CN109134555B - Anticoagulant pentasaccharide compound and preparation method and medical application thereof - Google Patents

Abstract

The invention relates to an anticoagulant drug, in particular to a synthetic pentasaccharide compound and salt thereof, a preparation method and pharmaceutical application thereof. The invention synthesizes an ionic pentasaccharide compound shown as a formula III or an acid form or a salt form thereof, and the ionic pentasaccharide compound has stronger anticoagulant factor Xa activity and longer elimination half-life than fondaparinux sodium. The pentasaccharide compound of the invention can be used for preparing medicines for preventing and treating diseases related to blood coagulation disorder, such as deep vein thrombosis, thrombophlebitis, arterial blockage caused by thrombosis or embolism, postoperative venous thrombosis or embolism and other diseases.

Description

Anticoagulant pentasaccharide compound and preparation method and medical application thereof

Technical Field

The invention relates to a pentasaccharide compound used as an anticoagulant, a preparation method thereof and a medical application thereof in anticoagulation.

Background

Thrombotic diseases are a group of diseases seriously harming human health, the incidence rate of which is the first of various diseases, and the incidence rate is increasing in recent years. It is mainly classified into arterial thrombosis and venous thrombosis. Venous thrombosis is usually found in deep veins, and is clinically manifested as local pain and swelling caused by thrombosis, distal blood backflow disorder and organ dysfunction caused by embolism after thrombus falls off. Arterial thrombosis is initiated by atherosclerotic lesions and platelet activation in arterial vessel walls, which can lead to serious cardiovascular diseases such as acute myocardial infarction, cerebral apoplexy, etc. The treatment method comprises anticoagulant therapy, antiplatelet therapy, thrombolysis therapy and the like, and the anticoagulant therapy is the core and the basis of clinical prevention and treatment of thrombotic diseases at present.

Heparin is a traditional anticoagulant drug and is a polysaccharide in the glycosaminoglycan family. Clinically used include plain heparin and low molecular weight heparin. Heparin induces a conformational change in Antithrombin (AT) by attaching to a specific binding domain of AT, which in turn inhibits the activity of factor Xa. Studies have shown that the smallest building block for heparin to bind AT and inhibit factor Xa is a unique pentasaccharide sequence.

To date, various documents have disclosed obtaining pentasaccharide compounds with antithrombotic and anticoagulant activities by total chemical synthesis. Sundaparinux sodium, which is the first generation synthetic analogue based on heparin antithrombin binding pentasaccharide sequences, is an indirect factor Xa inhibitor as reported in US patent No. 4818816, Carbohydrate Research 1987 (167). Fondaparinux sodium has a half-life of about 0.7 hours in rats and about 17 hours in humans. The product has been marketed in the United states since 2002, has been marketed in a number of countries and is clinically used for treating and preventing deep venous thromboembolic events, with the recommended dose being administered by subcutaneous injection once daily. Although fondaparinux sodium has excellent clinical performance, the synthesis difficulty is extremely high, and the production cost is high.

U.S. Pat. No. 5,5378829 reports a new class of pentasaccharides, such as edaravarin sodium (Idraparinux), formed by substituting an alkoxy group and an O-sulfonate group for the N-sulfonate group and the hydroxyl group in the fondaparinux sodium structure. Due to the introduction of alkoxy groups and O-sulfonate groups on the pentasaccharide units, the preparation mode is greatly simplified. Its half-life in rats was 9.2 hours (iv), in humans was extended to 120h, significantly increasing the risk of bleeding, thus stopping at the phase III clinical trial.

WO9936428 reports a class of pentasaccharide compounds prepared by replacing the O-alkyl group with an alkylene bridge and locking the configuration of the G unit L-iduronic acid, which compounds have potent anti-coagulation factor Xa activity and a strong affinity for AT.

WO9925720 discloses a pentasaccharide compound having 4-6 sulfate groups. The reduction of sulfate groups did not reduce antithrombotic activity nor did it result in heparin-induced thrombocytopenia.

WO0224754 discloses a class of pentasaccharide compounds covalently linked to biotin or a biotin derivative. The introduction of biotin or a derivative thereof does not alter the pharmaceutical activity of the pentasaccharide, and advantageously such compounds also have the advantage of being rapidly neutralized by an avidin lytic agent in an emergency state, thereby inhibiting its anticoagulant activity.

WO2012172104 discloses a class of highly active pentasaccharide compounds with short half-lives. The half-life of the alkylated/O-sulfated pentasaccharide is modulated by varying the substituents of the D unit.

The half-life of an anticoagulant drug is a very important pharmacokinetic parameter. In the clinic, it is desirable to switch off the anticoagulant effect as quickly as possible in the event of bleeding, and a long half-life would lead to an increased risk of bleeding. For certain thromboembolic disorders, such as pulmonary embolism, deep venous embolism, non-ST elevation myocardial infarction, and the like, it is more advantageous to extend the half-life of the anticoagulant appropriately.

On the other hand, the core of development of anticoagulant drugs is a safety problem, and how to effectively avoid bleeding tendency and reduce bleeding risk is a challenge faced by development of anticoagulant drugs.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a brand new synthesized pentasaccharide compound in an ionic form as shown in formula III:

wherein when R is₁When is H, R₂Is H or SO₃ ^-；

When R is₁Is SO₃ ^-Or C1-C4 alkyl, R₂Is H.

Preferably, the pentasaccharide compounds and salts thereof, in their anionic form, are selected from the following structures:

wherein R is₁Is C1-C4 alkyl, preferably methyl.

The invention includes the acid form of the synthetic pentasaccharide, in which-COO is present, and the salt forms thereof^-and-OSO₃ ^-The functional groups being respectively in the form of-COOH and-OSO₃Form H; the salt form of the present invention is selected from sodium, potassium, calcium or ammonium salts, preferably sodium or potassium salts.

Further, the present invention provides a method for preparing the pentasaccharide compound, comprising the steps of:

(a) reacting a disaccharide of formula DE with a trisaccharide of formula FGH to produce DEFGGH 0,

wherein R is₁Selected from Ac, Bn, C1-C4 alkyl, R₂Selected from Bn and Bz.

(b) Debenzylating DEFGH0 to produce DEFGH1,

wherein R is_aIs Ac, H, C1-C4 alkyl, R_bIs H, Bz；

(c) The compound of formula DEFGH1 is subjected to sulfation reaction to prepare DEFGH2,

wherein, R is_cIs Ac, SO₃ ^-C1-C4 alkyl, R_dIs SO₃ ^-，Bz；

(d) The compound shown as DEFGH2 is hydrolyzed to prepare the compounds shown as formulas III-1 to III-4.

The invention also provides an intermediate DE for preparing the pentasaccharide compound and the salt thereof,

wherein R is₁Is selected from Ac, Bn, C1-C4 alkyl.

And an intermediate of the formula,

wherein R is₂Selected from Bn and Bz.

And an intermediate of the formula,

wherein R is₁Selected from Ac, Bn, C1-C4 alkyl, R₂Selected from Bn and Bz.

And an intermediate of the formula,

wherein R is_aIs Ac, H, C1-C4 alkyl, R_bIs H, Bz.

The present invention also provides intermediates of the formula,

wherein R is_cIs Ac, SO₃ ^-C1-C4 alkyl, R_dIs SO₃ ^-，Bz。

In another aspect, the invention also relates to pharmaceutical compositions comprising a compound of the invention as an active ingredient, optionally together with pharmaceutically acceptable excipients.

The pentasaccharide compound represented by formula III of the present invention, an acid thereof or a salt thereof is 0.1 to 100mg, preferably 0.5 to 50mg per unit dose.

The pharmaceutical compositions may be administered by oral or parenteral routes including, but not limited to, intravenous, intramuscular, and subcutaneous injections.

For oral administration, the pharmaceutical compositions of the present invention are typically provided in the form of tablets, capsules, solutions. The tablet may comprise a pentasaccharide compound of formula III of the present invention, an acid or salt thereof and a pharmaceutically acceptable excipient. Such excipients include, but are not limited to, diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, or preservatives. The diluent is selected from starch and lactose; the disintegrating agent is selected from corn starch and alginic acid; the binder is selected from starch, polyvidone, and gelatin; the lubricant is selected from magnesium stearate, pulvis Talci, and sodium stearyl fumarate. The capsule includes hard capsule and soft capsule. The pentasaccharide compound represented by formula III of the present invention, an acid thereof or a salt thereof, is mixed with a diluent as an active ingredient in a hard capsule, and is mixed with water or an oil such as peanut oil or olive oil in a soft capsule.

For parenteral administration, the pharmaceutical composition of the present invention may be administered by intravenous injection, intramuscular injection or subcutaneous injection. It is usually provided as a sterile aqueous solution or suspension or lyophilized powder, with appropriate adjustment of pH and isotonicity. In addition, adjuvants such as mannitol, cellulose derivatives, lecithin may be optionally added. The preferred route of administration is subcutaneous injection.

In another aspect, the invention also relates to the use of a pentasaccharide compound of formula III, an acid or salt thereof, in the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with dyscoagulation mechanisms.

In yet another aspect, the present invention also relates to a method for preventing and/or treating diseases and disease states associated with blood coagulation disorders, which comprises administering a pentasaccharide compound represented by formula III of the present invention, an acid or a salt thereof, or a pharmaceutical composition of the present invention to a subject in need thereof.

In yet another aspect, the present invention also relates to a pentasaccharide compound represented by formula III, an acid or salt thereof, or a pharmaceutical composition of the present invention for use in the prevention and/or treatment of diseases and conditions associated with blood coagulation disorders.

Examples of such diseases or conditions associated with blood coagulation disorders include, but are not limited to, venous thrombosis, particularly deep vein thrombosis, arterial thrombosis, thrombophlebitis, pulmonary embolism, acute coronary syndrome, myocardial infarction or stroke, and arterial occlusion caused by thrombosis or embolism, arterial reocclusion during or after angioplasty or thrombolysis, post-operative venous thrombosis or embolism, acute or chronic arteriosclerosis, ST-elevation myocardial infarction, non-ST elevation. The compounds of the invention are also useful as anticoagulants in extracorporeal blood circulation, in hemodialysis and in surgery. The compounds of the invention may also be used for the prevention of ischemic reperfusion injury associated with solid organ transplantation.

The advantages of the invention are mainly reflected in that: the invention synthesizes a series of novel pentasaccharide compounds by changing the substituents of the E ring and the H ring, and surprisingly, the pentasaccharide compounds have unexpected technical effects. Firstly, the pentasaccharide compound has stronger anticoagulant factor Xa activity than fondaparinux sodium, and has smaller dosage, thereby having smaller side effect; secondly, the pentasaccharide compound of the invention has prolonged half-life period in vivo compared with fondaparinux sodium, can be administered once every 2-3 days, and is particularly suitable for treating patients with pulmonary embolism, deep venous embolism and non-ST-elevation myocardial infarction; in primary safety evaluation, the compound has almost no significant influence on bleeding tendency, and shows important potential application value; moreover, the preparation method of the compound is greatly simplified, the manufacturing cost is low, the development cost of the raw material medicine is obviously reduced, and the compound is very suitable for industrial mass production.

Detailed Description

Through intensive research, the inventors of the present application have synthesized the pentasaccharide compound shown in the general formula III, and have conducted biological experimental research, and found that the pentasaccharide compound of the present invention has high anti-coagulation factor Xa activity and a suitable half-life period for in vivo elimination, and is particularly suitable for use as an anticoagulant.

Definition of

Unless stated to the contrary, the following terms used in the specification and claims have the following meanings

"alkyl" refers to a saturated aliphatic hydrocarbon group, including straight or branched chain saturated monovalent hydrocarbon groups having the indicated number of carbon atoms. For example, the term "C1-C4 alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, and suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.

The abbreviations used in the claims and the specification have the following meanings:

Bz	benzoyl radical
		Bn	Benzyl radical
Ac	Acetyl group
		DMAP	4-dimethylaminopyridine
DBU	1, 8-diazabicycloundec-7-enes
		TEMPO	2,2,6, 6-tetramethylpiperidine-nitrogen-oxide
BAIB	Iodobenzene diethyl ester
		TCCA	Trichloroisocyanuric acid
TMSOTf	Trimethylsilyl trifluoromethanesulfonate
		Dowex	Ion exchange resin
Sephadex	Glucan gels

The preparation method comprises the following steps:

the process for producing the pentasaccharide compound of the present invention is more specifically described below, but these specific processes do not set any limit to the scope of the present invention. In addition, reaction conditions such as reactants, solvents, bases, amounts of compounds used, reaction temperature, reaction time and the like are not limited to the following examples.

The pentasaccharide compounds of the invention may also be conveniently prepared by optionally combining various synthetic methods described in the specification or known in the art, and such combinations may be readily carried out by those skilled in the art.

Synthesis of the first partial monosaccharide D Ring

The synthetic route is as follows:

a) dimethyl sulfate, KOH, acetonitrile, b) acetic acid, sulfuric acid, water, c) trichloroacetonitrile, DBU, dichloromethane

Preparation of compound D1: dissolving D0(748g) in tetrahydrofuran, adding excessive potassium hydroxide as alkali, dropwise adding dimethyl sulfate at 0 ℃ for reaction for 2h, quenching by using a potassium hydroxide aqueous solution for 4h after the reaction is finished, extracting by using ethyl acetate, separating liquid, and spin-drying to obtain D1(804g, yield 100%).

Preparation of compound D2: add D1(804g) to acetic acid: water: in 2000g sulfuric acid, 400g 98g sulfuric acid, reflux reaction for 1 hr, extraction with ethyl acetate and water, and evaporation of the organic phase to obtain D2(690g, 89% yield).

Preparation of compound D3: d2(388g) was dissolved in anhydrous dichloromethane, 288g of trichloroacetonitrile and 15g of DBU were added to the solution to react for 1 hour, and then dry column chromatography was performed to obtain D3(489g, 92% yield).

Preparation of the E Ring of the second partial monosaccharide

The synthetic route is as follows:

a) dimethyl sulfate, KOH, tetrahydrofuran, b) trifluoroacetic acid, water, c) pyridinium p-toluenesulfonate, benzaldehyde dimethyl acetal, d) ethyl acetate, acetic anhydride, triethylamine, DMAP, E) boron trifluoride diethyl etherate, p-methoxyphenol, f) potassium hydroxide, g) preparation E8: potassium hydroxide, dimethyl sulfate, h) acetic acid, water, i) TEMPO, BAIB, dichloromethane, water, j) iodomethane, potassium bicarbonate, acetonitrile

Preparation of Compounds E1-E4: reference is made to Tetrahedron,2012,68(36),7386 and 7399.

Preparation of compound E5:

e4(366g) and p-methoxyphenol 130g were dissolved in 366ml of anhydrous dichloromethane, 280g of boron trifluoride diethyl ether was added at room temperature, and after 2 hours of reaction, the mixture was diluted with 4L of dichloromethane, quenched with saturated aqueous sodium bicarbonate solution, then separated, and the organic phase was dried over anhydrous sodium sulfate to give E5(387g, yield 90%).

Preparation of compound E6:

dissolving the intermediate E5(86g) in 860ml methanol, adding sodium methoxide 2.7g, stirring at room temperature for reaction for 1h, adding glacial acetic acid for neutralization, decompressing and evaporating to dryness, adding dichloromethane 1L and water 1L to wash the residue, separating liquid, and spin-drying the organic phase to obtain E6(77.6g, yield 100%).

Preparation of compound E7:

dissolving E6(38.8g) in 400ml acetonitrile, adding potassium hydroxide 12g, adding benzyl bromide 18g dropwise at 10 ℃, reacting for 4h, adding methanol 50ml, refluxing for 1h, then performing rotary drying under reduced pressure, adding ethyl acetate 1L and water 1L into the residue, extracting and washing, performing rotary drying under reduced pressure to obtain crude product 50g, and recrystallizing the crude product with ethyl acetate 40ml and petroleum ether 120ml to obtain E7(45g, yield 94%).

Preparation of compound E8:

dissolving E6(38.8g) in 400ml acetonitrile, adding 23g potassium hydroxide, adding 18.9g dimethyl sulfate dropwise at 10 ℃, reacting for 4h, performing rotary drying under reduced pressure, adding ethyl acetate 1 and water 1L into the residue, extracting and washing, performing rotary drying under reduced pressure on the organic phase to obtain 48g crude product, and recrystallizing the crude product with 30ml ethyl acetate and 120ml petroleum ether to obtain E8(36.4g, yield 91%).

Preparation of compound E9:

dissolving E5(98.5g) in 430ml of acetic acid, adding 86g of water, stirring at 70 ℃ for reaction for 1h, then carrying out rotary evaporation at 55 ℃ under reduced pressure, adding 1L of water and 1L of ethyl acetate, adjusting the pH to 7 with sodium carbonate, then carrying out liquid separation to obtain an organic phase, carrying out vacuum evaporation on the organic phase to obtain 80g of crude E9, petroleum ether: the column chromatography is carried out with ethyl acetate 1:1 to obtain 70.5g of pure product with yield of 90%.

Preparation of compound E10:

dissolving E7(45g) in 450ml acetic acid, adding 90g of water, stirring at 70 ℃ for reaction for 1h, then carrying out rotary evaporation at 55 ℃ under reduced pressure to dry, adding 1L of water and 1L of ethyl acetate, adjusting the pH to 7 by using sodium carbonate, then carrying out liquid separation to obtain an organic phase, and carrying out vacuum evaporation to dry the organic phase to obtain 40g of crude E10, petroleum ether: column chromatography with ethyl acetate 1:1 gave 33.7g of pure product in 92% yield.

Preparation of compound E11:

dissolving E8(36g) in 450ml acetic acid, adding 90g of water, stirring at 70 ℃ for reaction for 1h, then carrying out rotary evaporation at 55 ℃ under reduced pressure to dry, adding 1L of water and 1L of ethyl acetate, adjusting the pH to 7 by using sodium carbonate, then carrying out liquid separation to obtain an organic phase, and carrying out vacuum evaporation to dry the organic phase to obtain 38g of crude E11, petroleum ether: column chromatography with ethyl acetate 1:1 gave 26.7g of pure product with 95% yield.

Preparation of compound E12:

e9(68.4g) and TEMPO 0.63g were added to 342ml of methylene chloride and 342ml of water, 86g of diethyl iodobenzene was added in one portion, reacted at room temperature for 45min with vigorous stirring, and then quenched with 200ml of 30% aqueous sodium sulfite solution. The aqueous phase was extracted 5 times with ethyl acetate (500 ml. times.5), and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give E12(57.6g, yield 80%).

Preparation of compound E13:

e10(33g) and TEMPO 0.27g were added to 330ml of methylene chloride and 330ml of water, 37g of diethyl iodobenzene was added in one portion, reacted at room temperature for 45min with vigorous stirring, and then quenched with 100ml of a 30% aqueous sodium sulfite solution. The aqueous phase was extracted 5 times with ethyl acetate (300 ml. times.5), and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give E13(28g, yield 81%).

Preparation of compound E14:

e11(26g) and TEMPO 0.27g were added to 260ml of methylene chloride and 260ml of water, 37g of diethyl iodobenzene was added in one portion, reacted at room temperature for 45min with vigorous stirring, and then quenched with 100ml of 30% aqueous sodium sulfite solution. The aqueous phase was extracted 5 times with ethyl acetate (300 ml. times.5), and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give E14(25g, yield 88%).

Preparation of compound E15:

e12(57.6g) was dissolved in 576ml of acetonitrile, and 33.5g of potassium carbonate and 34.5g of methyl iodide were added to the solution, and the mixture was stirred and reacted for 8 hours, and after completion of the reaction, 10ml of triethylamine was added to the reaction solution to quench the reaction solution, and the reaction solution was washed three times with saturated brine (200 ml. times.3) to adjust the pH to 7 to 8, and the organic phase was spin-dried under reduced pressure. Petroleum ether: the ethyl acetate 2:1 column chromatography purification yielded 50g, yield 85%.

Preparation of compound E16:

e13(28g) was dissolved in acetonitrile 280ml, potassium carbonate 9.6g and methyl iodide 10g were added, the mixture was stirred and reacted for 8 hours, triethylamine 5ml was added to the reaction product, the reaction product was quenched after completion of the reaction, the reaction product was washed three times with saturated saline (100 ml. times.3) to adjust pH to 7 to 8, and the organic phase was spin-dried under reduced pressure. Petroleum ether: purification by 2:1 column chromatography gave 25g, 87% yield.

Preparation of compound E17:

e14(25g) was dissolved in acetonitrile 250ml, potassium carbonate 10.5g and methyl iodide 11g were added, the mixture was stirred and reacted for 8 hours, triethylamine 5ml was added after the reaction was completed, the mixture was quenched, washed three times with saturated brine (100 ml. times.3) to adjust pH to 7 to 8, and the organic phase was spin-dried under reduced pressure. Petroleum ether: purification by 2:1 column chromatography gave 23.5g, 90% yield.

Preparation of the F and H rings of the third partial monosaccharide

The synthetic route is as follows:

a) sulfuric acid, benzaldehyde, DMF, b) benzyl bromide, potassium hydroxide, acetonitrile, c) triethylsilane, boron trifluoride diethyl etherate, dichloromethane, d) acetic acid, sulfuric acid, water, e) acetic anhydride, triethylamine, DMAP, ethyl acetate, f) piperidine, tetrahydrofuran, g) trichloroacetonitrile, DBU, dichloromethane, h) acetic acid, water, i) -40 ℃, benzoyl chloride, pyridine

Preparation of compound F1(H1) -F3 (H3): reference is made to Bioorganic & Medicinal Chemistry Letters,2009,19(14), 3875-.

Preparation of compound F4: f3(464g) was added to a mixed solution of 2.3kg of acetic acid and 464g of water, 46g of sulfuric acid was added, and the mixture was reacted at 100 ℃ for 1 hour. After completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure to give F4(427.5g, yield 95%).

Preparation of compound F5: dissolving F4(400g) in ethyl acetate, adding triethylamine 200ml, dropwise adding acetic anhydride 200ml, reacting for 1h, adding saturated sodium carbonate aqueous solution for neutralization, extracting with ethyl acetate, and evaporating to dryness under reduced pressure to obtain F5(475g, yield 100%).¹H NMR(300MHz,CDCl₃)δ7.35-7.29(m,15H),6.0(d,J＝3.6Hz,1H),4.9(dd,J＝9.2,3.4Hz,1H),4.7-4.6(m,6H),4.4(m,1H),4.27-4.18(m,2H),3.6(d,1H),3.4(d,1H),2.19(s,3H),2.14(s,3H).MS(ESI):557.3[M+Na]⁺

Preparation of compound F6: dissolving F5(450g) in tetrahydrofuran, adding piperidine 143g, reacting for 6h, neutralizing with acetic acid, rotary evaporating under reduced pressure, dissolving the residue with ethyl acetate, washing with water, and evaporating the organic phase under reduced pressure to obtain 373g, with a yield of 90%.

Preparation of compound F7: f6(350g) is added into dichloromethane to be dissolved, 400g of trichloroacetonitrile and 10g of DBU are added, after 1h of reaction, drying by spinning and column chromatography are carried out, and F7(406g, yield 90%) is obtained.

Preparation of compound H4: f2(463g) was added to a mixed solution of 2.3kg of acetic acid and 464g of water, and 46g of sulfuric acid was further added to react at 100 ℃ for 1 hour. After completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure to give H4(307g, yield 82%).

Preparation of compound H5: dissolving H4(187g) in pyridine, slowly adding 141g benzoyl chloride dropwise at-40 ℃ under cooling, reacting for 2H, adding saturated aqueous solution of sodium bicarbonate to precipitate a light yellow solid, washing, and drying to obtain H5(203.3g, yield 85%).

The fourth part: synthesis of the G Ring of the monosaccharide

The synthetic route is as follows:

a) dimethyl sulfate, KOH, tetrahydrofuran, b) phosphomolybdic acid, silica gel, water, acetonitrile, c) dipotassium hydrogenphosphate trihydrate, potassium dihydrogenphosphate, water, TEMPO, NaClO₂KBr, TCCA, d) iodomethane, potassium carbonate, acetonitrile, e) trifluoromethanesulfonic anhydride, pyridine, f) sodium trifluoroacetate, DMSO, water, g) trifluoroacetic acid, water, h) acetic anhydride, triethylamine, DMAP, ethyl acetate, j) neodymium trifluoromethanesulfonate,k) trichloroacetonitrile, DBU, dichloromethane

Preparation of compound G1: see synthesis of E1.

Preparation of compound G2: dissolving G1(1.5kg) in acetonitrile 9.72kg, adding silica gel 0.36kg, phosphomolybdic acid 0.036kg and water 0.8kg, stirring at room temperature for 16h, filtering off silica gel, neutralizing with sodium carbonate aqueous solution to pH 7, rotary evaporating the filtrate under reduced pressure, extracting the residue with water and ethyl acetate, and evaporating the filtrate under reduced pressure to obtain G2(1.15kg, yield 90%).

Preparation of compound G3: dissolving G2(1.15kg) in tetrahydrofuran 10L, adding dipotassium hydrogen phosphate trihydrate 500G, potassium dihydrogen phosphate 500G, water 1.5kg, TEMPO 20G, NaClO₂300G, 50G of KBr and 500G of TCCA, reacting for 1h, adding anhydrous sodium sulfite to quench, then spin-drying under reduced pressure, washing the residue with ethyl acetate, and spin-drying the organic phase under reduced pressure to obtain G3(1.1kg, yield 90%).

Preparation of compound G4: dissolving G3(1.1kg) in acetonitrile 6.0kg, adding 1.22kg potassium carbonate and methyl iodide 0.73kg, stirring at room temperature for 8h, after the reaction is finished, adding triethylamine to quench methyl iodide, filtering, and drying the filtrate under reduced pressure to obtain G4(929G, yield 80%).

Preparation of compound G5: adding pyridine 1L and acetonitrile 5L into G4(885G), dropwise adding trifluoromethanesulfonic anhydride 950G, reacting for 1h after the dropwise adding, then adding ice water for crystallization and suction filtration, dissolving the filter cake with dichloromethane, washing the dichloromethane phase with water, drying, and spin-drying under reduced pressure to obtain G5(1280G, yield 96%).

Preparation of compound G6: g5(1280G) and 1kg of sodium trifluoroacetate are dissolved in DMSO 10L and water 1L, stirred for 8h, then ethyl acetate is added for extraction, and the mixture is dried under reduced pressure to obtain G6(808G, yield 95%).

Preparation of compound G7: 2L of trifluoroacetic acid and 200ml of water were added to G6(808G), and the mixture was stirred for 1 hour and then spin-dried under reduced pressure to obtain G7(547G, yield 80%).

Preparation of compound G8: g7(547G) was dissolved in 5L of dichloromethane, and then DMAP 50G and triethylamine 1.15kg were added thereto, and then acetic anhydride 1.15kg was added dropwise at 0 ℃ to react for 0.5h, followed by washing with water and vacuum spin-drying of the organic phase to obtain G8(815G, yield 95%).

Preparation of compound G9: g8(810G) was added to 12L of methanol, and 60G of neodymium trifluoromethanesulfonate was added thereto, followed by stirring for 12 hours, concentration and column chromatography to give G9(605G, yield 85%).

Preparation of compound G10: g9(605G) was added to 6kg of dichloromethane, 1.2kg of trichloroacetonitrile and 45G of DBU, reacted for 1 hour, and then spin-dried and subjected to column chromatography to obtain G10(801G, yield 90%).

Synthesis of the fifth disaccharide DE

The synthetic route is as follows:

a) TMSOTf, dichloromethane, b) ceric ammonium nitrate, c) trichloroacetonitrile, DBU, dichloromethane

Preparation of compound DE 1: dissolving D3(50g) and E15(30g) in anhydrous dichloromethane, dropwise adding TMSOTf1g under the protection of nitrogen, reacting at-20 ℃ for 1h, adding triethylamine for quenching, performing reduced pressure spin drying, and performing column chromatography to obtain 51g of DE1 alpha isomer with the yield of 85%.

Preparation of compound DE 2: dissolving D3(55g) and E16(30g) in anhydrous dichloromethane, dropwise adding TMSOTf1g under the protection of nitrogen, reacting at-20 ℃ for 1h, adding triethylamine for quenching, performing reduced pressure spin drying, and performing column chromatography to obtain 46.9g of DE2 alpha isomer with the yield of 83%.

Preparation of compound DE 3: dissolving D3(50g) and E17(30g) in anhydrous dichloromethane, dropwise adding TMSOTf1g under the protection of nitrogen, reacting at-20 ℃ for 1h, adding triethylamine for quenching, performing reduced pressure spin drying, and performing column chromatography to obtain 50.8g of DE2 alpha isomer with the yield of 82%.

Preparation of compound DE 4: DE1(40g) was dissolved in a mixed solution of acetonitrile 0.4L \ toluene 0.2L \ water 0.2L, and 50g of ceric ammonium nitrate was added to react at room temperature for 1 hour, followed by extraction with ethyl acetate and spin-drying under reduced pressure to obtain DE4(30.9g, yield 90%).

¹H NMR(300MHz,CDCl₃)δ7.35-7.29(m,10H),6.29(d,J＝3.6Hz,1H),5.6(d,J＝3.4Hz,1H),4.97(dd,J＝9.2,3.4Hz,1H),4.65–4.54(m,5H),4.32-4.18(m,3H),4.00(m,1H),3.80(m,2H),3.70(s,3H),3.65–3.1(m,12H),2.02(s,3H).MS(ESI):657.3[M+Na]⁺

Preparation of compound DE 5: dissolving DE2(40g) in acetonitrile 0.4L \ toluene 0.2L \ water 0.2L mixed solution, adding cerium ammonium nitrate 45g, reacting at room temperature for 1h, extracting with ethyl acetate, and vacuum drying to obtain DE5(31.5g, yield 91%)

¹H NMR(300MHz,CDCl₃)δ7.36-7.26(m,15H),5.9(d,J＝3.6Hz,1H),5.6(dd,J＝9.2,3.4Hz,1H),4.65–4.54(m,7H),4.32(s,1H),4.23(m,1H),4.00(m,1H),3.80(m,3H),3.70(s,3H),3.65–3.1(m,10H).MS(ESI):705.3[M+Na]⁺

Preparation of compound DE 6: DE 340 g, adding acetonitrile 0.4L, toluene 0.2L, water 0.2L, adding ceric ammonium nitrate 50g, reacting at room temperature for 1h, extracting with ethyl acetate, and vacuum drying to obtain DE6(32g, yield 94%)¹H NMR(300MHz,CDCl₃)δ7.34-7.29(m,10H),5.90(d,J＝3.6Hz,1H),5.60(d,J＝3.6Hz,1H),4.65–4.61(m,5H),4.32(s,1H),4.23(d,1H),4.00(m,1H),3.80-3.77(m,3H),3.70(s,3H),3.61-3.60(m,2H),3.50(m,1H),3.41(m,12H).MS(ESI):629.3[M+Na]⁺

Preparation of compound DE 7: DE4(30g) was added to 0.6kg of dichloromethane, 40g of trichloroacetonitrile and 4g of DBU, reacted for 1 hour, and then spin-dried and subjected to column chromatography to obtain DE7(29.5g, yield 80%).

Preparation of compound DE 8: DE5(30g) was added to 0.6kg of dichloromethane, 40g of trichloroacetonitrile and 4g of DBU, reacted for 1 hour, and then spin-dried and subjected to column chromatography to obtain DE8(29.8g, yield 82%).

Preparation of compound DE 9: DE4(30g) was added to 0.6kg of dichloromethane, 40g of trichloroacetonitrile and 4g of DBU, reacted for 1 hour, and then spin-dried and subjected to column chromatography to obtain DE9(31.5g, yield 85%).

Synthesis of the sixth part of the trisaccharides FGH

The synthetic route is as follows:

a) TMSOTf, dichloromethane, b) potassium carbonate, methanol, c) TMSOTf, dichloromethane, d) trichloroacetimidobenzyl ester, trifluoromethanesulfonic acid, e) potassium carbonate, methanol

Preparation of compound GH 1: dissolving G10(631G) and H3(465G) in anhydrous dichloromethane, dropwise adding TMSOTf23G at 0 ℃, reacting for 1H, adding triethylamine for quenching, and performing column chromatography to obtain GH1(677G, yield 90%).

Preparation of compound GH 2: dissolving G10(631G) and H5(478G) in anhydrous dichloromethane, adding TMSOTf23G dropwise at 0 ℃, reacting for 1H, adding triethylamine for quenching, and performing column chromatography to obtain GH2(659G, yield 86%).

Preparation of compound GH 3: GH1(600g) and potassium carbonate (400g) are dissolved in absolute methanol, reacted at 25 ℃ for 6h, then water and ethyl acetate are added for extraction, spin-dried and subjected to column chromatography to obtain GH3(432g, yield 81%).

Preparation of compound GH 4: GH2(600g) and potassium carbonate (400g) are dissolved in absolute methanol, reacted at 25 ℃ for 6h, then water and ethyl acetate are added for extraction, spin-dried and subjected to column chromatography to obtain GH4(438g, yield 82%).

Preparation of compound FGH 1: GH3(400g) and F7(520g) are dissolved by adding anhydrous dichloromethane, TMSOTf15g is added dropwise at 0 ℃, after 1h of reaction, triethylamine is added for quenching, and column chromatography is carried out to obtain FGH1(561g, yield 82%).

Preparation of compound FGH 2: GH4(400g) and F7(530g) are dissolved by adding anhydrous dichloromethane, TMSOTf15g is added dropwise at 0 ℃, after 1h of reaction, triethylamine is added for quenching, and column chromatography is carried out to obtain FGH2(439g, yield 75%).

Preparation of compound FGH 3: dissolving FGH1(400g) and trichloroacetimidobenzyl ester (125g) in anhydrous dichloromethane, dropwise adding 10g of trifluoromethanesulfonic acid at 0 ℃, reacting for 1h, adding triethylamine for quenching, and performing column chromatography to obtain FGH3(388g, yield 90%). Preparation of compound FGH 4: dissolving FGH2(400g) and trichloroacetimidobenzyl ester (125g) in anhydrous dichloromethane, dropwise adding 10g of trifluoromethanesulfonic acid at 0 ℃, reacting for 1h, adding triethylamine for quenching, and performing column chromatography to obtain FGH4(397g, yield 92%). Preparation of compound FGH 5: dissolving FGH3(300g) and potassium carbonate (400g) in anhydrous methanol, reacting at 25 deg.C for 6h, extracting with water and ethyl acetate, spin-drying, and performing column chromatography to obtain FGH5(243g, yield 84%).

¹H NMR(300MHz,CDCl₃)δ7.32-7.30(m,35H),5.60(m,3H),4.88(s,1H),4.64–4.62(m,15H),4.49(m,1H),4.24-4.23(m,1H),4.00(m,1H),3.80-3.79(m,7H),3.70(m,2H),3.61-3.60(s,3H),3.50(d,1H),3.41-3.36(m,8H).MS(ESI):1213.5[M+Na]⁺

Preparation of compound FGH 6: dissolving FGH4(300g) and potassium carbonate (400g) in anhydrous methanol, reacting at 25 deg.C for 6h, extracting with water and ethyl acetate, spin-drying, and performing column chromatography to obtain FGH6(249g, yield 86%).

¹H NMR(300MHz,CDCl₃)δ8.06-7.55(m,5H),7.32-7.30(m,30H),5.60(m,3H),4.88(s,1H),4.64–4.62(m,13H),4.49(m,1H),4.24-4.23(m,2H),3.80-3.77(m,6H),3.61-3.60(m,2H),3.50(d,1H),3.41-3.40(d,6H).MS(ESI):1227.5[M+Na]⁺

EXAMPLE 1 preparation of pentasaccharide III-1 (sodium salt)

The synthetic route is as follows:

a) TMSOTf, dichloromethane, b) palladium on carbon, hydrogen, c) sulfur trioxide triethylamine salt, DMF, sodium bicarbonate, dowex-50x4, d)4N NaOH, sephadex g25

Preparation of compound DEFGH 10: DE5(29g) and FGH6(45g) were dissolved in anhydrous dichloromethane, TMSOTf1g was added dropwise at 0 ℃ and after 1h of reaction, triethylamine was added thereto for quenching, and column chromatography was performed to obtain DEFGH10(59g, yield 87%).¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),7.34-7.29(m,40H),5.99(d,1H),5.60(m,4H),5.17(dd,J＝9.0,3.4Hz,1H),4.64–4.62(m,18H),4.49-4.19(m,5H),4.00(m,2H),3.80-3.77(m,8H),3.70(s,6H),3.61(d,2H),3.50(d,3H),3.41-3.40(m,15H),3.36(m,2H),2.02(s,3H).MS(ESI):1843.8[M+Na]⁺

Preparation of compound DEFGH 11: DEFGH10(59g) is dissolved in absolute methanol, 10% palladium carbon is added, hydrogen is reduced for 24h under normal pressure, and then filtering and spin-drying are carried out to obtain DEFGH11(34.2g, yield 96%).¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),5.99(d,J＝3.6Hz,1H),5.40(m,4H),5.17(dd,J＝9.0,3.4Hz,1H),4.77(d,4H),4.71(d,2H),4.64–4.62(m,2H),4.49-4.19(m,6H),4.10(m,2H),3.94-3.80(m,8H),3.70(d,6H),3.60-3.50(m,7H),3.41-3.40(m,15H),3.30(m,2H),2.02(s,3H).MS(ESI):1123.4[M+Na]⁺

Preparation of compound DEFGH 12: DEFGH11(5.8g) is added into N, N-dimethylformamide to be dissolved, excess sulfur trioxide triethylamine salt is added, after reaction for 24 hours at 50 ℃, saturated sodium bicarbonate water solution is added for quenching, and the mixture is concentrated under reduced pressure. Gel column (Sephadex G-25) separation, eluent (water/acetonitrile 7/1, v/v). A sodium ion exchange resin column (DOWEX-50Na), rinsed with pure water, and concentrated under reduced pressure to give DEFGH12(9.0g, 89% yield) as a colorless solid.¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),5.99(d,J＝3.6Hz,1H),5.54(m,4H),5.30-5.17(m,7H),,4.64–4.62(m,2H),4.49-4.19(m,8H),3.96-3.94(m,4H),3.74(m,2H),3.70(d,6H),3.50-3.44(m,3H),3.41-3.40(m,15H),2.02(s,3H).MS(ESI):935.0[(M-2Na)/2]^-

Preparation of pentasaccharide III-1 (sodium salt): DEFGH12(5.2g) was added to a 4N aqueous NaOH solution, reacted at room temperature for 24 hours, and then concentrated under reduced pressure. Gel column (Sephadex G-25) separation, eluent (water/acetonitrile 4/1, v/v). A sodium ion exchange resin column (DOWEX-50Na), rinsed with pure water, and concentrated under reduced pressure to give a colorless solid (4.1g, 85% yield).

¹H NMR(300MHz,CDCl₃)δ5.54(m,4H),5.40(m,1H),5.30(m,2H),5.25-5.24(m,4H),4.77(m,1H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,1H),3.96-3.94(m,5H),3.80-3.74(m,3H),3.60(m,2H),3.51-3.40(m,19H).MS(ESI):870.0[(M-2Na)/2]^-

EXAMPLE 2 preparation of pentasaccharide III-2 (sodium salt)

The synthetic route is as follows:

referring to the method of example 1, DE9 and FGH6 are used as raw materials to react, and then the target compound is prepared by removing benzyl through hydrogenolysis reaction, sulfation reaction and hydrolysis reaction.

Compound DEFGH 20:

¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),7.34-7.29(m,45H),5.60(m,5H),4.64–4.62(m,20H),4.49-4.19(m,3H),4.00(m,2H),3.80-3.77(m,10H),3.70(s,6H),3.61(d,2H),3.50(d,3H),3.41-3.40(m,15H),3.36(m,2H),2.02(s,3H).MS(ESI):1891.8[M+Na]⁺

compound DEFGH 21:

¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),5.40(m,5H),4.77(d,5H),4.71(d,2H),4.64–4.62(m,2H),4.49-4.19(m,5H),4.10(m,3H),3.94-3.80(m,8H),3.70(d,6H),3.60-3.50(m,8H),3.41-3.40(m,15H),3.30(m,2H).MS(ESI):1081.4[M+Na]⁺

compound DEFGH 22:

¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),5.54(m,5H),5.30-5.24(m,7H),,4.64–4.62(m,2H),4.49-4.19(m,7H),3.96-3.94(m,4H),3.74(m,3H),3.70(d,6H),3.50-3.44(m,3H),3.41-3.40(m,15H).MS(ESI):964.9[(M-2Na)/2]^-

pentasaccharide III-2 (sodium salt):

¹H NMR(300MHz,CDCl₃)δ5.54(m,5H),5.40(m,1H),5.30(m,3H),5.25-5.24(m,3H),4.64–4.62(m,2H),4.23-4.21(m,4H),3.96-3.94(m,5H),3.80-3.74(m,4H),3.60-3.44(m,5H),3.41-3.40(m,15H).MS(ESI):920.9[(M-2Na)/2]^-

example 3 pentasaccharide III-3 (R)₁As methyl, sodium salt) preparation

The synthetic route is as follows:

referring to the method of example 1, DE6 and FGH6 are used as raw materials to react, and then the target compound is prepared by removing benzyl through hydrogenolysis reaction, sulfation reaction and hydrolysis reaction.

Compound DEFGH 30:

¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),7.34-7.29(m,40H),4.64–4.62(m,18H),4.49-4.19(m,4H),4.00(m,2H),3.80-3.77(m,10H),3.70(s,6H),3.61(d,2H),3.50(d,3H),3.41-3.40(m,18H),3.36(m,2H).MS(ESI):1815.8[M+Na]⁺

compound DEFGH 31:

¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),5.60(d,1H),5.40(m,4H),4.77(d,4H),4.71(d,2H),4.64–4.62(m,2H),4.49-4.19(m,5H),4.10(m,2H),3.94-3.80(m,10H),3.70(d,6H),3.60-3.50(m,7H),3.41-3.40(m,18H),3.30(m,2H).MS(ESI):1095.4[M+Na]⁺

compound DEFGH 32:

¹H NMR(300MHz,CDCl₃)δ8.05-7.55(m,5H),5.60(d,1H),5.54(m,4H),5.30-5.24(m,6H),4.64–4.62(m,2H),4.49-4.19(m,7H),3.96-3.94(m,4H),3.80-3.70(m,10H),3.50-3.44(m,3H),3.41-3.40(m,18H).MS(ESI):921.0[(M-2Na)/2]^-

pentasaccharide III-3 (sodium salt):

¹H NMR(300MHz,CDCl₃)δ5.60-5.54(m,5H),5.30-5.24(m,6H),4.64–4.62(m,2H),4.23-4.21(m,4H),3.96-3.94(m,5H),3.80-3.74(m,5H),3.60-3.44(m,5H),3.41-3.40(m,18H).MS(ESI):876.9[(M-2Na)/2]^-

EXAMPLE 4 preparation of pentasaccharide III-4 (sodium salt)

The synthetic route is as follows:

referring to the method of example 1, DE5 and FGH5 are used as raw materials to react, and then the target compound is prepared by removing benzyl through hydrogenolysis reaction, sulfation reaction and hydrolysis reaction.

Compound DEFGH 40:

¹H NMR(300MHz,CDCl₃)δ7.34-7.29(m,45H),5.60(d,1H),5.40(m,4H),5.17(m,1H),4.64–4.62(m,20H),4.49-4.19(m,3H),4.00(m,3H),3.80-3.77(m,8H),3.70(s,6H),3.61(d,3H),3.50(d,3H),3.41-3.40(m,15H),3.36(m,3H).MS(ESI):18298[M+Na]⁺

compound DEFGH 41:

¹H NMR(300MHz,CDCl₃)δ5.99(m,1H),5.60(d,1H),5.40(m,4H),5.17(m,1H),4.77(d,4H),4.71(d,2H),4.64–4.62(m,2H),4.49-4.19(m,3H),4.10(m,2H),3.94-3.80(m,10H),3.70(d,6H),3.60-3.50(m,7H),3.41-3.40(m,15H),3.30(m,2H),2.02(s,3H).MS(ESI):1019.4[M+Na]⁺

compound DEFGH 42:

¹H NMR(300MHz,CDCl₃)δ5.99(d,1H),5.54(m,4H),5.30-5.24(m,6H),5.17(m,1H),4.64–4.62(m,2H),4.49-4.19(m,6H),3.96-3.94(m,6H),3.74(m,2H),3.70(m,6H),3.50-3.44(m,3H),3.41-3.40(m,15H).MS(ESI):933.9[(M-2Na)/2]^-

pentasaccharide III-4 (sodium salt):

¹H NMR(300MHz,CDCl₃)δ5.60-5.54(m,4H),5.40(m,1H),5.30-5.24(m,6H),4.77(s,1H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,1H),3.96-3.94(m,6H),3.74(m,2H),3.60-3.44(m,4H),3.41-3.40(m,15H).MS(ESI):920.9[(M-2Na)/2]^-

example 5 biological assay

The biological activity test of the pentasaccharide compounds of the invention can be performed using methods well known to those skilled in the art. It is to be understood that the following test methods do not set any limit to the scope of the present invention.

AT III binding K_DValue determination

Fluorescence measurement is carried out by using a Perkin Elmer LS-50 type fluorescence spectrophotometer; excitation lambda 280nm and emission lambda 338 nm; a constant temperature sample chamber was prepared and stirred continuously at 37 ℃. The target pentasaccharide compounds prepared in examples 1 to 4 were added to 2mL Tris-HCl buffer (0.01M, pH 7, 0.15M NaCl, and 5 to 60nM human AT-III) tubes, respectively.

The proportion and concentration of the AT-III-pentasaccharide complex are calculated in a 1:1 reaction, with a dissociation constant K_DThe assay of (2) was performed using Scatchard analysis. The results are mean ± SEM, n is 3.

2. Determination of anticoagulant factor Xa Activity

The pentasaccharide compounds of the invention inhibit factor Xa by activating antithrombin AT III. anti-Xa activity was determined by the anti-Xa amidolytic AT III method (i.e., chromogenic substrate method). AT 37 ℃ factor Xa (7.5 nkat/ml in 20mM Tris/maleate buffer, pH 7.4, NaCl 150 mM; 100. mu.L), AT III (in 20mM Tris/maleate buffer)In the wash, the concentration was 0.5 unit/ml, pH 7.4, NaCl 150 mM; 100 μ L) and pentasaccharide of the invention (in 20mM Tris/maleate buffer, concentration 0.5 units/ml, pH 7.4, NaCl 150 mM; 100 μ L) for 2 minutes. Residual factor Xa was then measured by adding S-2222 medium (Bz-isoleucine-glutamate-glycine-arginine pNA; 1mM in 50mM Tris-HCl buffer, pH 8.4, 175mM NaCl, EDTA 27.5 mM; 100 μ L). After 2 minutes, the reaction was stopped by adding 50% aqueous acetic acid (100. mu.L), and the absorbance was measured at 405 nm. The percentage of inhibition was then calculated, formula: inhibition%₄₀₅A of the sample₄₀₅) Control buffer A₄₀₅(ii) a The activity of the compounds was determined using Excel 2015 software compared to calibrated standards. The result is the mean ± SEM, n is 3.

3. Half life (T)_1/2) Measurement of

The pharmacokinetics of the pentasaccharide compounds of the invention were studied in male Wistar rats after intravenous administration at a dose of 100 nmol/Kg. Blood samples were collected at various time points after dosing, 9 volumes of blood samples were mixed with 1 volume of 0.129M sodium citrate solution and immediately cooled in an ice bath, the samples were centrifuged at 3000Xg for 10 minutes at low temperature and frozen at-20 ℃. The concentration of the compound per ml of plasma was determined by the factor Xa activity. For each compound, the elimination half-life was calculated from the concentration versus time curve.

The results are shown in the following table:

compound (I)	AT III binding KDValue (nM)	anti-Xa activity (units/mg)	Half life (T)1/2,h)
				III-1	24±1	850±25	1.4±0.1
III-2	22±3	870±26	1.2±0.1
				III-3	20±2	920±30	1.3±0.1
III-4	24±1	1190±35	1.5±0.1
				Fondaparinux sodium	58±3	850±27	0.7±0.1

The data in the table show that the pentasaccharide compound has strong Xa resisting activity, and compared with fondaparinux sodium, the Xa resisting activity of the pentasaccharide compound is 1-1.4 times that of fondaparinux sodium; and the half-life period of the pentasaccharide compound is longer than that of fondaparinux sodium.

4. Bleeding impact test

Male Wistar rats (weighing 18-22g) were randomly divided into 4 test, blank and control groups of 12 rats each. Pentasaccharide III-1, III-2, III-3 and III-4 are respectively injected into 4 test groups, fondaparinux sodium is injected into a control group, and normal saline is injected into a blank group. Each group was subdivided into four sub-groups and administered at doses of 1mg/Kg, 3mg/Kg, 10mg/Kg and 15mg/Kg, respectively, of the pentasaccharide compound of the invention calculated as its sodium salt. After 1 hour of administration, the rat tail was cut off by about 5mm and immersed in 40mL of distilled water, and stirred at a constant temperature of 37 ℃ for 90 min. Measuring the amount of hemoglobin in water by spectrophotometry, detecting wavelength at 540nm, preparing standard curve, and calculating amount of hemorrhage. The results were averaged ± SD, with n being 3.

The results in the table above show that at high dosing doses, the pentasaccharide compounds of the invention have significantly less effect on bleeding tendency than the positive control fondaparinux sodium.

Claims (9)

1. Acids of the structures of formulae III-1, III-2, III-3 and III-4 or salts thereof,

wherein R is₁Is C1-C4 alkyl.

2. The acid or salt thereof according to claim 1, wherein R is₁Is methyl.

3. The acid or salt thereof according to any one of claims 1-2, wherein the salt is a sodium salt, a potassium salt, a calcium salt or an ammonium salt.

4. A process for preparing the acid or salt thereof of claim 1, comprising the steps of:

(a) reacting a disaccharide of formula DE with a trisaccharide of formula FGH to produce DEFGGH 0,

wherein R is₁Selected from Ac, Bn, C1-C4 alkyl, R₂Selected from Bn, Bz;

(b) debenzylating a compound shown as formula DEFGH0 to prepare DEFGH1,

wherein R is_aIs Ac, H, C1-C4 alkyl, R_bIs H, Bz;

(c) the compound shown as formula DEFGH1 is subjected to sulfation reaction to prepare DEFGH2,

wherein R is_cIs Ac, SO3^—C1-C4 alkyl, R_dIs SO3^—，Bz；

(d) The formula DEFGH2 is hydrolyzed to prepare pentasaccharides III-1 to III-4.

5. An acid having a structure represented by the following formula or a salt thereof

Wherein Rc is Ac, SO3^—C1-C4 alkyl, Rd is SO3^—，Bz。

6. A pharmaceutical composition comprising as an active ingredient an acid or salt thereof according to any one of claims 1 to 3 and optionally pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, which contains 0.1-100 mg of active ingredient.

8. Use of an acid or salt thereof according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 6 to 7 for the manufacture of a medicament for the prevention and/or treatment of diseases and conditions associated with blood coagulation disorders.

9. The use according to claim 8, the diseases and conditions being deep vein thrombosis, thrombophlebitis, arterial blockage by thrombosis or embolism, post-operative vein thrombosis or embolism, pulmonary embolism and non-ST elevation myocardial infarction.