WO2024046048A1 - 一种抗凝血肝素寡糖苯联二聚体及其制备方法与应用 - Google Patents

一种抗凝血肝素寡糖苯联二聚体及其制备方法与应用 Download PDF

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WO2024046048A1
WO2024046048A1 PCT/CN2023/111635 CN2023111635W WO2024046048A1 WO 2024046048 A1 WO2024046048 A1 WO 2024046048A1 CN 2023111635 W CN2023111635 W CN 2023111635W WO 2024046048 A1 WO2024046048 A1 WO 2024046048A1
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dimer
reaction
heparin
phenyl
anticoagulant
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PCT/CN2023/111635
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French (fr)
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刘纯慧
张桂姣
仇亚琪
李婧茹
杨开华
承艳真
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山东大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the invention relates to an anticoagulant heparin oligosaccharide phenyl-linked dimer and its preparation method and application, and belongs to the technical field of biomedicine.
  • Heparin is an ancient natural polysaccharide anticoagulant. It is still widely used for clinical indications such as thromboembolic disease, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, and hemodialysis. The global market size has reached 8 billion. US dollars and a growing trend.
  • Current commercialized natural heparin drugs mainly include unfractionated heparin (UFH) extracted from pig small intestinal mucosa and other low-molecular-weight heparins (low-molecular-weight heparins) obtained through partial depolymerization by chemical or enzymatic methods. LMWH).
  • LMWH drugs have gradually surpassed UFH to become the preferred clinical anticoagulant drug due to their long half-life in vivo, high bioavailability, small side effects, and safe use.
  • the anticoagulant activity is generally lower than that of UFH.
  • UFH and LMWH derived from animal tissues are not only contaminated by impurities and have a fragile raw material supply chain, but as a highly sulfated glycosaminoglycan family, their variable sulfation, isomerization and other modifications are unevenly distributed along the sugar chain, forming The highly complex and heterogeneous fine structure leads to insurmountable clinical limitations.
  • fondaparinux has excellent clinical anticoagulant therapeutic effects
  • its anti-Xa activity cannot be neutralized by protamine like animal-derived heparin
  • anticoagulant therapy cannot be terminated according to the treatment process to effectively avoid adverse reactions such as bleeding.
  • resulting in greater limitations in its clinical application Therefore, the development of new anticoagulant heparin molecules that can be neutralized by protamine has important clinical value.
  • protamine has a long history as a reversal agent for animal-derived heparin in clinical practice, its research is not systematic and in-depth enough. It is generally believed that the heparin chain length, charge density and distribution jointly determine the neutralization efficiency of protamine.
  • Professor Jian Liu from the United States used chemical enzymatic methods to synthesize a heparin dodecaose containing a single AT-binding pentasaccharide and four consecutive trisulfate disaccharides (IdoA2S-GlcNS6S). Its anticoagulant activity can be effectively neutralized by protamine.
  • the anticoagulant heparin dodecose containing four trisulfate disaccharides is the smallest heparin molecule that can be neutralized by protamine.
  • the synthesis steps of the above-mentioned neutralizing anticoagulant heparin dodecaccharide are 22 to 23, which are cumbersome, high cost, low overall yield, short half-life in vivo, and potential adverse reactions such as HIT.
  • the present invention provides an anticoagulant heparin oligosaccharide phenyl-linked dimer and its preparation method and application.
  • UDP-GlcNTFA Uridine diphosphate-N-trifluoroacetylglucosamine
  • UDP-GlcNAc Uridine diphosphate-N-acetylglucosamine
  • UDP-GlcA Uridine diphosphate-glucuronic acid
  • PAPS 3'-adenosine phosphate-5'-phosphosulfate.
  • KfiA Escherichia coli K5N-acetylglucosaminyltransferase
  • PmHS2 Pasteurella multocida Heparosan synthase 2
  • NST N-sulfatyltransferase
  • the first object of the present invention is to provide an anticoagulant heparin oligosaccharide phenyl dimer.
  • Anticoagulant heparin oligosaccharide phenyl dimer has a structure represented by general formula I:
  • R 1 is a sulfonyl group (-SO 3 H) or an acetyl group (-COCH 3 );
  • R 2 and R 3 are a sulfonyl group or hydrogen (-H);
  • n is an integer from 0 to 5.
  • n 0, 1, or 2.
  • the benzene ring connected to the heparin oligosaccharide can also be replaced by a substituted phenyl group, an aromatic heterocycle, a substituted aromatic heterocycle, a C 1- C 5 alkyl group or a cycloalkyl group.
  • the substituents of the substituted phenyl and substituted aromatic heterocycles are halogen, hydroxyl, nitro, trifluoromethyl, C 1- C 5 alkyl or cycloalkyl.
  • substituted phenyl group is selected from one of the following:
  • substituted aromatic heterocycle is selected from one of the following:
  • the C 1- C 5 alkyl group is
  • the cycloalkyl group is cyclohexane.
  • the anticoagulant heparin oligosaccharide phenyldimer is specifically one of the compounds of the following formula:
  • R 1 is a sulfonyl group (-SO 3 H) or an acetyl group (-COCH 3 ).
  • the anticoagulant heparin oligosaccharide phenyl-linked dimer molecule has significant anti-factor Xa activity and no obvious anti-factor IIa activity; its anti-factor Xa activity can be effectively neutralized by protamine. And, the neutralization rate is >70%; it has similar pharmacokinetic characteristics to short-chain heparin oligosaccharides, long half-life in vivo, and high bioavailability; it is not likely to cause HIT adverse reactions.
  • the second object of the present invention is to provide a method for preparing anticoagulant heparin oligosaccharide phenyl-linked dimer, which is carried out by adopting a chemical enzymatic synthesis strategy.
  • a method for preparing anticoagulant heparin oligosaccharide phenyl-linked dimers uses a chemically synthesized GlcA-containing dimer intermediate as a starting material, and repeats the following steps a and b glycosyltransferase catalyzed reactions at least once.
  • Step a catalyzed by N-acetylglucosaminyltransferase (KfiA) or Heparosan synthase 2 (PmHS2), UDP-GlcNTFA or UDP-GlcNAc is used as the glycosyl donor, and the GlcNTFA or GlcNAc residue is converted into ⁇ -1 ,4 glycosidic bonds are transferred to the two GlcA at the non-reducing end of the substrate phenylbisO-glucuronide or the oligosaccharide intermediate dimer with GlcA at the non-reducing end, forming an extended sugar chain in the middle of the dimer. product;
  • KfiA N-acetylglucosaminyltransferase
  • PmHS2 Heparosan synthase 2
  • Step b under the catalysis of PmHS2 enzyme, UDP-GlcA is used as the glycosyl donor reaction, and the GlcA residue is connected to the two glucosamine GlcNTFA or GlcNAc at the non-reducing end of the substrate through ⁇ -1,4 glycosidic bonds to generate Intermediates for sugar chain elongation;
  • step c the heparin oligosaccharide dimer intermediate is dissolved in an alkaline aqueous solution and placed on ice, so that all GlcNTFA residues of the sugar chain are detrifluoroacetyl (TFA) and converted into GlcNH 2 , and then N- It is converted into GlcNS under the catalysis of sulfate transferase (NST) to obtain the N-sulfated heparin oligosaccharide dimer intermediate product;
  • TFA detrifluoroacetyl
  • NST sulfate transferase
  • Step d under the joint catalysis of C 5 -isomerase (C 5 -epi) and 2-O-sulfotransferase (2OST), all GlcA residues located between the two GlcNS in the dimer sugar chain are The radicals are converted into 2-O-sulfated iduronic acid (IdoA2S), Obtain IdoA2S-containing heparin oligosaccharide dimer intermediate product;
  • Step e under the sole catalysis of 2OST, all specific GlcA residues located between two GlcNS in the substrate are converted into 2-O-sulfated gluconic acid (GlcA2S);
  • Step f under the joint catalysis of 6-O-sulfotransferase 1 and 6-O-sulfotransferase 3 (6OST1 and 6OST3), 6-OH of all GlcNS or GlcNAc residues of the substrate sugar chain is generated Sulfation modification produces an intermediate product containing 6-O-sulfated heparin oligosaccharide dimer;
  • Step g under the catalytic action of 3-O-sulfatyltransferase 1 (3OST1), the 3-OH of GlcNS6S between GlcA and IdoA2S in the substrate sugar chain is sulfated (GlcNS6S3S) to obtain anticoagulant heparin oligo Sugar phenyldimer target compound.
  • 3-O-sulfatyltransferase 1 3-O-sulfatyltransferase 1
  • the GlcA-containing dimer intermediate is phenyl bis O-gluconoside or substituted phenyl bis O-gluconoside, aromatic heterocyclic bis O-gluconoside, substituted aromatic Heterocyclyl bis-O-glucuronide, C 1- C 5 alkyl bis-O-gluconoside or cycloalkyl bis-O-gluconoside.
  • the starting material phenylbis-O-glucuronide is prepared as follows:
  • acetyl hydrazide is used to remove the terminal acetyl group to obtain an intermediate.
  • the exposed anomeric hydroxyl group reacts with trichloroacetonitrile (CNCCl 3 ) to obtain glycosyl trichloroacetimide ester, and then With the participation of an accelerator, a glycosidation reaction occurs with p-diphenol to generate an intermediate, followed by alcohol detachment and deacetylization to obtain phenyl bis O-glucoside.
  • PhI(OAc) 2 and Tempo jointly selectively oxidize C 6 -OH to obtain the target product.
  • the reaction solvent for removing the terminal acetyl group of fully acetylated glucose 1 using acetylhydrazine is anhydrous DMF, and the reaction is carried out at room temperature for 1 hour.
  • the exposed anomeric hydroxyl group of intermediate 2 reacts with trichloroacetonitrile (CNCCl 3 ) at less than 0° C. for more than 3 hours, and the reaction solvent is a mixture of DBU and anhydrous DCM.
  • CNCCl 3 trichloroacetonitrile
  • the mixing ratio of the two can be arbitrary.
  • the reaction between glycosyl trichloroacetimide ester 3 and p-diphenol is to use anhydrous DCM containing molecular sieves as the solvent, BF 3 -Et 2 O as the accelerator, and react at -5°C for 1 hour.
  • the reaction solution was extracted with EA, dried in vacuum, and recrystallized from methanol to obtain intermediate 4; intermediate 4 was treated with anhydrous MeOH-sodium methoxide at room temperature for more than 1 hour to remove the acetyl group, and then the solution was neutralized with acidic resin to neutrality and filtered. , vacuum drying and crystallization to obtain intermediate 5.
  • the dosage of each reagent is determined according to the current technology in this field.
  • intermediate 5 uses PhI(OAc) 2 and Tempo to jointly selectively oxidize C 6 -OH.
  • the solvent used is t-BuOH, DCM and H 2 O with a volume ratio of 4:4:1, and the reaction is overnight at room temperature, and then recrystallized from acetonitrile to obtain the target product phenylbis-O-glucuronide 6.
  • each reagent is determined according to the current technology in this field.
  • the preferred preparation method of the anticoagulant heparin oligosaccharide phenyl dimer of the present invention uses phenyl bis O-glucuronide 6 as raw material and adopts the methods of steps a, b and steps c, d, f and g.
  • the target compound I-1 is prepared, and the synthesis route is shown in Formula III:
  • the chemical enzymatic synthesis route of I-1 can be abbreviated as: a ⁇ b ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ c or none ⁇ f ⁇ g.
  • I-2 a ⁇ b ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ c or none ⁇ f ⁇ g;
  • I-3 a ⁇ b ⁇ a ⁇ b ⁇ c ⁇ e ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ c or none ⁇ f ⁇ g;
  • I-4 a ⁇ b ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ b ⁇ c ⁇ d ⁇ a ⁇ c or none ⁇ f ⁇ g;
  • the enzymes KfiA and PmHS2 are respectively derived from Escherichia coli K5 and Pasteurella multocida, and are recombinantly expressed in Escherichia coli.
  • the buffer used in the enzymatic sugar chain elongation reaction is 50 mmol/L.
  • the addition amount of enzyme and substrate and the reaction time are carried out according to the existing technology.
  • the heparinase modifying enzymes NST, C 5 -epi, 2OST, 6OST1, 6OST3, and 3OST1 are obtained by recombinant expression using E. coli, yeast or insect cells, and the NST, 2OST, 6OST1, 6OST3,
  • MES 2-(N-morpholino)ethanesulfonic acid
  • PAPS 3'- Adenosine monophosphate-5'-phosphate sulfate
  • PAPS 3'- Adenosine monophosphate
  • the addition amount of enzyme and substrate and the reaction time are carried out according to the existing technology.
  • the third object of the present invention is to provide the application of heparin oligosaccharide phenyl-linked dimers containing AT-binding sequences, which have specific anti-factor Xa activity, no obvious anti-IIa activity, and their anticoagulant activity can be neutralized by protamine. , has better pharmacokinetics and is used to prepare potent and safe anticoagulant and antithrombotic drugs.
  • An anticoagulant and antithrombotic pharmaceutical composition including the above-mentioned AT-binding sequence-containing heparin oligosaccharide phenyldimer molecule and one or more pharmaceutically acceptable carriers or excipients.
  • the ratio of heparin oligosaccharide phenyl dimer to carrier or excipient is arbitrary.
  • this invention uses two molecules of glucuronic acid covalently connected to the same benzene ring as starting materials and uses chemical enzymatic methods to successfully synthesize anticoagulant heparin oligosaccharide phenyl dimers containing AT binding sequences. It not only has strong It has effective anticoagulant activity. The most important thing is that protamine has neutralizing properties similar to long-chain heparin molecules, ideal pharmacokinetic characteristics similar to short-chain molecules, and has a low risk of inducing adverse reactions such as HIT. There are few preparation steps, with the minimum being only 9 steps. It can be developed into a new generation of safer and higher-quality innovative anticoagulant drugs with independent intellectual property rights, and has important application value.
  • the present invention has confirmed for the first time that different heparin oligosaccharides containing AT-binding sequences (6 sugar, octasaccharide, deca-saccharide) dimer molecules.
  • the novel AT-binding sequence-containing heparin oligosaccharide phenylconjugate dimer of the present invention has significant anti-factor
  • the two AT-binding sequences in the dimer can activate AT without interfering with each other, and its half inhibitory molar concentration (IC 50 ) for inhibiting factor Xa is significantly lower than the positive control fondaparinux and long-chain heparin 10 reported by Jian Liu. disaccharide molecule.
  • the specific anti-Xa factor activity of the novel AT-binding sequence-containing heparin oligosaccharide phenyl-linked dimer of the present invention can be efficiently neutralized by protamine, with a neutralization rate of >70%, which is comparable to long-chain unfractionated heparin UFH and already The reported heparin dodecaccharide is equivalent, but the anti-factor Protamine is neutralized, so the heparin oligosaccharide phenyl-linked dimer of the present invention has similar characteristics to heparin dodecose and above long-chain heparin molecules, and can produce a higher affinity interaction with protamine. And be neutralized.
  • the heparin oligosaccharide phenyldimer of the present invention has ideal pharmacokinetic characteristics.
  • the in vivo half-life of compound I-1-1 is equivalent to that of commercially available fondaparinux sodium, and is significantly better than animal-derived heparin and current heparin.
  • dodecasaccharide molecules for heparin There are reported dodecasaccharide molecules for heparin.
  • sugar chains and modified groups outside the AT binding sequence in the heparin oligosaccharide phenyldimer structure of the present invention have little impact on the anti-Xa activity, but have an impact on the protamine neutralization efficiency and pharmacokinetic characteristics. more obvious.
  • the heparin oligosaccharide phenyl dimer of the present invention can be used to prepare cost-effective, more high-quality and safer potent anticoagulant and antithrombotic drugs.
  • Figure 1 is the 1 H NMR spectrum of phenylbisO-gluconoside 6 prepared in Example 1;
  • Figure 2 is a high performance liquid chromatogram (A) and a mass spectrum (B) of the heparin hexasaccharide phenyldimer I-1-1 prepared in Example 5;
  • Figure 3 is the 1 H NMR (A) and HSQC (B) spectra of heparin hexasaccharide phenyl dimer I-1-1 prepared in Example 5;
  • Figure 4 is the in vitro anti-Xa factor of heparin hexasaccharide phenyldimer I-1-1 prepared in Example 5;
  • Figure 5 shows the neutralizing effect of protamine on the anticoagulant activity of heparin hexasaccharide phenyldimer I-1-1 in Example 5 in vitro.
  • Figure 6 is the in vivo pharmacokinetic characteristics of heparin hexasaccharide phenyldimer I-1-1 prepared in Example 5.
  • Example 4 Chemical enzymatic synthesis of heparin pentasaccharide phenyl dimer 10 containing IdoA2S
  • Pentasaccharide dimer 10 was used as the substrate, and the KfiA enzyme was used to catalyze the elongation of the sugar chain as described in Example 1.
  • the reaction solution was purified with a Q-Sepharose strong anion column (1cm ⁇ 20cm).
  • the obtained product was subjected to chemical enzymatic method according to Example 4.
  • N-sulfation modification yields heparin hexasaccharide phenyl dimer 11.
  • ESI-MS determined its molecular weight to be 2772.41Da, which is consistent with the theoretical value.
  • Test Example 1 Determination of in vitro anticoagulant activity of heparin hexasaccharide phenyldimer I-1-1
  • the IC 50 value of the anti-FXa activity of the heparin hexasaccharide phenyldimer I-1-1 prepared in Example 5 was measured using the chromogenic substrate method to be 7.2 nmol/L (28.15 ng/mL). Measured under the same conditions The IC 50 values of unfractionated heparin (UFH) and fondaparinux were 378.3ng/mL and 10.8nmol/L (18.63ng/mL) respectively, that is, the IC of I-1-1 anti-FXa activity based on molar concentration. The value of 50 is significantly lower than fondaparinux, indicating that it is a potent Xa inhibitor, as shown in Figure 4.
  • the heparin hexasaccharide phenyl dimer I-1-1 prepared by the present invention is a specific inhibitor of factor Xa, and because it contains two AT-binding sequences that can activate AT without interfering with each other, it exhibits excellent For fondaparinux containing a single AT-binding sequence and the heparin dodecaose reported by Jian Liu, the half molar inhibitory concentration (IC 50 ) was significantly lower than the positive control fondaparinux and the long-chain heparin dodecaose reported by Jian Liu. Sugar molecules.
  • Test Example 2 Determination of the neutralizing effect of protamine on the anticoagulant activity of heparin hexasaccharide phenyldimer I-1-1
  • the chromogenic substrate method was used to determine the effect of adding different concentrations of protamine on the anti-FXa activity of the heparin hexasaccharide phenyldimer I-1-1 of Example 5.
  • the results are shown in Figure 5. It can be seen from Figure 5 that with Similar to UFH, the in vitro anti-FXa activity of heparin hexasaccharide phenyldimer I-1-1 was almost completely reversed by protamine. Therefore, the heparin hexasaccharide phenyldimer I-1-1 prepared in the present invention is a new heparin analog whose anticoagulant activity can be neutralized by protamine.
  • Test Example 3 In vivo pharmacokinetic characteristics of heparin hexasaccharide phenyldimer I-1-1
  • heparin standard as an example, a standard curve was established, and the titers of fondaparinux sodium and heparin hexasaccharide phenyl dimer I-1-1 were measured to be 1290.9IU/mg, respectively. 1121.5IU/mg.
  • Fondaparinux and heparin hexasaccharide phenyldimer I-1-1 were injected subcutaneously at a dose of 300IU/kg into Wister male rats (n ⁇ 3) weighing 200 ⁇ 20g.
  • Rat blood samples were collected from the jugular vein at 0, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 8, 12, and 24 hours (subject to the actual blood collection time). After the blood sample was centrifuged at 5000 r/min for 15 minutes, the supernatant was taken and the anti-factor Xa activity was measured using the above method. The remaining concentrations of fondaparinux sodium and heparin hexasaccharide phenyldimer I-1-1 in the rat body were calculated and drawn. The drug concentration-time curve is shown in Figure 6.

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Abstract

本发明涉及一种抗凝血肝素寡糖苯联二聚体及其制备方法与应用，具有如通式I所示的结构，苯环可替换为取代苯基、芳杂环或取代芳杂环。本发明所述肝素寡糖苯联二聚体合成步数少、总产率显著高，具有抗凝血酶依赖的强效特异抗Xa因子活性、无明显抗IIa活性，抗凝活性可被鱼精蛋白高效中和，并具有优异的药代动力学特征，因此适合用于制备具成本优势的、更加优质安全的新型抗凝血抗血栓药物。

Description

一种抗凝血肝素寡糖苯联二聚体及其制备方法与应用 技术领域
本发明涉及一种抗凝血肝素寡糖苯联二聚体及其制备方法与应用，属于生物医药技术领域。
背景技术
肝素是一种古老的天然多糖抗凝剂，至今仍广泛用于血栓栓塞性疾病、心肌梗死、心血管手术、心脏导管检查、体外循环、血液透析等临床适应症，全球市场规模已达80亿美元且呈不断增长的趋势。当前商品化天然肝素类药物主要包括提取自猪小肠黏膜等的未分级肝素(unfractionated heparin，UFH)及其经化学或酶法部分解聚制得的不同低分子量肝素(low-molecular-weight heparins,LMWH)。LMWH药物因具有体内半衰期长、生物利用度高，副作用小及使用安全等优点，逐渐超越UFH成为首选临床抗凝药物，但抗凝活性普遍低于UFH。动物组织来源的UFH、LMWH，除存在杂质污染、原料供应链脆弱外，作为高度硫酸化的糖胺聚糖家族，其可变的硫酸化、异构化等修饰沿糖链不均匀分布，形成高度复杂、不均一的精细结构，导致其无法克服的临床局限性。
研究证实，UFH及LMWH发挥抗凝作用共同的结构基础是肝素链中与抗凝血酶(antithrombin，AT)特异性结合的独特五糖序列(简写为：GlcNS/Ac6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S)，其甲基糖苷衍生物——磺达肝癸钠(商品名)于2001年成功上市，是迄今唯一批准上市的全合成的肝素类单一化合物。然而，尽管磺达肝癸钠具有出色的临床抗凝治疗效果，但是其抗Xa活性无法像动物源肝素一样被鱼精蛋白中和，不能根据治疗进程结束抗凝治疗，有效避免出血等不良反应，导致其在临床应用中受到较大限制，因此研发可被鱼精蛋白中和的抗凝肝素新分子具有重要的临床价值。
然而，尽管临床上鱼精蛋白作为动物源肝素的逆转剂有较长的历史，但对其研究不够系统和深入，一般认为肝素链长、电荷密度及分布共同决定鱼精蛋白的中和效率。例如，美国Jian Liu教授采用化学酶法合成一种含单一AT结合五糖、4个连续三硫酸双糖(IdoA2S-GlcNS6S)的肝素十二糖，其抗凝活性能够被鱼精蛋白有效地中和，并由此被广泛认为含4个三硫酸双糖的抗凝肝素十二糖是鱼精蛋白可中和的最小肝素分子。然而，上述可中和抗凝肝素十二糖合成步数为22～23步、步骤繁琐、成本高、总产率较低，而且其体内半衰期短，并存在潜在的HIT等不良反应。
发明内容
针对现有技术的不足，本发明提供一种抗凝血肝素寡糖苯联二聚体及其制备方法与应用。
术语说明：
AT：抗凝血酶
UDP-GlcNTFA：尿苷二磷酸-N-三氟乙酰葡糖胺
UDP-GlcNAc：尿苷二磷酸-N-乙酰葡糖胺
UDP-GlcA：尿苷二磷酸-葡糖醛酸
PAPS：3'-磷酸腺苷-5'-磷酸硫酸。
KfiA：大肠杆菌K5N-乙酰氨基葡糖基转移酶
PmHS2：多杀巴斯德菌Heparosan合酶2
NST：N-硫酸基转移酶
C5-epi：C5-异构化酶
2OST：2-O-硫酸基转移酶
6OST：6-O-硫酸基转移酶
3OST：3-O-硫酸基转移酶
本发明的技术方案如下：
本发明的第一个目的是提供一种抗凝血肝素寡糖苯联二聚体。
抗凝血肝素寡糖苯联二聚体，或其药学上可接受的盐，具有通式I所示的结构：
其中，R1为磺酰基(-SO3H)或乙酰基(-COCH3)；R2、R3为磺酰基或氢(-H)；n为0-5的整数。
根据本发明优选的，n＝0、1、或2。
根据本发明优选的，连接肝素寡糖的苯环还能替换为取代苯基、芳杂环、取代芳杂环、C1-C5的烷基或环烷基。
根据本发明优选的，所述取代苯基、取代芳杂环的取代基为卤素、羟基、硝基、三氟甲基、C1-C5的烷基或环烷基。
进一步优选的，取代苯基选自如下之一：
进一步优选的，取代芳杂环选自如下之一：
进一步优的，C1-C5的烷基为
进一步优选的，环烷基为环己烷。
根据本发明优选的，抗凝血肝素寡糖苯联二聚体具体为如下式化合物之一：


式I-1-式I-5中，R1为磺酰基(-SO3H)或乙酰基(-COCH3)。
根据本发明优选的，所述的抗凝血肝素寡糖苯联二聚体分子具有显著的抗Xa因子活性、无明显的抗IIa因子活性；其抗Xa因子活性可以被鱼精蛋白有效地中和，中和率>70％；其具有与短链肝素寡糖类似的药代动力学特征，体内半衰期长、生物利用度高；其不容易导致HIT不良反应。
本发明的第二个目的是提供抗凝血肝素寡糖苯联二聚体的制备方法，采用化学酶法合成策略进行。
一种抗凝血肝素寡糖苯联二聚体的制备方法，该方法以化学合成含GlcA的二聚体中间体为起始原料，为如下步骤a、b糖基转移酶催化反应至少重复一次与步骤c、d、e、f、g化学酶法修饰反应中其中四步或五步组合的方法；
步骤a，在N-乙酰氨基葡糖基转移酶(KfiA)或Heparosan合酶2(PmHS2)催化下，以UDP-GlcNTFA或UDP-GlcNAc为糖基供体，GlcNTFA或GlcNAc残基以α-1,4糖苷键转移至底物苯基双O-葡糖酸苷或非还原末端为GlcA的寡糖中间体二聚体的非还原末端的两个GlcA上，生成糖链延长的二聚体中间产物；
步骤b，在PmHS2酶催化下，以UDP-GlcA为糖基供体反应，GlcA残基以β-1,4糖苷键连接至底物非还原末端的两个葡糖胺GlcNTFA或GlcNAc上，生成糖链延长的中间产物；
步骤c，肝素寡糖二聚体中间体溶于碱性水溶液中并静置于冰上，以使糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，随后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化肝素寡糖二聚体中间产物；
步骤d，在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，二聚体糖链中所有位于两个GlcNS之间的GlcA残基均被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)， 得到含IdoA2S肝素寡糖二聚体中间产物；
步骤e，在2OST的单独催化下，底物中所有位于两个GlcNS之间的特定GlcA残基均被转变为2-O-硫酸化葡糖酸(GlcA2S)；
步骤f，在6-O-硫酸基转移酶1和6-O-硫酸基转移酶3(6OST1和6OST3)的共同催化作用下，底物糖链的全部GlcNS或GlcNAc残基的6-OH发生硫酸化修饰，生成含6-O-硫酸化肝素寡糖二聚体中间产物；
步骤g，在3-O-硫酸基转移酶1(3OST1)的催化作用下，底物糖链中GlcA与IdoA2S之间的GlcNS6S的3-OH发生硫酸化(GlcNS6S3S)，得到抗凝血肝素寡糖苯联二聚体目标化合物。
根据本发明优选的，含GlcA的二聚体中间体为苯基双O-葡糖酸苷或取代苯基双O-葡糖酸苷、芳杂环基双O-葡糖酸苷、取代芳杂环基双O-葡糖酸苷、C1-C5的烷基双O-葡糖酸苷或环烷基双O-葡糖酸苷。
根据本发明优选的，起始原料苯基双O-葡糖酸苷是按如下方法制得：
以全乙酰化葡萄糖为起始原料，利用乙酰肼脱除端基乙酰基得中间体，裸露的异头位羟基与三氯乙腈(CNCCl3)反应得糖基三氯乙酰亚胺酯，然后在促进剂参与下与对二苯酚发生糖苷化反应生成中间体，接着醇解脱去乙酰基得到苯基双O-葡糖苷，最后由PhI(OAc)2和Tempo共同选择性氧化C6-OH得目标产物。
合成路线如下式Ⅱ所示：
根据本发明优选的，全乙酰化葡萄糖1利用乙酰肼脱除端基乙酰基的反应溶剂为无水DMF，在常温下反应1h。
根据本发明优选的，中间体2裸露的异头位羟基与三氯乙腈(CNCCl3)在小于0℃下反应3h以上，反应溶剂为DBU和无水DCM的混合液。两者混合比例任意。
根据本发明优选的，糖基三氯乙酰亚胺酯3与对二苯酚的反应是以含分子筛的无水DCM为溶剂、BF3-Et2O为促进剂，在-5℃下反应1h，反应液经EA萃取、真空干燥、甲醇重结晶得中间体4；中间体4经无水MeOH-甲醇钠于常温下处理1h以上脱去乙酰基，然后用酸性树脂中和溶液至中性，过滤、真空干燥及结晶得中间体5。各试剂的用量按本领域现有技术进行。
根据本发明优选的，中间体5利用PhI(OAc)2和Tempo共同选择性氧化C6-OH，所用溶剂为体积比为4:4:1的t-BuOH、DCM和H2O，反应于常温下过夜，后经乙腈重结晶得目标产物苯基双O-葡糖酸苷6。
各试剂的用量按本领域现有技术进行。
本发明优选的抗凝血肝素寡糖苯联二聚体的制备方法，以苯基双O-葡糖酸苷6为原料，采用步骤a、b与步骤c、d、f、g的方法，制备目标化合物I-1，合成路线如式Ⅲ所示：
I-1的化学酶法合成路线可简记为：a→b→a→b→c→d→a→c或无→f→g。
根据本发明优选的，式I-2-式I-5的合成路线如下：
I-2：a→b→a→b→c→d→a→b→c→d→a→c或无→f→g；
I-3：a→b→a→b→c→e→a→b→c→d→a→c或无→f→g；
I-4：a→b→a→b→c→d→a→b→c→d→a→b→c→d→a→c或无→f→g；
I-5：a→b→a→b→c→d→f→a→b→a→b→c→d→a→c或无→f→g；
根据本发明优选的，所述酶KfiA、PmHS2分别来源于大肠杆菌K5、多杀巴斯德菌(Pasteurella multocida)，以大肠杆菌重组表达，酶促糖链延长反应所用的缓冲液为50mmol/L Tris-HCl，pH＝7.0～7.5，反应温度20℃～37℃，酶促反应液利用反相C18或阴离子交换柱层析纯化得产物。
酶与底物的加入量、反应时间按现有技术进行。
根据本发明优选的，所述肝素酶修饰酶NST、C5-epi、2OST、6OST1、6OST3、3OST1是利用大肠杆菌、酵母或昆虫细胞重组表达得到，所述NST、2OST、6OST1、6OST3、3OST1各修饰酶催化反应的缓冲液为50mmol/L 2-(N-吗啉代)乙烷磺酸(MES)，pH＝7.0～7.5，反应温度20℃～37℃，并均以3'-磷酸腺苷-5'-磷酸硫酸(PAPS)为硫酸基供体，酶促反应液利用阴离子交换柱层析纯化得产物。
酶与底物的加入量、反应时间按现有技术进行。
本发明的第三个目的是提供含AT结合序列的肝素寡糖苯联二聚体的应用，具特异抗Xa因子活性、无明显抗IIa活性，且其抗凝活性可被鱼精蛋白中和，药代动力学更佳，用于制备强效安全抗凝抗血栓药物。
一种抗凝抗血栓药物组合物，包括上述含AT结合序列的肝素寡糖苯联二聚体分子和一种或多种药学上可接受载体或赋形剂。
肝素寡糖苯联二聚体与载体或赋形剂的比例任意。
本发明首次以共价连接于同一苯环的两分子葡糖醛酸为起始原料，采用化学酶法成功合成含AT结合序列的抗凝血肝素寡糖苯联二聚体，其不仅具有强效抗凝活性，最主要的是表现出与长链肝素分子类似的鱼精蛋白可中和特性，与短链分子相似的理想药代动力学特征，且其诱导HIT等不良反应的风险低，制备步骤少，最少的仅有9步，可开发成为具有自主知识产权的新一代更加安全优质的抗凝创新药物，具有重要的应用价值。
本发明的技术特点及优点：
1.本发明首次证实，以含2个GlcA的苯基双O-葡糖酸苷为起始原料，经9～18步化学酶法反应成功制得含AT结合序列的不同肝素寡糖(六糖、八糖、十糖)二聚体新分子。
2.经生色底物法测定，本发明的新型含AT结合序列的肝素寡糖苯联二聚体具有显著的抗Xa因子活性、无明显的抗IIa因子活性，首次证实肝素寡糖苯联二聚体中的两个AT结合序列能够互不干扰地激活AT，其抑制Xa因子的半数抑制摩尔浓度(IC50)显著低于阳性对照磺达肝癸钠和Jian Liu报道的长链肝素十二糖分子。
3.本发明的新型含AT结合序列的肝素寡糖苯联二聚体的特异抗Xa因子活性可以被鱼精蛋白高效中和，中和率>70％，与长链未分级肝素UFH和已报道的肝素十二糖相当，而市售磺达肝癸钠的抗Xa因子活性几乎完全不能被鱼精蛋白中和，现有已报道的抗凝肝素六糖、八糖、十糖均不能被鱼精蛋白中和，因此本发明的肝素寡糖苯联二聚体具有与肝素十二糖及以上的长链肝素分子相似的特性，能够与鱼精蛋白之间产生较高的亲和相互作用而被中和。
4.本发明的肝素寡糖苯联二聚体具有理想的药代动力学特征，如化合物I-1-1的体内半衰期与市售磺达肝癸钠相当，显著优于动物源肝素及现有已报道的肝素十二糖分子。
5.本发明的肝素寡糖苯联二聚体结构中AT结合序列之外的糖链及修饰基团部分对抗Xa活性影响较小，但对鱼精蛋白中和效率、药代动力学特征影响较明显。
6.本发明的肝素寡糖苯联二聚体可用于制备具成本优势的、更加优质安全的强效抗凝血抗血栓药物。
附图说明
图1是实施例1制备的苯基双O-葡糖酸苷6的1H NMR谱图；
图2是实施例5制备的肝素六糖苯联二聚体I-1-1的高效液相色谱图(A)和质谱图(B)；
图3是实施例5制备的肝素六糖苯联二聚体I-1-1的1H NMR(A)和HSQC(B)谱图；
图4是实施例5制备的肝素六糖苯联二聚体I-1-1的体外抗Xa因子；
图5是鱼精蛋白体外对实施例5的肝素六糖苯联二聚体I-1-1抗凝活性的中和作用。
图6是实施例5制备的肝素六糖苯联二聚体I-1-1的体内药代动力学特征。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此，下列实例中的目标化合物的编号与表1相同。实施例中涉及的药品及试剂，若无特殊说明，均为普通市售产品。
实施例1：苯基双O-葡糖酸苷6的化学法合成
将化合物1(1g，2.6mmol)和AcOH/NH2-NH2(1.4equiv.3.6mmol，330mg)溶解于5ml无水DMF，常温下反应1h。反应结束后，用EA稀释反应液，分别用H2O、饱和食盐水洗涤有机层，真空干燥得滤渣，经硅胶柱层析(PE:EA＝2:1)纯化得752mg无色油状液体2，产率84.3％。
将化合物2(752mg，2.16mmol)溶解于5ml无水DCM中，低温下依次加入CNCCl3(3equiv.6.5mmol，0.65ml)和DBU(0.2equiv.0.43mmol)，在该温度下反应3h。反应结束后，将反应液直接进行真空干燥得滤渣，经硅胶柱层析(PE:EA＝4:1)纯化得893mg无色油状液体3，产率83.8％。
将化合物3(893mg，1.8mmol，2.2equiv.)和对二苯酚(0.82mmol，83mg)溶解于含有适量分子筛的10ml无水DCM中，-5℃下加入BF3-Et2O(4equiv.3.28mmol，0.4ml)，在该温度下继续反应1h。反应结束后，用EA稀释反应液，依次用饱和碳酸氢钠溶液和水，饱和食盐水依次洗涤有机层，将洗涤过后的有机层真空干燥，所得的残渣经甲醇重结晶后得到462mg白色固体化合物4，产率73％。1H NMR(400MHz,Chloroform-d)δ6.91(d,J＝4.6Hz,4H),5.30–5.11(m,6H),4.98(dd,J＝7.4,3.6Hz,2H),4.26(dd,J＝12.4,5.2Hz,2H),4.15(dt,J＝12.2,2.8Hz,2H),3.80(ddt,J＝10.0,5.2,2.4Hz,2H),2.04(dt,J＝13.2,4.8Hz,24H)。
将化合物4(462mg，0.6mmol)和甲醇钠(0.1equiv.3mg)溶解于10ml无水MeOH中，常温下反应1h。反应结束后，加入酸性树脂搅拌10min，将反应液中和至中性后，过滤，真空干燥大部分溶剂后将剩余反应液放入冰箱里析出，得247mg白色固体化合物5，产率95％。
将化合物5(247mg，0.57mmol)、PhI(OAc)2(8equiv.4.6mmol，1.5g)和Tempo(4equiv.2.3mmol，355mg)溶解于18ml混合溶剂中(t-BuOH:DCM:H2O＝4:4:1)，常温下反应反应过夜。反应结束后，EA稀释反应液，水洗涤有机层，将水层旋干，真空干燥的残渣，乙腈重结晶，得162mg白色固体化合物6，产率62％。
合成路线如下式Ⅱ所示：
化合物6的1H NMR谱图见图1。
重要的1H NMR(400MHz,D2O)数据为δ7.13(dd,J＝2.8,1.8Hz,4H),5.16–5.06(m,2H),4.10–4.00(m,2H),3.72–3.56(m,6H)。
实施例2：肝素五糖骨架二聚体8的酶法合成
称取50mg苯基双O-葡糖酸苷6溶于～50mL 50mmol/LTris-HCl缓冲液(含6mmol/L MnCl2，pH＝7.2)，加入底物2.2倍当量的UDP-GlcNTFA、2.5mL KfiA酶，室温搅拌过夜，反应用PAMN-HPLC检测，色谱条件为在45min内以0→100％KH2PO4梯度洗脱，流速为0.5mL/min，检测波长为280nm。待反应产率≥99％，用三氟乙酸(TFA)调pH至2-3中止反应，用C18层析柱(3×50cm)进行纯化，以含0.1％TFA的甲醇-水洗脱，收到目标组分。得到的产物置于50mL上述相同缓冲液，同时加入2.2倍当量的UDP-GlcA、3mL PmHS2酶，室温搅拌过夜。PAMN-HPLC检测反应至产率≥99％，以C18层析柱纯化得中间体7。重复上述KfiA、PmHS2酶反应，得到肝素五糖骨架二聚体8，ESI测得其分子量与理论相符。
合成路线如下式Ⅳ所示：
实施例4：含IdoA2S的肝素五糖苯联二聚体10的化学酶法合成
取五糖骨架二聚体8溶于100mL去离子水，置于冰上，逐滴加入0.5mol/L LiOH溶液至pH＝12，继续置于冰浴中2h，PAMN-HPLC检测反应进程；反应结束后，以冰醋酸调节pH至中性，加入适量1mol/L MES溶液(pH＝7.4)使其终浓度为50mmol/L，同时加入4.4倍当量的PAPS、3mL NST酶，室温搅拌过夜，利用PAMN-HPLC检测反应；反应产率>95％时醋酸调pH至4-5终止反应，用Q Sepharose层析柱(1×20cm)纯化，流速为3mL/min，以0→100％含1mol/L NaCl、50mmol/L NaAc缓冲液(pH＝5)梯度洗脱，检测波长为260nm和280nm，收集目标组分、脱盐、干燥得N-硫酸化产物9。ESI-MS测得其分子量2130.22Da。
取上述产物9于pH＝7.0～7.4、50mmol/L的MES缓冲液中加入2mmol/L CaCl2及适量酶C5-epi，调整反应体积为100mL，于37℃水浴反应2h。然后加入约2.5倍当量的PAPS、额外C5-epi和足量2OST酶，室温反应过夜。PAMN-HPLC检测反应，并根据需要补加酶或PAPS，至反应结束。反应液用Q-Sepharose强阴离子柱(1×20cm)纯化得到肝素五糖二聚体10。ESI-MS测定其分子量为2290.20Da。
合成路线如下式V所示：
实施例5：肝素六糖苯联二聚体I-1-1的化学酶法制备
以五糖二聚体10为底物，参照实施例1所述KfiA酶催化延长糖链，反应液以Q-Sepharose强阴离子柱(1cm×20cm)纯化，所得产物参照实施例4进行化学酶法N-硫酸化修饰，得肝素六糖苯联二聚体11。ESI-MS测定其分子量为2772.41Da，与理论值相符。
将六糖苯联二聚体11置于pH＝7.0～7.5、50mmol/L的MES缓冲液中，加入7倍当量PAPS、4mL的6OST1和4mL的6OST3酶，调整反应体积为140mL，37℃水浴反应过夜。利用SAX-HPLC检测反应进程，根据需要补加酶或PAPS。色谱条件为：流速为1mL/min，以0→100％洗脱液B(50mmol/L NaAc+2mol/L NaCl，pH＝5)梯度洗脱为，检测波长为260nm和280nm。待反应产率>99％，用稀醋酸调pH＝4-5终止反应，-20℃冰箱冻融除酶，无需纯化。调节溶液pH＝7.0～7.5，并加入约2.5倍当量PAPS、5mL的3OST1酶，调整反应体积为200mL，37℃水浴反应过夜。PAMN-HPLC检测反应至底物修饰率为99％以上，反应液用稀醋酸调pH＝4-5后，用Q-Sepharose强阴离子柱(1cm×10cm)纯化得到目标肝素六糖苯联二聚体I-1-1,其纯度达92％以上(图2A)。ESI-MS测定其分子量3411.99Da(图2B)，与理论值相符。采用1H NMR和HSQC(图3)表征其结构。
合成路线如下式VI所示：
试验例1：肝素六糖苯联二聚体I-1-1体外抗凝活性测定
采用生色底物法测得实施例5制备得到的肝素六糖苯联二聚体I-1-1抗FⅩa活性的IC50值为7.2nmol/L(28.15ng/mL)，同样条件下测得未分级肝素(UFH)、磺达肝癸钠的IC50值分别为378.3ng/mL、10.8nmol/L(18.63ng/mL)，即以摩尔浓度计I-1-1抗FⅩa活性的IC50值显著低于磺达肝癸钠，表明其为强效Xa抑制剂，如图4所示。同时经生色底物法测定，所述目标化合物I-1-1与磺达肝癸钠类似，无显著的抗IIa因子活性(数据略)。因此，本发明制备得到的肝素六糖苯联二聚体I-1-1为Xa因子的特异抑制剂，且由于其所含两个AT结合序列能够互不干扰地激活AT，从而表现出优于含单一AT结合序列的磺达肝癸钠和Jian Liu报道的肝素十二糖，半数抑制摩尔浓度(IC50)显著低于阳性对照磺达肝癸钠和Jian Liu报道的长链肝素十二糖分子。
试验例2：鱼精蛋白对肝素六糖苯联二聚体I-1-1抗凝活性的中和作用测定
采用生色底物法，测定加入不同浓度的鱼精蛋白对实施例5的肝素六糖苯联二聚体I-1-1抗FⅩa活性的影响，结果如图5，由图5可知，与UFH类似，肝素六糖苯联二聚体I-1-1的体外抗FⅩa活性几乎完全被鱼精蛋白逆转。因此，本发明制备得到的肝素六糖苯联二聚体I-1-1为抗凝活性可被鱼精蛋白中和的新型肝素类似物。
试验例3：肝素六糖苯联二聚体I-1-1的体内药代动力学特征
采用生色底物法以低分子量肝素标准品为例，建立标准曲线，测得磺达肝癸钠及肝素六糖苯联二聚体I-1-1的效价分别为1290.9IU/mg、1121.5IU/mg。磺达肝癸钠及肝素六糖苯联二聚体I-1-1均以300IU/kg的剂量皮下注射给体重为200±20g的Wister雄性大鼠(n≥3)。分别于0，0.5，0.75，1，1.25，1.5，1.75，2，2.5，3，4，8，12，24h颈静脉采集大鼠血样(以实际采血时间为准)。血样经5000r/min离心15分钟后取上清利用上述方法测定抗Xa因子活性，计算磺达肝癸钠及肝素六糖苯联二聚体I-1-1在大鼠体内的剩余浓度，绘制药物浓度-时间曲线，见图6，可以看出磺达肝癸钠与本发明的肝素六糖苯联二聚体I-1-1的曲线非常相似。进一步利用DAS 2.0处理数据，可得肝素六糖苯联二聚体I-1-1的t1/2达2.269h，略长于磺达肝癸钠的t1/2为2.166h，表明肝素六糖苯联二聚体I-1-1的半衰期极佳。

Claims (11)

  1. 抗凝血肝素寡糖苯联二聚体，或其药学上可接受的盐，具有通式I所示的结构：
    其中，R1为磺酰基(-SO3H)或乙酰基(-COCH3)；R2、R3为磺酰基或氢(-H)；n为0-5的整数。
  2. 根据权利要求1所述的抗凝血肝素寡糖苯联二聚体，或其药学上可接受的盐，其特征在于，n＝0、1、或2，连接肝素寡糖的苯环可替换为取代苯基、芳杂环、取代芳杂环、C1-C5的烷基或环烷基，所述取代苯基、取代芳杂环的取代基为卤素、羟基、硝基、三氟甲基、C1-C5的烷基或环烷基。
  3. 根据权利要求2所述的抗凝血肝素寡糖苯联二聚体，或其药学上可接受的盐，其特征在于，取代苯基选自如下之一：
    取代芳杂环选自如下之一：
    C1-C5的烷基为
    环烷基为环己烷。
  4. 根据权利要求1所述的抗凝血肝素寡糖苯联二聚体，或其药学上可接受的盐，其特征在于，抗凝血肝素寡糖苯联二聚体具体为如下式化合物之一：
    式I-1-式I-5中，R1为磺酰基(-SO3H)或乙酰基(-COCH3)。
  5. 权利要求1所述的抗凝血肝素寡糖苯联二聚体的制备方法，该方法以化学合成含GlcA的二聚体中间体为起始原料，为如下步骤a、b糖基转移酶催化反应至少重复一次与步骤c、d、e、f、g化学酶法修饰反应中其中四步或五步组合的方法；
    步骤a，在N-乙酰氨基葡糖基转移酶(KfiA)或Heparosan合酶2(PmHS2)催化下，以UDP-GlcNTFA或UDP-GlcNAc为糖基供体，GlcNTFA或GlcNAc残基以α-1,4糖苷键转移至底物苯基双O-葡糖酸苷或非还原末端为GlcA的寡糖中间体二聚体的非还原末端的两个GlcA上，生成糖链延长的二聚体中间产物；
    步骤b，在PmHS2酶催化下，以UDP-GlcA为糖基供体反应，GlcA残基以β-1,4糖苷键连接至底物非还原末端的两个葡糖胺GlcNTFA或GlcNAc上，生成糖链延长的中间产物；
    步骤c，肝素寡糖二聚体中间体溶于碱性水溶液中并静置于冰上，以使糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，随后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化肝素寡糖二聚体中间产物；
    步骤d，在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，二聚体糖链中所有位于两个GlcNS之间的GlcA残基均被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)，得到含IdoA2S肝素寡糖二聚体中间产物；
    步骤e，在2OST的单独催化下，底物中所有位于两个GlcNS之间的特定GlcA残基均被转变为2-O-硫酸化葡糖酸(GlcA2S)；
    步骤f，在6-O-硫酸基转移酶1和6-O-硫酸基转移酶3(6OST1和6OST3)的共同催化作用下，底物糖链的全部GlcNS或GlcNAc残基的6-OH发生硫酸化修饰，生成含6-O-硫酸化肝素寡糖二聚体中间产物；
    步骤g，在3-O-硫酸基转移酶1(3OST1)的催化作用下，底物糖链中GlcA与IdoA2S之间的GlcNS6S的3-OH发生硫酸化(GlcNS6S3S)，得到抗凝血肝素寡糖苯联二聚体目标化合物。
  6. 根据权利要求5所述的制备方法，其特征在于，含GlcA的二聚体中间体为苯基双O-葡糖酸苷或取代苯基双O-葡糖酸苷、芳杂环基双O-葡糖酸苷、取代芳杂环基双O-葡糖酸苷、C1-C5的烷基双O-葡糖酸苷或环烷基双O-葡糖酸苷；
    起始原料苯基双O-葡糖酸苷是按如下方法制得：
    以全乙酰化葡萄糖为起始原料，利用乙酰肼脱除端基乙酰基得中间体，裸路的异头位羟基与三氯乙腈(CNCCl3)反应得糖基三氯乙酰亚胺酯，然后在促进剂参与下与对二苯酚发生糖苷化反应生成中间体，接着醇解脱去乙酰基得到苯基双O-葡糖苷，最后由PhI(OAc)2和Tempo共同选择性氧化C6-OH得目标产物。
  7. 根据权利要求6所述的制备方法，其特征在于，全乙酰化葡萄糖1利用乙酰肼脱除端基乙酰基的反应溶剂为无水DMF，在常温下反应1h；
    中间体裸露的异头位羟基与三氯乙腈(CNCCl3)在小于0℃下反应3h以上，反应溶剂为DBU和无水DCM的混合液；
    糖基三氯乙酰亚胺酯3与对二苯酚的反应是以含分子筛的无水DCM为溶剂、BF3-Et2O为促进剂，在-5℃下反应1h，反应液经EA萃取、真空干燥、甲醇重结晶得中间体4；中间体4经无水MeOH-甲醇钠于常温下处理1h以上脱去乙酰基，然后用酸性树脂中和溶液至中性，过滤、真空干燥及结晶得中间体5；
    中间体5利用PhI(OAc)2和Tempo共同选择性氧化C6-OH，所用溶剂为体积比为4:4:1的t-BuOH、DCM和H2O，反应于常温下过夜，后经乙腈重结晶得目标产物苯基双O-葡糖酸苷6。
  8. [根据细则91更正 08.11.2023]
    根据权利要求7所述的制备方法，其特征在于，以苯基双O-葡糖酸苷6为原料，采用步骤a、b与步骤c、d、f、g的方法，制备目标化合物I-1，合成路线如式Ⅲ所示：
  9. [根据细则91更正 08.11.2023]
    根据权利要求5所述的制备方法，其特征在于，式I-2-式I-5的合成路线如下：
    I-2：a→b→a→b→c→d→a→b→c→d→a→c或无→f→g；
    I-3：a→b→a→b→c→e→a→b→c→d→a→c或无→f→g；
    I-4：a→b→a→b→c→d→a→b→c→d→a→b→c→d→a→c或无→f→g；
    I-5：a→b→a→b→c→d→f→a→b→a→b→c→d→a→c或无→f→g；
  10. [根据细则91更正 08.11.2023]
    根据权利要求5所述的制备方法，其特征在于，所述酶KfiA、PmHS2分别来源于大肠杆菌K5、多杀巴斯德菌(Pasteurella multocida)，以大肠杆菌重组表达，酶促糖链延长反应所用的缓冲液为50mmol/L Tris-HCl，pH＝7.0～7.5，反应温度20℃～37℃，酶促反应液利用反相C18或阴离子交换柱层析纯化得产物；
    所述肝素酶修饰酶NST、C5-epi、2OST、6OST1、6OST3、3OST1是利用大肠杆菌、酵母或昆虫细胞重组表达得到，所述NST、2OST、6OST1、6OST3、3OST1各修饰酶催化反应的缓冲液为50mmol/L 2-(N-吗啉代)乙烷磺酸(MES)，pH＝7.0～7.5，反应温度20℃～37℃，并均以3'-磷酸腺苷-5'-磷酸硫酸(PAPS)为硫酸基供体，酶促反应液利用阴离子交换柱层析纯化得产物。
  11. [根据细则91更正 08.11.2023]
    权利要求1所述的含AT结合序列的肝素寡糖苯联二聚体的应用，具特异抗Xa因子活性、无明显抗IIa活性，且其抗凝活性可被鱼精蛋白中和，药代动力学更佳，用于制备强效抗凝抗血栓药物。
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