phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and their derivatives , alginate, gelatin, polyvinylpyrrolidone, glycerin,
  • the pharmaceutical dosage form of the invention is an injection.
  • the content of the compound A in the unit preparation is from 1.5 mg to 6.5 mg.
  • Another object of the invention is to provide a pharmaceutical use of Compound A.
  • the thrombotic diseases of the present invention are mainly classified into arterial thrombosis and venous thrombotic diseases.
  • the invention also provides methods for determining the chemical structure and purity of Compound A.
  • the method for determining the purity of the compound A of the present invention comprises the following steps:
  • the invention also provides a structural assay for Compound A.
  • the present invention identifies the structure of Compound A by nuclear magnetic and mass spectrometry. From the nuclear magnetic resonance spectrum, compared with fondaparinux, a methyl peak was added at 2.74, and the hydrogen chemical shift of the H sugar 2 position shifted from 3.25 to the low field of 3.66, and the hydrogen of the H sugar position shifted from 3.62. 3.93, other locations have changed little. From the nuclear magnetic spectrum, compared with fondaparinux, there is an additional carbon peak at 32.76, and the carbon at the 2nd position of H sugar moves from 60.37 to 62.85. The carbons at the 1 and 3 positions of H sugar also have different degrees of change. Other locations have changed little.
  • the exact molecular weight of the compound was estimated by high resolution mass spectrometry to be: 1740.7864, and the molecular formula was C 32 H 45 N 3 Na 10 O 49 S 8 .
  • mass spectrometry it can be inferred that the hydrogen on the amino group linked to the 2-position of the H sugar is replaced by a methyl group, so that the chemical shift of the hydrogen at the 2-position of the H-saccharide shifts to the lower field, and the 1-position and the 3-position of the H-saccharide also change.
  • the invention measures the anti-Xa factor (FXa) activity of the compound A according to the national drug standard WS1-(X-147)-2005Z in the low-molecular weight heparin calcium issued by the State Food and Drug Administration.
  • the anti-Xa factor titer of Compound A was determined to be 612 IU/mg, the fondaparinux sodium titer was 400 IU/mg, and the low molecular weight heparin calcium titer was 105 IU/mg.
  • Compound A belongs to a new compound and has not been reported at present in domestic and foreign literature patents.
  • its anti-Xa titer is 612 IU/mg, which is about 1.5 times that of fondaparinux. It exhibits better anti-Xa factor activity and may become a sulfidamide in the future. Upgrading or upgrading of hepatic sodium.
  • Compound A may have other potential pharmacological effects, and in the long run, it will have higher value through more in-depth research.
  • the compound A of the present invention has the characteristics of strong biological activity and few side effects. For example, it has stronger biological activity than fondaparinux and low molecular weight heparin calcium; compound A belongs to a single compound and has fewer side effects than low molecular weight heparin calcium belonging to the mixture.
  • the compounds D1, EF, G, and H of the present invention are existing products and are commercially available.
  • TBSOTf Trimethylsilyl triflate
  • TMSOTf trimethylsilyl trifluoromethanesulfonate
  • Figure 1 Liquid chromatogram of Compound A of the present invention
  • GH1 (16.5 g, 15.8 mmol) was dissolved in 150 ml of anhydrous dichloromethane, and triethylamine (55 ml, 294.7 mmol) was added at room temperature for 3 h, and passed through a silica gel column to give product GH (11.8 g, 14.0 mmol). .
  • the sugar gel LH-20 column was desalted, and the collected product liquid was distilled under reduced pressure, and applied to a 732 sodium type strong acid cation exchange resin chromatography column, and the collected product liquid was concentrated to dryness under reduced pressure.
  • the obtained sample 360 ml of purified water and 10.8 g of 10% palladium carbon were placed in a hydrogenation vessel, and the mixture was hydrogenated to 1.5 MPa in the autoclave for 68 hours. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was transferred to a reaction kettle, and purified water was added thereto. Sulfur trioxide pyridine complex (9.5 g, 59.7 mmol) was added to the reaction kettle in portions and reacted for 7 hours.
  • the column was Dionex CarboPac TM PA1 (250 ⁇ 4 mm), the flow rate was 1.0 ml/min, the detection wavelength was 210 nm, the column temperature was 35 ° C, and water was used as mobile phase A (about 10 ⁇ l of dimethyl sulfoxide was added to 1000 ml of water), 116.9
  • the g/l sodium chloride solution is mobile phase B, and the gradient is as shown in the table:
  • Example 3 Structural identification of compound A by nuclear magnetic resonance and mass spectrometry
  • test data is as follows:
  • Tris-hydroxymethylaminomethane-sodium chloride buffer (pH 7.4): Dissolve 6.08 g of trishydroxymethylaminomethane and 8.77 g of sodium chloride plus 500 ml of water, add 10 g of bovine serum albumin, adjust the pH with hydrochloric acid to 7.4, dilute to 1000ml with water.
  • Tris-hydroxymethylaminomethane-ethylenediaminetetraacetic acid disodium buffer (pH 8.4): Take 5.12 g of sodium chloride, 3.03 g of trishydroxymethylaminomethane and 1.4 g of disodium edetate, and add 250 ml of water. Dissolve, adjust the pH to 8.4 with hydrochloric acid, and dilute to 500 ml with water.
  • LMWH standard and test solution Dilute standard (S) and test (T) into 4 different concentrations of solution with tris buffer (pH 7.4).
  • the ratio of the agent to each dose is generally 1:0.7 to 1:0.6.
  • the concentration should be within the linear range of the log dose-reaction. Generally, it contains 0.025 IU to 0.2 per 1 ml. IU.
  • Antithrombin (AT III) solution A solution containing 1 IU per 1 ml was prepared in a buffer of tris (sodium chloride)-sodium chloride (pH 7.4).
  • the chromogenic substrate s-2765 (or other FXa-specific chromogenic substrate): the solution is made into a solution of 0.003 mol/L in water, and the trishydroxymethylaminomethane-ethylenediaminetetraacetic acid disodium buffer is used immediately before use. (pH 8.4) was diluted to 0.0005 mol/L.
  • Xa factor (FXa) solution Prepare FXa solution with tris buffer (pH 7.4) and adjust its concentration to replace the anti-Xa factor of LMWH with 0.9% sodium chloride solution. Among them, the absorption value at a wavelength of 405 nm is 0.6 to 0.7.
  • T 1 , T 2 , T 3 , T 4 and S 1 , S 2 , S 3 , and S 4 were respectively labeled.
  • Two tubes were made in parallel for each concentration. Add 4 concentrations of test sample (T) or standard (S) dilution 50 ⁇ l to each tube, and 50 ⁇ l of antithrombin solution, mix (no air bubbles).
  • T test sample
  • S standard
  • S 1 , S 2 , S 3 , S 4 T 1 , T 2 , T 3 , T 4 , T 1 , T 2 , T 3 , T 4 , S 1 , S 2 , S 3 , S 4 , equilibrate for 1 minute at 37 °C.
  • the anti-Xa factor titer of Compound A was 612 IU/mg, the sulfa heparin sodium titer was 400 IU/mg, and the low molecular weight heparin calcium titer was 105 IU/mg as determined by the above method.
  • Compound A has an anti-Xa factor titer of about 1.5 times that of fondaparinux.
  • Step 1 under aseptic conditions, take a prescription amount of 80% water for injection, add a prescription amount of sodium chloride to dissolve, adjust the pH to 5.0-8.0 with 0.1M sodium hydroxide or hydrochloric acid solution;
  • Step 2 under sterile conditions, take a prescribed amount of Compound A into the above solution, stir to dissolve;
  • Step 3 under sterile conditions, adding water for injection to the above obtained solution to the full amount;
  • Step 4 under aseptic conditions, adding 0.1% medicinal charcoal to the above obtained solution, keeping warm, stirring for 30 minutes;
  • step 5 the above obtained solution is dispensed into a glass ampoule under aseptic conditions to obtain a finished product.

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Abstract

一种具有抗Xa因子活性的磺酸化五糖化合物及其制备方法和用途，该化合物为具有抗凝血作用的五糖化合物的衍生物，具有显著的抗Xa因子活性。

Description

一种新的具有抗Xa因子活性的磺酸化五糖化合物 技术领域
本发明属于医药技术领域，涉及一种新的化合物，具体涉及一种新的具有抗Xa因子活性的磺酸化五糖化合物。
技术背景
血栓性疾病是严重危害人类健康的疾病，根据血栓形成部位、条件与性质，主要分为动脉血栓与静脉血栓。动脉血栓形成是从动脉血管壁动脉粥样硬化病变与血小板激活开始，其导致的严重临床疾病主要为急性心肌梗死、脑卒中；静脉血栓由静脉血管中多种原因诱发形成，可导致静脉血栓栓塞，其主要临床表现为深静脉血栓形成和肺栓塞。大规模临床试验证据显示，抗凝治疗可阻止血栓的蔓延和复发，并进一步降低脑卒中、肺栓塞等的发生率和死亡率。因此，抗凝治疗已成为目前临床预防和治疗血栓栓塞性疾病的核心和基础，而抗凝药物的研发也始终是新药研发的热点。
目前，正在研发或已经上市的新型抗凝药物主要包括直接凝血酶抑制剂、Xa因子抑制剂、IX因子抑制剂、组织因子抑制剂以及新型维生素K拮抗剂。其中，直接凝血酶抑制剂和Xa因子抑制剂是最具代表性的抗凝新药。
磺达肝癸钠是一种人工合成的选择性活化因子(Xa)抑制剂，通过与AT III的选择性结合，增强了AT III对因子Xa固有的中和作用，因子Xa的中和作用可干扰凝血级联系统，并能同时抑制凝血酶形成和血栓发展，其化学结构式如下(用D、E、F、G、H分别表示从左至右5个单糖)：
Figure PCTCN2016095464-appb-000001
发明内容
本发明目的在于提供一种具有抗Xa因子活性的磺酸化五糖化合物(以下简称为化合物A)。
本发明所述的化合物A，经测定其具有非常好的抗Xa因子活性，其抗Xa效价为612IU/mg，约是磺达肝癸钠的1.5倍，具有极高的药用价值和显著的治疗效果。
本发明所述的化合物A，其化学结构式如下(用D、E、F、G、H分别表示从左至 右5个单糖)：
Figure PCTCN2016095464-appb-000002
本发明的另一个目的在于提供化合物A的制备方法。
具体的，本发明的化合物A的合成路线如下：
Figure PCTCN2016095464-appb-000003
本发明所述的化合物A的制备方法，包括以下步骤：
步骤1，D的制备：
将D1以适量无水二氯甲烷溶解，加入三氯乙腈，加入DBU，室温反应0.5h，得产品D；
步骤2，DEF的制备：
,将D和EF溶于适量无水二氯甲烷中，加入适量干燥4A分子筛，室温下搅拌30分钟后，于氮气保护、-20℃下加入三氟甲磺酸三甲基硅酯的二氯甲烷溶液，反应30分钟，得DEF1。将DEF1溶于适量乙酸酐中，于氮气保护、0℃下加入三氟甲磺酸三甲基硅酯的二氯甲烷溶液，室温反应3小时；再加入氨水，室温反应4小时；最后加入适量三氯乙腈和DBU，氮气保护，室温反应3小时，得产品DEF；
步骤3，GH的制备：
,将G、BSP和4A分子筛溶于无水二氯甲烷，氮气保护下室温搅拌30min，降温至-60℃，缓慢滴加Tf2O，滴毕后，温度升至-40℃，加入用无水二氯甲烷溶解稀释后的H，-40℃反应1h，得产品GH1。将GH1溶于适量无水二氯甲烷中，在室温下加入三乙胺，反应3h，得产品GH；
步骤4，DEFGH的制备：
,将DEF和GH溶于适量无水二氯甲烷中，加入适量干燥4A分子筛，室温搅拌30分钟后，于氮气保护、-20℃下加入适量三氟甲磺酸三甲基硅酯的二氯甲烷溶液，反应1小时，加入适量三乙胺，搅拌30分钟，过滤，滤液浓缩至干，加入适量二氯甲烷溶解，依次用水洗，无水硫酸钠干燥，过滤，滤液浓缩至干，过硅胶柱，得产品DEFGH；
步骤5，化合物A的制备
,将DEFGH溶于THF，加入纯化水，之后滴加NaOH溶液，反应18小时，滴加盐酸中和，然后加入三氯甲烷萃取，取有机相，减压浓缩至干。所得样品溶于DMF，加入三氧化硫三甲胺络合物，氮气保护，反应18小时，减压浓缩，滤液上样至葡聚糖凝胶LH-20层析柱除盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干。将所得样品、纯化水和10％钯碳加入氢化釜中，釜内加氢至1.5MPa，反应68小时，反应结束后，过滤，将滤液减压浓缩。将浓缩剩余物移至反应釜中，加入纯化水，将三氧化硫吡啶络合物分批加入反应釜中，反应7小时，至溶液澄清透明后，过滤，上样至葡聚糖凝胶G-25层析柱脱盐， 将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干，经制备纯化后得化合物A。
优选的，本发明化合物A的制备方法，包括以下步骤：
步骤1，D的制备：
将D1(18.0g，42.1mmol)溶于90ml无水二氯甲烷中，加入三氯乙腈(21ml，209.4mmol)，加入DBU(1.3ml，8.7mmol)，室温反应0.5h。减压浓缩至干，过硅胶柱，得产品D(22.6g，39.6mmol)。
步骤2，DEF的制备：
将D(22.6g，39.6mmol)和EF(17.5g，29.2mmol)溶于200ml无水二氯甲烷中，加入40g干燥4A分子筛，室温下搅拌30分钟后，于氮气保护、-20℃下加入TBSOTf(2.7ml，11.8mmol)的二氯甲烷溶液，反应30分钟，过硅胶柱，得DEF1(19.0g，18.8mmol)。将DEF1(19.0g，18.8mmol)溶于95ml乙酸酐中，于氮气保护、0℃下加入TBSOTf(0.9ml，3.9mmol)的二氯甲烷溶液，室温反应3小时；再加入氨水2ml，室温反应4小时；最后加入三氯乙腈(11.3ml，113.0mmol)和DBU(0.4ml，2.7mmol)，氮气保护，室温反应3小时，过硅胶柱，得产品DEF(11.1g，9.2mmol)。
步骤3，GH的制备：
将G(30.0g，41.8mmol)、BSP(11.4g，54.6mmol)和30g干燥的4A分子筛溶于150ml无水二氯甲烷，氮气保护下室温搅拌30min，降温至-60℃，缓慢滴加Tf2O(9.6ml，58.5mmol)，滴毕后，温度升至-40℃，加入用无水二氯甲烷溶解稀释后的H(23.1g，50.4mmol)，-40℃反应1h，过硅胶柱，得产品GH1(16.5g，15.8mmol)。将GH1(16.5g，15.8mmol)溶于150ml无水二氯甲烷中，在室温下加入三乙胺(55ml，294.7mmol)，反应3h，过硅胶柱，得产品GH(11.8g，14.0mmol)。
步骤4，DEFGH的制备：
将DEF(11.1g，9.2mmol)和GH(9.3g，11.0mmol)溶于100ml无水二氯甲烷中，加入20g干燥的4A分子筛，室温搅拌30分钟后，于氮气保护、-20℃下加入TMSOTf(0.7ml，3.9mmol)的二氯甲烷溶液，反应1小时，加入适量三乙胺，搅拌30分钟，过滤，滤液浓缩至干，加入适量二氯甲烷溶解，依次用水洗，无水硫酸钠干燥，过滤，滤液浓缩至干，过硅胶柱，得产品DEFGH(12.2g，6.4mmol)。
步骤5，化合物A的制备：
将DEFGH(12.2g，6.4mmol)溶于150ml四氢呋喃中，加入80ml纯化水，之后滴 加1N氢氧化钠溶液120ml，室温反应18小时，滴加1N盐酸中和，然后加入三氯甲烷萃取，取有机相，减压浓缩至干。所得样品溶于100ml N,N-二甲基甲酰胺中，加入三氧化硫三甲胺络合物(15.5g，112.0mmol)，氮气保护，60℃反应18小时，减压浓缩，滤液上样至葡聚糖凝胶LH-20层析柱除盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干。将所得样品、360ml纯化水和10.8g 10％钯碳加入氢化釜中，釜内加氢至1.5MPa，30℃反应68小时，反应结束后，过滤，将滤液减压浓缩。将浓缩剩余物移至反应釜中，加入纯化水，将三氧化硫吡啶络合物(9.5g，59.7mmol)分批加入反应釜中，10℃反应7小时，至溶液澄清透明后，过滤，上样至葡聚糖凝胶G-25层析柱脱盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干，经制备纯化后得化合物A(2.6g，1.5mmol)。
本发明的另一个目的在于提供一种含有化合物A的药物组合物。
本发明的药物组合物中的药物活性物质为本发明的化合物，其在制剂中所占重量百分比可以是0.01-99.99％，其余为药物可接受的载体。
本发明的药物组合物，在使用时可以根据需要制备成任何药物制剂形式，如口服制剂形式，注射剂形式等。
本发明的药物组合物在制备成药物制剂时，根据需要可以加入药学上可接受的载体。
本发明的药物组合物，以单位剂量的制剂形式存在，所述单位剂量是指制剂的单位，如片剂的每片，胶囊的每粒胶囊，口服液的每瓶，颗粒剂每袋，注射剂的每支等。
本发明的药物组合物可以是任何可药用的剂型，这些剂型包括：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂、滴丸剂、贴剂。
本发明的药物组合物，其口服给药的制剂可含有常用的赋形剂，诸如粘合剂、填充剂、稀释剂、压片剂、润滑剂、崩解剂、着色剂、调味剂和湿润剂，必要时可对片剂进行包衣。
适用的填充剂包括纤维素、甘露糖醇、乳糖和其它类似的填充剂。适宜的崩解剂包括淀粉、聚乙烯吡咯烷酮和淀粉衍生物，例如羟基乙酸淀粉钠。适宜的润滑剂包括，例如硬脂酸镁。适宜的药物可接受的湿润剂包括十二烷基硫酸钠。
本发明可通过混合，填充，压片等常用的方法制备固体口服组合物。进行反复混合 可使活性物质分布在整个使用大量填充剂的那些组合物中。
口服液体制剂的形式例如可以是水性或油性悬浮液、溶液、乳剂、糖浆剂或酏剂，或者可以是一种在使用前可用水或其它适宜的载体复配的干燥产品。这种液体制剂可含有常规的添加剂，诸如悬浮剂，例如山梨醇、糖浆、甲基纤维素、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶或氢化食用脂肪，乳化剂，例如卵磷脂、脱水山梨醇一油酸酯或阿拉伯胶；非水性载体(它们可以包括食用油)，例如杏仁油、分馏椰子油、诸如甘油的酯的油性酯、丙二醇或乙醇；防腐剂，例如对羟基苯甲酯或对羟基苯甲酸丙酯或山梨酸，并且如果需要，可含有常规的香味剂或着色剂。
对于注射剂，制备的液体单位剂型含有本发明的活性物质和无菌载体。根据载体和浓度，可以将此化合物悬浮或者溶解。溶液的制备通常是通过将活性物质溶解在一种载体中，在将其装入一种适宜的小瓶或安瓿前过滤消毒，然后密封。辅料例如一种局部麻醉剂、防腐剂和缓冲剂也可以溶解在这种载体中。为了提高其稳定性，可在装入小瓶以后将这种组合物冰冻，并在真空下将水除去。
本发明的药物组合物，在制备成药剂时可选择性的加入适合的药物可接受的载体，所述药物可接受的载体选自：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、土温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β－环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
优选的，本发明药物剂型为注射剂。
本发明所述注射剂，单位制剂中化合物A的含量为1.5mg-6.5mg。
本发明的另一个目的在于提供化合物A的药物用途。
本发明所述的化合物A在在制备具有抗Xa因子活性的药物中的应用。
本发明所述的化合物A在制备治疗血栓性疾病的药物中的应用。
本发明所述血栓性疾病主要分为动脉血栓与静脉血栓性疾病。
本发明还提供化合物A化学结构和纯度的测定方法。
本发明所述化合物A的纯度测定方法，包括以下步骤：
采用高效液相色谱仪，以季铵阴离子薄壳树脂为填充剂的聚合阴离子交换柱，水和 氯化钠溶液为流动相，检测化合物A的纯度。
本发明的具体检测方法请见说明书实施例2.
本发明还提供化合物A的结构测定。
本发明通过核磁和质谱鉴定化合物A的结构。从核磁氢谱看，与磺达肝癸钠相比，2.74处多了一个甲基峰，H糖2位的氢化学位移由3.25移向低场3.66，H糖3位的氢由3.62移向3.93，其他位置变化不大。从核磁碳谱看，与磺达肝癸钠相比，32.76处多了一个碳峰，H糖2位的碳由60.37移向62.85，H糖1位和3位的碳也有不同程度的变化，其他位置变化不大。通过高分辨质谱检测，推断该化合物的精确分子量为：1740.7864，分子式为C32H45N3Na10O49S8。结合质谱可以推断与H糖2位相连的氨基上的氢被甲基取代，使得H糖2位的氢的化学位移移向低场，同时，H糖1位与3位也发生了变化。
本发明按照国家食品药品监督管理局颁布的，低分子量肝素钙的国家药品标准WS1-(X-147)-2005Z中效价测定法测定化合物A的抗Xa因子(FXa)活性。测定化合物A的抗Xa因子效价为612IU/mg，磺达肝癸钠效价为400IU/mg，低分子量肝素钙效价为105IU/mg。
化合物A属于新的化合物，目前国内外文献专利未对其进行报导。通过对化合物A的抗Xa因子活性进行测定，其抗Xa效价为612IU/mg，大约是磺达肝癸钠的1.5倍，其表现出更好的抗Xa因子活性，未来有可能成为磺达肝癸钠的升级或更新换代品种。
此外，作为新的化合物，化合物A可能还具有其它潜在的药理方面的作用，从长远考虑，经过更加深入的研究，其还会有更高的价值。
本发明的化合物A与现有产品相比较，具有生物活性强，副作用少等特点。如比磺达肝癸钠和低分子肝素钙具有更强的生物活性；化合物A属于单一化合物，比属于混合物的低分子肝素钙的副作用少。
本发明所述化合物D1，EF、G,、H，属于现有产品，可以在市场上购买到。
对文中所述的名词作进一步的解释：
DBU：1,8-二氮杂二环十一碳-7-烯
TBSOTf：三氟甲磺酸三甲基硅酯
TMSOTf：三氟甲磺酸三甲基硅脂
BSP：1-(苯基亚硫酰基)哌啶
上述成分属于现有产品可以在市场上买到。
附图说明
图1：本发明化合物A的液相色谱图
具体实施方式
通过以下具体实施例对本发明作进一步的解释，但不作为本发明的限制。
实施例1：化合物A的制备
步骤1，D的制备：
将D1(18.0g，42.1mmol)溶于90ml无水二氯甲烷中，加入三氯乙腈(21ml，209.4mmol)，加入DBU(1.3ml，8.7mmol)，室温反应0.5h。减压浓缩至干，过硅胶柱，得产品D(22.6g，39.6mmol)。
步骤2，DEF的制备：
将D(22.6g，39.6mmol)和EF(17.5g，29.2mmol)溶于200ml无水二氯甲烷中，加入40g干燥4A分子筛，室温下搅拌30分钟后，于氮气保护、-20℃下加入TBSOTf(2.7ml，11.8mmol)的二氯甲烷溶液，反应30分钟，过硅胶柱，得DEF1(19.0g，18.8mmol)。将DEF1(19.0g，18.8mmol)溶于95ml乙酸酐中，于氮气保护、0℃下加入TBSOTf(0.9ml，3.9mmol)的二氯甲烷溶液，室温反应3小时；再加入氨水2ml，室温反应4小时；最后加入三氯乙腈(11.3ml，113.0mmol)和DBU(0.4ml，2.7mmol)，氮气保护，室温反应3小时，过硅胶柱，得产品DEF(11.1g，9.2mmol)。
步骤3，GH的制备：
将G(30.0g，41.8mmol)、BSP(11.4g，54.6mmol)和30g干燥的4A分子筛溶于150ml无水二氯甲烷，氮气保护下室温搅拌30min，降温至-60℃，缓慢滴加Tf2O(9.6ml，58.5mmol)，滴毕后，温度升至-40℃，加入用无水二氯甲烷溶解稀释后的H(23.1g，50.4mmol)，-40℃反应1h，过硅胶柱，得产品GH1(16.5g，15.8mmol)。将GH1(16.5g，15.8mmol)溶于150ml无水二氯甲烷中，在室温下加入三乙胺(55ml，294.7mmol)，反应3h，过硅胶柱，得产品GH(11.8g，14.0mmol)。
步骤4，DEFGH的制备：
将DEF(11.1g，9.2mmol)和GH(9.3g，11.0mmol)溶于100ml无水二氯甲烷中，加入20g干燥的4A分子筛，室温搅拌30分钟后，于氮气保护、-20℃下加入TMSOTf(0.7ml，3.9mmol)的二氯甲烷溶液，反应1小时，减压浓缩至干，过硅胶柱，得产品DEFGH(12.2g，6.4mmol)。
步骤5，化合物A的制备：
将DEFGH(12.2g，6.4mmol)溶于150ml四氢呋喃中，加入80ml纯化水，之后滴加1N氢氧化钠溶液120ml，反应18小时，滴加1N盐酸中和，然后加入三氯甲烷萃取，取有机相，减压浓缩至干。所得样品溶于100ml N,N-二甲基甲酰胺中，加入三氧化硫三甲胺络合物(15.5g，112.0mmol)，氮气保护，反应18小时，减压浓缩，滤液上样至葡聚糖凝胶LH-20层析柱除盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干。将所得样品、360ml纯化水和10.8g 10％钯碳加入氢化釜中，釜内加氢至1.5MPa，反应68小时，反应结束后，过滤，将滤液减压浓缩。将浓缩剩余物移至反应釜中，加入纯化水，将三氧化硫吡啶络合物(9.5g，59.7mmol)分批加入反应釜中，反应7小时，至溶液澄清透明后，过滤，上样至葡聚糖凝胶G-25层析柱脱盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干，经制备纯化后得化合物A(2.6g，1.5mmol)。
实施例2通过高效液相色谱仪器检测化合物A
色谱柱为Dionex CarboPacTM PA1(250×4mm)，流速为1.0ml/min，检测波长为210nm，柱温为35℃，以水为流动相A(1000ml水中加入约10μl二甲亚砜)，116.9g/l氯化钠溶液为流动相B，梯度如表所示：
Figure PCTCN2016095464-appb-000004
化合物A的纯度检测结果如附图1所示：保留时间为16.304min，纯度为99.73％。
实施例3：通过核磁和质谱对化合物A进行结构鉴定
仪器检测条件：Agilent 6520Q-TOF LCMS高分辨质谱仪，流速1ml/min，进样1μl，离子源ESI；BRUKER 400MHz核磁共振仪；溶剂：D2O；内标：TMS。检测数据如下：
表1氢谱与碳谱数据
Figure PCTCN2016095464-appb-000005
Figure PCTCN2016095464-appb-000006
表2高分辨质谱数据
Figure PCTCN2016095464-appb-000007
Figure PCTCN2016095464-appb-000008
从核磁氢谱看，如表1所示，与磺达肝癸钠相比，2.74处多了一个甲基峰，H糖2位的氢化学位移由3.25移向低场3.66，H糖3位的氢由3.62移向3.93，其他位置变化不大。从核磁碳谱看，与磺达肝癸钠相比，32.76处多了一个碳峰，H糖2位的碳由60.37移向62.85，H糖1位和3位的碳也有不同程度的变化，其他位置变化不大。通过高分辨质谱检测，如表2所示，推断该化合物的精确分子量为：1740.7864，分子式为C32H45N3Na10O49S8。结合质谱可以推断与H糖2位相连的氨基上的氢被甲基取代，使得H糖2位的氢的化学位移移向低场，同时，H糖1位与3位也发生了变化。由此确定，化合物A的结构式如下：
Figure PCTCN2016095464-appb-000009
实施例4：化合物A的Xa因子(FXa)活性检测
按照国家食品药品监督管理局颁布的，低分子量肝素钙的国家药品标准WS1-(X-147)-2005Z中效价测定法测定，即体外通过抗凝血酶III(AT III)与低分子肝素(LMWH)标准品比较以测定供试品加速抑制Xa因子(FXa)的活性。具体方法如下：
一、溶液配制
三羟甲基氨基甲烷-氯化钠缓冲液(pH 7.4)：取三羟甲基氨基甲烷6.08g和氯化钠8.77g加水500ml使溶解，加牛血清白蛋白10g，用盐酸调节pH值至7.4，加水稀释至1000ml。
三羟甲基氨基甲烷-乙二胺四醋酸二钠缓冲液(pH 8.4)：取氯化钠5.12g、三羟甲基氨基甲烷3.03g和乙二胺四醋酸二钠1.4g，加水250ml使溶解，用盐酸调节pH值至8.4，加水稀释至500ml。
LMWH标准品及供试品溶液的配制：用三羟甲基氨基甲烷-氯化钠缓冲液(pH 7.4)分别将标准品(S)和供试品(T)稀释成4个不同浓度的溶液，各剂量间的剂距比一般为1:0.7～1:0.6该浓度应在log剂量-反应的线性范围内。一般为每1ml中含0.025IU～0.2 IU。
抗凝血酶(AT III)溶液：以三羟甲基氨基甲烷-氯化钠缓冲液(pH 7.4)制成每1ml中含1IU的溶液。
发色底物s-2765(或其他FXa特异性发色底物)：溶液用水制成0.003mol/L的溶液，临用前用三羟甲基氨基甲烷-乙二胺四醋酸二钠缓冲液(pH 8.4)稀释至0.0005mol/L。
Xa因子(FXa)溶液：用三羟甲基氨基甲烷-氯化钠缓冲液(pH 7.4)，配制FXa溶液，调试其浓度，使之在用0.9％氯化钠溶液代替LMWH的抗Xa因子实验中，在405nm的波长处的吸收值在0.6～0.7。
二、测定法
取小试管16支，分别标记T1、T2、T3、T4及S1、S2、S3、S4。各浓度平行做两管。每管分别加人4种浓度的供试品(T)或标准品(S)稀释液50μl，及抗凝血酶溶液50μl，混匀(不得产生气泡)。按S1、S2、S3、S4、T1、T2、T3、T4、T1、T2、T3、T4、S1、S2、S3、S4顺序排列，37℃平衡1分钟。每管加FXa溶液100μl，混匀，37℃平衡1分钟，加发色底物溶液250μl，混合，37℃准确保温4分钟后，各加30％醋酸溶液375μl，终止反应。用1cm光程的半微量比色池，以三羟甲基氨基甲烷-氯化钠缓冲液(pH 7.4)为空白，在405nm的波长处测定每管的吸光度。以三轻甲基氨基甲烷-氯化钠缓冲液(pH 7.4)代替供试品溶液(平行做两管)同法操作作为空白对照管，在16支管开始和结尾时，分别测定空白对照管的吸光度。两者的吸光度不得有显著性差异。以吸光度为纵坐标，标准品溶液(或供试品溶液)浓度的对数值为横坐标分别作线性回归，按生物检定统计法(中国药典2005年版二部附录X IV)中的量反应平行线原理4×4法实验设计，计算效价及实验误差。平均可信限率(FL％)不得大于15％。
三、试验结果
经上述方法测定，化合物A的抗Xa因子效价为612IU/mg，磺达肝癸钠效价为400IU/mg,低分子量肝素钙效价为105IU/mg。与磺达肝癸钠相比，化合物A的抗Xa因子效价约为其1.5倍。
实施例5化合物A注射液
处方：
Figure PCTCN2016095464-appb-000010
Figure PCTCN2016095464-appb-000011
制备工艺：
步骤1，在无菌条件下，取处方量80％注射用水，加入处方量氯化钠溶解，以0.1M氢氧化钠或盐酸溶液调节pH至5.0-8.0；
步骤2，在无菌条件下，取处方量化合物A加入上述溶液中，搅拌，使溶解；
步骤3，在无菌条件下，向上述所得溶液中加入注射用水至全量；
步骤4，在无菌条件下，向上述所得溶液加入0.1％药用炭，保温，搅拌30分钟；
步骤5，在无菌条件下，将上述所得溶液分装至玻璃安瓿中，即得成品。

Claims (10)

  1. 一种具有抗Xa因子活性的磺酸化五糖化合物A，其结构式如下：
    Figure PCTCN2016095464-appb-100001
  2. 权利要求1所述的化合物A，其特征在于，该化合物抗Xa因子效价为612IU/mg
  3. 权利要求1所述的化合物A的制备方法，其特征在于，包括以下步骤：
    步骤1，D的制备：
    将D1以适量无水二氯甲烷溶解，加入三氯乙腈，加入DBU，室温反应0.5h，得产品D；
    步骤2，DEF的制备：
    ,将D和EF溶于适量无水二氯甲烷中，加入适量干燥4A分子筛，室温下搅拌30分钟后，于氮气保护、-20℃下加入三氟甲磺酸三甲基硅酯的二氯甲烷溶液，反应30分钟，得DEF1。将DEF1溶于适量乙酸酐中，于氮气保护、0℃下加入三氟甲磺酸三甲基硅酯的二氯甲烷溶液，室温反应3小时；再加入氨水，室温反应4小时；最后加入适量三氯乙腈和DBU，氮气保护，室温反应3小时，得产品DEF；
    步骤3，GH的制备：
    ,将G、BSP和4A分子筛溶于无水二氯甲烷，氮气保护下室温搅拌30min，降温至-60℃，缓慢滴加Tf2O，滴毕后，温度升至-40℃，加入用无水二氯甲烷溶解稀释后的H，-40℃反应1h，得产品GH1。将GH1溶于适量无水二氯甲烷中，在室温下加入三乙胺，反应3h，得产品GH；
    步骤4，DEFGH的制备：
    ,将DEF和GH溶于适量无水二氯甲烷中，加入适量干燥4A分子筛，室温搅拌30分钟后，于氮气保护、-20℃下加入适量三氟甲磺酸三甲基硅酯的二氯甲烷溶液，反应1小时，加入适量三乙胺，搅拌30分钟，过滤，滤液浓缩至干，加入适量二氯甲烷溶解，依次用水洗，无水硫酸钠干燥，过滤，滤液浓缩至干，过硅胶柱，得产品DEFGH；
    步骤5，化合物A的制备
    ,将DEFGH溶于THF，加入纯化水，之后滴加NaOH溶液，反应18小时，滴加盐酸 中和，然后加入三氯甲烷萃取，取有机相，减压浓缩至干。所得样品溶于DMF，加入三氧化硫三甲胺络合物，氮气保护，反应18小时，减压浓缩，滤液上样至葡聚糖凝胶LH-20层析柱除盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干。将所得样品、纯化水和10％钯碳加入氢化釜中，釜内加氢至1.5MPa，反应68小时，反应结束后，过滤，将滤液减压浓缩。将浓缩剩余物移至反应釜中，加入纯化水，将三氧化硫吡啶络合物分批加入反应釜中，反应7小时，至溶液澄清透明后，过滤，上样至葡聚糖凝胶G-25层析柱脱盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干，经制备纯化后得化合物A。
  4. 权利要求1所述的化合物A的制备方法，其特征在于，包括以下步骤：
    步骤1，D的制备：
    将D1(18.0g，42.1mmol)溶于90ml无水二氯甲烷中，加入三氯乙腈(21ml，209.4mmol)，加入DBU(1.3ml，8.7mmol)，室温反应0.5h。减压浓缩至干，过硅胶柱，得产品D(22.6g，39.6mmol)。
    步骤2，DEF的制备：
    将D(22.6g，39.6mmol)和EF(17.5g，29.2mmol)溶于200ml无水二氯甲烷中，加入40g干燥4A分子筛，室温下搅拌30分钟后，于氮气保护、-20℃下加入TBSOTf(2.7ml，11.8mmol)的二氯甲烷溶液，反应30分钟，过硅胶柱，得DEF1(19.0g，18.8mmol)。将DEF1(19.0g，18.8mmol)溶于95ml乙酸酐中，于氮气保护、0℃下加入TBSOTf(0.9ml，3.9mmol)的二氯甲烷溶液，室温反应3小时；再加入氨水2ml，室温反应4小时；最后加入三氯乙腈(11.3ml，113.0mmol)和DBU(0.4ml，2.7mmol)，氮气保护，室温反应3小时，过硅胶柱，得产品DEF(11.1g，9.2mmol)。
    步骤3，GH的制备：
    将G(30.0g，41.8mmol)、BSP(11.4g，54.6mmol)和30g干燥的4A分子筛溶于150ml无水二氯甲烷，氮气保护下室温搅拌30min，降温至-60℃，缓慢滴加Tf2O(9.6ml，58.5mmol)，滴毕后，温度升至-40℃，加入用无水二氯甲烷溶解稀释后的H(23.1g，50.4mmol)，-40℃反应1h，过硅胶柱，得产品GH1(16.5g，15.8mmol)。将GH1(16.5g，15.8mmol)溶于150ml无水二氯甲烷中，在室温下加入三乙胺(55ml，294.7mmol)，反应3h，过硅胶柱，得产品GH(11.8g，14.0mmol)。
    步骤4，DEFGH的制备：
    将DEF(11.1g，9.2mmol)和GH(9.3g，11.0mmol)溶于100ml无水二氯甲烷中，加入 20g干燥的4A分子筛，室温搅拌30分钟后，于氮气保护、-20℃下加入TMSOTf(0.7ml，3.9mmol)的二氯甲烷溶液，反应1小时，加入适量三乙胺，搅拌30分钟，过滤，滤液浓缩至干，加入适量二氯甲烷溶解，依次用水洗，无水硫酸钠干燥，过滤，滤液浓缩至干，过硅胶柱，得产品DEFGH(12.2g，6.4mmol)。
    步骤5，化合物A的制备：
    将DEFGH(12.2g，6.4mmol)溶于150ml四氢呋喃中，加入80ml纯化水，之后滴加1N氢氧化钠溶液120ml，室温反应18小时，滴加1N盐酸中和，然后加入三氯甲烷萃取，取有机相，减压浓缩至干。所得样品溶于100ml N,N-二甲基甲酰胺中，加入三氧化硫三甲胺络合物(15.5g，112.0mmol)，氮气保护，60℃反应18小时，减压浓缩，滤液上样至葡聚糖凝胶LH-20层析柱除盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干。将所得样品、360ml纯化水和10.8g 10％钯碳加入氢化釜中，釜内加氢至1.5MPa，30℃反应68小时，反应结束后，过滤，将滤液减压浓缩。将浓缩剩余物移至反应釜中，加入纯化水，将三氧化硫吡啶络合物(9.5g，59.7mmol)分批加入反应釜中，10℃反应7小时，至溶液澄清透明后，过滤，上样至葡聚糖凝胶G-25层析柱脱盐，将收集的产品液减压蒸馏，并上样至732钠型强酸性阳离子交换树脂层析柱，将收集的产品液减压浓缩至干，经制备纯化后得化合物A(2.6g，1.5mmol)。
  5. 权利要求1所述的化合物A在制备具有抗Xa因子活性的药物中的应用。
  6. 权利要求1所述的化合物A在制备治疗血栓性疾病的药物中的应用，所述血栓性疾病主要分为动脉血栓与静脉血栓性疾病。
  7. 含有权利要求1所述化合物A的药物组合物。
  8. 根据权利要求7所述的药物组合物，其特征在于，可以制备成任何药用剂型。
  9. 根据权利要求7所述的药物组合物，其特征在于，所述药物组合物为注射剂，单位制剂中化合物A的含量为1.5mg-6.5mg。
  10. 根据权利要求9所述的药物组合物，其特征在于，注射剂的制备方法包括以下步骤：
    步骤1，在无菌条件下，取处方量80％注射用水，加入处方量氯化钠溶解，以0.1M氢氧化钠或盐酸溶液调节pH至5.0-8.0；
    步骤2，在无菌条件下，取处方量化合物A加入上述溶液中，搅拌，使溶解；
    步骤3，在无菌条件下，向上述所得溶液中加入注射用水至全量；
    步骤4，在无菌条件下，向上述所得溶液加入0.1％药用炭，保温，搅拌30分钟；
    步骤5，在无菌条件下，将上述所得溶液分装至玻璃安瓿中，即得成品。
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