WO2020114418A1 - 抗凝血的五糖类化合物、组合物及其制备方法和医药用途 - Google Patents

抗凝血的五糖类化合物、组合物及其制备方法和医药用途 Download PDF

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WO2020114418A1
WO2020114418A1 PCT/CN2019/122954 CN2019122954W WO2020114418A1 WO 2020114418 A1 WO2020114418 A1 WO 2020114418A1 CN 2019122954 W CN2019122954 W CN 2019122954W WO 2020114418 A1 WO2020114418 A1 WO 2020114418A1
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methyl
compound
alkanoyl
compound according
add
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PCT/CN2019/122954
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English (en)
French (fr)
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张林林
冯海威
赵建良
吴有智
吴舰
马昌友
柴雨柱
王华萍
徐丹
朱春霞
田舟山
吴明一
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南京正大天晴制药有限公司
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Priority to CN201980075677.2A priority Critical patent/CN113056472B/zh
Publication of WO2020114418A1 publication Critical patent/WO2020114418A1/zh

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    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • 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
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings

Definitions

  • the invention relates to a pentasaccharide compound and composition used as an anticoagulant, a preparation method thereof and its medical use in anticoagulation.
  • thromboembolic diseases are a class of diseases that seriously endanger human health, and its incidence is the highest among various diseases. In recent years, there is an increasing trend. It is mainly divided into arterial thrombosis and venous thrombosis. Venous thrombosis is common in deep veins, and the clinical manifestations are local pain and swelling of thrombosis, obstruction of distal blood return and embolism caused by thrombectomy resulting in organ dysfunction. Arterial thrombosis begins with atherosclerotic lesions and platelet activation in the arterial wall, which can lead to serious cardiovascular diseases such as acute myocardial infarction and stroke. Treatment methods include anticoagulant therapy, antiplatelet therapy, and thrombolytic therapy. Anticoagulant therapy is currently the core and foundation for the prevention and treatment of thromboembolic diseases.
  • Heparin is a traditional anticoagulant drug and a polysaccharide in the glycosaminoglycan family. Clinically used include unfractionated heparin and low molecular weight heparin. Heparin induces a change in the conformation of AT by attaching to a specific binding domain of antithrombin (AT), thereby inhibiting the activity of coagulation factor Xa (FXa). Studies have shown that the smallest structural unit in which heparin binds AT and inhibits factor Xa is a unique pentasaccharide sequence.
  • the present invention provides a pentasaccharide compound whose anion form is represented by the following formula (A):
  • R 1 is selected from H, SO 3 -, C1 ⁇ C4 alkanoyl, or C1 ⁇ C4 alkyl;
  • R 2 is selected from H, SO 3 -, C1 ⁇ C4 alkanoyl, or C1 ⁇ C4 alkyl;
  • R 3 is selected from H or C1-C4 alkanoyl
  • R 4 is selected from H or SO 3 - ;
  • the condition is that when R 3 is H, R 1 and R 2 are not methyl at the same time.
  • the present invention provides a pentasaccharide compound whose anion form is represented by the following formula I:
  • R 1 is selected from H, SO 3 -, C1 ⁇ C4 alkanoyl, or C1 ⁇ C4 alkyl;
  • R 2 is selected from H, SO 3 -, C1 ⁇ C4 alkanoyl, or C1 ⁇ C4 alkyl;
  • R 3 is selected from H or C1-C4 alkanoyl
  • R 3 is H
  • R 1 and R 2 are not methyl groups at the same time.
  • R 1 is selected from H, SO 3 - , formyl, acetyl, methyl, or ethyl; in some typical embodiments, R 1 is selected from H, SO 3 - , acetyl, or methyl In some more typical embodiments, R 1 is selected from H or methyl.
  • R 2 is selected from H, SO 3 ⁇ , formyl, acetyl, methyl or ethyl; in some typical embodiments, R 2 is selected from H, SO 3 ⁇ , acetyl or methyl In some more typical embodiments, R 2 is selected from H, SO 3 - or methyl.
  • R 3 is selected from H, formyl, or acetyl; in some typical embodiments, R 3 is H or acetyl; in some more typical embodiments, R 3 is H; In some more typical embodiments, R 3 is acetyl.
  • the present invention provides pentasaccharide compounds in anionic form as shown below:
  • the present invention provides pentasaccharide compounds in anionic form as shown below:
  • the compound is present in acid form or in salt form, in the acid form, -COO - and -SO 3 - form functional groups are -COOH and -SO 3 H form; in a salt form, the The salt is selected from sodium salt or potassium salt.
  • the present invention provides pentasaccharide compounds as shown below:
  • the present invention provides pentasaccharide compounds as shown below:
  • the present invention provides a method for preparing a pentasaccharide compound whose anionic form is represented by Formula I, including the following steps:
  • R a and R b are each independently selected from C1-C4 alkanoyl, Bn or C1-C4 alkyl, and R g is C1-C4 alkanoyl;
  • R c and Rd are independently selected from C1-C4 alkanoyl, H or C1-C4 alkyl, and R g is C1-C4 alkanoyl;
  • R e and R f are each independently selected from C1-C4 alkanoyl, SO 3 - or C1-C4 alkyl, and R g is C1-C4 alkanoyl;
  • R a, R b are each independently selected from Ac, Bn or methyl; In some exemplary embodiments, R a is selected from Ac or methyl, R b is selected from Ac, Bn or methyl .
  • R c and Rd are each independently selected from Ac, H, or methyl; in some typical embodiments, R c is selected from Ac or methyl, and R d is selected from Ac, H, or methyl .
  • R e, R f are each independently selected from Ac, SO 3 - or methyl; In some exemplary embodiments, R e is selected from methyl or Ac, R f is selected from Ac, SO 3 - Or methyl.
  • the above preparation method may optionally further include step (4):
  • the present invention also provides compounds of formula DEFGH0:
  • R a and R b are each independently selected from C1-C4 alkanoyl, Bn or C1-C4 alkyl, and R g is C1-C4 alkanoyl.
  • R a, R b are each independently selected from Ac, Bn or methyl, R g is Ac.
  • R a is selected from Ac or methyl
  • R b is selected from Ac, Bn or methyl
  • R g is Ac.
  • the present invention also provides the following compounds:
  • the invention also provides the use of the formula DEFGH0 in the preparation of compounds whose anionic form is represented by formula I.
  • the present invention provides the use of a compound of formula DEFGH10, a compound of formula DEFGH20, a compound of formula DEFGH30, or a compound of formula DEFGH40 in the preparation of a compound whose anionic form is represented by formula I.
  • the present invention also provides compounds of formula DEFGH1:
  • R c and Rd are independently selected from C1-C4 alkanoyl, H or C1-C4 alkyl, and R g is C1-C4 alkanoyl.
  • R c and Rd are each independently selected from Ac, H, or methyl, and R g is Ac.
  • R c is selected from Ac or methyl
  • R d is selected from Ac, H or methyl
  • R g is Ac.
  • the present invention also provides the following compounds:
  • the present invention also provides the use of the compound of formula DEFGH1 in the preparation of the compound whose anionic form is represented by formula I.
  • the present invention provides the use of a compound of formula DEFGH11, a compound of formula DEFGH21, a compound of formula DEFGH31, or a compound of formula DEFGH41 in the preparation of a compound whose anionic form is represented by formula I.
  • the present invention also provides compounds whose anionic form is represented by the formula DEFGH2:
  • R e and R f are each independently selected from C1 to C4 alkanoyl, SO 3 - or C1 to C4 alkyl, and R g is C1 to C4 alkanoyl.
  • R e and R f are each independently selected from Ac, SO 3 - or methyl, and R g is Ac.
  • R e is selected from Ac or methyl
  • R f is selected from Ac, SO 3 - or methyl
  • R g is Ac.
  • the present invention also provides compounds whose anionic form is shown below:
  • the compound whose anionic form is represented by the formula DEFGH2 exists in an acid form or a salt form, and in the acid form, the -COO - and -SO 3 - functional groups are -COOH form and -SO 3 H form, respectively;
  • salt form the salt is selected from sodium or potassium salts.
  • the present invention also provides the following compounds:
  • the present invention also provides the use of a compound whose anionic form is represented by formula DEFGH2 in the preparation of a compound whose anionic form is represented by formula I.
  • the present invention also provides the use of a compound represented by the formula DEFGH22-1, a compound represented by the formula DEFGH32-1, or a compound represented by the formula DEFGH42-1 in the preparation of a compound whose anionic form is represented by the formula I.
  • the present application also includes compounds of the present application which are the same as those described herein, but one or more atoms are replaced by an isotope labeled with an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature.
  • isotopes that can be bound to the compounds of the present application include isotopes of hydrogen, carbon, oxygen, and sulfur, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 O, 17 O, 18 O, and 35 S, respectively Wait.
  • Certain isotopically-labeled compounds of the present application can be used in the analysis of compound and/or substrate tissue distribution.
  • Deuterated (ie 2 H) and carbon-14 (ie 14 C) isotopes are particularly preferred for their ease of preparation and detectability.
  • Positron emission isotopes such as 15 O and 11 C, can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • PET positron emission tomography
  • the isotopically labeled compounds of the present application can generally be prepared by isotopically labeled reagents instead of unisotopically labeled reagents by the following procedures similar to those disclosed in the schemes and/or examples below.
  • deuterium substitution may be partial or complete, and partial deuterium substitution means that at least one hydrogen is replaced by at least one deuterium.
  • deuterated compounds include, but are not limited to:
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising, as an active ingredient, a pentasaccharide compound whose anionic form is represented by Formula I.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising, as an active ingredient, a pentasaccharide compound whose anionic form is represented by Formula I and one or more pharmaceutical excipients.
  • the pentasaccharide compound of the present invention is 0.1 to 100 mg, preferably 0.5 to 50 mg.
  • the pharmaceutical composition can be administered orally or parenterally.
  • Parenteral administration includes but is not limited to intravenous injection, intramuscular injection and subcutaneous injection.
  • the pharmaceutical composition of the present invention is usually provided in the form of tablets, capsules, and solutions.
  • the tablet may contain the pentasaccharide compound of the present invention or a salt thereof and a pharmaceutically acceptable excipient.
  • the excipient includes, but is not limited to, at least one of a diluent, a disintegrant, a binder, a lubricant, a sweetener, a flavoring agent, a coloring agent, or a preservative.
  • Capsules include hard capsules and soft capsules.
  • the pharmaceutical composition of the present invention can be administered by intravenous injection, intramuscular injection, or subcutaneous injection. It is usually supplied as a sterile aqueous solution or suspension or lyophilized powder, and the appropriate pH and isotonicity are adjusted.
  • the preferred route of administration is subcutaneous injection.
  • the present invention also provides the use of a pentasaccharide compound whose anionic form is represented by Formula I for the preparation of a medicament for preventing and/or treating diseases or disease states related to coagulopathy.
  • the present invention also provides a method for preventing and/or treating diseases and disease states associated with coagulopathy, which comprises administering to an individual in need thereof the anionic form of the present invention as shown in Formula I Sugar compound or the pharmaceutical composition of the present invention.
  • the present invention also provides the pentasaccharide compound of the present invention or the pharmaceutical composition of the present invention for preventing and/or treating diseases and disease states associated with coagulopathy.
  • diseases or disease states related to coagulation dysfunction include, but are not limited to, venous thrombosis, arterial thrombosis, and thrombophlebitis.
  • the inventors of the present application conducted in-depth studies to synthesize pentasaccharide compounds whose anionic form is represented by general formula I, and conducted biological experimental studies, and found that the pentasaccharide compounds of the present invention have high anticoagulant factor Xa activity and It can meet the clinical needs of the elimination half-life in the body, especially suitable for use as an anticoagulant.
  • the preparation method of the pentasaccharide compound of the invention is greatly simplified, the manufacturing cost is low, the development cost of the raw material medicine is significantly reduced, and it is suitable for large-scale industrial production.
  • Figure 1 is the 1 H NMR spectrum of compound I-1-1;
  • FIG. 3 is the COSY spectrum of compound I-1-1
  • Figure 4 is the HSQC spectrum of compound I-1-1
  • Alkyl refers to a saturated aliphatic hydrocarbon group, including a linear or branched saturated monovalent hydrocarbon group having the indicated number of carbon atoms.
  • C1-C4 alkyl includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl.
  • Suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl , Isobutyl, tert-butyl;
  • Alkanoyl refers to a group having an RC(O)- structure, R is H or a saturated aliphatic hydrocarbon group, including a linear or branched saturated monovalent hydrocarbon group.
  • C1-C4 alkanoyl includes C1 alkanoyl, C2 alkanoyl, C3 alkanoyl, C4 alkanoyl.
  • Suitable alkanoyl groups include formyl, acetyl, n-propionyl, isopropyl, n-butyryl, Isobutyryl, tert-butyryl.
  • Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride
  • BSA bovine serum albumin
  • Tris NaCl EDTA PEG buffer Tris NaCl EDTA PEG buffer
  • reaction conditions such as reactants, solvents, bases, amounts of compounds used, reaction temperature, reaction time, etc. are not limited to the following examples.
  • the pentasaccharide compound of the present invention can also be optionally prepared by combining various synthetic methods described in this specification or known in the art, and such a combination can be easily performed by those skilled in the art.
  • Dissolve D4 (464.0g) by adding 2.3kg of acetic acid, add 230ml of 1M sulfuric acid aqueous solution, reflux for 1.5h and neutralize by adding 460ml of 2N sodium hydroxide aqueous solution, then spin dry under reduced pressure, and extract and wash the residue with ethyl acetate and water The organic phase was spin-dried under reduced pressure and subjected to silica gel column chromatography to obtain D8 (360.0g).
  • D8 (225.0g) was dissolved in 2.2L of ethyl acetate, 260ml of triethylamine was added, 200ml of acetic anhydride was added dropwise. After reaction at room temperature for 1h, water was added to quench, the reaction solution was separated, and the organic phase was evaporated to dryness under reduced pressure to obtain D9 (133.5 g).
  • D13 (36.3g) was added to 363ml of 0.1M sulfuric acid aqueous solution, and barium carbonate (14.2g) was added after reacting at 80°C for 2h. After stirring for 2h, it was filtered, and the reaction solution was dried under reduced pressure to obtain D14 (25.9g).
  • D14 (25.9g) was dissolved in 260ml of ethyl acetate, 80ml of triethylamine was added, 60ml of acetic anhydride was added dropwise. After reaction at room temperature for 1h, water was added to quench, the reaction liquid was separated, and the organic phase was evaporated to dryness under reduced pressure to obtain D15 (48.3g ).
  • D21 (38.1g) was added to 380ml of acetonitrile and 78ml of water, and 80.0g of cerium ammonium nitrate was added. After reaction at room temperature for 1h, saturated sodium sulfite solution was added to quench. The reaction solution was evaporated to dryness under reduced pressure, and the residue was subjected to column chromatography to obtain D22 (24.3g).
  • acetic anhydride triethylamine, methylene chloride
  • TMSOTf p-methoxyphenol, methylene chloride
  • sodium methoxide methanol
  • tetrabutylammonium iodide benzyl bromide, dibutyl Tin oxide, N,N-diisopropylethylamine
  • acetic anhydride triethylamine
  • f) cerium ammonium nitrate acetonitrile
  • g) trichloroacetonitrile, DBU dichloromethane
  • Dissolve D1 38.8g
  • 380ml of dichloromethane add 85.0g of triethylamine
  • 85.0g of acetic anhydride dropwise, react at room temperature for 1h, quench with water, separate the reaction solution, wash with water, and dry the organic phase under reduced pressure Get D24 (72.4g).
  • D28 (41.2g) was added to a mixed solvent of 380ml of acetonitrile and 78ml of water, 82.2g of cerium ammonium nitrate was added, reacted at room temperature for 1h, and quenched by adding saturated sodium sulfite solution. The reaction solution was evaporated to dryness under reduced pressure, and the residue was subjected to column chromatography to obtain D29 (26.7g).
  • E2 (293.0g) was dissolved in 2L of anhydrous dichloromethane, triethylsilane (300.0g) was added, boron trifluoride etherate (30.0g) was added dropwise at 5°C, and the reaction was completed at 5°C for 2h.
  • G1 (1.15kg) was dissolved in a mixed solvent of pyridine 5L and dichloromethane 5L, pivaloyl chloride (600g) was added dropwise at 0°C, naturally warmed to room temperature, reacted for 8h, and then methanesulfonyl chloride (600g) was added dropwise at 0°C ), diluted with 5L of dichloromethane after 3 hours of reaction, washed with 5% hydrochloric acid and saturated sodium bicarbonate solution, and then the dichloromethane phase was dried over anhydrous sodium sulfate, and rot-dried under reduced pressure to obtain G2 (1.75kg), which was used directly In the next step.
  • G2 (1.75kg) was dissolved in a mixed solvent of 10L dichloromethane and 2L tert-butanol, potassium tert-butoxide (1.49kg) was added at 0°C, and the temperature was naturally raised to room temperature for 8h. It was then washed with water, and the methylene chloride phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain G3 (859 g).
  • G3 (859.0 g) was added to 4.3 L of 0.1 M sulfuric acid aqueous solution and reacted at 60° C. for 2 h, then 127.0 g of barium carbonate was added to neutralize for 2 h, filtered, and the filtrate was spin-dried under reduced pressure to obtain G4 (771.0 g).
  • G9 (957.0g) was added to a mixed solvent of 5L of acetonitrile and 2L of water, and cerium ammonium nitrate (2.74kg) was added at 0°C. After 2 hours of reaction, it was diluted with 5L of ethyl acetate, washed with saturated sodium sulfite solution, and dried over anhydrous sodium sulfate , Concentrate under reduced pressure, and obtain G10 (670.0g) by column chromatography.
  • DE6 (12.6g) was added to 120ml of dichloromethane, trichloroacetonitrile (6.7g) and DBU (0.12g) were added, the reaction was spin-dried after 1h, and the residue was subjected to column chromatography to obtain DE10 (13.3g).
  • DE7 (11.4g) was added to 120ml of dichloromethane, trichloroacetonitrile (6.7g) and DBU (0.12g) were added, the reaction was spin-dried after 1h, and the residue was subjected to column chromatography to obtain DE11 (12.3g).
  • DE8 (11.8g) was added to 120ml of dichloromethane, trichloroacetonitrile (6.7g) and DBU (0.12g) were added, the reaction was spin-dried after 1h, and the residue was subjected to column chromatography to obtain DE12 (12.7g).
  • TMSOTf dichloromethane
  • b 80% acetic acid aqueous solution
  • dipotassium hydrogen phosphate trihydrate potassium dihydrogen phosphate, water, TEMPO, NaClO 2 , KBr, TCCA
  • potassium bicarbonate benzyl bromide, Acetonitrile
  • e TMSOTf, methylene chloride
  • f potassium bicarbonate, methanol
  • GH1 (369.0g) was added to 2L of 80% acetic acid aqueous solution. After reaction at 80°C for 3h, the residue was spin-dried under reduced pressure. The residue was washed with petroleum ether, then dissolved with ethyl acetate and washed with saturated sodium bicarbonate solution. Dry GH2 (268.0g).
  • GH2 (292.0g) to tetrahydrofuran 2.4L, add dipotassium hydrogen phosphate trihydrate (100g), potassium dihydrogen phosphate (100.0g), water (150g), TEMPO (1.6g), NaClO 2 (24g), KBr( 4g) and TCCA (40.0g). After 2h of reaction, quench with anhydrous sodium sulfite, then spin dry under reduced pressure. The residue was washed with ethyl acetate and the organic phase was spin dried under reduced pressure to give GH3 (223.0g).
  • GH3 (223.0g) was added to 1.8L of acetonitrile, potassium bicarbonate (60.0g) was added, and benzyl bromide (62.0g) was added dropwise at 10°C. After 1 hour of reaction, 2L of ethyl acetate and 1L of water were added for extraction and washing. Autoclave to dryness, and obtain GH4 (213.0g) by column chromatography.
  • FGH2 (131.0g) was added to 1.3L of methanol, potassium bicarbonate (15g) was added, the reaction was spin-dried after 8h, ethyl acetate and water were added for extraction and washing, and the organic phase was evaporated to dryness to obtain FGH2 (88.7g) by column chromatography.
  • the residue was desalted by a gel column (Sephadex G-25) (deionized water as the eluent), and then treated by 732 sodium type cation exchange resin to a sodium salt, which was purified by GE QFF anion exchange resin (0.5M ⁇ 2M NaCl aqueous solution gradient elution), and finally desalted by gel column (Sephadex G-25), lyophilized to obtain the target product (3.2g).
  • DEFGH20 (6.2g) was dissolved in 61ml of anhydrous methanol, and 300mg of 10% palladium carbon was added. After reduction under normal pressure of hydrogen for 24h, the filtrate was spin-dried. The residue was stirred and beaten with 15ml of ethyl acetate, and then suction filtered, and the filter cake was dried. Dried DEFGH21 (2.4g).
  • DEFGH21 (1.96g) was added to 10ml of N,N-dimethylformamide to dissolve, and 8.4g of sulfur trioxide trimethylamine salt was added. After reaction at 50°C for 24h, it was quenched with saturated sodium bicarbonate aqueous solution and concentrated under reduced pressure.
  • the residue is desalted by a gel column (Sephadex G-25) (deionized water is the eluent), and then treated by 732 sodium type cation exchange resin to a sodium salt, which is purified by GE QFF anion exchange resin (0.5M ⁇ 2M NaCl aqueous solution gradient elution), and finally desalted by gel column (Sephadex G-25), lyophilized to obtain white flake solid DEFGH32-1 (3.1g).
  • the residue is desalted by a gel column (Sephadex G-25) (deionized water is the eluent), and then treated by 732 sodium type cation exchange resin to a sodium salt, which is purified by GE QFF anion exchange resin (0.5M ⁇ 2M NaCl aqueous solution gradient elution), and finally desalted by gel column (Sephadex G-25), lyophilized to obtain white flake solid DEFGH42-1 (3.1g).
  • the biological activity test of the pentasaccharide compound of the present invention can be measured by a method known to those skilled in the art. It can be understood that the following test methods do not limit the scope of the present invention.
  • APTT reagent MDC Hemostasis, TECO GmbH (Germany), Lot: 20002467;
  • CaCl 2 (0.02M): MDC Hemostasis, TECO GmbH (Germany), Lot:031N-G187A;
  • PT reagent (containing rabbit brain powder and CaCl 2 ), MDC Hemostasis, TECO GmbH (Germany), Lot: 10002518;
  • TT reagent (with thrombin), MDC Hemostasis, TECO GmbH (Germany), Lot: 30002569;
  • Tris-HCl purity >99.5%, Amresco (USA), Lot: 20110723.
  • LMWH Low Molecular Weight Heparin: Enoxaparin Sodium Injection (0.6ml: 6000AxaIU), Sanofi-Aventis (France), Lot: 5SK26;
  • Fpx Fondaparinux injection (2.5mg/0.5ml), GlaxoSmithkline (UK), Lot: 6181A;
  • Vortex oscillator SCIENTIFIC INDUSTRIES (USA), model VortexGenie;
  • Pipette gun (10 ⁇ l, 100 ⁇ l, 200 ⁇ l, 1000 ⁇ l range), Eppendorf;
  • Tris-HCl buffer solution (0.02M Tris-HCl, pH 7.40): Weigh 3.125g of Tris-HCl, dissolve it in 800ml of purified water, adjust the pH value to 7.40 with 0.5M NaOH solution, and dissolve it in 1000ml volumetric flask, Store at 4°C for use;
  • Sample solution The weighed samples I-1-1 (37mg), I-2-1 (26mg), I-3-1 (17mg) and I-4-1 (19mg) were dissolved in pure water to prepare 40mg/ml stock solution, take appropriate amount of stock solution before experiment and dilute to 1280 ⁇ g/ml with Tris-HCl buffer, and then use Tris-HCl buffer to dilute to the concentration required for the experiment.
  • APTT, PT, TT reagent solution and plasma solution prepared according to the instructions.
  • APTT detection Experiment according to the kit instructions: (1) Accurately take 5 ⁇ l series concentration of sample solution, positive control solution, Tris-HCl buffer solution into the pre-warmed test tube at 37°C, and then add 45 ⁇ l normal coagulation quality Control plasma and incubate at 37°C for 2min; (2) Add 50 ⁇ l of preheated APTT reagent at 37°C to the detection tube and incubate the mixture at 37°C for 3min; (3) Transfer the detection tube from the incubation well to the detection well and add 50 ⁇ l 37 Preheat 0.02M CaCl 2 at °C, meanwhile start counting and record the clotting time.
  • PT detection Experiment according to the kit instructions: (1) Accurately measure 5 ⁇ l series concentration of sample solution, positive control solution and Tris-HCl buffer solution into the pre-warmed test tube at 37°C, then add 45 ⁇ l normal coagulation Control the plasma and incubate at 37°C for 2 min; (2) Transfer the detection tube from the incubation well to the detection well, add 100 ⁇ l of PT reagent preheated at 37°C, and start counting and recording the clotting time.
  • TT test Experiment according to the kit instructions: (1) Accurately take 10 ⁇ l series concentration of sample solution, positive control solution, Tris-HCl buffer solution into the 37°C pre-warmed test tube, and then add 90 ⁇ l normal coagulation Control the plasma and incubate at 37°C for 2 min; (2) Transfer the detection tube from the incubation well to the detection well, add 50 ⁇ L of 37°C preheated TT reagent, and start counting and recording the clotting time.
  • Tris-HCl buffer control well that is, no sample exists, its four APTT detection results were 33.9s, 33.3s, 33.2s and 34.5s, with an average value of 33.725s and SD of 0.60.
  • the APTT experiment results are shown in the table below.
  • the final concentration of the sample ( ⁇ g/ml) and the average value of APTT (s) were linearly fitted, and the concentration of the sample required to extend the coagulation quality control plasma by twice the APTT was calculated from the fitting equation.
  • Tris-HCl buffer blank control wells that is, in the absence of samples, the four PT detection results were 13.4s, 13.5s, 13.4s and 13.5s, with an average value of 13.45s and SD of 0.06.
  • the PT experiment results are shown in the table below.
  • Tris-HCl buffer blank control well that is, in the absence of sample, its four TT detection results were 10.2s, 9.2s, 9.2s and 10.1s, with an average value of 9.675s and SD of 0.55.
  • the TT test results are shown in Tables 4-15 to 4-20.
  • ATIII-dependent anti-factor Xa FXa detection kit (BIOPHEN ANTI-Xa): containing R1 (ATIII), R2 (FXa) and R3 (FXa-specific chromogenic substrate (CS-11 (65))), HYPHEN BioMed (France), Lot: F171100232;
  • LMWH Low Molecular Weight Heparin: Enoxaparin Sodium Injection (0.6ml: 6000AxaIU), Sanofi-Aventis (France), Lot: 5SK26;
  • Fpx Fondaparinux injection (2.5mg/0.5ml), GlaxoSmithkline (UK), Lot: 6181A;
  • Vortex oscillator SCIENTIFIC INDUSTRIES (USA), model VortexGenie;
  • Pipette gun (10 ⁇ l, 100 ⁇ l, 200 ⁇ l, 1000 ⁇ l range), Eppendorf;
  • Tris-HCl buffer (0.02M Tris-HCl, pH 7.40): Same as above;
  • Sample solution and reference substance solution Use Tris-HCl buffer solution, prepared from the sample stock solution or reference substance to a concentration of 128000ng/ml, and then gradient dilution to the required concentration;
  • R1, R2 and R3 reagent solutions prepared according to the instructions.
  • the experiment was carried out according to the instructions in the kit: (1) After adding a series concentration of 30 ⁇ l sample solution, reference solution and Tris-HCl buffer solution to each well in a 96-well plate, add 30 ⁇ l R1 (1IU/ml ATIII) and place In the microplate reader, mix the shaker plate and incubate at 37°C for 1 min; (2) Take out the 96-well plate, add 30 ⁇ l R2 (8 ⁇ g/ml FXa solution), place in the microplate reader, mix the shaker plate at 37°C Precise incubation for 1 min; (3) Take out the 96-well plate, add 30 ⁇ l of preheated R3 (1.2 mM FXa specific chromogenic substrate), place it in a microplate reader, and measure the absorbance at 405 nm (OD 405 nm ) for 30 s Continuous detection for intervals of 4.5min.
  • the average value of OD 405nm of repeated detection is taken as the detection value of each concentration sample and reference substance.
  • the detection result is analyzed by software Excel (2007).
  • the detection value is linearly fitted with time.
  • the slope of the fitted straight line is the rate of change of absorbance ⁇ OD 405nm /min, this value corresponds to the activity of FXa.
  • FXa activity of the negative control bladenk control well without inhibitor
  • B IC 50 n / ⁇ IC 50 n +[I] n ⁇
  • B is the percentage of FXa activity
  • [I] is the concentration of the inhibitor
  • IC 50 is the half inhibitory concentration, that is, the concentration of the compound required to inhibit FXa activity by 50%
  • n is the Hill coefficient.
  • the FXa activity of the blank control well (using the Tris-HCl buffer instead of the sample solution) is defined as 1 (100%), calculate the relative FXa activity (%) in the presence of the sample, and then use the Origin8 software to determine the sample concentration -FXa percent active nonlinear fitting results shown in Figure 5.
  • the 50% inhibition concentration of the IC 50 values the results of the following Table.
  • ATIII Antithrombin III: HYPHEN BioMed (France), Lot: F1700099;
  • Amine-PEG3-Biotin (Amine-PEG3-Biotin): Thermo SCIENTIFIC (USA), Lot: A167761A;
  • Bovine serum albumin (Albumin Bovine, BSA): LIFE SCIENCE, Lot:0905C473;
  • Heparin Heparin standard product, China National Institute for the Control of Pharmaceutical and Biological Products, Lot: 15050-200912;
  • Disodium hydrogen phosphate Na 2 HPO 4 ⁇ 12H 2 O: analytically pure, Tianjin Damao Chemical Reagent Factory, Lot: 20100515.
  • LMWH Low Molecular Weight Heparin: Enoxaparin Sodium Injection (0.6ml: 6000AxaIU), Sanofi-Aventis (France), Lot: 5SK26;
  • Fpx Fondaparinux injection (2.5mg/0.5ml), GlaxoSmithkline (UK), Lot: 6181A;
  • Vortex oscillator SCIENTIFIC INDUSTRIES (USA), model VortexGenie;
  • PBS buffer Loading buffer: phosphate buffer containing 0.15M NaCl, pH 7.30; weigh 9g NaCl, 1.4g NaH 2 PO 4 ⁇ 2H 2 O, 15.4g Na 2 HPO 4 ⁇ 12H 2 O, set In a beaker, add about 800ml of pure water to dissolve, set the volume in a 1000ml volumetric flask, and store at 4°C until use;
  • PBSB buffer (Running buffer): the above PBS buffer containing 0.2% BSA, pH 7.30; add appropriate amount of BSA powder to PBS buffer before use to make it contain 0.2% BSA;
  • Regeneration buffer an aqueous solution of 4M NaCl
  • Ligand solution Dilute biotinylated heparin with PBS buffer to the desired concentration
  • Sample solution After preparing the stock solution, dilute to the required concentration with PBS buffer;
  • ATIII solution Prepare 1.5mg/ml ATIII solution with pure water, store in -20°C refrigerator, and set aside. Remove before use and dilute with PBSB to the desired concentration.
  • Heparin biotinylation reaction Reference method (Siska Cochran, et al., A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins. Glycoconj J, 2009, 26:577-587; Chuang Xiao, et al.
  • Macromolecular interaction (compound-ATIII) detection On the Octet Red 96 instrument, 200 ⁇ l of sample solution (each well contains 100 ⁇ l of the same concentration of ATIII and 100 ⁇ l of different concentration samples) is added to a flat-bottom black 96-well plate, and the solution in the well is rotated Speed 1000r/min, experiment temperature is 30°C. The experiment was carried out according to the procedure described in Table 29. First, biotinylated heparin was coupled to the SA sensor, and then a series of sample solutions were used to competitively bind to ATIII with immobilized heparin. The sample solution contained 500 nM ATIII.
  • mice Using the tail bleeding method of mice, observe the effect of the pentasaccharide compound of the present invention on the hemostatic function of mice at the same dose, and compare the safety advantages of the pentasaccharide compound of the present invention with pharmacodynamics.
  • Vortex oscillator model VortexGenie, American SCIENTIFIC INDUSTRIES company
  • Constant temperature water bath model HH-4, Jintan Fuhua Co., Ltd.;
  • Pipette gun (10 ⁇ l, 100 ⁇ l, 200 ⁇ l, 1000 ⁇ l range), eppendorf.
  • Enoxaparin Sodium (LMWH): Enoxaparin Sodium Injection, specification 0.4ml: 4000AxaIU, Sanofi-Aventis;
  • mice were randomly divided into 6 groups, 6 in each group, half male and female, respectively:
  • mice in each group were injected with the corresponding drugs subcutaneously (Sc) in the back, the administration volume was 0.1 mL/10 g, and the experiment was conducted 60 minutes after the injection of the drugs.
  • SPSS16.0 statistical analysis software to sort and analyze the data, with mean ⁇ standard deviation Said.
  • One-Sample KS test is used for the normality test of data in different groups, and Levene test is used for the homogeneity of variance test. If the data conforms to the normal distribution, One-Way ANOVA is used to judge its significance; if the data of each group does not conform to the non-normal Distribution, the Cruskal-Wallis H method was used to test the difference between multiple groups, and the Mann-Whiteny U method was used to compare the two groups.
  • Microplate reader Thermo Sientific Multiskan FC, Thermo Sientific, USA;
  • Vortex Oscillator VortexGenie, American SCIENTIFIC INDUSTRIES company
  • Constant temperature water bath HH-4, Jintan Fuhua Co., Ltd.;
  • Pipette gun (10 ⁇ l, 100 ⁇ l, 200 ⁇ l, 1000 ⁇ l range), Eppendorf, Germany;
  • ATIII-dependent anti-factor Xa detection kit BIOPHEN ANTI-Xa: containing R1 (ATIII), R2 (FXa) and R3 (FXa-specific chromogenic substrate (CS-11 (65))), HYPHEN BioMed ( France), Lot: 1800033P4;
  • One-time automatic quantitative blood collection tube (sodium citrate anticoagulation tube), blood collection volume 4mL (containing sodium citrate), Wuhan Zhiyuan Technology Co., Ltd., batch number 20171104.
  • Test article pentasaccharide compound solution Weigh precisely 5.00 mg pentasaccharide compound, add 1ml Buffer to dissolve and mix well, which is the test article mother liquor. Take 100 ⁇ L of the mother liquor and successively dilute 10 pentasaccharide compound solutions with Buffer and set aside.
  • R1, R2 and R3 reagent solutions prepared according to the instructions.
  • Plasma After SD rats were anesthetized by intraperitoneal injection with chloral hydrate (3mL/kg), blood was collected from the abdominal aorta and collected in sodium citrate anticoagulation tube for about 4mL; 1800g ⁇ 10min was centrifuged to take the supernatant.
  • Plasma sample of positive control drug with gradient concentration 25 ⁇ L of plasma + 75 ⁇ L of positive control drug, vortex to mix for 10 s and place in 1.5 mL EP tube for use;
  • the experiment was carried out according to the instructions in the kit: (1) After adding a serial concentration of 30 ⁇ L of sample solution or control solution (Tris-HCl buffer) to each well in a 96-well plate, add 30 ⁇ L of R1 (1IU/mL ATIII) and place in the enzyme In the standard instrument, shake the plate for 15 s and incubate at 37°C for 2 min; (2) Take out the 96-well plate, add 30 ⁇ L R2 (8 ⁇ g/ml FXa solution), place it in the microplate reader, mix the shake plate for 15 s and mix at 37 Precise incubation at °C for 2min; (3) Take out the 96-well plate, add 30 ⁇ L of preheated R3 (FXa-specific chromogenic substrate), place in a microplate reader, mix the plate for 15s, and measure the absorbance at 405nm (OD405nm ), continuous detection for 7min at 20s intervals.
  • sample solution or control solution Tris-HCl buffer
  • the absorbance value (Optical density, OD 405 ) was measured at 405 nm, the plate was read every 20 seconds to detect the absorbance, and the test was continued 22 times.
  • the above method is used for re-hole measurement, and the average value is used when calculating data.
  • Dilution linearity study the mixed plasma of three rats was used as the standard curve, and it was also used as the blank matrix for dilution. Separately, a single rat plasma was mixed with the drug candidate to obtain a high-concentration quality control sample (final drug candidate concentrations are 1500, 2000, and 3000 ng/mL), and each concentration of the sample was diluted to a quantitative range with a blank matrix (candidate drug final (Concentration is 15, 375, 600ng/mL), 5 samples for each concentration of quality control, double well detection. The concentration of the quality control sample is calculated back according to the standard curve and dilution factor, and the accuracy and precision are calculated.
  • Plasma stability investigation Take the mixed plasma of three rats as the blank matrix, and mix the quality control sample with the blank matrix (the final concentration of the candidate drug is 15, 750ng/mL).
  • the blank matrix is placed immediately after preparation at room temperature for 4h, 4 Placed at °C for 12h, -20°C for 24h, and -20°C for 48h. Re-hole detection
  • Plasma-quality control sample stability investigation take the mixed plasma of three rats as the blank matrix, and mix the quality control sample with the blank matrix (the final concentration of the candidate drug is 15,750 ng/mL). Immediately, 4h at room temperature, 12h at 4°C, 24h at -20°C, 48h at -20°C, double well detection.
  • ATIII-dependent anti-factor Xa detection kit BIOPHEN ANTI-Xa: containing R1 (ATIII), R2 (FXa) and R3 (FXa-specific chromogenic substrate (CS-11 (65)), HYPHEN BioMed (France ), Lot: 1800033P5;
  • Buffer Tris NaCl EDTA PEG buffer (pH 8.40), HYPHEN BioMed (France), Lot: F171200766;
  • One-time automatic quantitative blood collection tube sodium citrate anticoagulation tube
  • blood collection volume 4mL containing sodium citrate
  • Wuhan Zhiyuan Technology Co., Ltd. batch number 20161104;
  • Microplate reader Thermo Scientific, Multiskan FC, Thermo Scientific, USA;
  • Vortex oscillator VortexGenie, American SCIENTIFIC INDUSTRIES company
  • FPX Fondaparinux injection (0.5 mL: 2.5 mg), GlaxoSmithkline (UK), Lot: 6497;
  • I-1-1 molecular weight 2019;
  • I-3-1 molecular weight 1875;
  • I-4-1 molecular weight 1861;
  • R1, R2 and R3 reagent solutions R1, R2 are placed at room temperature for 30min, add 1mL of purified water to stabilize at room temperature for 30min, shaking occasionally, then R1, R2 and then add 4mL buffer to mix; R3 directly add 1.6mL of purified water and mix.
  • Blank control group 3 SD rats were randomly selected and injected subcutaneously with physiological saline (Sc);
  • FPX Positive control group
  • test group
  • Group I-1-1 I-1-1 was administered subcutaneously to 6 SD rats at a dose of 0.2019 mg/kg (100 nmol/kg);
  • Group I-2-1 I-2-1 was subcutaneously injected into 6 SD rats at a dose of 0.1963 mg/kg (100 nmol/kg);
  • Group I-3-1 I-2-1 was administered subcutaneously to 6 SD rats at a dose of 0.1875 mg/kg (100 nmol/kg);
  • Group I-4-1 I-4-1 was administered subcutaneously to 6 SD rats at a dose of 0.1861 mg/kg (100 nmol/kg).
  • SD rats were collected plasma at the following time points: control 15 minutes before injection, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours, and 32 hours after injection.
  • the blood collection method is tail vein blood collection.
  • the total volume of each blood collection is 100 ⁇ L, which contains 10% sodium citrate as an anticoagulant (control is 200 ⁇ L).
  • the supernatant is collected by centrifugation at 1800 g for 10 min at room temperature, and immediately enter the downstream test or- Store at 20°C.
  • the pharmacokinetic indexes of the four pentasaccharide compounds at peak time, peak concentration, half-life, and area under the time-blood concentration curve are significantly higher than the control FPX, while clearing The rate is significantly lower than FPX.
  • the above indicators indicate that anticoagulant pentasaccharide has greater blood exposure and longer drug exposure time than FPX.
  • the elimination rate was significantly lower than that of the control FPX.
  • the volume of distribution was slightly lower than FPX except for I-4-1, which was significantly lower than FPX.
  • Reagents chloroformaldehyde hydrate, Tianjin Kemeiou Chemical Reagent Co., Ltd., batch number 20090630; reagents such as gauze, iodophor, alcohol, etc., are all commercially available and are of hygienic level.
  • Group I-1-1 100 nmol/kg
  • Group I-3-1 100 nmol/kg
  • the animals were randomly divided into 16 groups, each of which was divided into 2 groups, and the inferior vena cava thrombosis experiment was performed after 1h or 3h administration respectively.
  • An equal volume of 1ml/kg subcutaneous injection (Sc) in the back was given a set dose of drug or blank control saline.
  • the rabbits were killed by injection of air into the ear vein. Immediately take the brain and place it on ice. Carefully separate and remove the attached blood vessels and meninges. Rinse it with physiological saline. Cut the brain into small pieces with ophthalmic scissors and move it to the test tube. Add 4 Double volume of acetone. Ultrasonic cell disruptor (American SONICS company, VC130PB type) to break the rabbit brain tissue (60KHz, each 3-5 seconds, repeated 2-3 times), centrifuged at 4 °C 4000rpm x 10min to remove the supernatant; repeat adding 4 Double volume of acetone, ultrasonic treatment, centrifugation, a total of 6 to 7 times to remove water and fat.
  • Ultrasonic cell disruptor American SONICS company, VC130PB type
  • brain tissue was laid flat on sulfuric acid paper, dried in a 37°C oven for 30 minutes to obtain brain tissue powder, placed in a cryotube, and stored at -20°C until use. Before use, a 2% concentration suspension was prepared with physiological saline.
  • Rats fasted for 12 hours in advance. Rats were given peritoneal ligation 1h or 3h after administration. Intraperitoneal anesthesia (10% chloral hydrate 0.3ml/100g), cut the abdominal wall longitudinally along the white line of the abdomen, remove the internal organs, separate the inferior vena cava and its branches, and pass a ligature at the lower edge of the left renal vein of the inferior vena cava Line, ligating the inferior vena cava branch below the left renal vein. Rabbit brain powder leachate (2% rabbit brain powder leachate, 1ml/kg) was injected from the femoral vein for 20 seconds and the ligation line of the lower margin of the left renal vein was ligated.
  • Intraperitoneal anesthesia (10% chloral hydrate 0.3ml/100g)
  • Rabbit brain powder leachate 2% rabbit brain powder leachate, 1ml/kg
  • the blood vessel was clamped with a hemostatic forceps 2 cm below the ligature line, the blood vessel was longitudinally cut, the thrombus was removed, the length of the thrombus was measured and the wet weight of the thrombus was measured, and the dry weight was weighed after drying at 50°C for 24 hours, and the inhibition rate was calculated:
  • SPSS16.0 statistical analysis software to sort and analyze the data, with mean ⁇ standard deviation Said.
  • One-Sample KS test was used for the normality test of data between groups, and t test was used for the homogeneity test of variance. P ⁇ 0.05 was considered statistically significant.
  • This test uses a human quality-controlled plasma system to examine the effect of the compound of the present invention on the generation of thrombin in plasma to analyze the inhibitory activity of the compound on thrombin in the plasma system.
  • Thrombin generation analyzer ThrombinoSCOPE: Calibrated automatic thrombin detection system (CAT, Calibrated Automated ), Stago.
  • Thrombin generation experiment reagents Thrombin, Calibrator, Cat#86192. Flu CaKit, Cat#86197.PPP Reagent, Cat#86193, provided by Stago.
  • FPX control group take 40 ⁇ L of FPX with a concentration of 50 ⁇ g/ml and dilute 1:1 with physiological saline to obtain 5 concentration gradients of 25 ⁇ g/ml, 12.5 ⁇ g/ml, 6.25 ⁇ g/ml, 3.125 ⁇ g/ml, 1.562 ⁇ g/ml, mix and reserve;
  • Compound group Take 40 ⁇ L of four compounds with a concentration of 100 ⁇ g/ml, respectively, and dilute 1:1 with physiological saline to obtain 5 concentration gradients: 50 ⁇ g/ml, 25 ⁇ g/ml, and 12.5 ⁇ g /ml, 6.25 ⁇ g/ml, 3.125 ⁇ g/ml, mix well and set aside.
  • Quality control plasma was added with 1ml of deionized water, left to dissolve, and left at room temperature for 30min.
  • Fluo-Buffer at 37°C for at least 5 minutes.
  • Platelet-poor plasma is a trigger for thrombin generation and contains tissue factors and phospholipids. Add 1ml deionized water, dissolve and let stand for 30min.
  • Blank control wells Take 8 ⁇ L of normal saline and 72 ⁇ L of quality control plasma + 20 ⁇ L of PPP in one test well.
  • Calibration hole Take 8 ⁇ L of normal saline + 72 ⁇ L of quality control plasma + 20 ⁇ L Thrombin Calibrator in a detection hole, and each sample is simultaneously calibrated with a fixed amount of thrombin and a thrombin generation experiment to exclude The error caused by the bottom deviation.
  • the thrombin generation analyzer is connected to a computer equipped with automatic analysis software, and the instrument is preheated at 37°C. Put the 96-well plate into the device, operate the instrument according to the software manual, the reaction time is 1 hour, and the detection interval is 20s. Add Fluo-substrate reagent to the automatic sample loading system, add 20 ⁇ l of each Fluo-substrate to the test sample automatically, shake and mix for 50 seconds, the software automatically records thrombin generation data. 8.4 Statistics

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Abstract

本发明涉及其阴离子形式如式（A）所示的抗凝血化合物、其制备方法以及其在制备预防和/或治疗与血液凝固障碍相关的疾病的药物中的用途。

Description

抗凝血的五糖类化合物、组合物及其制备方法和医药用途
本申请要求于2018年12月5日向国家知识产权局提交的发明专利申请CN201811478647.X(发明名称为：抗凝血的五糖类化合物及其制备方法和医药用途)的优先权。
技术领域
本发明涉及用作抗凝血剂的五糖类化合物和组合物，其制备方法以及其在抗凝血方面的医药用途。
背景技术
血栓栓塞性疾病是一类严重危害人类健康的疾病，其发病率高居各种疾病之首，近年来还有渐增之势。其主要分为动脉血栓和静脉血栓。静脉血栓常见于深静脉，临床表现为血栓形成的局部疼痛肿胀、远端血液回流障碍以及血栓脱落后引起的栓塞导致脏器功能障碍。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的，可导致严重的心血管疾病如急性心肌梗死、脑卒中等。治疗方法包括抗凝血治疗、抗血小板治疗及溶栓治疗等，而抗凝血治疗是目前临床上预防和治疗血栓栓塞性疾病的核心和基础。
肝素是传统的抗凝血药物，是糖胺聚糖家族中的一种多糖。临床上使用的包括普通肝素和低分子量肝素。肝素通过附着至抗凝血酶(AT)的特定结合域而诱发AT的构象发生变化，进而抑制凝血因子Xa(FXa)的活性。研究表明，肝素结合AT并抑制凝血因子Xa的最小结构单元是一个独特的五糖序列。
到目前为止，已有多篇文献公开了通过化学全合成制备得到具有抗血栓形成和抗凝血活性的五糖化合物。如美国专利US4818816、Carbohydrate Research 1987(167):67-75报道的磺达肝癸钠，其是基于肝素抗凝血酶结合五糖序列的第一代合成类似物，属于间接凝血因子Xa抑制剂。磺达肝癸钠在大鼠体内的半衰期约为0.7小时(iv)，在人体内的半衰期约为17小时。该品自2002年在美国上市以来，已在多个国家上市，临床上用于治疗和预防深部静脉血栓栓塞事件发生，推荐剂量为2.5mg，每日一次，皮下注射给药。尽管磺达肝癸钠临床表现出色，但是其合成难度极大，生产成本高昂，从而增加患者的经济负担。
因此，仍需要新的抗凝血五糖类化合物以及制备该类化合物的温和的方法。
发明内容
一方面，本发明提供了其阴离子形式如下式(A)所示的五糖化合物：
Figure PCTCN2019122954-appb-000001
其中，R 1选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
R 2选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
R 3选自H或C1～C4烷酰基；
R 4选自H或SO 3 -；
条件是当R 3为H时，R 1和R 2不同时为甲基。
一方面，本发明提供了其阴离子形式如下式I所示的五糖化合物：
Figure PCTCN2019122954-appb-000002
其中，R 1选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
R 2选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
R 3选自H或C1～C4烷酰基；
当R 3为H时，R 1和R 2不同时为甲基。
在一些实施方案中，R 1选自H、SO 3 -、甲酰基、乙酰基、甲基或乙基；在一些典型的实施方案中，R 1选自H、SO 3 -、乙酰基或甲基；在一些更为典型的实施方案中，R 1选自H或甲基。
在一些实施方案中，R 2选自H、SO 3 -、甲酰基、乙酰基、甲基或乙基；在一些典型的实施方案中，R 2选自H、SO 3 -、乙酰基或甲基；在一些更为典型的实施方案中，R 2选自H、SO 3 -或甲基。
在一些实施方案中，R 3选自H、甲酰基或乙酰基；在一些典型的实施方案中，R 3为H或乙酰基；在一些更为典型的实施方案中，R 3为H；在一些更为典型的实施方案中，R 3为乙酰基。
在一些实施方案中，本发明提供了其阴离子形式如下列所示的五糖化合物：
Figure PCTCN2019122954-appb-000003
Figure PCTCN2019122954-appb-000004
在一些典型的实施方案中，本发明提供了其阴离子形式如下列所示的五糖化合物：
Figure PCTCN2019122954-appb-000005
在一些实施方案中，所述化合物以酸形式或盐形式存在，在酸形式中，-COO -和-SO 3 -官能团分别是-COOH形式和-SO 3H形式；在盐形式中，所述盐选自钠盐或钾盐。
在一些更为典型的实施方案中，本发明提供了下列所示的五糖化合物：
Figure PCTCN2019122954-appb-000006
Figure PCTCN2019122954-appb-000007
在一些更为典型的实施方案中，本发明提供了下列所示的五糖化合物：
Figure PCTCN2019122954-appb-000008
另一方面，本发明提供了一种制备其阴离子形式如式I所示的五糖化合物的方法，包括以下步骤：
(1)将式DE所示的二糖与式FGH2所示的三糖反应制备式DEFGH0，
Figure PCTCN2019122954-appb-000009
其中，R a、R b各自独立地选自C1～C4烷酰基、Bn或C1～C4烷基，R g为C1～C4烷酰基；
(2)将式DEFGH0脱除苄基以制备式DEFGH1，
Figure PCTCN2019122954-appb-000010
其中，R c、R d各自独立地选自C1～C4烷酰基、H或C1～C4烷基，R g为C1～C4烷酰基；
(3)将式DEFGH1发生硫酸化反应制备其阴离子形式如式DEFGH2所示的五糖化合物，
Figure PCTCN2019122954-appb-000011
其中，R e、R f各自独立地选自C1～C4烷酰基、SO 3 -或C1～C4烷基，R g为C1～C4烷酰基；
在一些实施方案中，R a、R b各自独立地选自Ac、Bn或甲基；在一些典型的实施方案中，R a选自Ac或甲基，R b选自Ac、Bn或甲基。
在一些实施方案中，R c、R d各自独立地选自Ac、H或甲基；在一些典型的实施方案中，R c选自Ac或甲基，R d选自Ac、H或甲基。
在一些实施方案中，R e、R f各自独立选自Ac、SO 3 -或甲基；在一些典型的实施方案中，R e选自Ac或甲基，R f选自Ac、SO 3 -或甲基。
上述制备方法可以任选地进一步包括步骤(4)：
(4)将步骤(3)的产物水解。
另一方面，本发明还提供了式DEFGH0化合物：
Figure PCTCN2019122954-appb-000012
其中，R a、R b各自独立地选自C1～C4烷酰基、Bn或C1～C4烷基，R g为C1～C4烷酰基。
在一些实施方案中，R a、R b各自独立地选自Ac、Bn或甲基，R g为Ac。
在一些典型的实施方案中，R a选自Ac或甲基，R b选自Ac、Bn或甲基，R g为Ac。
在一些实施方案中，本发明还提供了如下化合物：
Figure PCTCN2019122954-appb-000013
本发明还提供了式DEFGH0在制备其阴离子形式如式I所示化合物中的用途。
在一些实施方案中，本发明提供了式DEFGH10化合物、式DEFGH20化合物、式DEFGH30化合物或式DEFGH40化合物在制备其阴离子形式如式I所示的化合物中的用途。
另一方面，本发明还提供了式DEFGH1化合物：
Figure PCTCN2019122954-appb-000014
其中，R c、R d各自独立地选自C1～C4烷酰基、H或C1～C4烷基，R g为C1～C4烷酰基。
在一些实施方案中，R c、R d各自独立地选自Ac、H或甲基，R g为Ac。
在一些典型的实施方案中，R c选自Ac或甲基，R d选自Ac、H或甲基，R g为Ac。
在一些实施方案中，本发明还提供了如下化合物：
Figure PCTCN2019122954-appb-000015
本发明还提供了式DEFGH1化合物在制备其阴离子形式如式I所示的化合物中的用途。
在一些实施方案中，本发明提供了式DEFGH11化合物、式DEFGH21化合物、式DEFGH31化合物或式DEFGH41化合物在制备其阴离子形式如式I所示的化合物中的用途。
另一方面，本发明还提供了其阴离子形式如式DEFGH2所示的化合物：
Figure PCTCN2019122954-appb-000016
其中，R e、R f各自独立选自C1～C4烷酰基、SO 3 -或C1～C4烷基，R g为C1～C4烷酰基。
在一些实施方案中，R e、R f各自独立选自Ac、SO 3 -或甲基，R g为Ac。
在一些典型的实施方案中，R e选自Ac或甲基，R f选自Ac、SO 3 -或甲基，R g为Ac。
在一些更典型的实施方案中，本发明还提供了其阴离子形式如下列所示的化合物：
Figure PCTCN2019122954-appb-000017
Figure PCTCN2019122954-appb-000018
在一些实施方案中，其阴离子形式如式DEFGH2所示的化合物以酸形式或盐形式存在，在酸形式中，-COO -和-SO 3 -官能团分别是-COOH形式和-SO 3H形式；在盐形式中，所述盐选自钠盐或钾盐。
在一些更为典型的实施方案中，本发明还提供了如下化合物：
Figure PCTCN2019122954-appb-000019
本发明还提供了其阴离子形式如式DEFGH2所示的化合物在制备其阴离子形式如式I所示的化合物中的用途。
在一些实施方案中，本发明还提供了如式DEFGH22-1化合物、式DEFGH32-1化合物或式DEFGH42-1所示的化合物在制备其阴离子形式如式I所示的化合物中的用途。
另一方面，本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氧、硫的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 15O、 17O、 18O、 35S等。某些同位素标记的本申请化合物(例如用 3H及 14C标记的那些)可用于化合物和/或底物组织分布分析中。氘化(即 2H)和碳-14(即 14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如 15O、 11C可用于正电子发射断层扫描(PET)研究测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。
此外，用较重同位素(诸如氘( 2H))取代可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的，其中氘取代可以是部分或完全的，部分氘取代是指至少一个氢被至少一个氘取代。氘代化合物的非限制性实例包括但不限于：
Figure PCTCN2019122954-appb-000020
另一方面，本发明还提供了一种药物组合物，其包含作为活性成分的其阴离子形式如式I所示的五糖化合物。
在一些实施方案中，本发明还提供了一种药物组合物，其包含作为活性成分的其阴离子形式如式I所示的五糖化合物以及一种或多种药用辅料。
在每个单位剂量中，本发明的五糖化合物为0.1～100mg，优选0.5～50mg。
所述药物组合物可通过口服或胃肠道外途径给药，胃肠道外途径给药包括但不限于静脉内注射、肌内注射和皮下注射。
对于口服途径给药，本发明的药物组合物通常以片剂、胶囊剂、溶液的形式提供。片剂可以包含本发明的五糖化合物或其盐以及药学上可接受的赋形剂。所述赋形剂包括但不限于稀释剂、崩解剂、粘合剂、润滑剂、甜味剂、矫味剂、着色剂或防腐剂中的至少一种。胶囊剂包括硬胶囊剂和软胶囊剂。
对于胃肠道外途径给药，本发明的药物组合物可以通过静脉内注射、肌内注射或皮下注射给药。其通常以无菌水溶液或混悬液或冻干粉末提供，并调节合适的pH和等渗性。优选的给药途径是皮下注射给药。
另一方面，本发明还提供了其阴离子形式如式I所示的五糖化合物在制备用于预防和/或治疗与凝血功能障碍相关的疾病或疾病状态的药物中的用途。
另一方面，本发明还提供了用于预防和/或治疗与凝血功能障碍相关的疾病和疾病状态的方法，其包括向有需要的个体给予本发明的其阴离子形式如式I所示的五糖化合物或本发明的药物组合物。
另一方面，本发明还提供了用于预防和/或治疗与凝血功能障碍相关的疾病和疾病状态的本发明的五糖化合物或本发明的药物组合物。
所述与凝血功能障碍相关的疾病或疾病状态的实例包括但不限于静脉血栓形成、动脉血栓形成和血栓性静脉炎等。
本申请的发明人经过深入研究，合成了其阴离子形式如通式I所示的五糖化合物，并进行了生物学实验研究，发现本发明的五糖化合物具有高的抗凝血因子Xa活性以及能够满足临床需求的体内消除半衰期，特别适合用作抗凝血剂。本发明五糖化合物的制备方法大大简化，制造成本低，显著降低了原料药的开发成本，适合于大规模的工业化生产。
附图说明
图1为化合物I-1-1的 1H NMR谱图；
图2为化合物I-1-1的 13C-NMR谱图；
图3为化合物I-1-1的COSY谱图；
图4为化合物I-1-1的HSQC谱图；
图5为不同样品依赖ATIII抑制FXa的活性(n＝4，平均±SD)；
图6为不同样品对b-UFH与ATIII结合的影响(n＝3，平均±SD)。
具体实施方式
定义
除非有相反陈述，下列用在说明书和权利要求书中的术语具有下述含义：
“烷基”指饱和的脂族烃基团，包括直链或支链的饱和一价烃基，所述烃基具有所示出的碳原子数。如术语“C1～C4烷基”包括C1烷基、C2烷基、C3烷基、C4烷基，合适的烷基基团包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基；
“烷酰基”指具有RC(O)-结构的基团，R为H或饱和的脂族烃基团，包括直链或支链的饱和一价烃基。如“C1～C4烷酰基”包括C1烷酰基、C2烷酰基、C3烷酰基、C4烷酰基，合适的烷酰基基团包括甲酰基、乙酰基、正丙酰基、异丙酰基、正丁酰基、异丁酰基、叔丁酰基。
权利要求书和说明书中所使用的简称其含义如下：
Bn：苄基
Piv：特戊酰基
Ms：甲磺酰基
Ac：乙酰基
DMAP：4-二甲氨基吡啶
DBU：1,8-二氮杂二环十一碳-7-烯
TEMPO：2,2,6,6-四甲基哌啶-氮-氧化物
BAIB：碘苯二乙酯
TCCA：三氯异氰尿酸
TMSOTf：三氟甲磺酸三甲基硅酯
Sephadex：葡聚糖凝胶
h：小时
min:分
M：mol/L
nM：nmol/L
APTT：活化部分凝血活酶时间
PT：凝血酶原时间
TT：凝血酶时间
Tris-HCl：三(羟甲基)氨基甲烷盐酸盐
Lot：批号
ATIII：抗凝血酶III
Amine-PEG3-Biotin：胺-PEG3-生物素
Albumin Biovine(BSA)：牛血清白蛋白
UFH：肝素
BLI：生物膜层干涉
Tris NaCl EDTA PEG buffer：Tris NaCl EDTA PEG缓冲液
制备方法：
下面更具体地描述本发明的五糖化合物的制备方法，但这些具体的制备方法不对本发明的范围构成任何限制。此外，反应条件如反应物、溶剂、碱、所用化合物的量、反应温度、反应时间等不限于下面的实例。
本发明的五糖化合物还可以任选地将在本说明书中描述的或本领域已知的各种合成方法组合起来而制得，这样的组合可由本领域的技术人员容易地进行。
第一部分 单糖D环的合成
合成路线一：
Figure PCTCN2019122954-appb-000021
a)硫酸，苯甲醛，DMF；b)苄溴，氢氧化钾，乙腈；c)三乙基硅烷，三氟化硼乙醚，二氯甲烷；d)氢氧化钾，硫酸二甲酯，四氢呋喃；e)醋酸，硫酸，水；f)三氯乙腈，DBU，二氯甲烷
化合物D1-D4的制备：
参考Bioorganic&Medicinal Chemistry Letters,2009,19(14),3875-3879。
化合物D5的制备：
将D4(46.4g)溶于460ml四氢呋喃，加氢氧化钾23.0g，10℃下滴加硫酸二甲酯18.9g，反应4h后加水搅拌淬灭5h，加乙酸乙酯、水萃取洗涤，有机相减压旋干得D5(43.0g)。
化合物D6的制备：
将D5(23.9g)溶于240ml醋酸，加1M的硫酸水溶液24ml，回流反应1.5h后加2N氢氧化钠水溶液24ml中和，然后减压旋干，残余物加乙酸乙酯、水萃取洗涤，有机相减压旋干后经硅胶柱层析得D6(18.6g)。
化合物D7的制备：
将D6(18.6g)加入二氯甲烷190ml溶解，加入三氯乙腈30.0g和DBU 2.0g，反应1h后旋干，柱层析得D7(20.0g)。
合成路线二：
Figure PCTCN2019122954-appb-000022
a)醋酸，硫酸，水；b)醋酐，三乙胺，DMAP，乙酸乙酯；c)吗啡啉，四氢呋喃；g)三氯乙腈，DBU，二氯甲烷
化合物D8的制备：
将D4(464.0g)加入醋酸2.3kg溶解，加1M的硫酸水溶液230ml，回流反应1.5h后加2N氢氧化钠水溶液460ml中和，然后减压旋干，残余物加乙酸乙酯、水萃取洗涤，有机相减压旋干后经硅胶柱层析得D8(360.0g)。
化合物D9的制备：
将D8(225.0g)加入乙酸乙酯2.2L溶解，加入三乙胺260ml，滴加醋酐200ml，室温反应1h后加水淬灭，将反应液分液，有机相减压蒸干得D9(133.5g)。
化合物D10的制备：
将D9(45.0g)加入四氢呋喃350ml溶解，加入吗啡啉143.0g，室温反应6h后加醋酸中和，减压旋蒸，残余物用乙酸乙酯、水萃取洗涤，减压蒸干有机相得D10(33.2g)。
化合物D11的制备：
将D10(33.2g)加入二氯甲烷330ml溶解，加入三氯乙腈50.0g和DBU 2.0g，室温反应1h后旋干，柱层析得D11(36.5g)。
合成路线三：
Figure PCTCN2019122954-appb-000023
a)硫酸二甲酯，KOH，四氢呋喃；b)硫酸，水，碳酸钡；c)乙酸乙酯，醋酐，三乙胺，DMAP；d)TMSOTf，对甲氧基苯酚；e)甲醇钠，甲醇；f)对甲苯磺酸吡啶盐，苯甲醛二甲缩醛；g)氢氧化钾，苄溴；h)三乙基硅烷，三氟乙酸，二氯甲烷；i)乙酸乙酯，醋酐，三乙胺；j)硝酸铈铵，乙腈，水；k)三氯乙腈，DBU，二氯甲烷
化合物D13的制备：
将D12(52.2g)溶于520ml四氢呋喃，加氢氧化钾30g，10℃下滴加硫酸二甲酯50g，反应4h后加水搅拌淬灭5h，加乙酸乙酯、水萃取洗涤，有机相减压旋干得D13(49.9g)。
化合物D14的制备：
将D13(36.3g)加入363ml 0.1M硫酸水溶液中，80℃反应2h后加碳酸钡(14.2g)，搅拌2h后过滤，反应液减压旋干得D14(25.9g)。
化合物D15的制备：
将D14(25.9g)加入乙酸乙酯260ml溶解，加入三乙胺80ml，滴加醋酐60ml，室温反应1h后加水淬灭，将反应液分液，有机相减压蒸干得D15(48.3g)。
化合物D16的制备：
将D15(48.3g)加入250ml无水二氯甲烷溶解，加入对甲氧基苯酚(19.9g)，滴加TMSOTf(6.0g)，室温反应18h后加水淬灭，将反应液分液，有机相减压蒸干，残余物经石油醚/乙酸乙酯2:1重结晶得D16(45.5g)。
化合物D17的制备：
将D16(42.6g)加入340ml无水甲醇溶解，10℃下加入甲醇钠(5.4g)，反应1h后加浓盐酸中和，将反应液减压蒸干得D17粗品(38.0g)，直接投下一步。
化合物D18的制备：
将D17粗品(38.0g)加入380ml N,N-二甲基甲酰胺中溶解，加入苯甲醛二甲缩醛(18.3g)，加对甲苯磺酸吡啶盐调节pH至3，60℃反应3h后加30％氢氧化钠中和。然后将反应液减压蒸干，残余物加饱和碳酸氢钠水溶液搅拌1h后抽滤，滤饼烘干后用石油醚/乙酸乙酯2:1重结晶得D18(35.0g)
化合物D19的制备：
将D18(35g)加入380ml乙腈中溶解，加入苄基溴(17.0g)、氢氧化钾(15.1g)，室温反应8h后加 甲醇回流2h淬灭。然后将反应液减压蒸干，残余物经柱层析得D19(38.8g)。
化合物D20的制备：
将D19(38.8g)加入388ml无水二氯甲烷中溶解，加入三乙基硅烷(14.0g)、三氟乙酸(28.0g)，0℃反应2h，加饱和碳酸钠溶液中和。分液，将有机相减压蒸干，残余物经柱层析得D20(35.0g)。
化合物D21的制备：
将D20(35.0g)加入350ml乙酸乙酯溶解，加入三乙胺80ml，滴加醋酐60ml，室温反应1h，加水淬灭，将反应液分液，有机相减压蒸干得D21(38.1g)。
化合物D22的制备：
将D21(38.1g)加入乙腈380ml、水78ml中，加入硝酸铈铵80.0g，室温反应1h后加饱和亚硫酸钠溶液淬灭。将反应液减压蒸干，残余物经柱层析得D22(24.3g)。
化合物D23的制备：
将D22(24.3g)加入240ml二氯甲烷溶解，加入三氯乙腈(18.0g)和DBU(0.7g)，室温反应1h后旋干，残余物经柱层析得D23(27.8g)。
合成路线四：
Figure PCTCN2019122954-appb-000024
a)醋酐，三乙胺，二氯甲烷；b)TMSOTf，对甲氧基苯酚，二氯甲烷；c)甲醇钠，甲醇；d)四丁基碘化铵，苄基溴，二丁基氧化锡，N,N-二异丙基乙基胺；e)醋酐，三乙胺；f)硝酸铈铵，乙腈；g)三氯乙腈，DBU，二氯甲烷
化合物D24的制备：
将D1(38.8g)加入380ml二氯甲烷溶解，加入三乙胺85.0g，滴加醋酐85.0g，室温反应1h，加水淬灭，将反应液分液、水洗涤，有机相减压蒸干得D24(72.4g)。
化合物D25的制备：
将D24(72.4g)加入600ml无水二氯甲烷溶解，加入对甲氧基苯酚29.8g，滴加TMSOTf 4.4g，室温反应18h，加水淬灭，将反应液分液，有机相减压蒸干，残余物经石油醚/乙酸乙酯2:1重结晶得D25(72.7g)。
化合物D26的制备：
将D25(45.4g)加入340ml无水甲醇溶解，10℃下加入甲醇钠5.4g，反应1h，加浓盐酸中和，将反应液减压蒸干得D26粗品34.0g，直接投下一步。
化合物D27的制备：
将D26粗品(34.0g)加入105.0g N,N-二异丙基乙胺中，加入苄基溴51.0g、二丁基氧化锡2.5g、四丁基碘化铵3.7g，110℃反应6h后加甲醇回流2h淬灭苄基溴。然后将反应液减压蒸干，残余物经柱层析得D27(35.0g)。
化合物D28的制备：
将D27(35.0g)加入350ml乙酸乙酯溶解，加入三乙胺16.0g，滴加醋酐16.0g，室温反应1h，加水 淬灭，将反应液分液、水洗涤，有机相减压蒸干得D28(41.2g)。
化合物D29的制备：
将D28(41.2g)加入乙腈380ml和水78ml的混合溶剂中，加入硝酸铈铵82.2g，室温反应1h，加饱和亚硫酸钠溶液淬灭。将反应液减压蒸干，残余物经柱层析得D29(26.7g)。
化合物D30的制备：
将D29(22.2g)加入220ml二氯甲烷溶解，加入三氯乙腈21.7g和DBU 0.4g，室温反应1h，旋干，残余物经柱层析得D30(25.0g)。
第二部分 单糖E环的制备
合成路线：
Figure PCTCN2019122954-appb-000025
a)对甲苯磺酸吡啶盐，苯甲醛二甲缩醛；b)乙酸乙酯，醋酐，三乙胺，DMAP；c)三乙基硅烷，三氟化硼乙醚，二氯甲烷；d)TEMPO，BAIB，二氯甲烷，水；e)苄基溴，碳酸氢钾，乙腈
化合物E1的制备：
将D14(194.0g)溶于N,N-二甲基甲酰胺1L，加对甲苯磺酸吡啶盐调至pH 3～4，加苯甲醛二甲缩醛(200g)，60℃反应2h后加1M NaOH水溶液调pH至中性。反应液减压旋干，用乙酸乙酯4L搅拌洗涤，析出固体得E1(226.0g)。
化合物E2的制备
将E1(226.0g)溶于乙酸乙酯2L，加三乙胺(100.0g)、DMAP(1.0g)，15℃下滴加(100.0g)醋酐，滴完15℃反应6h后分液。乙酸乙酯相用1L水洗涤，减压旋干，用1L乙酸乙酯重结晶得E2(293.0g)。
化合物E3的制备
将E2(293.0g)溶于无水二氯甲烷2L，加三乙基硅烷(300.0g)，5℃下滴加(30.0g)三氟化硼乙醚，滴完5℃反应2h。将反应液滴入碳酸氢钠饱和溶液淬灭至中性，减压旋干后用石油醚/乙酸乙酯＝0.6L:0.9L重结晶得E3(200.0g)。
化合物4的制备
将E3(200.0g)溶于二氯甲烷1L和水1L的混合溶剂，加入TEMPO(2.0g)，5℃下分4批加入BAIB(463.0g)，每次间隔15min，加完5℃反应1h。将反应液滴入过量亚硫酸钠饱和溶液淬灭，减压旋干后用乙酸乙酯1L重结晶得E4(179.0g)。
化合物E5的制备：
将E4(100.0g)溶于1.5L乙腈，加碳酸氢钾75.0g，室温下加入溴苄(98.0g)，反应4h后过滤，滤液减压旋干，所得残余物加乙酸乙酯、水萃取洗涤，有机相减压旋干得E5粗品，粗品用石油醚/乙酸乙酯1:1重结晶得E5(152.0g)。
第三部分 单糖F环和H环的制备
F环的合成同D11，H环的合成同D4
第四部分：单糖G环的合成
合成路线：
Figure PCTCN2019122954-appb-000026
a)磷钼酸，硅胶，水，乙腈；b)(1)特戊酰氯，吡啶，(2)甲磺酰氯,吡啶；c)叔丁醇钾,叔丁醇；d)硫酸(0.1M)，碳酸钡；e)醋酐，三乙胺，乙酸乙酯；f)对甲氧基苯酚,TMSOTf，二氯甲烷；g)甲醇钠，甲醇；h)苯甲醛二甲缩醛，对甲苯磺酸吡啶盐；i)氢氧化钾，苄基溴；j)硝酸铈铵,乙腈/水；k)三氯乙腈，DBU，二氯甲烷
化合物G1的制备：
将D13(1.5kg)溶于乙腈9.72kg，加硅胶(0.36kg)、磷钼酸(0.036kg)和水(0.8kg)，室温搅拌16h，滤去硅胶，用碳酸钠水溶液中和至pH＝7，滤液减压旋蒸，残余物加水、乙酸乙酯萃取，减压蒸干得G1(1.15kg)。
化合物G2的制备：
将G1(1.15kg)溶于吡啶5L和二氯甲烷5L的混合溶剂，0℃下滴加特戊酰氯(600g)，自然升温至室温，反应8h，然后0℃下滴加甲磺酰氯(600g)，反应3h后加5L二氯甲烷稀释，用5％的盐酸和饱和碳酸氢钠溶液洗涤，然后二氯甲烷相用无水硫酸钠干燥，减压旋干得G2(1.75kg)，直接用于下一步。
化合物G3的制备：
将G2(1.75kg)溶解于10L二氯甲烷和2L叔丁醇的混合溶剂中，0℃下加入叔丁醇钾(1.49kg)，自然升至室温反应8h。然后加水洗涤，二氯甲烷相用无水硫酸钠干燥，减压浓缩得G3(859g)。
化合物G4的制备：
将G3(859.0g)加入0.1M硫酸水溶液4.3L，60℃下反应2h，然后加碳酸钡127.0g中和2h，过滤，滤液减压旋干得G4(771.0g)。
化合物G5的制备：
将G4(25.9g)加入260ml乙酸乙酯溶解，加入三乙胺80ml，滴加醋酐60ml，室温反应1h后加水淬灭，将反应液分液，有机相减压蒸干得G5(48.3g)。
化合物G6的制备：
将G5(48.3g)加入250ml无水二氯甲烷溶解，加入对甲氧基苯酚(19.9g)，滴加TMSOTf(6g)，室温反应18h后加水淬灭，将反应液分液，有机相减压蒸干，残余物经石油醚/乙酸乙酯2:1重结晶得G6(45.5g)。
化合物G7的制备：
将G6(42.6g)加入340ml无水甲醇溶解，10℃下加入甲醇钠(5.4g)反应1h后加浓盐酸中和，将反应液减压蒸干得G7粗品(38g)，直接投入下一步。
化合物G8的制备：
将G7粗品(38g)加入380ml N,N-二甲基甲酰胺中溶解，加入苯甲醛二甲缩醛(18.3g)，加对甲苯磺酸吡啶盐调节pH至3，60℃反应3h后加30％氢氧化钠中和。然后将反应液减压蒸干，残余物加饱和碳酸氢钠水溶液搅拌1h后抽滤，滤饼烘干后用石油醚/乙酸乙酯2:1重结晶得G8(35g)。
化合物G9的制备：
将G8(35g)加入380ml乙腈中溶解，加入苄基溴(17g)、氢氧化钾(15.1g)，室温反应8h后加甲醇回流2h淬灭。然后将反应液减压蒸干，残余物经柱层析(石油醚：乙酸乙酯＝5:1)得G9(38.8g)。
化合物G10的制备：
将G9(957.0g)加入乙腈5L和水2L的混合溶剂中，0℃下加入硝酸铈铵(2.74kg)，反应2h后加乙酸乙酯5L稀释，加饱和亚硫酸钠溶液洗涤，无水硫酸钠干燥，减压浓缩，经柱层析得G10(670.0g)。
化合物G11的制备：
将G10(372.0g)加入二氯甲烷(4.0kg)，加入三氯乙腈(289.0g)和DBU(7.6g)，反应1h后旋干，柱层析得G11(465.0g)。
第五部分 二糖DE的合成
合成路线：
Figure PCTCN2019122954-appb-000027
a)TMSOTf，二氯甲烷；b)吗啡啉，四氢呋喃；c)三氯乙腈，DBU，二氯甲烷
化合物DE1的制备：
取D7(15.2g)和E5(8.7g)溶于180ml无水二氯甲烷，在氮气保护下滴加TMSOTf(0.28g)，-20℃反应1h后加三乙胺淬灭，减压旋干，柱层析(石油醚：乙酸乙酯＝5：1)得1,4-α糖苷键异构体DE1(Rf＝0.45，TLC条件为石油醚：乙酸乙酯＝5：1，16.0g)。
化合物DE2的制备：
取D11(15.9g)和E5(8.7g)溶于180ml无水二氯甲烷，在氮气保护下滴加TMSOTf(0.28g)，-20℃反应1h后加三乙胺淬灭，减压旋干，柱层析(石油醚：乙酸乙酯＝5：1)得1,4-α糖苷键异构体DE2(Rf＝0.40，TLC条件为石油醚：乙酸乙酯＝5：1，16.6g)。
化合物DE3的制备：
取D23(14.0g)和E5(8.7g)溶于180ml无水二氯甲烷，在氮气保护下滴加TMSOTf(0.28g)，-20℃反应1h后加三乙胺淬灭，减压旋干，柱层析(石油醚：乙酸乙酯＝5：1)得1,4-α糖苷键异构体DE3(Rf＝0.45，TLC条件为石油醚：乙酸乙酯＝5：1，15.1g)。
化合物DE4的制备：
取D30(14.7g)和E5(8.7g)溶于180ml无水二氯甲烷，在氮气保护下滴加TMSOTf(0.28g)，-20℃反应1h后加三乙胺淬灭，减压旋干，柱层析(石油醚：乙酸乙酯＝5：1)得1,4-α糖苷键异构体DE4(Rf＝0.35，TLC条件为石油醚：乙酸乙酯＝5：1，15.6g)。
化合物DE5的制备：
将DE1(16.0g)加入160ml四氢呋喃溶解，加入吗啡啉(8.4g)，室温反应6h后加盐酸中和，用乙酸乙酯、水萃取洗涤，有机相减压旋蒸，残余物经石油醚/乙酸乙酯1:1重结晶得DE5(12.2g)。MS(ESI):809.9[M+Na] +
化合物DE6的制备：
将DE2(16.6g)加入166ml四氢呋喃溶解，加入吗啡啉(8.4g)，室温反应6h后加盐酸中和，用乙酸乙酯、水萃取洗涤，有机相减压旋蒸，残余物经石油醚/乙酸乙酯1:1重结晶得DE6(12.6g)。MS(ESI):837.9[M+Na] +
化合物DE7的制备：
将DE3(14g)加入140ml四氢呋喃溶解，加入吗啡啉(8.4g)，室温反应6h后加盐酸中和，用乙酸乙酯、水萃取洗涤，有机相减压旋蒸，残余物经石油醚/乙酸乙酯1:1重结晶得DE7(11.4g)。MS(ESI):761.8[M+Na] +
化合物DE8的制备：
将DE4(14.7g)加入147ml四氢呋喃溶解，加入吗啡啉(8.4g)，室温反应6h后加盐酸中和，用乙酸乙酯、水萃取洗涤，有机相减压旋蒸，残余物经石油醚/乙酸乙酯1:1重结晶得DE8(11.8g)。MS(ESI):789.8[M+Na] +
化合物DE9的制备：
将DE5(12.2g)加入二氯甲烷120ml，加入三氯乙腈(6.7g)和DBU(0.12g)，反应1h后旋干，残余物经柱层析(石油醚：乙酸乙酯＝8：1)得DE9(13.0g)。
化合物DE10的制备：
将DE6(12.6g)加入二氯甲烷120ml，加入三氯乙腈(6.7g)和DBU(0.12g)，反应1h后旋干，残余物经柱层析得DE10(13.3g)。
化合物DE11的制备：
将DE7(11.4g)加入二氯甲烷120ml，加入三氯乙腈(6.7g)和DBU(0.12g)，反应1h后旋干，残余物经柱层析得DE11(12.3g)。
化合物DE12的制备：
将DE8(11.8g)加入二氯甲烷120ml，加入三氯乙腈(6.7g)和DBU(0.12g)，反应1h后旋干，残余物经柱层析得DE12(12.7g)。
第六部分 三糖FGH的合成
合成路线：
Figure PCTCN2019122954-appb-000028
a)TMSOTf，二氯甲烷；b)80％醋酸水溶液；c)三水合磷酸氢二钾、磷酸二氢钾、水、TEMPO、NaClO 2、KBr、TCCA；d)碳酸氢钾，苄基溴，乙腈；e)TMSOTf，二氯甲烷；f)碳酸氢钾，甲醇
化合物GH1的制备：
将G11(310.0g)和D4(232.0g)加入4L无水二氯甲烷中，加入烘干的
Figure PCTCN2019122954-appb-000029
分子筛，-20℃下滴加TMSOTf(11.0g)，反应4h后加三乙胺淬灭，反应液减压旋干后经柱层析得GH1(369.0g)。
化合物GH2的制备：
将GH1(369.0g)加入80％的醋酸水溶液2L，80℃反应3h后减压旋干，残余物加石油醚洗涤，再加乙酸乙酯溶解、饱和碳酸氢钠溶液洗涤，有机相减压旋干得GH2(268.0g)。
化合物GH3的制备：
将GH2(292.0g)加入四氢呋喃2.4L，加入三水合磷酸氢二钾(100g)、磷酸二氢钾(100.0g)、水(150g)、TEMPO(1.6g)、NaClO 2(24g)、KBr(4g)和TCCA(40.0g)，反应2h后加无水亚硫酸钠淬灭，然后减压旋干，残余物用乙酸乙酯洗涤，有机相减压旋干得GH3(223.0g)。
化合物GH4的制备：
将GH3(223.0g)加入乙腈1.8L，加入碳酸氢钾(60.0g)，10℃下滴加苄基溴(62.0g)，反应1h后加乙酸乙酯2L、水1L萃取洗涤，残余物减压蒸干，经柱层析得GH4(213.0g)。
化合物FGH1的制备：
将GH4(167.0g)和D11(153.0g)加入无水二氯甲烷1.7L，加入烘干的
Figure PCTCN2019122954-appb-000030
分子筛，-20℃下滴加TMSOTf(4.4g)，反应4h后加三乙胺淬灭，反应液过滤后减压旋干，经柱层析得FGH1(230.0g)。
化合物FGH2的制备：
将FGH2(131.0g)加入甲醇1.3L，加入碳酸氢钾(15g)，反应8h后旋干，加乙酸乙酯、水萃取洗涤，有机相蒸干经柱层析得FGH2(88.7g)。
1H NMR(500MHz,CDCl 3)δ7.50–7.19(m,45H),5.39(d,J＝6.8Hz,1H),5.27(d,J＝3.5Hz,1H),5.15(d,J＝12.3Hz,1H),5.01(d,J＝2.1Hz,1H),4.99(d,J＝3.6Hz,1H),4.95(d,J＝11.5Hz,1H),4.85(dd,J＝11.6,7.4Hz,2H),4.81–4.77(m,3H),4.68(d,J＝5.4Hz,1H),4.67(dd,J＝3.0,1.8Hz,2H),4.66–4.59(m,3H),4.58–4.55(m,2H),4.51(d,J＝12.1Hz,1H),3.98(dd,J＝8.6,1.6Hz,1H),3.96–3.94(m,1H),3.93(d,J＝3.6Hz,1H),3.92–3.85(m,2H),3.83–3.75(m,2H),3.73–3.68(m,1H),3.68–3.60(m,3H),3.57(dt,J＝9.5,3.5Hz,2H),3.54(s,4H),3.44(s,3H),3.35(t,J＝7.3Hz,1H).
13C NMR(126MHz,CDCl 3)δ169.07,139.28,139.17,138.50,138.44,138.34,138.31,138.14,135.34,128.53,128.49,128.37,128.32,128.26,128.22,128.19,128.12,128.09,128.07,127.81,127.76,127.70,127.63,127.54,127.50,127.35,127.21,127.07,126.99,99.92,99.07,98.34,81.66,81.36,80.85,80.24,80.04,79.37,77.32,77.07,76.81,76.74,76.42,75.39,74.83,74.51,73.59,73.53,73.33,72.82,72.51,72.31,70.59,70.26,68.52,68.13,66.75,60.74,55.28.
实施例1 五糖I-1-1的制备
合成路线：
Figure PCTCN2019122954-appb-000031
化合物DEFGH10的制备：
将DE9(9.3g)和FGH2(6.3g)加150ml无水二氯甲烷溶解，加入烘干的
Figure PCTCN2019122954-appb-000032
分子筛，-20℃下滴加TMSOTf(0.22g)，反应4h后加三乙胺淬灭，反应液过滤后减压旋干，经柱层析(石油醚：乙酸乙酯＝5:1)得DEFGH10(7.6g)。
1H NMR(500MHz,DMSO-d 6)δ7.41–7.17(m,60H),5.43(d,J＝3.4Hz,1H),5.27(d,J＝3.5Hz,1H),5.14–5.01(m,3H),4.97(d,J＝12.4Hz,1H),4.90(d,J＝12.7Hz,1H),4.86–4.54(m,23H),4.51(dd,J＝12.1,4.2Hz,3H),4.46–4.33(m,3H),4.05(q,J＝7.8,5.5Hz,2H),3.98(t,J＝9.1Hz,1H),3.82–3.24(m,29H),2.02(s,3H).
13C NMR(126MHz,DMSO)δ169.45,169.09,168.34,139.56,139.46,139.22,138.95,138.85,138.81,138.66,138.57,138.55,135.91,135.29,128.81,128.79,128.77,128.67,128.66,128.62,128.60,128.56,128.50,128.39,128.25,128.23,128.10,128.06,127.99,127.93,127.90,127.87,127.81,127.78,127.36,100.16,100.03,97.29,96.67,96.33,83.17,81.16,79.96,79.69,79.53,79.39,79.32,78.96,77.01,76.06,75.00,74.74,74.65,74.28,74.21,74.12,73.52,73.01,72.95,72.92,72.72,72.16,72.05,71.69,71.09,70.71,70.32,68.58,67.99,67.34,66.57,60.62,59.51,59.48,55.01,20.97.
化合物DEFGH11的制备：
将DEFGH10(6.1g)溶于无水甲醇61ml，加入10％钯碳(300mg)，氢气常压下还原24h后过滤，滤液旋干，残余物用15ml乙酸乙酯搅拌打浆，然后抽滤，滤饼烘干得DEFGH11(2.3g)。
1H NMR(500MHz,DMSO-d 6)δ5.18–5.02(m,3H),5.00–4.79(m,4H),4.80–4.23(m,9H),4.06(q,J＝7.1Hz,1H),3.95(d,J＝11.9Hz,2H),3.82(t,J＝8.9Hz,1H),3.73–3.13(m,34H),3.05(t,J＝9.5Hz,1H),2.12(s,3H).
13C NMR(126MHz,DMSO)δ170.99,170.80,169.69,101.48,99.95,98.95,97.35,84.12,78.99,78.50,77.95,76.90,74.65,74.48,73.07,72.93,72.81,72.42,67.31,60.47,60.21,60.02,59.94,59.83,59.26,58.02,54.85,21.15.MS(ESI):977.4[M+Na] +,953.4[M-H] -
化合物I-1-1的制备：
将DEFGH11(1.9g)加入N,N-二甲基甲酰胺10ml溶解，加入三氧化硫三甲胺盐(8.4g)，50℃反应24h后，加饱和碳酸氢钠水溶液淬灭，减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得到目标产物(3.2g)。
1H NMR(500MHz,D 2O)δ5.66–5.54(m,1H),5.20(d,J＝3.7Hz,2H),4.97(q,J＝8.0,7.6Hz,1H),4.91(d,J＝7.3Hz,2H),4.55(d,J＝11.2Hz,2H),4.49(dd,J＝9.0,4.7Hz,1H),4.40(m,6H),4.34–4.29(m,3H),4.26(d,J＝12.1Hz,3H),4.10–3.98(m,5H),3.92(d,J＝9.6Hz,1H),3.88–3.82(m,2H),3.65(m,7H),3.60(m,3H),3.52(m,4H),2.33(s,3H).
13C NMR(126MHz,D 2O)δ173.26,99.73,99.34,97.29,96.76,94.18,83.95,80.92,77.53,77.45,77.40,77.37,76.20,75.48,75.38,75.06,74.99,74.92,74.04,73.49,72.06,71.68,69.61,69.58,69.23,69.09,66.24,66.12,65.53,60.47,60.12,59.09,55.41,55.35,20.87.MS(ESI):2018.7[M+H +] +,2040.7[(M+Na +] +,1960.7[M-SO 3+Na +] +
实施例2 五糖I-2-1的制备
合成路线：
Figure PCTCN2019122954-appb-000033
制备方法：
化合物DEFGH20的制备：
将DE10(9.6g)和FGH2(6.3g)加150ml无水二氯甲烷溶解，加入烘干的
Figure PCTCN2019122954-appb-000034
分子筛，-20℃下滴加TMSOTf 0.22g，反应4h后加三乙胺淬灭，反应液过滤后减压旋干，经柱层析(石油醚：乙酸乙酯＝5:1)得 DEFGH20(7.7g)。
1H NMR(300MHz,CDCl 3)δ7.63–6.81(m,60H),5.93(d,J＝3.9Hz,1H),5.63–4.99(m,8H),4.99–4.26(m,24H),4.26–3.16(m,31H),2.21(d,3H),1.80(d,3H).
13C NMR(75MHz,CDCl 3)δ169.64,169.56,169.45,169.32,139.16,139.11,138.55,138.43,138.41,138.35,138.20,138.16,138.14,137.84,137.78,135.33,135.16,128.63,128.58,128.52,128.48,128.46,128.39,128.36,128.30,128.21,128.18,128.14,128.07,128.04,128.00,127.93,127.88,127.79,127.73,127.64,127.61,127.57,127.52,127.38,127.23,123.25,99.87,98.80,98.31,97.40,95.75,81.23,80.64,80.17,79.97,79.50,79.33,79.24,79.17,78.91,78.67,77.55,77.33,77.12,76.91,76.70,76.48,76.27,75.85,75.46,75.36,75.12,75.06,74.40,73.72,73.63,73.56,73.45,73.42,73.30,72.98,72.83,72.54,72.21,71.63,70.97,70.25,70.16,69.36,69.23,68.51,68.00,67.30,67.11,66.67,60.72,57.53,55.31,53.80,20.89,20.85.
化合物DEFGH21的制备：
将DEFGH20(6.2g)溶于无水甲醇61ml，加入10％钯碳300mg，氢气常压下还原24h后过滤，滤液旋干，残余物用15ml乙酸乙酯搅拌打浆，然后抽滤，滤饼烘干得DEFGH21(2.4g)。 1H NMR(500MHz,DMSO-d 6)δ5.21(d,J＝3.8Hz,1H),5.12–4.93(m,4H),4.88(s,2H),4.81(s,1H),4.78–4.67(m,4H),4.65(s,1H),4.55(d,J＝3.6Hz,1H),4.52–4.44(m,1H),4.06(q,J＝7.1Hz,3H),3.95(s,1H),3.87(t,J＝9.0Hz,1H),3.64(d,J＝12.3Hz,2H),3.55(m,9H),3.47(s,4H),3.39(s,6H),3.38–3.32(m,3H),3.32–3.24(m,9H),3.20(s,1H),2.02(d,J＝9.0Hz,6H). 13C NMR(126MHz,DMSO)δ171.04,169.80,169.77,169.71,101.52,99.94,98.77,84.10,76.84,74.60,73.05,72.40,72.32,72.04,71.38,71.26,70.81,70.43,67.99,67.28,60.48,60.20,59.89,59.30,57.99,54.85,21.42,21.17.MS(ESI):1005.4[M+Na] +,981.3[M-H] -
化合物DEFGH22-1的制备：
将DEFGH21(1.96g)加入N,N-二甲基甲酰胺10ml溶解，加入8.4g三氧化硫三甲胺盐，50℃反应24h后，加饱和碳酸氢钠水溶液淬灭，减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得到白色片状固体DEFGH22-1(3.3g)。 1H NMR(500MHz,D 2O)δ5.71(s,1H),5.59(s,1H),5.38–5.13(m,2H),5.11–4.87(m,3H),4.55(m,4H),4.50–4.19(m,15H),4.19–3.77(m,9H),3.66(m,7H),3.56(s,4H),2.37(s,3H),2.22(s,4H).
13C NMR(126MHz,D 2O)δ173.42,172.83,99.83,97.32,96.82,94.24,83.84,80.35,76.99,76.40,75.93,75.57,75.43,75.38,75.00,74.76,74.45,74.16,73.56,72.35,69.66,69.08,68.11,67.95,66.30,65.67,65.59,60.22,59.07,55.49,20.94,20.79..MS(ESI):999.8[(M-2Na)/2] -
五糖I-2-1的制备：
将DEFGH22-1(2.05g)加入1N NaOH水溶液，0℃反应1h后加乙酸铵中和。反应液减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得目标化合物(1.7g)。
1H NMR(500MHz,D 2O)δ5.64-5.55(m,1H),5.28–5.13(m,2H),4.97(s,1H),4.41(m,20H),4.15–3.95(m,7H),3.86(m,4H),3.67(m,4H),3.51(s,3H).
13C NMR(126MHz,D 2O)δ101.12,97.28,96.39,93.83,85.28,80.99,78.13,77.72,76.19,75.93,75.36,74.89,74.58,74.53,73.41,72.49,71.82,69.94,69.71,69.04,68.09,66.21,66.15,65.99,59.86,59.10,55.37,23.29.MS(ESI):1962.7[M+H] +,1984.6[M+Na] +
实施例3 五糖I-3-1的制备
合成路线：
Figure PCTCN2019122954-appb-000035
制备方法：
化合物DEFGH30的制备：
将DE11(8.83g)和FGH2(6.3g)加140ml无水二氯甲烷溶解，加入烘干的
Figure PCTCN2019122954-appb-000036
分子筛，-20℃下滴加TMSOTf 0.22g，反应4h后加三乙胺淬灭，反应液过滤后减压旋干，经柱层析(石油醚：乙酸乙酯＝5:1)得DEFGH30(7.46g)。
1H NMR(300MHz,CDCl 3)δ7.32(m,55H),5.88(d,J＝3.7Hz,1H),5.35(d,J＝6.6Hz,1H),5.27–5.00(m,6H),5.00–4.88(m,2H),4.88–4.40(m,20H),4.35(q,J＝3.8Hz,1H),4.25–3.15(m,31H),3.01(d,3H),2.04(s,3H),1.98(s,3H).
13C NMR(75MHz,CDCl 3)δ169.93,169.60,169.54,169.35,169.10,139.17,139.09,138.84,138.48,138.44,138.41,138.37,138.25,138.20,138.15,138.07,137.80,137.62,135.34,135.21,135.16,128.87,128.61,128.58,128.54,128.51,128.45,128.40,128.36,128.31,128.29,128.24,128.21,128.17,128.14,128.11,128.07,128.02,127.93,127.86,127.81,127.78,127.62,127.60,127.57,127.52,127.38,127.23,123.22,122.94,104.18,99.87,99.07,98.85,98.32,97.42,96.84,95.84,83.27,81.24,80.66,80.51,80.18,79.96,79.33,79.24,78.83,76.28,75.89,75.67,75.47,75.37,74.51,74.40,73.92,73.63,73.57,73.41,73.30,72.90,72.83,72.55,72.20,71.96,71.64,70.99,70.39,70.17,69.30,68.48,68.01,67.26,67.14,66.68,60.72,60.45,57.88,57.50,55.31,21.01,20.94.
化合物DEFGH31的制备：
将DEFGH30(6.0g)溶于无水甲醇61ml，加入10％钯碳(300mg)，氢气常压下还原24h后过滤，滤 液旋干，残余物用15ml乙酸乙酯搅拌打浆，然后抽滤，滤饼烘干得DEFGH31(2.4g)。
1H NMR(500MHz,DMSO-d 6)δ5.33–5.01(m,4H),4.95(t,J＝4.1Hz,1H),4.92–4.59(m,8H),4.55(d,J＝3.6Hz,2H),4.52–4.08(m,4H),4.02(d,J＝9.4Hz,1H),3.94(s,2H),3.86(t,J＝8.9Hz,1H),3.80–3.60(m,5H),3.55(m,6H),3.43(m,12H),3.29(m,6H),3.18(m,2H),2.13(s,3H),2.05(s,3H).
13C NMR(126MHz,DMSO)δ170.96,169.77,169.71,169.56,101.48,101.44,100.29,99.94,98.93,97.39,84.02,80.87,78.54,77.87,76.90,74.68,74.50,73.07,72.85,72.42,72.39,72.28,71.93,71.86,71.39,71.30,71.06,70.42,70.38,69.51,67.95,67.28,60.46,59.95,59.81,59.52,59.24,58.03,54.85,21.27,21.14.MS(ESI):1019.3[M+Na] +,995.3[M-H] -
化合物DEFGH32-1的制备：
将DEFGH31(1.99g)加入N,N-二甲基甲酰胺10ml溶解，加入三氧化硫三甲胺盐(8.4g)，50℃反应24h后，加饱和碳酸氢钠水溶液淬灭，减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得到白色片状固体DEFGH32-1(3.1g)。
1H NMR(500MHz,D 2O)δ5.66(d,J＝3.6Hz,1H),5.61(d,J＝3.5Hz,2H),5.27(dd,J＝11.1,4.0Hz,3H),5.15(q,,2H),5.09–4.92(m,5H),4.69–4.52(m,6H),4.52–4.37(m,2H),4.37–4.19(m,3H),4.19–4.10(m,4H),4.10–3.97(m,4H),3.97–3.78(m,4H),3.76–3.55(m,12H),2.13(s,3H).
13C NMR(126MHz,D 2O)δ173.41,173.08,172.90,99.79,99.40,99.25,97.36,96.90,84.06,80.41,78.59,78.46,77.53,76.98,76.72,76.34,75.68,75.43,75.07,74.56,74.48,74.20,73.69,72.23,71.38,69.71,69.13,68.01,66.32,65.85,65.62,60.56,60.30,59.07,58.00,55.58,21.01,20.65.MS(ESI):955.8[(M-2Na)/2] -
化合物I-3-1的制备：
将DEFGH32-1(1.96g)加入1N NaOH水溶液，0℃反应1h后加乙酸铵中和。反应液减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得目标产物I-3(1.6g)。
1H NMR(500MHz,D 2O)δ5.74–5.49(m,2H),5.42(t,J＝9.8Hz,1H),5.31–5.17(m,3H),5.12–4.82(m,4H),4.53(dt,J＝12.5,9.0Hz,4H),4.47–4.28(m,8H),4.27–4.16(m,3H),4.16–3.75(m,10H),3.66(s,3H),3.62(s,2H),3.58(m,2H),3.53(s,3H).
13C NMR(126MHz,D 2O)δ173.44,172.61,99.71,97.29,96.83,94.18,84.15,80.47,76.37,75.57,75.36,74.96,74.66,74.50,73.68,72.35,70.75,69.64,69.05,67.83,67.58,66.27,65.54,60.46,59.08,55.43,20.88,20.50,20.29.
MS(ESI):913.8[(M-2Na)/2] -
实施例4 五糖I-4-1的制备
合成路线：
Figure PCTCN2019122954-appb-000037
制备方法：
化合物DEFGH40的制备：
将DE12(9.11g)和FGH2(6.3g)加150ml无水二氯甲烷溶解，加入烘干的
Figure PCTCN2019122954-appb-000038
分子筛，-20℃下滴加TMSOTf(0.22g)，反应4h后加三乙胺淬灭，反应液过滤后减压旋干，经柱层析(石油醚：乙酸乙酯＝5:1)得DEFGH40(7.6g)。
1H NMR(500MHz,CDCl 3)δ7.52–7.17(m,55H),5.93–5.83(m,1H),5.52(s,1H),5.41(d,J＝3.6Hz,1H),5.32(s,1H),5.25(s,1H),5.20–5.16(m,2H),5.16–4.94(m,10H),4.94–4.88(m,3H),4.88–4.78(m,7H),4.78–4.36(m,16H),4.14(d,J＝9.2Hz,1H),4.03–3.08(m,16H),3.00–2.81(m,2H),2.19–1.82(m,9H).
13C NMR(126MHz,CDCl 3)δ170.32,169.55,169.08,168.99,167.56,139.30,139.26,138.52,138.41,138.33,138.18,137.82,137.76,137.52,135.57,134.83,128.96,128.85,128.70,128.60,128.56,128.54,128.53,128.50,128.47,128.40,128.37,128.31,128.29,128.27,128.21,128.19,128.10,128.09,128.07,128.04,128.02,128.00,127.98,127.89,127.83,127.78,127.74,127.73,127.71,127.64,127.62,127.58,127.44,127.37,127.18,126.96,124.38,123.48,119.06,118.91,100.30,99.93,99.08,98.23,97.34,83.00,81.26,80.90,80.18,79.52,79.35,78.73,75.39,75.02,73.55,73.53,73.32,73.15,72.96,72.82,72.46,71.97,70.55,70.29,69.25,68.97,67.44,66.75,60.68,59.39,55.25,31.93,31.52,30.36,30.18,29.70,29.36,22.69,20.92,20.84,20.61.
化合物DEFGH41的制备：
将DEFGH40(6.0g)溶于无水甲醇61ml，加入10％钯碳(300mg)，氢气常压下还原24h后过滤，滤 液旋干，残余物用15ml乙酸乙酯搅拌打浆，然后抽滤，滤饼烘干得DEFGH41(2.5g)。
1H NMR(500MHz,DMSO-d 6)δ5.27(d,J＝3.8Hz,1H),5.23–4.19(m,15H),4.16–3.79(m,4H),3.79–3.03(m,31H),2.30–1.88(m,9H).
13C NMR(126MHz,DMSO)δ171.00,170.38,170.15,169.75,169.72,169.53,101.48,99.95,98.80,97.34,83.91,77.95,76.87,75.44,75.26,74.46,73.36,73.14,72.80,72.42,72.30,71.91,71.40,71.02,70.04,69.60,69.31,68.49,67.96,67.27,60.47,60.21,60.04,59.50,59.26,58.02,54.85,40.48,40.32,40.15,39.98,39.81,39.65,39.48,21.17,20.99,14.54..MS(ESI):1047.3[M+Na] +,1023.4[M-H] -
化合物DEFGH42-1的制备：
将DEFGH41(1.99g)加入N,N-二甲基甲酰胺10ml溶解，加入三氧化硫三甲胺盐(8.4g)，50℃反应24h后，加饱和碳酸氢钠水溶液淬灭，减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得到白色片状固体DEFGH42-1(3.1g)。
1H NMR(500MHz,D 2O)δ5.66–5.55(m,2H),5.42(t,J＝9.8Hz,1H),5.32–5.13(m,3H),5.05–4.88(m,4H),4.53(dt,J＝12.5,9.0Hz,4H),4.47–4.16(m,10H),4.16–3.71(m,7H),3.71–3.55(m,6H),3.53(s,3H),2.17(d,J＝10.1Hz,6H).
13C NMR(126MHz,D 2O)δ173.44,172.61,99.71,97.29,96.83,94.18,84.15,80.47,76.37,75.57,75.36,74.96,74.66,74.50,73.68,72.35,70.75,69.64,69.05,67.83,67.58,66.27,65.54,60.46,59.08,55.43,20.88,20.50,20.29.MS(ESI):970.0[(M-2Na)/2] -
化合物I-4-1的制备：
将DEFGH42-1(1.96g)加入1N NaOH水溶液，0℃反应1h后加乙酸铵中和。反应液减压浓缩。残余物经凝胶柱(Sephadex G-25)除盐(去离子水为洗脱剂)，再经732钠型阳离子交换树脂处理成钠盐，该钠盐经GE QFF阴离子交换树脂纯化(0.5M～2M NaCl水溶液梯度洗脱)，最后经凝胶柱(Sephadex G-25)脱盐，冻干得目标产物(1.5g)。
1H NMR(500MHz,D 2O)δ5.61(m,1H),5.19(brs,1H),5.18(d,J＝2.9Hz,1H),4.97(m,3H),4.56-4.30(m,14H),4.29-3.75(m,12H),3.64(m,8H),3.50(s,3H). 13C NMR(126MHz,D 2O)δ101.11,97.28,96.21,93.94,85.72,80.77,77.33,76.31,75.99,75.35,74.88,74.75,74.47,73.96,73.62,72.50,72.12,70.31,69.92,69.68,69.15,69.04,68.83,66.25,66.01,60.05,59.12,55.39,23.34,21.61.
13C NMR(126MHz,D 2O)δ174.30,172.59,101.11,97.28,96.21,93.94,85.72,80.77,77.33,76.31,75.99,75.35,74.88,74.75,74.47,73.96,73.62,72.50,72.12,70.31,69.92,69.68,69.15,69.04,68.83,66.25,66.01,60.05,59.12,55.39,23.34,21.61..MS(ESI):1904.6[M+2Na-H] -。
实施例7生物学实验
本发明的五糖化合物的生物学活性测试可以采用本领域技术人员公知的方法进行测定。可以理解的是，下述测试方法对本发明的范围不构成任何限制。
1.凝血时间检测
1.1试剂
正常凝血质控血浆，MDC Hemostasis，TECO GmbH公司(德国)，Lot:047U-G142A(APTT实验)，Lot：047F-G211A(PT和TT实验)；
APTT试剂：MDC Hemostasis，TECO GmbH公司(德国)，Lot:20002467；
CaCl 2(0.02M)：MDC Hemostasis，TECO GmbH公司(德国)，Lot:031N-G187A；
PT试剂(含兔脑粉和CaCl 2)，MDC Hemostasis，TECO GmbH公司(德国)，Lot:10002518；
TT试剂(含凝血酶)，MDC Hemostasis，TECO GmbH公司(德国)，Lot:30002569；
NaOH:分析纯，天津市大茂化学试剂厂，Lot:20180502；
Tris-HCl：纯度>99.5％，Amresco公司(美国)，Lot:20110723。
1.2对照品
LMWH(低分子肝素)：依诺肝素钠注射液(0.6ml:6000AxaIU)，Sanofi-Aventis公司(法国)，Lot: 5SK26；
Fpx(磺达肝癸钠)：磺达肝癸钠注射液(2.5mg/0.5ml)，GlaxoSmithkline公司(英国)，Lot:6181A；
1.3实验器材
血液凝固分析仪，TECO GmbH公司(德国)，型号MC-4000；
涡旋振荡器，SCIENTIFIC INDUSTRIES公司(美国)，型号VortexGenie；
电子天平，METTLER TOLEDO公司(美国)，型号XS 105DU；
pH计，METTLER TOLEDO公司(美国)，型号FE20；
移液枪(10μl、100μl、200μl、1000μl量程)，Eppendorf；
标准型血凝杯，TECO GmbH公司(德国)，规格：500T；
1.4溶液配制
Tris-HCl缓冲液(0.02M Tris-HCl，pH 7.40)：称量Tris-HCl 3.125g，将其溶解于800ml纯净水中，用0.5M NaOH溶液调pH值至7.40，定溶于1000ml容量瓶，于4℃保存备用；
样品溶液：将已称量样品I-1-1(37mg)、I-2-1(26mg)、I-3-1(17mg)和I-4-1(19mg)分别用纯水溶解配制为40mg/ml储备液，实验前取适量储备液用Tris-HCl缓冲液稀释为1280μg/ml，再用Tris-HCl缓冲液梯度稀释至实验所需浓度。
阳性对照品溶液：实验前取磺达肝癸钠注射液(2.5mg/0.5ml)用Tris-HCl缓冲液稀释为1280μg/ml，再用Tris-HCl缓冲液梯度稀释至实验所需浓度。
APTT、PT、TT试剂溶液及血浆溶液：按各说明书方法配制。
1.5实验过程
APTT检测：按试剂盒说明书方法进行实验：(1)精确量取5μl系列浓度的样品溶液、阳性对照品溶液、Tris-HCl缓冲液加入37℃预温的检测管中，然后加入45μl正常凝血质控血浆，于37℃孵育2min；(2)取50μl 37℃预热的APTT试剂加入检测管中，于37℃孵育混合物3min；(3)将检测管从孵育孔转移至检测孔，加入50μl 37℃预热的0.02M CaCl 2，同时开始计时并记录凝血时间。
PT检测：按试剂盒说明书方法进行实验：(1)精确量取5μl系列浓度的样品溶液、阳性对照品溶液、Tris-HCl缓冲液加入37℃预温的检测管中，然后加入45μl正常凝血质控血浆，于37℃孵育2min；(2)将检测管从孵育孔转移至检测孔，加入100μl 37℃预热的PT试剂，同时开始计时并记录凝血时间。
TT检测：按试剂盒说明书方法进行实验：(1)精确量取10μl系列浓度的样品溶液、阳性对照品溶液、Tris-HCl缓冲液加入37℃预温的检测管中，然后加入90μl正常凝血质控血浆，于37℃孵育2min；(2)将检测管从孵育孔转移至检测孔，加入50μL 37℃预热的TT试剂，同时开始计时并记录凝血时间。
1.6实验结果
实验结果采用软件Excel(2007)分析，以血浆样品终浓度对检测的各凝血时间(4次重复检测的均值)作图，得样品浓度-凝血时间关系方程，并根据该方程计算使血浆凝血时间延长1倍，即倍增APTT、PT或TT所需的化合物终浓度。
(1)APTT检测结果
Tris-HCl缓冲液对照孔，即无样品存在，其四次APTT检测结果分别为33.9s、33.3s、33.2s和34.5s，均值为33.725s，SD为0.60。不同样品存在下，APTT实验结果见下列表所示。
表1 不同浓度LMWH存在下APTT检测结果
Figure PCTCN2019122954-appb-000039
表2 不同浓度Fpx存在下APTT检测结果
Figure PCTCN2019122954-appb-000040
表3 不同浓度I-1-1存在下APTT检测结果
Figure PCTCN2019122954-appb-000041
表4 不同浓度I-2-1存在下APTT检测结果
Figure PCTCN2019122954-appb-000042
表5 不同浓度I-3-1存在下APTT检测结果
Figure PCTCN2019122954-appb-000043
表6 不同浓度I-4-1存在下APTT检测结果
Figure PCTCN2019122954-appb-000044
对样品终浓度(μg/ml)与APTT均值(s)进行线性拟合，并由拟合方程计算使凝血质控血浆延长一倍APTT所需样品浓度，其结果见下表所示。
表7 不同化合物抗凝血活性APTT测试结果
Figure PCTCN2019122954-appb-000045
a使凝血质控血浆APTT时间延长一倍所需的样品浓度；
x：样品终浓度(μg/ml)；
y:APTT时间(s)。
(2)PT检测结果
Tris-HCl缓冲液空白对照孔，即无样品存在下，其四次PT检测结果分别为13.4s、13.5s、13.4s和13.5s，均值为13.45s，SD为0.06。不同样品存在下，PT实验结果见下表所示。
表8 不同浓度LMWH存在下PT检测结果
Figure PCTCN2019122954-appb-000046
表9 不同浓度Fpx存在下PT检测结果
Figure PCTCN2019122954-appb-000047
表10 不同浓度I-1-1存在下PT检测结果
Figure PCTCN2019122954-appb-000048
表11 不同浓度I-2-1存在下PT检测结果
Figure PCTCN2019122954-appb-000049
Figure PCTCN2019122954-appb-000050
表12 不同浓度I-3-1存在下PT检测结果
Figure PCTCN2019122954-appb-000051
表13 不同浓度I-4-1存在下PT检测结果
Figure PCTCN2019122954-appb-000052
对样品终浓度(μg/ml)与PT值(s)进行线性拟合，并由拟合方程计算延长一倍PT所需样品浓度，其结果见下表。
表14 不同化合物抗凝血活性PT测试结果
Figure PCTCN2019122954-appb-000053
a使血浆PT时间延长一倍所需的样品浓度；
b拟合方程线性差(R 2<0.9)，无显著的量效关系。
(3)TT检测结果
Tris-HCl缓冲液空白对照孔，即无样品存在下，其四次TT检测结果分别10.2s、9.2s、9.2s和10.1s，均值为9.675s，SD为0.55。不同样品存在下，TT实验结果见表4-15至4-20。
表15 不同浓度LMWH存在下TT检测结果
Figure PCTCN2019122954-appb-000054
表16 不同浓度Fpx存在下TT检测结果
Figure PCTCN2019122954-appb-000055
Figure PCTCN2019122954-appb-000056
表17 不同浓度I-1-1存在下TT检测结果
Figure PCTCN2019122954-appb-000057
表18 不同浓度I-2-1存在下TT检测结果
Figure PCTCN2019122954-appb-000058
表19 不同浓度I-3-1存在下TT检测结果
Figure PCTCN2019122954-appb-000059
表20 不同浓度I-4-1存在下TT检测结果
Figure PCTCN2019122954-appb-000060
对样品终浓度(μg/ml)与TT值(s)进行线性拟合，并由拟合方程计算延长一倍TT所需样品浓度，其结果见下表。
表21 不同化合物抗凝血活性TT测试结果
Figure PCTCN2019122954-appb-000061
Figure PCTCN2019122954-appb-000062
a使血浆TT时间延长一倍所需的样品浓度；
b拟合方程线性差(R 2<0.9)，无显著的量效关系。
2.ATIII依赖的FXa抑制活性实验
2.1试剂
ATIII依赖的抗因子Xa(FXa)检测试剂盒(BIOPHEN ANTI-Xa)：含R1(ATIII)、R2(FXa)和R3(FXa特异性生色底物(CS-11(65)))，HYPHEN BioMed公司(法国)，Lot:F171100232；
Tris NaCl EDTA PEG缓冲液-pH 8.40，HYPHEN BioMed公司(法国)，Lot:F1700291。
2.2对照品
LMWH(低分子肝素)：依诺肝素钠注射液(0.6ml:6000AxaIU)，Sanofi-Aventis公司(法国)，Lot:5SK26；
Fpx(磺达肝癸钠)：磺达肝癸钠注射液(2.5mg/0.5ml)，GlaxoSmithkline公司(英国)，Lot:6181A；
2.3实验器材
酶标仪，Bio-Tek公司(美国)，型号Microplate Reader(ELx 808)；
涡旋振荡器，SCIENTIFIC INDUSTRIES公司(美国)，型号VortexGenie；
恒温水浴锅，金坛市富华有限公司，型号HH-4；
移液枪(10μl、100μl、200μl、1000μl量程)，Eppendorf；
96孔微孔板，Nest公司(中国)，Lot:110517A005；
2.4溶液配制
Tris-HCl缓冲液(0.02M Tris-HCl，pH 7.40)：同上；
样品溶液及对照品溶液：使用Tris-HCl缓冲液，由样品储备液或对照品配制为128000ng/ml浓度，再梯度稀释至所需浓度；
R1、R2和R3试剂溶液：按各说明书方法配制。
2.5实验过程
按试剂盒说明书方法进行实验：(1)96孔板中各孔分别加入系列浓度的30μl样品溶液、对照品溶液液、Tris-HCl缓冲液后，加入30μl R1(1IU/ml ATIII)，置于酶标仪中，振板混匀并于37℃孵育1min；(2)取出96孔板，加入30μl R2(8μg/ml FXa溶液)，置于酶标仪中，振板混匀并于37℃精确孵育1min；(3)取出96孔板，加入30μl预热的R3(1.2mM FXa特异性生色底物)，置于酶标仪中，检测405nm处的吸光值(OD 405nm)，以30s为间隔连续检测4.5min。
2.6数据处理
取重复检测的OD 405nm均值作为各浓度样品和对照品的检测值，检测结果采用软件Excel(2007)分析，检测值与时间线性拟合，拟合直线的所得斜率即为吸光值的变化率ΔOD 405nm/min，此值对应于FXa的活性。将阴性对照(无抑制剂存在的空白对照孔)的FXa活性定义为1(100％)，计算抑制剂存在下FXa活性的相对值，并以该值与抑制剂浓度作图，并通过Origin8软件进行非线性拟合分析(B＝IC 50 n/{IC 50 n+[I] n})，计算IC 50值。拟合方程式中，B为FXa活性百分比，[I]为抑制剂的浓度，IC 50为半数抑制浓度，即为抑制FXa活性达50％时所需化合物浓度，n为Hill系数。
2.7实验结果
如上所述，以空白对照孔(以溶媒Tris-HCl缓冲液代替样品溶液)的FXa活性定义为1(100％)，计算样品存在下的相对FXa活性(％)，再通过Origin8软件对样品浓度-FXa活性百分数进行非线性拟合，结果见图5，计算半数抑制浓度IC 50值，结果见下列表。
表22 不同浓度LMWH的依赖ATIII抑制FXa的活性结果
Figure PCTCN2019122954-appb-000063
表23 不同浓度Fpx的依赖ATIII抑制FXa的活性结果
Figure PCTCN2019122954-appb-000064
表24 不同浓度I-1-1的依赖ATIII抑制FXa的活性结果
Figure PCTCN2019122954-appb-000065
表25 不同浓度I-2-1的依赖ATIII抑制FXa的活性结果
Figure PCTCN2019122954-appb-000066
表26 不同浓度I-3-1的依赖ATIII抑制FXa的活性结果
Figure PCTCN2019122954-appb-000067
表27 不同浓度I-4-1的依赖ATIII抑制FXa的活性结果
Figure PCTCN2019122954-appb-000068
表28 不同化合物依赖ATIII的抗FXa活性
Figure PCTCN2019122954-appb-000069
3.与肝素竞争结合ATIII活性实验
3.1试剂
ATIII(抗凝血酶III)：HYPHEN BioMed(法国),，Lot:F1700099；
氰基硼氢化钠(NaBH 3CN)：阿拉丁公司，Lot:40509；
胺-PEG3-生物素(Amine-PEG3-Biotin)：Thermo SCIENTIFIC(美国)，Lot：A167761A；
牛血清白蛋白(Albumin Bovine，BSA)：LIFE SCIENCE，Lot:0905C473；
肝素(UFH)：肝素标准品，中国药品生物制品检定所，Lot:15050-200912；
磷酸二氢钠(NaH 2PO 4·2H 2O)：分析纯，天津市大茂化学试剂厂，Lot:20130407；
磷酸氢二钠(Na 2HPO 4·12H 2O)：分析纯，天津市大茂化学试剂厂，Lot:20100515。
氯化钠(NaCl)：分析纯，天津市大茂化学试剂厂，Lot:20180301；
3.2对照品
LMWH(低分子肝素)：依诺肝素钠注射液(0.6ml:6000AxaIU)，Sanofi-Aventis公司(法国)，Lot:5SK26；
Fpx(磺达肝癸钠)：磺达肝癸钠注射液(2.5mg/0.5ml)，GlaxoSmithkline公司(英国)，Lot:6181A；
3.3实验仪器
生物大分子相互作用仪，Pall公司(美国)，型号Octet RED96；
链酶亲和素生物传感器(SA传感器，Streptavidin Biosensors)，Pall公司(美国)，Lot:1709281；
离心机(Spectrafuge,24D)，Labnet公司(美国)，型号D907386；
涡旋振荡器，SCIENTIFIC INDUSTRIES公司(美国)，型号VortexGenie；
电子天平，METTLER TOLEDO公司(美国)，型号XS 105DU；
pH计，METTLER TOLEDO公司(美国)，型号FE20；
96孔板(黑色，平底)，greiner bio-one公司(德国)，Lot:E16083KW；
3.4溶液配制
PBS缓冲液(Loading buffer)：含0.15M NaCl的磷酸盐缓冲液，pH 7.30；分别称取9g NaCl，1.4g NaH 2PO 4·2H 2O，15.4g Na 2HPO 4·12H 2O，置于烧杯，加纯水约800ml溶解后，定容于1000ml容量瓶，于4℃保存备用；
PBSB缓冲液(Running buffer)：含0.2％BSA的上述PBS缓冲液，pH 7.30；临用前加入适量BSA粉末至PBS缓冲液使之含0.2％BSA；
再生缓冲液(Regeneration buffer)：为4M NaCl的水溶液；
配基溶液：将生物素化肝素用PBS缓冲液稀释至所需浓度；
样品溶液：配制储备液后，用PBS缓冲液稀释至所需浓度；
ATIII溶液：用纯水配制1.5mg/ml ATIII溶液，贮存于-20℃冰箱中，备用。使用前取出并用PBSB稀释至所需的浓度。
3.5实验方法
肝素生物素化反应：参考文献方法(Siska Cochran,et al.,A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins.Glycoconj J,2009,26:577-587；Chuang Xiao,et al.,Interactions between depolymerized fucosylated glycosaminoglycan and coagulation proteases or inhibitors.Thromb Res,2016,146:59-68；Boyangzi Li,et al.,Oversulfated chondroitin sulfate interaction with heparin-binding proteins:new insights into adverse reactions from contaminated heparins.Biochem Pharmacol,2009,78:292-300.)，称取约5mg肝素，用水稀释为10mg/ml溶液；加入Amine-PEG3-Biotin混合(与样品的摩尔比约1.5:1)，70℃水浴24h后，加入NaBH 3CN(与样品的摩尔比约1000:1)，70℃继续水浴24h。所得产物采用Zeba TM Spin Desalting Columns(2ml)(>7,000Da)离心脱盐柱脱盐，获得生物素化肝素，于4℃保存备用。
大分子相互作用(化合物-ATIII)检测：在Octet Red 96仪器上，将200μl样品溶液(每孔含100μl相同浓度ATIII及100μl不同浓度的样品)加入到平底黑色96孔板中，孔内溶液旋速1000r/min，实验温度为30℃。按照如表29所述步骤进行实验。先将生物素化肝素偶联到SA传感器上，再通过系列浓度的样品溶液与固定化肝素进行竞争性结合ATIII，其中样品溶液中含有500nM ATIII。
表29 化合物与蛋白相互作用基本实验步骤
Figure PCTCN2019122954-appb-000070
Figure PCTCN2019122954-appb-000071
3.6数据处理及实验结果
检测结果通过软件ForteBio Data Analysis 7.0分析，并计算出相关动力学参数。以化合物浓度为横坐标，用相应动力学曲线的相对响应值(绝对相应值占对照响应值(Control,无样品存在下)的百分比)为纵坐标，并按方程B＝IC 50 n/{IC 50 n+[I] n}进行拟合，得到对应的IC 50值。式中，B为特异性结合信号的百分比，[I]为抑制剂的浓度，IC 50为半数抑制浓度，即为抑制50％BLI信号所需化合物的浓度，n为Hill系数。
读取b-UFH与ATIII结合的响应值，以对照孔即无样品存在下b-UFH与ATIII的结合响应值为100％，计算各个样品的相对响应值(％)，结果见表30至35所示。采用Origin8软件对样品浓度-响应值百分数进行非线性拟合，结果见图6，计算半数抑制浓度IC50值，结果见表36。
表30 不同浓度LMWH存在下b-UFH与ATIII结合的响应值
Figure PCTCN2019122954-appb-000072
表31 不同浓度Fpx存在下b-UFH与ATIII结合的响应值
Figure PCTCN2019122954-appb-000073
表32 不同浓度I-1-1存在下b-UFH与ATIII结合的响应值
Figure PCTCN2019122954-appb-000074
表33 不同浓度I-2-1存在下b-UFH与ATIII结合的响应值表
Figure PCTCN2019122954-appb-000075
Figure PCTCN2019122954-appb-000076
表34 不同浓度I-3-1存在下b-UFH与ATIII结合的响应值
Figure PCTCN2019122954-appb-000077
表35 不同浓度I-4-1存在下b-UFH与ATIII结合的响应值
Figure PCTCN2019122954-appb-000078
表36 不同化合物对b-UFH与ATIII结合的影响
Figure PCTCN2019122954-appb-000079
a由浓度-相对相应值按方程(B＝IC 50 n/{IC 50 n+[I] n})拟合的结果。
4.出血风险评估
4.1实验目的
采用小鼠断尾出血法，观察本发明的五糖化合物在相同剂量下对小鼠止血功能的影响，比较本发明的五糖化合物与药效学相关的安全性优势特征。
4.2实验材料
仪器：
电子天平，型号XS 105DU，美国METTLER TOLEDO公司；
漩涡振荡器，型号VortexGenie，美国SCIENTIFIC INDUSTRIES公司；
电热恒温水浴锅，型号DZKW-D-1，北京市永光明医疗仪器有限公司，；
微型台式高速离心机，型号Spectrafuge 24D，美国Labnet公司；
恒温水浴锅，型号HH-4，金坛市富华有限公司；
移液枪(10μl、100μl、200μl、1000μl量程)，eppendorf。
对照品：
磺达肝癸钠(Fpx)：磺达肝癸钠注射液，规格0.5ml:2.5mg，GSK公司(英国)；
依诺肝素钠(LMWH)：依诺肝素钠注射液，规格0.4ml:4000Axa IU,赛诺菲安万特；
动物：
昆明种小鼠，体重18～22g，雌雄兼用，由昆明医科大学提供，SCXK(滇)2011-0004。
4.3实验方法
分组及给药：
小鼠随机分为6组，每组6只，雌雄各半，分别是：
(1)空白对照(NS)组；
(2)磺达肝癸钠：3.25mg/kg组(10倍的人体等效剂量)；
(3)依诺肝素钠：52mg/kg组(10倍的人体等效剂量)；
(4)4个抗凝五糖化合物：剂量均为3.25mg/kg，4组。
各组小鼠经背部皮下注射(Sc)相应药物，给药体积均为0.1mL/10g，药物注射后60min进行实验。试验过程：
将小鼠置于小鼠固定器中，利用剪尾法(参考文献：Wang JP,Hsu MF,Hsu TP,et al.Antihemostatic and antithrombotic effects of capsaicin in comparison with aspirin and indomethacin.Throm.Res.,1985,37:669-679；Zancan P,
Figure PCTCN2019122954-appb-000080
PAS.Venous and arterial thrombosis in rat models:dissociation of the antithrombotic effects of glycosaminoglycans.Blood Coagulation and Fibrinolysis 2002,15:45-54.)剪去小鼠尾尖5mm,将鼠尾浸入盛有40mL纯净水(37℃)的烧杯中，从剪断鼠尾流出第1滴血起开始计时，并不停搅拌，60min时，取下烧杯放置60min后用紫外分光光度计测吸光度(A540)，并做标准曲线，计算出血量。
统计方法：
应用SPSS16.0统计分析软件整理、分析数据，以均数±标准差
Figure PCTCN2019122954-appb-000081
表示。不同组的数据正态性检验采用One-Sample K-S检验，方差齐性检验采用Levene检验，若数据符合正态分布，则应用One-Way ANOVA判断其显著性；若各组数据不符合非正态分布，多组间差异检验用Cruskal-Wallis H法，两组间比较采用Mann-Whiteny U法。
5生物样品定量分析方法的建立
5.1仪器：
酶标仪：Thermo Sientific Multiskan FC，美国Thermo Sientific公司；
漩涡振荡器：VortexGenie，美国SCIENTIFIC INDUSTRIES公司；
电热恒温水浴锅：DZKW-D-1，北京市永光明医疗仪器有限公司；
微型台式高速离心机：Eppendrof Certrifyge 5804R，德国Eppendorf公司；
恒温水浴锅：HH-4，金坛市富华有限公司；
移液枪(10μl、100μl、200μl、1000μl量程)，德国Eppendorf公司；
96孔微孔板，Nest公司(中国)，Lot:110517A005。
5.2试剂：
ATIII依赖的抗因子Xa检测试剂盒(BIOPHEN ANTI-Xa)：含R1(ATIII)、R2(FXa)和R3(FXa特异性生色底物(CS-11(65)))，HYPHEN BioMed公司(法国)，Lot:1800033P4；
Tris NaCl EDTA PEG buffer-pH 8.40，HYPHEN BioMed公司(法国)，Lot:F1700766P2；
一次性使用自动定量静脉采血管(枸橼酸钠抗凝管)，采血量4mL(含枸橼酸钠)，武汉致远科技有限公司，批号20171104.
5.3受试品：
本发明的五糖化合物
5.4对照品：
磺达肝癸钠(Fpx)：磺达肝癸钠注射液(0.5ml：2.5mg)，GlaxoSmithkline公司(英国)，Lot:6497
5.5动物：
SD大鼠，雄性，体重250～350g，湖南斯莱克景达实验动物有限公司，许可证号：SCXK(湘)2016-0002
5.6实验方法
试剂配制:
受试品五糖化合物溶液：精密称取五糖化合物5.00mg，加1ml Buffer溶解混匀，即为受试品母液。取母液100μL依次用Buffer稀释10个五糖化合物溶液，备用。
R1、R2和R3试剂溶液：按各说明书方法配制。
血浆处理:
a)血浆：SD大鼠用水合氯醛(3mL/kg)腹腔注射麻醉后，腹主动脉取血收集在枸橼酸钠抗凝管中约4mL；1800g×10min离心取上清。
b)加药物：取稀释后系列浓度候选药物溶液和Buffer按照以下方式加入到血浆中：
(1)梯度浓度阳性对照药物的血浆样品：25μL血浆+75μL阳性对照药物，旋涡混匀10s后置于1.5mL EP管备用；
(2)梯度浓度候选药物的血浆样品：25μL血浆+75μL候选药物溶液，旋涡混匀10s后置于1.5mL EP管备用；
(3)空白对照样品(2份)：25μL血浆+75μL buffer，旋涡混匀10s置于1.5mL EP管备用。
生色底物法检测:
按试剂盒说明书方法进行实验：(1)96孔板中各孔分别加入系列浓度的30μL样品溶液或对照液(Tris-HCl缓冲液)后，加入30μL R1(1IU/mL ATIII)，置于酶标仪中，振板混匀15s并于37℃孵育2min；(2)取出96孔板，加入30μL R2(8μg/ml FXa溶液)，置于酶标仪中，振板混匀15s并于37℃精确孵育2min；(3)取出96孔板，加入30μL预热的R3(FXa特异性生色底物)，置于酶标仪中，振板混匀15s，检测405nm处的吸光值(OD405nm)，以20s为间隔连续检测7min。在405nm处检测吸光值(Optical density,OD 405)，每20秒读板检测一次吸光度，连续检测22次。上述方法复孔测定，计算数据时取平均值。
5.7数据处理
以复孔检测的OD 405平均值为纵坐标(OD 405)，检测时间为横坐标(t)，进行线性拟合，斜率为OD 405的变化率(ΔOD 405/min)反映出FXa的剩余量，此值对应于FXa的活性。将阴性对照(无抑制剂存在的空白对照孔)的FXa活性定义为1(100％)，计算抑制剂存在下FXa活性的相对值，并以该值与抑制剂浓度作图，并通过Origin8软件进行非线性拟合分析(B＝IC 50 n/{IC 50 n+[I] n})，计算IC 50值。拟合方程式中，B为FXa活性百分比，[I]为抑制剂的浓度，IC 50为半数抑制浓度，即为抑制FXa活性达50％时所需化合物浓度，n为Hill系数。
此外还采用(1)Excel2003进行y＝a ln(x)+b拟合；(2)酶免助手3.7进行y＝(A+B)/[1+(x/C)^D]-B拟合。选择拟合度R 2较高及质控样品回算百分率最符合80～120％的拟合方式进行报告。
5.8方法学的建立内容
混合血浆作标准曲线：随机取三只大鼠，腹主动脉取血后分离血浆并(1:1:1)混合；用buffer配制系列浓度的候选药物浓度；分别取75μL候选药物样品与25μL血浆混匀，轻轻振荡使混合均匀(候选药物终浓度为0、6.25、12.5、25、50、100、200、400、800ng/mL)，复孔检测。将候选药物与药物活性的相对值线性拟合得到标准曲线。
精密度与准确度考察：考察批内精密度与准确度，与混合血浆作标准曲线同步进行。用buffer配制系列浓度的候选药物浓度(8.33、20、500、800ng/mL)；分别取75μL候选药物样品与25μL血浆混匀，轻轻振荡使混合均匀(候选药物终浓度为6.25、15、375、600ng/mL)，每个质控的浓度做5个样品，复孔检测。按相应的混合血浆所作标准曲线回算候选药物的浓度，计算批内的精密度与准确度。
稀释线性考察：取三只大鼠的混合血浆作标准曲线，同时也作为稀释用空白基质。另取单只大鼠血浆与候选药物混合得到高浓度的质控样品(候选药物终浓度为1500、2000、3000ng/mL)，用空白基质将每一浓度的样品稀释至定量范围(候选药物终浓度为15、375、600ng/mL)，每个质控的浓度做5个样品，复孔检测。根据标准曲线和稀释倍数回算质控样品浓度，计算准确度和精密度。
血浆稳定性考察：取三只大鼠的混合血浆作为空白基质，质控样品与空白基质混合(候选药物终浓度为15、750ng/mL)，空白基质分别在配制后即刻、室温放置4h、4℃放置12h、-20℃放置24h、-20℃放置48h，复孔检测。
血浆-质控样品稳定性考察：取三只大鼠的混合血浆作为空白基质，质控样品与空白基质混合(候选药物终浓度为15、750ng/mL)，血浆-质控样品分别在配制后即刻、室温放置4h、4℃放置12h、-20℃放 置24h、-20℃放置48h，复孔检测。
6.大鼠体内药代动力学研究
6.1试剂
ATIII依赖的抗因子Xa检测试剂盒(BIOPHEN ANTI-Xa)：含R1(ATIII)、R2(FXa)和R3(FXa特异性生色底物(CS-11(65))，HYPHEN BioMed公司(法国)，Lot:1800033P5；
Buffer：Tris NaCl EDTA PEG缓冲液(pH 8.40)，HYPHEN BioMed公司(法国)，Lot:F171200766；
一次性使用自动定量静脉采血管(枸橼酸钠抗凝管)，采血量4mL(含枸橼酸钠)，武汉致远科技有限公司，批号20161104；
96孔微孔板：Nest公司，Lot:20160170902B。
6.2仪器
酶标仪：Thermo Scientific Multiskan FC，美国Thermo Scientific公司；
涡旋振荡器：VortexGenie，美国SCIENTIFIC INDUSTRIES公司；
电子天平：ME104E，瑞士METTLER TOLEPO公司；
高速离心机：Eppendrof Centrifyge 5804R，德国Eppendorf公司；
恒温水浴槽：DK-8AXX，上海一恒科学仪器有限公司；
移液枪(10μL、100μL、200μL和1000μL量程)，德国Eppendorf公司；
6.3受试品：
磺达肝癸钠(FPX)：磺达肝癸钠注射液(0.5mL：2.5mg)，GlaxoSmithkline公司(英国)，Lot:6497；
以下受试品由南京正大天晴制药有限公司提供，批号无:
I-1-1：分子量2019；
I-2-1：分子量1963；
I-3-1：分子量1875；
I-4-1：分子量1861；
6.4动物：
SD大鼠，雄性，体重250～300g，湖南斯莱克景达实验动物有限公司，许可证号：SCXK(湘)2016-0002。6.5实验过程
6.5.1试剂配制
R1、R2和R3试剂溶液：R1、R2在室温下放置30min，加1mL纯净水室温下稳定30min，间或摇动，然后R1、R2再加4mL buffer混匀；R3直接加1.6mL纯净水混匀。
6.5.2试验分组：
空白对照组：随机选取3只SD大鼠，使用生理盐水进行皮下注射(Sc)；
阳性对照组(FPX)：将FPX按0.1728mg/kg的剂量(100nmol/kg)分别对6只SD大鼠进行皮下注射给药；
实验组：
(1)I-1-1组：将I-1-1按0.2019mg/kg的剂量(100nmol/kg)分别对6只SD大鼠进行皮下注射给药；
(2)I-2-1组：将I-2-1按0.1963mg/kg的剂量(100nmol/kg)分别对6只SD大鼠进行皮下注射给药；
(3)I-3-1组：将I-2-1按0.1875mg/kg的剂量(100nmol/kg)分别对6只SD大鼠进行皮下注射给药；
(4)I-4-1组：将I-4-1按0.1861mg/kg的剂量(100nmol/kg)分别对6只SD大鼠进行皮下注射给药。
6.5.3血浆采集
SD大鼠分别按以下时间点进行血浆采集：注射前对照15min，注射后30min、1h、2h、3h、4h、6h、8h、10h、24h、32h。
取血方式为尾静脉采血，每次采血总体积为100μL，其中含10％的枸橼酸钠作为抗凝剂(对照为200μL)，室温下1800g离心10min取上清，立即进入下游试验或-20℃保存。
6.5.4生色底物法检测
(1)96孔板中各孔分别加入系列浓度的30μL样品溶液或对照液后，加入30μL R1(1IU/mL ATIII)，置于酶标仪中，振板混匀并于37℃孵育1min；(2)取出96孔板，加入30μL R2(8μg/mL FXa溶液)，置于酶标仪中，振板混匀并于37℃精确孵育1min；(3)取出96孔板，加入30μL预热的R3(FXa特异性生色底物)，置于酶标仪中，检测405nm处的吸光值(OD 405nm)，以30s为间隔连续检测7min。复孔检测，计算数据时取平均值。
其中I-3-1组和I-4-1组以及FPX组的一只大鼠是采用20μL体系检测(即样品和R1，R2，R3加样体积均为20μL)，其余条件均无变化。
6.5.5数据处理
(1)以检测的OD405为纵坐标(OD405)，检测时间为横坐标(t)，取7个点进行线性拟合，复孔检测的平均斜率值为OD405的变化率(ΔOD405/min)反映出FXa的剩余量，此值对应于FXa的活性。将阴性对照(注射药物前的血浆)的FXa活性定义为1(100％)，计算抑制剂存在下FXa活性的相对值，并以该值与抑制剂浓度作图，并通过Origin8软件进行非线性拟合分析(B＝IC 50 n/{IC 50 n+[I] n})，计算IC 50值。拟合方程式中，B为FXa活性百分比，[I]为抑制剂的浓度，IC 50为半数抑制浓度，即为抑制FXa活性达50％时所需化合物浓度，n为Hill系数。
(2)将检测得到的血药浓度和相应的时间导入DAS 2.0软件，通过药代动力学的智能化分析获取每只大鼠的各项药代动力学参数。然后对每组各只大鼠的评价值进行各项药代动力学数值进行统计分析。
6.6实验结果
对以上检测的对照样品(磺达肝癸钠)以及受试样品(4种不同的抗凝五糖)的各项大鼠体内药代动力学指标统计学分析结果进行整合如表37。
表37 皮下注射FPX及受试五糖化合物后各组评价值的药代动力学参数汇总
Figure PCTCN2019122954-appb-000082
从表37中可以看出，4种五糖化合物在达峰时间、达峰浓度、半衰期以及时间-血药浓度曲线下面积等各项药代动力学指标均显著高于对照品FPX，而清除速率则显著低于FPX。以上指标说明抗凝五糖与FPX相比，有更大血液暴露量及更长的药物暴露时间。而消除率均显著低于对照FPX，分布容积除I-4-1显著低于FPX外，其余三个化合物略低于FPX。
7.本发明化合物对大鼠下腔静脉血栓形成的影响
7.0实验材料
阳性对照：磺达肝癸钠注射液：缩写为FPX，0.5ml:5mg/ml，GSK公司(英国)，批号：6497。
试剂：水合三氯乙醛，天津市科密欧化学试剂有限公司，批号20090630；纱布，碘伏，酒精等试剂，均为市售，卫生级别。
动物：SD大鼠，雄性，重量220～250g，由湖南斯莱克景达实验动物有限公司提供，SCXK(湘)2016-0002。
7.1分组与处理
(1)生理盐水组：1ml/kg；
(2)FPX组：100nmol/kg；
(3)I-1-1组：100nmol/kg；
(4)I-2-1组：100nmol/kg；
(5)I-3-1组：100nmol/kg；
(6)I-4-1组：100nmol/kg。
动物随机分组，每个药物16只，各均分为2组，分别给药1h或3h后做下腔深静脉血栓实验。等容量1ml/kg背部皮下注射(Sc)给予设定剂量的药物或空白对照生理盐水。
7.2家兔脑粉混悬液制备
家兔经耳缘静脉注射空气处死，立即取脑，置于冰上，仔细分离去除附着的血管和脑膜，用生理盐水冲洗干净，用眼科剪将脑剪成小块移至试管中，加入4倍体积丙酮。超声波细胞破碎仪(美国SONICS公司，VC130PB型)将兔脑组织打碎(60KHz,每次3-5秒,反复2-3次)，4℃下4000rpm x 10min离心去上清液；重复加入4倍体积丙酮、超声波处理、离心，共6～7次，以除去水分及脂肪。最后将脑组织平铺在硫酸纸上，置37℃烘箱中烘干30min得脑组织粉末，置于冻存管内，-20℃储存备用。使用前，用生理盐水配制成浓度2％的混悬液。
7.3兔脑粉浸出液诱导下腔静脉血栓形成
大鼠提前禁食12h。大鼠给药1h或3h后，行腹腔静脉结扎术。腹腔麻醉(10％水合氯醛0.3ml/100g)，沿腹白线纵向剪开腹壁，移开内脏，分离下腔静脉及其分支，于下腔静脉的左肾静脉下缘穿过一根结扎线，结扎左肾静脉之下的下腔静脉分支。自股静脉注射兔脑粉浸出液(2％兔脑粉浸出液，1ml/kg)循环20s后结扎左肾静脉下缘结扎线。结扎20min，在结扎线下2cm处用止血钳夹闭血管，纵向剖开血管，取出血栓，测量血栓长度并称量血栓湿重，50℃烘干24h后称取干重，并计算抑制率：
Figure PCTCN2019122954-appb-000083
7.4统计分析
应用SPSS16.0统计分析软件整理、分析数据，以均数±标准差
Figure PCTCN2019122954-appb-000084
表示。组间的数据正态性检验采用One-Sample K-S检验，方差齐性检验采用t检验显著性。以p<0.05为差异有统计学意义。
7.5实验结果
表38 化合物给药1h后对下腔静脉血栓形成的影响(n＝8)
Figure PCTCN2019122954-appb-000085
Figure PCTCN2019122954-appb-000086
Figure PCTCN2019122954-appb-000087
与空白对照组相比， ***P<0.001； **P<0.01； *P<0.05。
表39 化合物给药3h后对下腔静脉血栓形成的影响(n＝8)
Figure PCTCN2019122954-appb-000088
Figure PCTCN2019122954-appb-000089
与空白对照组相比， ***P<0.001； **P<0.01； *P<0.05。
8.本发明化合物对血浆中凝血酶生成的抑制活性
本试验采用人质控血浆系统，考察本发明化合物对血浆中凝血酶生成量的影响，以分析化合物对血浆系统中凝血酶的抑制活性。
8.1实验仪器
凝血酶生成分析仪(ThrombinoSCOPE)：校正的自动凝血酶检测系统(CAT，Calibrated Automated
Figure PCTCN2019122954-appb-000090
)，Stago公司。
8.2试剂
阳性对照：磺达肝癸钠注射液：缩写为FPX，0.5ml:5mg/ml，GSK公司(英国)，批号：6497
质控血浆，Bedford公司提供，NO：889958；
凝血酶生成实验试剂：Thrombin Calibrator,Cat#86192.Flu Ca Kit,Cat#86197.PPP Reagent,Cat#86193，Stago公司提供。
8.3实验方法
溶液配制：
FPX对照组：取浓度为50μg/ml的FPX 40μL,用生理盐水按1：1梯度稀释，得到5个浓度梯度分别为25μg/ml,12.5μg/ml,6.25μg/ml,3.125μg/ml,1.562μg/ml，混匀备用；
化合物组：分别取浓度为100μg/ml的四种化合物各40μL，用生理盐水按1:1梯度稀释，均得到5个浓度梯度的待测液分别为：50μg/ml,25μg/ml,12.5μg/ml,6.25μg/ml,3.125μg/ml，混匀备用。
实验步骤：
(1)试剂准备：
质控血浆加入1ml去离子水，静置溶解，室温放置30min。
在37℃预热Fluo-Buffer至少5分钟以上。在Fluo-Buffer中加入Fluo-Substrate试剂，两者比例为40:1。混匀，置于37℃水浴锅备用。
在Thrombin Calibrator管中加入1ml去离子水轻摇溶解，室温静置放置30min。
贫血小板血浆(PPP)为促使凝血酶生成的触发剂，含有组织因子及磷脂。加1ml去离子水，溶解静置30min。
(2)加样分析
1)按下述方案构建120μL的反应体系：
质控血浆72μL
待测受试品或生理盐水8μL
PPP reagent high激活剂或校正液20μL
Flu-substrate含Ca 2+缓冲液(1:40)20μL
2)样本设置
空白对照孔：取8μL生理盐水与72μL质控血浆+20μL PPP于一个检测孔。
定标孔：取8μL生理盐水+72μL的质控血浆+20μL Thrombin Calibrator于一个检测孔，对每个样本分别同时用固定含量的凝血酶进行定标及进行凝血酶生成实验，排除由于样本血浆本底偏差所造成的误差。
FPX对照孔：取8μL样品+72μL质控血浆+20μL PPP于一个检测孔(FPX:质控血浆＝1:9)，共五个浓度的样品，每个浓度梯度均按此法加样。
样品孔：每个化合物分别按不同的浓度取8μL+72μL质控血浆+20μL PPP于一个检测孔(化合物:质控血浆＝1:9),一共五个浓度的样品，每个浓度梯度均按此法加样。
(3)仪器操作
将凝血酶生成分析仪与一台装有自动分析软件的电脑相连，仪器37℃预热。将96孔板放入设备中，按照软件说明书操作仪器，反应时间1小时，检测间隔：20s。将Fluo-substrate试剂加入到自动加样系统，按每个孔20μl Fluo-substrate自动加入到受试样品中，震荡混匀50秒后，软件自动记录凝血酶生成数据。8.4数据统计
每个分析样品相同浓度下，在同一条件下复孔检测3次，取平均值。数据用凝血酶生成分析仪自带软件实时记录凝血酶生成曲线，软件计算曲线下积分面积(ETP，nM·min，内源性凝血酶生成潜力值,反映每分钟凝血酶生成的量)，结果如表40、41所示。
表40 FPX抑制人质控血浆凝血酶生成的ETP(nM·min)数据(n＝3，X±SD)
Figure PCTCN2019122954-appb-000091
表41 本发明化合物抑制人质控血浆凝血酶生成的ETP(nM·min)数据(n＝3，X±SD)
Figure PCTCN2019122954-appb-000092
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (28)

  1. 化合物，其阴离子形式具有如下式(A)所示结构，
    Figure PCTCN2019122954-appb-100001
    其中，R 1选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
    R 2选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
    R 3选自H或C1～C4烷酰基；
    R 4选自H或SO 3 -；
    条件是当R 3为H时，R 1和R 2不同时为甲基。
  2. 根据权利要求1所述的化合物，其阴离子形式具有如下式(I)所示结构：
    Figure PCTCN2019122954-appb-100002
    其中，R 1选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
    R 2选自H、SO 3 -、C1～C4烷酰基或C1～C4烷基；
    R 3选自H或C1～C4烷酰基；
    当R 3为H时，R 1和R 2不同时为甲基。
  3. 根据权利要求1～2任一项所述的化合物，其特征在于，R 1选自H、SO 3 -、甲酰基、乙酰基、甲基或乙基；优选H、SO 3 -、乙酰基或甲基；更优选H或甲基。
  4. 根据权利要求1～2任一项所述的化合物，其特征在于，R 2选自H、SO 3 -、甲酰基、乙酰基、甲基或乙基；优选H、SO 3 -、乙酰基或甲基；更优选H、SO 3 -或甲基。
  5. 根据权利要求1～2任一项所述的化合物，其特征在于，R 3选自H、甲酰基或乙酰基；优选 H或乙酰基；更优选为H；更优选为乙酰基。
  6. 根据权利要求1～2任一项所述的化合物，其阴离子形式选自下列结构：
    Figure PCTCN2019122954-appb-100003
  7. 根据权利要求1～2任一项所述的化合物，其阴离子形式选自下列结构：
    Figure PCTCN2019122954-appb-100004
  8. 根据权利要求1～7任一项所述的化合物，其以酸形式或盐形式存在，所述盐选自钠盐或钾盐。
  9. 根据权利要求1所述的化合物，选自下列化合物：
    Figure PCTCN2019122954-appb-100005
    Figure PCTCN2019122954-appb-100006
  10. 根据权利要求1所述的化合物，选自下列化合物，
    Figure PCTCN2019122954-appb-100007
  11. 制备权利要求2所述化合物的方法，包括以下步骤：
    (1)将式DE所示的二糖与式FGH2所示的三糖反应制备式DEFGH0，
    Figure PCTCN2019122954-appb-100008
    其中，R a、R b各自独立地选自C1～C4烷酰基、Bn或C1～C4烷基，R g为C1～C4烷酰基；
    (2)将式DEFGH0脱除苄基以制备式DEFGH1，
    Figure PCTCN2019122954-appb-100009
    其中，R c，R d各自独立地选自C1～C4烷酰基、H或C1～C4烷基，R g为C1～C4烷酰基；
    (3)将式DEFGH1发生硫酸化反应制备其阴离子形式如式DEFGH2所示的五糖化合物，
    Figure PCTCN2019122954-appb-100010
    其中，R e，R f各自独立选自C1～C4烷酰基、SO 3 -或C1～C4烷基，R g为C1～C4烷酰基；
    任选地进一步包括步骤(4)：
    (4)将步骤(3)的产物水解。
  12. 根据权利要求11所述的方法，其特征在于，R a、R b各自独立地选自Ac、Bn或甲基；优选R a为Ac或甲基，Rb为Ac、Bn或甲基。
  13. 根据权利要求11所述的方法，其特征在于，R c、R d各自独立地选自Ac、H或甲基；优选R c为Ac或甲基，R d为Ac、H或甲基。
  14. 根据权利要求11所述的方法，其特征在于，R e、R f各自独立选自Ac、SO 3 -或甲基；优选R e为Ac或甲基，R f为Ac、SO 3 -或甲基。
  15. 下式化合物，
    Figure PCTCN2019122954-appb-100011
    其中，R a、R b各自独立地选自C1～C4烷酰基、Bn或C1～C4烷基，R g为C1～C4烷酰基。
  16. 根据权利要求15所述化合物，其特征在于，R a、R b各自独立地选自Ac、Bn或甲基，R g为Ac；优选的，R a选自Ac或甲基，R b选自Ac、Bn或甲基，R g为Ac。
  17. 下列化合物，
    Figure PCTCN2019122954-appb-100012
  18. 下式化合物，
    Figure PCTCN2019122954-appb-100013
    其中，R c、R d各自独立地选自C1～C4烷酰基、H或C1～C4烷基，R g为C1～C4烷酰基。
  19. 根据权利要求18所述的化合物，其特征在于，R c、R d各自独立地选自Ac、H或甲基，R g为Ac；优选的，R c选自Ac或甲基，R d选自Ac、H或甲基，R g为Ac。
  20. 下列化合物，
    Figure PCTCN2019122954-appb-100014
    Figure PCTCN2019122954-appb-100015
  21. 化合物，其阴离子形式具有如下式结构所示：
    Figure PCTCN2019122954-appb-100016
    其中，R e、R f各自独立选自C1～C4烷酰基、SO 3 -或C1～C4烷基，R g为C1～C4烷酰基。
  22. 根据权利要求21所述的化合物，其特征在于，R e、R f各自独立选自Ac、SO 3 -或甲基，R g为Ac；优选的，R e选自Ac或甲基，R f选自Ac、SO 3 -或甲基，R g为Ac。
  23. 根据权利要求21-22任一项所述化合物，其以酸形式或盐形式存在，所述盐选自钠盐或钾盐。
  24. 下列化合物，
    Figure PCTCN2019122954-appb-100017
  25. 药物组合物，其包含作为活性成分的权利要求1～10中任一项所述的化合物及任选的药学上可接受的辅料。
  26. 权利要求1～10中任一项所述的化合物或权利要求25所述的药物组合物在制备用于预防和/或治疗与血液凝固障碍相关的疾病和疾病状态的药物中的用途，所述疾病和疾病状态优选为静脉血栓形成、动脉血栓形成或血栓性静脉炎。
  27. 用于预防和/或治疗与凝血功能障碍相关的疾病和疾病状态的方法，其包括向有需要的个体给予权利要求1～10中任一项所述的化合物或权利要求25所述的药物组合物，所述疾病和疾病状态优选为静脉血栓形成、动脉血栓形成或血栓性静脉炎。
  28. 用于预防和/或治疗与凝血功能障碍相关的疾病和疾病状态的权利要求1～10中任一项所述的化合物或权利要求25所述的药物组合物，所述疾病和疾病状态优选为静脉血栓形成、动脉血栓形成或血栓性静脉炎。
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