Fucosylated chondroitin sulfate oligosaccharide derivative and use thereof

Abstract

The present invention relates to a fucosylated chondroitin sulfate oligosaccharide derivative, which has the following technical advantages: the preparation process does not involve the use of controlled chemicals such as sodium azide and is more suitable for industrial production. The fucosylated chondroitin sulfate oligosaccharide derivative can selectively inhibit iFXase, has a weak impact on the activity of FXa and FIIa, has significant APTT-prolonging activity, has no significant impact on PT in human quality control plasma, has a weak impact on TT, has potent in-vivo efficacy against venous thrombosis, and has an extremely low bleeding risk. This indicates that the fucosylated chondroitin sulfate oligosaccharide derivative provided in the present invention has better anticoagulant activity and also has better safety potential, realizes separation of the anticoagulant activity from the bleeding tendency, and shows superior antithrombotic activity and safety compared with the prior art.

Description

A fucosylated chondroitin sulfate oligosaccharide derivative and its application Technical Field

[0001] The present invention relates to the field of chondroitin sulfate, and in particular to a fucosylated chondroitin sulfate oligosaccharide derivative, an intermediate compound, a preparation process and applications thereof. Background Art

[0002] With the aging of the population and changes in people's lifestyles and habits, thromboembolic diseases are becoming an increasingly major global health problem and have become the leading cause of death worldwide. Currently, unfractionated heparin and low molecular weight heparins are used clinically as anticoagulants for the prevention and treatment of thromboembolic diseases. However, these glycosaminoglycans can only show anticoagulant and antithrombotic activities after intravenous or subcutaneous parenteral administration (Reference 1: Hull RD, Raskob GE, Pineo GF, Green D, Trowbridge AA, Elliott CG, Lerner RG, Hall J, Sparling T, Brettell HR, et al. Subcutaneous low-molecular-weight heparin compared with continuous intravenous heparin in the treatment of proximal-vein thrombosis. N Engl J Med. 1992 Apr 9; 326(15): 975-82.). Their biological activity depends on endogenous antithrombin, with large individual differences and is accompanied by a serious risk of bleeding (Reference 2: van Rein N, Biedermann JS, van der Meer FJM, Cannegieter SC, Wiersma N, Vermaas HW, Reitsma PH, Kruip MJHA, Lijfering WM. Major bleeding risks of different low-molecular-weight heparin agents: a cohort study in 12 934patients treated for acute venous thrombosis. J Thromb Haemost. 2017Jul;15(7):1386-1391.), some newly developed oral anticoagulants (such as dabigatran, rivaroxaban and apixaban, etc.), although they have predictable pharmacokinetics, still act on the "common coagulation pathway" of the "coagulation cascade" (i.e., selectively inhibiting human coagulation factor Xa or coagulation factor IIa), so these drugs still have potential bleeding risks in clinical application (Reference 3: Cohen AT, Spiro TE, Spyropoulos AC; MAGELLAN Steering Committee. Rivaroxaban for thromboprophylaxis in acutely ill medical patients. N Engl J Med. 2013 May 16; 368(20): 1945-6.).

[0003] Fucosylated chondroitin sulfate (FuCS) is a glycosaminoglycan extracted from sea cucumbers. It has unique sulfated fucose branches that can effectively inhibit endogenous coagulation factor Xase (iFXase), thus having good anticoagulant activity. However, due to its relatively high molecular weight (Mw) and the fact that its sulfated fucose branches are partially hydrolyzed in gastric acid, its absorption rate in the intestine is low. Therefore, oral administration of FuCS polysaccharide requires very high doses to show pharmacological effects (Reference 4: Fonseca RJ, PA. Fucosylated chondroitin sulfate as a new oral antithrombotic agent. Thromb Haemost. 2006 Dec;96(6):822-9.). Although gastric acid-resistant tablets (GR) prepared with FuCS polysaccharide overcome the problem of FuCS being destroyed by acidic gastric juice, FuCS polysaccharide has been observed to have a bleeding tendency due to its action on multiple targets in the coagulation pathway in plasma (Reference 5: Fonseca RJ, Santos GR, PA. Effects of polysaccharides enriched in 2,4-disulfated fucose units on coagulation, thrombosis and bleeding. Practical and conceptual implications. Thromb Haemost. 2009 Nov; 102(5): 829-36.), therefore, structural modification based on FuCS has gradually attracted attention.

[0004] Chinese patent publication number CN110724209B discloses a fucosylated chondroitin sulfate with an azide terminal group and a preparation process thereof. Although its anticoagulant activity test shows that the nona saccharide can significantly prolong plasma APTT (activated partial thromboplastin time) and has no significant effect on PT (prothrombin time) and TT (thrombin time), achieving an in vitro anticoagulant effect comparable to low molecular weight heparin, its preparation process inevitably requires the use of sodium azide (see Examples 1-14 of the CN110724209B specification). Sodium azide is a highly toxic agent that can block the cytochrome electron transport system and has a certain degree of explosiveness. It is a controlled chemical and is therefore not suitable for large-scale industrial production. Summary of the Invention

[0005] To address the above-mentioned problems, the present invention provides a fucosylated chondroitin sulfate oligosaccharide derivative with a novel structure, which has better anticoagulant activity, significantly prolongs plasma APTT, and has no significant effect on PT and TT, thus achieving the separation of anticoagulant activity and bleeding tendency. More importantly, its preparation process does not involve the use of controlled chemicals such as sodium azide, making it more suitable for industrial production.

[0006] Specifically, the present invention provides such a fucosylated chondroitin sulfate oligosaccharide derivative, wherein the fucosylated chondroitin sulfate oligosaccharide derivative is a compound having a structure as shown in formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof:

[0007] in:

[0008] Said R1 is selected from H,

[0009] The R2 is selected from Ac,

[0010] Said R3 is selected from H,

[0011] The R4 is selected from H,

[0012] The R5 is selected from H, alkali metal, alkaline earth metal;

[0013] The m is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0014] Wavy lines are covalent attachment sites.

[0015] Furthermore, the R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium.

[0016] Furthermore, R1, R2, R3, R4, and R5 in formula (I) can be selected independently, and when R1 or R2 or R3 or R5 appear simultaneously in formula (I), they can all be selected independently.

[0017] In some preferred embodiments, R1, R2, R3, and R4 are selected from the following combinations, and R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium:

[0018] In some more preferred embodiments, R1 is H, R2 is Ac, R3 is H, R4 is H, and R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium.

[0019] In some more preferred embodiments, the R1 is The R2 is Ac, the R3 is H, the R4 is H, and the R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium.

[0020] In some more preferred embodiments, the R1 is The R2 is Ac, the R3 is The R4 is The R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium.

[0021] In some more preferred embodiments, the R1 is H, the R2 is The R3 is H, the R4 is H, and the R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium.

[0022] In some more preferred embodiments, the R1 is The R2 is The R3 is The R4 is The R5 is selected from H, sodium, lithium, potassium, calcium, and magnesium.

[0023] In some specific embodiments, the fucosylated chondroitin sulfate oligosaccharide derivative is a compound selected from the following structures or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt:

[0024] In some preferred embodiments, R5 in any of the above items is H; in other preferred embodiments, R5 in any of the above items is sodium; in other preferred embodiments, R5 in any of the above items is lithium; in other preferred embodiments, R5 in any of the above items is potassium; in other preferred embodiments, R5 in any of the above items is calcium; in other preferred embodiments, R5 in any of the above items is magnesium. In some more preferred embodiments, R5 in any of the above items is sodium.

[0025] In some specific embodiments, R5 is sodium, R1 is H, R2 is Ac, R3 is H, and R4 is H.

[0026] In some more preferred embodiments, the R5 is sodium, and the R1 is The R2 is Ac, the R3 is H, and the R4 is H.

[0027] In some more preferred embodiments, the R5 is sodium, and the R1 is The R2 is Ac, the R3 is The R4 is

[0028] In some more preferred embodiments, the R5 is sodium, the R1 is H, and the R2 is The R3 is H, and the R4 is H.

[0029] In some more preferred embodiments, the R5 is sodium, and the R1 is The R2 is The R3 is The R4 is

[0030] In some more specific embodiments, the fucosylated chondroitin sulfate oligosaccharide derivative is a compound selected from the following structures or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt:

[0031] Furthermore, m in any of the above items is an integer selected from 1, 2, 3, and 4; in some more preferred embodiments, m in any of the above items is an integer selected from 1, 2, and 3; in some specific embodiments, m in any of the above items is 1; in other specific embodiments, m in any of the above items is 2; in other specific embodiments, m in any of the above items is 3. In some more preferred embodiments, m is preferably 2.

[0032] In some more specific embodiments, the fucosylated chondroitin sulfate oligosaccharide derivative is a compound selected from the following structures or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt:

[0033] The present invention also relates to a pharmaceutical composition, and the pharmaceutical composition comprises any one of the above-mentioned fucosylated chondroitin sulfate oligosaccharide derivatives.

[0034] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or additive.

[0035] The present invention also relates to the use of any of the aforementioned fucosylated chondroitin sulfate oligosaccharide derivatives or a pharmaceutical composition comprising any of the aforementioned fucosylated chondroitin sulfate oligosaccharide derivatives in the preparation of drugs for preventing and treating thromboembolic diseases.

[0036] The present invention also relates to the use of any of the aforementioned fucosylated chondroitin sulfate oligosaccharide derivatives or a pharmaceutical composition comprising any of the aforementioned fucosylated chondroitin sulfate oligosaccharide derivatives in the prevention and treatment of thromboembolic diseases.

[0037] Furthermore, the thromboembolic disease is selected from venous thromboembolic disease and arterial thromboembolic disease. Furthermore, the venous thromboembolic disease includes but is not limited to pulmonary thromboembolism (PTE) and deep vein thrombosis (DVT); the arterial thromboembolic disease includes but is not limited to acute coronary syndrome (ACS), atrial fibrillation (i.e., AF), arterial ischemic attack, stroke, etc.

[0038] The present invention also relates to a process for preparing any of the above-mentioned fucosylated chondroitin sulfate oligosaccharide derivatives.

[0039] Specifically, the present invention relates to a process for preparing any one of the above-mentioned fucosylated chondroitin sulfate oligosaccharide derivatives (I-1), (I-2), (I-3), (I-4), or (I-5).

[0040] More specifically, the present invention relates to a process for preparing any one of the above-mentioned fucosylated chondroitin sulfate oligosaccharide derivatives (I-1'), (I-2'), (I-3'), (I-4'), or (I-5').

[0041] In some specific embodiments, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-1') comprises the following steps (i.e., step A-1):

[0042] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-1') further comprises the following steps (i.e., step A-2):

[0043] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-1') further comprises the following steps (i.e., step A-3):

[0044] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-1') further comprises the following steps (i.e., step A-4):

[0045] In some specific embodiments, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-2') comprises the following steps (i.e., step B-1):

[0046] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-2') further comprises the following steps (i.e., step B-2):

[0047] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-2') further comprises the following steps (i.e., step B-3):

[0048] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-2') further comprises the following steps (i.e., step B-4):

[0049] In some specific embodiments, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-3') comprises the following steps (i.e., step C-1):

[0050] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-3') further comprises the following steps (i.e., step C-2):

[0051] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-3') further includes the aforementioned step A-3.

[0052] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-3') further includes the aforementioned step A-4.

[0053] In some specific embodiments, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-4') comprises the following steps (i.e., step D-1):

[0054] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-4') further comprises the following steps (i.e., step D-2):

[0055] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-4') further comprises the following steps (i.e., step D-3):

[0056] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-4') further comprises the following steps (i.e., step D-4):

[0057] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-4') further comprises the following steps (i.e., step D-5):

[0058] In some specific embodiments, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-5') comprises the following steps (i.e., step E-1):

[0059] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-5') further comprises the following steps (i.e., step E-2):

[0060] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-5') further comprises the following steps (i.e., step E-3):

[0061] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-5') further comprises the following steps (i.e., step E-4):

[0062] Furthermore, the preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative (I-5') further comprises the following steps (i.e., step E-5):

[0063] In any of the above structural formulas, Lev is levulinic acid, Bz is benzoyl, Ac is acetyl, Ph is phenyl, and PMB is p-methoxybenzyl.

[0064] The present invention also relates to any of the aforementioned intermediate compounds.

[0065] Specifically, the present invention also relates to the following intermediate compounds:

[0066] The present invention also relates to a process for preparing the intermediate compound described in any one of the above.

[0067] The present invention also relates to the use of any of the aforementioned intermediate compounds in the preparation of fucosylated chondroitin sulfate oligosaccharide derivatives.

[0068] Specifically, the present invention also relates to the use of Compound 1, Compound 2, Compound 3, and / or Compound 4 in the preparation of Compound (I-1').

[0069] Specifically, the present invention also relates to the use of Compound 4, Compound 5, Compound 6, and / or Compound 7 in the preparation of Compound (I-2').

[0070] Specifically, the present invention also relates to the use of Compound 2, Compound 3, Compound 4, and / or Compound 8 in the preparation of Compound (I-3').

[0071] Specifically, the present invention also relates to the use of Compound 9, Compound 10, Compound 11, Compound 12, and / or Compound 13 in the preparation of Compound (I-4').

[0072] Specifically, the present invention also relates to the use of Compound 13, Compound 14, Compound 15, Compound 16, and / or Compound 17 in the preparation of Compound (I-5').

[0073] The fucosylated chondroitin sulfate oligosaccharide derivatives provided herein have the following technical advantages: the preparation process does not involve the use of controlled chemicals such as sodium azide, making them more suitable for industrial production. They selectively inhibit iFXase, have minimal effects on FXa and FIIa activity, exhibit significant APTT prolongation activity, have no significant effect on the PT of human control plasma, and minimally affect the TT. They possess potent anti-venous thrombotic activity with a very low risk of bleeding. This suggests that the fucosylated chondroitin sulfate oligosaccharide derivatives provided herein have the potential for enhanced safety while exhibiting enhanced anticoagulant activity. This separation of anticoagulant activity and bleeding tendency is achieved, demonstrating superior antithrombotic activity and safety compared to existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG1 shows the effects of compound (I-1′) on thrombus wet weight at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0075] FIG2 shows the effects of compound (I-1′) on thrombus dry weight at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0076] FIG3 shows the effect of compound (I-1′) on thrombus length at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0077] FIG4 shows the effects of compound (I-1′) on plasma APTT at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0078] FIG5 shows the effects of compound (I-1′) on plasma PT at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0079] Figure 6 shows the effects of compound (I-1') on plasma TT at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0080] FIG7 shows the effects of compound (I-3') on thrombus wet weight at doses of 1.25 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0081] FIG8 shows the effects of compound (I-3') on thrombus dry weight at doses of 1.25 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0082] FIG9 shows the effect of compound (I-3′) on thrombus length at doses of 1.25 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0083] FIG10 shows the effects of compound (I-3′) on plasma APTT at doses of 1.25 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0084] FIG11 shows the effects of compound (I-3′) on plasma PT at doses of 1.25 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0085] Figure 12 shows the effects of compound (I-3') on plasma TT at doses of 1.25 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg.

[0086] FIG13 shows the effects of the experimental group compound (I-1′) and the experimental group compound (I-3′) as well as the positive control group compound D-1 on the wet weight of thrombus at a dose of 5.0 mg / kg.

[0087] FIG14 shows the effects of the experimental group compound (I-1′) and the experimental group compound (I-3′) as well as the positive control group compound D-1 on the dry weight of thrombus at a dose of 5.0 mg / kg.

[0088] FIG15 shows the effects of the experimental group compound (I-1′) and the experimental group compound (I-3′) as well as the positive control group compound D-1 on thrombus length at a dose of 5.0 mg / kg.

[0089] FIG16 shows the survival and death of animals in the experimental group compound (I-1′) and the experimental group compound (I-3′) as well as the positive control group compound D-1 at a dose of 5.0 mg / kg.

[0090] FIG17 shows the bleeding volume of the experimental group compound (I-3') and enoxaparin in the mouse tail-cut bleeding model. DETAILED DESCRIPTION

definition

[0091] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0092] Preferably, the terms used herein are defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Klb1, Hb, eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0093] Unless the context requires otherwise, throughout the specification and the claims that follow, the word "comprise" and its variations such as "comprising" and "containing" will be understood to implicitly include the stated integers or steps, or groups of integers or steps, but not to exclude any other integers or steps, or groups of integers or steps. In the following paragraphs, the same aspects of the invention will be defined in more detail. Each aspect so defined can be combined with any other aspect or aspects unless there is a clear indication to the contrary. In particular, any feature that is optional, preferred or advantageous can be combined with any other feature or features that are optional, preferred or advantageous.

[0094] Throughout this specification, some documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, operating instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to such disclosures as prior inventions. Certain documents cited herein are identified as "incorporated by reference." In the event that a definition or teaching in such an incorporated reference conflicts with a definition or teaching described in this specification, the text of this specification shall prevail.

[0095] The term "pharmaceutically acceptable" as used herein refers to compounds or agents that are compatible with the compounds of the present invention and do not interfere with and / or substantially reduce their diagnostic or prophylactic and therapeutic activity. Pharmaceutically acceptable carriers preferably have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the subject to be prevented or treated.

[0096] The term "pharmaceutically acceptable salts" as used herein can be prepared by conventional methods, such as by reacting any free base and / or acid of the fucosylated chondroitin sulfate oligosaccharide derivatives according to the present invention with at least a stoichiometric amount of a desired salt-forming acid or base, respectively. Pharmaceutically acceptable salts of the present invention include salts with inorganic cations such as sodium, potassium, calcium, magnesium, zinc, and ammonium, and salts with organic bases.

[0097] The "pharmaceutical composition" of the present invention is a single active ingredient formulation comprising the fucosylated chondroitin sulfate oligosaccharide derivative of the present invention. It is understood that the pharmaceutical composition may also contain necessary, pharmaceutically acceptable carriers, excipients, diluents, and / or additives. Each of the "carriers, excipients, diluents, and / or additives" may exhibit different functional effects. Those skilled in the art can generally select appropriate pharmaceutically acceptable carriers, diluents, and / or additives based on the formulation of the pharmaceutical composition / formulation, including but not limited to fillers, bulking agents, carriers, disintegrants, binders, lubricants, glidants, coatings, solvents and cosolvents, buffers, preservatives, adjuvants, antioxidants, wetting agents, defoaming agents, thickeners, sweeteners, flavorings, and humectants. The "pharmaceutical composition" of the present invention may also be understood as a combination pharmaceutical composition, in which the fucosylated chondroitin sulfate oligosaccharide derivative of the present invention is one of the important active ingredients.

[0098] The elements of the present invention are described below. These elements are listed together with specific embodiments. However, it should be understood that they can be combined in any manner and in any number to form other embodiments. The various described embodiments and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments explicitly described. This description should be understood to support and cover solutions that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. In addition, it should be considered that any arrangement and combination of all elements described in this application is disclosed in the specification of this application, unless the context indicates otherwise. [Specific Examples]

[0099] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0100] Example 1 Preparation of Compound 4

[0101] The preparation route of compound 4 is as follows:

[0102] Preparation of compound b: Compound a (1 g) was dissolved in anhydrous methanol (50 ml), cooled to 0°C, and acetyl chloride (1.75 ml) was slowly added dropwise. The mixture was stirred for 15 min, and the mixture was naturally warmed to room temperature and the reaction was continued for 16 h. After the reaction was completed, the mixture was quenched with triethylamine and concentrated to remove the solvent to obtain a white solid. The white solid was dissolved in 10 ml of anhydrous dichloromethane, and 10 ml of triethylamine and 100 mg of 4-DMAP were added in sequence. The mixture was cooled to 0°C, and 8 ml of acetic anhydride was slowly added dropwise. The mixture was reacted at room temperature for 2 h and monitored by TLC. After the reaction was completed, the reaction solution was dissolved in dichloromethane and extracted with saturated sodium bicarbonate, saturated sodium thiosulfate, and saturated brine in sequence. The mixture was dried over anhydrous sodium sulfate and subjected to column chromatography (gradient elution) to obtain the hexasaccharide fully acetylated product compound b (white solid).

[0103] Preparation of compound c: Compound b (1.5 g, 853 μmol) was dissolved in a THF / H₂O (3:1, 30 mL) mixture, cooled to -5°C, and a 1M LiOH(aq)-35% H₂O (2:1, 12 mL) mixture was added dropwise. The mixture was allowed to react at -5°C for 1 h, then at room temperature for 8 h. MeOH (8 mL) was added under an ice bath, followed by the slow addition of 4M NaOH aqueous solution (18 mL), and the mixture was allowed to warm to room temperature for 10 h. The reaction solution was neutralized with IR-120 cation exchange resin to pH 7, filtered, concentrated, purified with Sephadex LH-20, and freeze-dried to yield compound c (white solid, 850 mg, 90% for 2 steps from 2-2).

[0104] Preparation of Compound d: Under argon, compound c (844 mg, 721 μmol) and (+)-CSA (690 mg, 2.88 mmol, 4.0 equiv.) were dissolved in anhydrous DMF (15 mL). PhCH(OMe)2 (2.7 mL, 18 mmol, 25 equiv.) was added and the mixture was heated to 45°C and reacted under reduced pressure for 5 h. After completion of the reaction as determined by TLC, saturated sodium bicarbonate solution was added dropwise to quench the reaction. The reaction solution was directly purified over Sephadex LH-20 and freeze-dried to afford compound d (a white solid, 870 mg, 84%).

[0105] Preparation of Compound 4: Compound d (870 mg, 0.607 mmol) and Bz2O (4.0 g, 18.2 mmol, 30 equiv.) were dissolved in anhydrous DMF (40 ml) and reacted at 85°C for 16 h. The mixture was cooled to room temperature, and pyridine (8 ml) and DMAP (120 mg) were added, followed by 36 h at room temperature. After completion of the reaction as monitored by TLC, MeOH (8 ml) and NaOAc (118 mg, 0.91 mmol, 1.5 eq.) were added sequentially, and the reaction was allowed to proceed at room temperature overnight. The reaction solution was concentrated to remove methanol, and a large amount of anhydrous ether was added to precipitate a white, slightly yellowish solid. The filtrate was filtered and purified by column chromatography (DCM / MeOH = 30:1) to afford Compound 4 (white solid, 60%).

[0106] Example 2 Preparation of Compound 13

[0107] The preparation route of compound 13 is as follows:

[0108] Take compound a (10g, 8.65mmol) and dissolve it in anhydrous methanol (300mL), cool it to 0℃, slowly add acetyl chloride (10.5mL), stir for 15 minutes, remove the ice bath, naturally warm to room temperature and continue to react for 12 hours. After the reaction is completed, triethylamine (21mL) is added dropwise to quench the reaction, and the reaction solution is concentrated to remove methanol to obtain a white solid (i.e., compound e). Compound e is dissolved in anhydrous dichloromethane (40mL), and triethylamine (66mL), 4-dimethylaminopyridine (660mg), and acetic anhydride (40mL) are added in sequence under ice bath conditions. After stirring for 15 minutes, the ice bath is removed, and the reaction is naturally warmed to room temperature and continued to react for 2 hours. After the reaction is completed by TLC monitoring, the reaction solution is diluted with dichloromethane, washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, concentrated, and the solvent is dried with toluene to obtain a white solid (i.e., compound f). Compound f was dissolved in anhydrous pyridine (108 mL), and trifluoroacetic anhydride (36 mL, 0.260 mol, 10 equiv. / acetamido) was slowly added dropwise. The mixture was heated at reflux for 1.5 hours at 135°C. After completion of the reaction as monitored by TLC, the mixture was cooled to room temperature, quenched with methanol, and concentrated to remove the solvent. The mixture was diluted with dichloromethane and washed sequentially with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (dichloromethane / methanol 50:1) to afford compound g (5.02 g, 30%) as a pale yellow solid.

[0109] Preparation of compound h: Compound g (800 mg, 0.42 mmol) was dissolved in a tetrahydrofuran / water (6.6:2.2 mL) mixture, cooled to -5°C, and a freshly prepared 1 M lithium hydroxide solution-30% hydrogen peroxide (2:1, 4.5 mL) mixture was added dropwise. The mixture was allowed to react at -5°C for 1 hour, then brought to room temperature and allowed to react overnight. Methanol (3.0 mL) was added under ice-bath conditions, followed by a slow dropwise addition of a 4 M aqueous sodium hydroxide solution (3.0 mL), and the mixture was allowed to react naturally at room temperature for 10 hours. The reaction solution was neutralized with IR-120 cation exchange resin to pH = 7, filtered, and the filtrate was concentrated and purified using Sephadex LH-20 with water as the eluent. The mixture was freeze-dried to obtain compound h (white solid, 392 mg, 85%).

[0110] Preparation of compound i: Compound h (510 mg, 0.46 mmol), imidazolesulfonyl azide hydrochloride (577 mg, 2.76 mmol, 2 equiv. / amino group), potassium carbonate (762 mg, 5.51 mmol, 12 equiv.), and copper sulfate pentahydrate (2 mg, 0.01 mmol, catalytic) were dissolved in water (5 mL) and reacted at room temperature for 24 hours. After the reaction was completed, the reaction solution was neutralized with IR-120 cation exchange resin to pH = 7, filtered, and the filtrate was concentrated and purified using Sephadex LH-20 with water as the eluent. The product was freeze-dried to obtain compound i (white solid, 402 mg, 70%).

[0111] Preparation of Compound J: Under argon, compound i (550 mg, 0.44 mmol) and (+)-camphorsulfonic acid (518 mg, 1.56 mmol, 3.5 equiv.) were dissolved in anhydrous N,N-dimethylformamide (6.7 mL). Benzaldehyde dimethyl acetal (2.0 mL, 13.35 mmol, 30 equiv.) was added and reacted at 45°C under reduced pressure for 5 hours. After completion of the reaction as monitored by TLC, saturated sodium bicarbonate solution was added dropwise to quench the reaction. The mixture was concentrated, purified on Sephadex LH-20 using water as the eluent, and freeze-dried to afford compound j (432 mg, 67%) as a white solid.

[0112] Preparation of Compound 13: Under argon, compound j (376 mg, 0.26 mmol) and benzoic anhydride (1.76 g, 7.77 mmol, 30 equiv.) were dissolved in anhydrous N,N-dimethylformamide (13 mL). The mixture was heated at 85°C for 10 hours. After completion of the reaction, the mixture was cooled to room temperature. 4-Dimethylaminopyridine (63 mg, 0.52 mmol, 2 equiv.) and anhydrous pyridine (6.5 mL) were added, and the reaction was continued at room temperature for 36 hours. After completion of the reaction, methanol (6.5 mL) and sodium acetate (32 mg, 0.39 mmol, 1.5 equiv.) were added sequentially, and the reaction was allowed to proceed at room temperature overnight. After completion of the reaction, the reaction solution was concentrated, diluted with dichloromethane, and washed sequentially with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (dichloromethane / methanol 60:1) to give compound 13 (white solid, 320 mg, 67%).

[0113] Example 3 Preparation of Compound (I-1')

[0114] The preparation route of compound (I-1') is as follows:

[0115] Preparation of compound 3: Under argon protection, compound k (748.2 mg, 1.36 mmol) and compound 4 (129 mg, 68 μmol) were dissolved in anhydrous CH2Cl2 / DMF (5 mL / 2.5 mL) and added Molecular sieves (750 mg) were added, stirred at room temperature for 2 h, cooled to 0°C, and NIS (359 mg, 26 eq) and AgOTf (48 mg, 3 eq) were added sequentially. The temperature was maintained for 2.5 h, then the temperature was slowly raised to room temperature and the reaction was allowed to proceed overnight. After completion of the reaction, triethylamine was added to quench the reaction, and the molecular sieves were removed by filtration through celite. The filtrate was evaporated to dryness to obtain an oily liquid, which was dissolved in a mixed solvent of acetic acid / acetic anhydride (2 mL / 6 mL) and reacted at 70°C for 3 h. The solvent was dried over toluene and separated by column chromatography (DCM / MeOH = 35:1) to afford compound 3 (159 mg, 70%) as a white solid with Rf = 0.3 (DCM / MeOH = 20:1).

[0116] Preparation of compound 2: Compound 3 (100 mg, 29 μmol) was dissolved in MeOH / CHCl3 / AcOH (2 mL: 2 mL: 0.05 mL), Pa(OH)2 (600 mg) was added, catalytic hydrogenation was carried out for 2 h, triethylamine was quenched, Pa(OH)2 was filtered off by diatomaceous earth, the filtrate was concentrated, and directly purified by Sephadex LH-20 (MeOH / CH2Cl2=1:1), and then concentrated and purified by silica gel column chromatography to give compound 2 (50 mg, 80%) as a white solid, Rf=0.3 (EA:MeOH:H2O=4:1:0.8).

[0117] Preparation of Compound 1: Compound 2 (50 mg, 21 μmol) and sulfur trioxide-triethylamine complex (913 mg, 5.04 mmol) were dissolved in anhydrous DMF (2 mL) and reacted at 60°C for 72 h. After completion of the reaction, the product was quenched with triethylamine and methanol and directly purified on Sephadex LH-20 (MeOH / CH2Cl2 = 1:1) to afford Compound 1 (41 mg, 60%).

[0118] Preparation of Compound (I-1'): Compound 1 (41 mg) was dissolved in a THF / H2O (3:1, 4 mL) mixture, cooled to -5°C, and a 1M LiOH(aq)-35% H2O (2:1, 1.5 mL) mixture was added dropwise. The mixture was allowed to react at -5°C for 1 h, then transferred to room temperature for 8 h. MeOH (1 mL) was added under an ice bath, followed by the slow addition of a 4M aqueous NaOH solution (2 mL), and the mixture was allowed to warm to room temperature for 10 h. The reaction solution was neutralized with IR-120 cation exchange resin to pH 7, filtered, concentrated, purified with Sephadex LH-20, and freeze-dried to afford Compound (I-1') (35 mg, 90%) as a white solid.

[0119] Example 4 Preparation of Compound (I-2')

[0120] The preparation route of compound (I-2') is as follows:

[0121] Preparation of compound 7: Under argon protection, compound m (748.2 mg, 1.38 mmol) and compound 4 (129 mg, 68 μmol) were dissolved in anhydrous CH2Cl2 / DMF (5 mL / 2.5 mL) and added Molecular sieves (750 mg) were added, stirred at room temperature for 2 h, cooled to 0°C, and NIS (359 mg, 26 eq) and AgOTf (48 mg, 3 eq) were added sequentially. The temperature was maintained for 2.5 h, then the temperature was slowly raised to room temperature and the reaction was allowed to proceed overnight. After completion of the reaction, triethylamine was added to quench the reaction, and the molecular sieves were removed by filtration through celite. The filtrate was evaporated to dryness to obtain an oily liquid, which was dissolved in a mixed solvent of acetic acid / acetic anhydride (2 mL / 6 mL) and reacted at 70°C for 3 h. The solvent was dried over toluene and separated by column chromatography (DCM / MeOH = 40:1) to afford compound 7 (159 mg, 70%) as a white solid with Rf = 0.3 (DCM / MeOH = 25:1).

[0122] Preparation of compound 6: Compound 7 (100 mg, 29 μmol) was dissolved in MeOH / CHCl3 / AcOH (2 mL: 2 mL: 0.05 mL), Pd(OH)2 (600 mg) was added, catalytic hydrogenation was performed for 2 h, triethylamine was quenched, Pd(OH)2 was filtered off with celite, the filtrate was concentrated, and directly purified by Sephadex LH-20 (MeOH / CH2Cl2 = 1:1), and then purified by silica gel column chromatography to obtain compound 6 (50 mg, 70%) as a white solid.

[0123] Preparation of Compound 5: Compound 6 (50 mg, 21 μmol) and sulfur trioxide-triethylamine complex (913 mg, 5.09 mmol) were dissolved in anhydrous DMF (2 mL) and reacted at 60°C for 72 h. After completion of the reaction, the product was quenched with triethylamine and methanol and directly purified on Sephadex LH-20 (MeOH / CH2Cl2 = 1:1) to obtain Compound 5 (42 mg, 60%).

[0124] Preparation of Compound (I-2'): Compound 5 (42 mg) was dissolved in a THF / H2O (3:1, 4 mL) mixture, cooled to -5°C, and a 1M LiOH(aq)-35% H2O (2:1, 1.5 mL) mixture was added dropwise. The mixture was allowed to react at -5°C for 1 h, then at room temperature for 8 h. MeOH (1 mL) was added under an ice bath, followed by the slow addition of a 4M aqueous NaOH solution (2 mL), and the mixture was allowed to warm to room temperature for 10 h. The reaction solution was neutralized with IR-120 cation exchange resin to pH 7, filtered, concentrated, purified with Sephadex LH-20, and freeze-dried to afford Compound (I-2') (36 mg, 90%) as a white solid.

[0125] Example 5 Preparation of Compound (I-3')

[0126] The preparation route of compound (I-3') is as follows:

[0127] The preparation of compound 2 and compound 3 refers to the aforementioned Example 3 and will not be described in detail.

[0128] Preparation of Compound 8: Compound 2 (60 mg) was dissolved in a THF / H₂O (3:1, 4 mL) mixture, cooled to -5°C, and a 1 M LiOH(aq)-35% H₂O (2:1, 1.5 mL) mixture was added dropwise. The mixture was allowed to react at -5°C for 1 h, then transferred to room temperature for 8 h. MeOH (1 mL) was added under ice-cooling conditions, followed by the slow addition of a 4 M aqueous NaOH solution (2 mL), and the mixture was allowed to warm to room temperature for 10 h. The reaction solution was neutralized with IR-120 cation exchange resin to pH 7, filtered, concentrated, purified with Sephadex LH-20, and freeze-dried to afford Compound 8 (45 mg, 90%) as a white solid.

[0129] Preparation of Compound (I-3'): Compound 8 (45 mg, 26 μmol) and sulfur trioxide-triethylamine complex (913 mg, 5.09 mmol) were dissolved in anhydrous DMF (2 mL) and reacted at 60°C for 72 h. After completion of the reaction, the product was quenched with triethylamine and methanol, directly purified on Sephadex LH-20, and lyophilized to obtain a white flocculent product. The product was then dissolved in DMF, and sulfur trioxide-triethylamine complex (913 mg, 5.09 mmol) and DMF (2 mL) were added. The product was reacted at 60°C for 4 d. After completion of the reaction, the product was quenched with triethylamine and methanol, directly purified on Sephadex LH-20, and lyophilized to obtain compound (I-3') (50 mg, 60%) as a white flocculent product.

[0130] Example 6 Preparation of Compound (I-4')

[0131] The preparation route of compound (I-4') is as follows:

[0132] Preparation of compound 12: Under argon protection, compound k (i.e., tolyl 2,4-bis-O-(4-methoxybenzyl)-3-O-acetylpropyl-1-thio-β-L-pyranose, 544 mg, 0.894 mmol, 15 equiv.) and compound 13 (110 mg, 0.060 mmol) were dissolved in anhydrous dichloromethane / N, N-dimethylformamide (2:1, 6 mL) mixed solvent and added. Molecular sieves (600 mg) were added and stirred at room temperature for 1 hour. N-iodosuccinimide (402 mg, 1.789 mmol, 30 equiv.) and trifluoromethanesulfonic acid (16 uL, 0.179 mmol, 3 equiv.) were then added sequentially and allowed to react at room temperature overnight. After completion of the reaction, monitored by TLC, the reaction was quenched with triethylamine, the molecular sieves were removed by filtration through celite, and the filtrate was concentrated and separated by column chromatography (dichloromethane / methanol = 100:1) to afford compound 12 (122 mg, 62%) as a white solid.

[0133] Preparation of Compound 11: Compound 12 (62 mg) was dissolved in a mixture of dichloromethane and hexafluoroisopropanol (3.1 / 3.1 mL) and 0.1 M hydrochloric acid solution (310 μL) was added. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the product was directly purified using Sephadex LH-20 using dichloromethane / methanol (1 / 1) as the eluent and concentrated under reduced pressure to afford Compound 11 (27 mg, 62%) as a white solid.

[0134] Preparation of compound 10: Under argon protection, compound 11 (30 mg, 13.0 μmol) was dissolved in anhydrous N,N-dimethylformamide (3.0 mL), and sulfur trioxide trimethylamine complex (847 mg, 4.67 mmol, 30 eq / hydroxyl) was added. The reaction was carried out at 60 ° C. in a microwave synthesizer for 5 hours, cooled to room temperature, and triethylamine (400 μL) and methanol (600 μL) were added in sequence to quench the reaction. Water was directly used as the eluent, and the product was purified using Sephadex LH-20 and freeze-dried to obtain compound 10 (25 mg, 60%) as a white solid.

[0135] Preparation of compound 9: Compound 10 (36 mg) was dissolved in a mixed solvent of tetrahydrofuran / water (1.5 / 0.5 mL), cooled to -5°C, and a freshly prepared 1 M lithium hydroxide aqueous solution-30% hydrogen peroxide (2:1, 0.75 mL) was added dropwise. The mixture was reacted at -5°C for 1 hour and then moved to room temperature for overnight reaction. Methanol (0.5 mL) was added under ice bath conditions, and 4 M sodium hydroxide aqueous solution (1.3 mL) was slowly added dropwise. The mixture was naturally warmed to room temperature and reacted for 10 hours. The reaction solution was neutralized with IR-120 cation exchange resin to pH = 7, filtered, and the resin was washed with distilled water. The filtrate was concentrated and purified using Sephadex LH-20 with water as the eluent. The mixture was freeze-dried to obtain compound 9 (30 mg, 70%) as a white solid.

[0136] Preparation of Compound (I-4'): Compound 9 (17 mg, 6.15 μmol) was dissolved in a mixture of pyridine and water (490 μL / 123 μL). Propanedithiol (93 μL, 0.920 mmol, 150 equiv.) and triethylamine (46 μL) were added and allowed to react overnight at room temperature in the dark. After completion of the reaction, the mixture was monitored by TLC, filtered through a pad of Celite, and the filtrate was concentrated and dried with toluene. The resulting solid was dissolved in a mixture of anhydrous N,N-dimethylformamide and ultra-dry pyridine (1.8 mL / 0.6 mL). Sulfur trioxide-pyridine complex (29 mg, 0.185 mmol, 10 equiv. / amino group) was added and allowed to react at room temperature for 15 hours. After the reaction was completed as monitored by TLC, triethylamine (25 μL) and methanol (37.5 μL) were added sequentially to quench the reaction. Water was used as the eluent and the product was purified using Sephadex LH-20. The product was freeze-dried to obtain compound (I-4') (11 mg, 60%) as a white solid.

[0137] Example 7 Preparation of Compound (I-5')

[0138] The preparation route of compound (I-5') is as follows:

[0139] Preparation of compound 17: Under argon protection, compound n (i.e. p-tolyl 2,3,4-tri-O-(4-methoxybenzyl)-1-thio-β-L-pyranose, 564 mg, 0.894 mmol, 15 equiv.) and compound 13 (110 mg, 0.060 mmol) were dissolved in anhydrous dichloromethane / N,N-dimethylformamide (2:1, 6 mL) mixed solvent and added Molecular sieves (600 mg) were added and stirred at room temperature for 1 hour. N-iodosuccinimide (402 mg, 1.789 mmol, 30 equiv.) and trifluoromethanesulfonic acid (16 uL, 0.179 mmol, 3 equiv.) were then added sequentially and allowed to react at room temperature overnight. After completion of the reaction, monitored by TLC, the reaction was quenched with triethylamine, the molecular sieves were removed by filtration through celite, and the filtrate was concentrated and separated by column chromatography (dichloromethane / methanol = 100:1) to afford compound 17 (128 mg, 64%) as a white solid.

[0140] Preparation of Compound 16: Compound 17 (50 mg) was dissolved in a mixture of dichloromethane and hexafluoroisopropanol (2.5 / 2.5 mL) and 0.1 M hydrochloric acid solution (250 μL) was added. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the product was directly purified using Sephadex LH-20 using dichloromethane / methanol (1 / 1) as the eluent and concentrated under reduced pressure to afford compound 16 (18 mg, 58%) as a white solid.

[0141] Preparation of Compound 15: Compound 16 (35 mg) was dissolved in a mixture of tetrahydrofuran and water (1.5 / 0.5 mL), cooled to -5°C, and a fresh mixture of 1 M aqueous lithium hydroxide and 30% hydrogen peroxide (2:1, 0.75 mL) was added dropwise. The mixture was allowed to react at -5°C for 1 hour, then brought to room temperature and allowed to react overnight. Methanol (0.5 mL) was added under an ice bath, followed by the slow dropwise addition of 4 M aqueous sodium hydroxide (1.3 mL), and the mixture was allowed to warm to room temperature and react for 10 hours. The reaction mixture was neutralized with IR-120 cation exchange resin to pH 7, filtered, and washed with distilled water. The filtrate was concentrated and purified using Sephadex LH-20 with water as the eluent. The mixture was then freeze-dried to afford Compound 15 (20 mg, 70% for 2 steps) as a white solid.

[0142] Preparation of Compound 14: Under argon, compound 15 (10 mg, 6.15 μmol) was dissolved in anhydrous N,N-dimethylformamide (1.5 mL). Sulfur trioxide trimethylamine complex (636 mg, 3.50 mmol, 30 eq / hydroxyl group) was added and reacted at 60°C for 5 days. After completion of the reaction, monitored by TLC, the mixture was cooled to room temperature and quenched by the addition of triethylamine (200 μL) and methanol (300 μL). The product was then purified directly using Sephadex LH-20 with water as the eluent and freeze-dried to afford compound 14 (9.5 mg, 42%) as a white solid.

[0143] Preparation of Compound (I-5'): Compound 14 (44 mg, 12.3 μmol) was dissolved in a mixture of pyridine and water (980 μL / 246 μL). Propanedithiol (186 μL, 1.840 mmol, 150 equiv.) and triethylamine (92 μL) were added and allowed to react overnight at room temperature in the dark. After completion of the reaction, the mixture was monitored by TLC, filtered through a pad of Celite, and the filtrate was concentrated and dried with toluene. The resulting solid was dissolved in a mixture of anhydrous N,N-dimethylformamide and ultra-dry pyridine (3.6 mL / 1.2 mL). Sulfur trioxide-pyridine complex (58 mg, 0.370 mmol, 10 equiv. / amino group) was added and allowed to react at room temperature for 15 hours. After the reaction was completed as monitored by TLC, triethylamine (50 μL) and methanol (75 μL) were added sequentially to quench the reaction. Water was used as the eluent and the product was purified using Sephadex LH-20. The product was freeze-dried to obtain compound (I-5') (28 mg, 60% for 2 steps) as a white solid.

[0144] Example 8 Preparation of other compounds

[0145] Other fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention were prepared with reference to the preparation routes and / or methods of Examples 1-7. Those skilled in the art can replace and / or eliminate the corresponding raw materials based on / inspired by Examples 1-7 for preparation, and the specific preparation methods are not described in detail here.

[0146] The general structural formula of other fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention is:

[0147] Wherein, R1, R2, R3, R4, R5 and m are selected from the following combinations:

[0148] Example 9 Preparation of control compound D-1

[0149] The reference compound D-1 was prepared according to the preparation method provided in the publication number CN110724209B, which will not be described in detail.

[0150] Example 10 Detection of Selective Inhibitory Activity of iFXase, FXa and FIIa

[0151] iFXase activity assay

[0152] The iFXase activity detection system was constructed by combining FVIII reagents and a FVIII detection kit for clinical use (BIOPHEN FVIII: C). The BIOPHEN FVIII: C kit contains R1 (FX), R2 (activation reagent, containing FIXa-FIIa-Ca 2+ -phospholipids) and R3 (FXa-specific chromogenic substrate SXa-11).

[0153] (1) Detection method: Add 30 μL of sample solution or reference solution of a series of concentrations, 30 μL of FVIII solution (2 IU / mL), and 30 μL of R2 solution to each well of a 96-well plate, shake the plate to mix, and incubate at 37°C for 2 min. Add 30 μL of R1 solution, shake the plate to mix, incubate at 37°C for 2 min, and then add 30 μL of R3 solution. Measure the absorbance at 405 nm using a microplate reader, and measure continuously for 2 min at 10 s intervals. Set up a blank control well, in which an equal volume of solvent is added to the reaction system instead of the sample solution.

[0154] (2) Data processing: The rate of change of absorbance value (ΔOD / min) of each detection well was calculated. The ΔOD / min of blank control was set as 100%, and the relative activity of iFXase in the presence of compound (% of control) was calculated. Graphpad Prism was used to perform a four-parameter fitting of log(inhibitor) vs. response--variable slope for the logarithmic concentration of compound-iFXase relative activity, and the IC was calculated. 50 .

[0155] FXa activity assay

[0156] The AT-dependent anti-FXa activity of the compounds was detected using an Anti-Xa kit containing R1 (AT), R2 (FXa), and R3 (FXa-specific chromogenic substrate CS-01 (38)).

[0157] (1) Detection method: Add 30 μL of sample solution or reference solution of a series of concentrations, 30 μL of R1 solution, and 30 μL of R2 solution to each well of a 96-well plate, shake the plate to mix, and incubate at 37°C for 2 min; add 30 μL of R3 solution, and measure the absorbance at 405 nm (kinetic method, 10 s / time, 8 times).

[0158] (2) Data processing: The rate of change of absorbance value (ΔOD / min) of each detection well was calculated. The ΔOD / min of blank control was set as 100%, and the relative activity of FXa in the presence of compound (% of control) was calculated. Graphpad Prism was used to perform a four-parameter fitting of log(inhibitor) vs. response--variable slope on the logarithmic concentration of compound-relative activity of FXa, and the IC was calculated. 50 .

[0159] FIIa activity detection

[0160] The AT-dependent anti-FIIa activity of the compound was detected using an Anti-IIa kit containing R1 (AT), R2 (FIIa), and R3 (FIIa-specific chromogenic substrate CS-11 (65)).

[0161] (1) Detection method: Add 30 μL of sample solution or reference solution of a series of concentrations, 30 μL of R1 solution, and 30 μL of R2 solution to each well of a 96-well plate, shake the plate to mix, and incubate at 37°C for 2 min; add 30 μL of R3 solution, and measure the absorbance at 405 nm (kinetic method, 10 s / time, 8 times).

[0162] (2) Data processing: The rate of change of absorbance value (ΔOD / min) of each detection well was calculated. The ΔOD / min of blank control was set as 100%, and the relative activity of FIIa in the presence of compound (% of control) was calculated. Graphpad Prism was used to perform a four-parameter fitting of log(inhibitor) vs. response--variable slope on the logarithmic concentration of compound-relative activity of FIIa, and the IC was calculated. 50 .

[0163] In this example, compounds (I-1'), (I-2'), (I-3'), (I-4'), and (I-5') were used as experimental groups, and enoxaparin was used as a positive control group. The selective inhibitory activity of iFXase, FXa, and FIIa was tested according to the above method. Table 1 IC values ​​for selective inhibitory activity of iFXase, FXa, and FIIa 50 value

[0164] The results showed (see Table 1): The experimental group compounds could potently inhibit the activity of iFXase in a concentration-dependent manner at 4-4096 ng / mL. The IC 50 The value was 93.1 ng / mL, and the IC value of compound (I-2') was 50 The value is 171.5 ng / mL, and the IC of compound (I-3') 50 The value is 10.5 ng / mL, and the IC of compound (I-4') 50 The value is 12.4 ng / mL, and the IC of compound (I-5') 50 The value was 32.9 ng / mL. The experimental group compounds had no antithrombin (AT)-dependent coagulation factor inhibitory activity. In the presence of AT, the inhibitory activity against FXa (IC 50 value greater than 1000ng / ml) and FIIa (IC 50 The inhibitory activity of the positive control group, enoxaparin, was weak at 4-4096 ng / mL, and its IC 50 In addition, the positive control group enoxaparin could strongly inhibit FXa and FIIa in the presence of AT, with IC50 values ​​of 58.43 ng / mL and 48.35 ng / mL, respectively.

[0165] The conclusion shows that the anticoagulant target of the fucosylated chondroitin sulfate oligosaccharide derivative provided by the present invention is different from that of enoxaparin. Taking compound (I-1'), compound (I-2'), compound (I-3'), compound (I-4'), and compound (I-5') as examples, they can selectively inhibit iFXase and have little effect on the activity of FXa and FIIa, while enoxaparin has strong inhibitory activity on iFXase, FXa, and FIIa. Prior art studies have shown that when drugs selectively inhibit human coagulation factor Xa or coagulation factor IIa, these drugs often bring potential bleeding risks in clinical practice (Document 3: Cohen AT, Spiro TE, Spyropoulos AC; MAGELLAN Steering Committee. Rivaroxaban for thromboprophylaxis in acutely ill medical patients. N Engl J Med. 2013 May 16; 368(20): 1945-6.). However, the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention have little effect on the activity of FXa and FIIa, which also indicates that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention have good safety potential in clinical practice.

[0166] Example 11 Anticoagulant Activity Study

[0167] APTT test

[0168] Add 45 μL of human coagulation quality control plasma and 5 μL of sample solution, reference solution or solvent to the test tube preheated at 37°C and incubate at 37°C for 2 minutes; add 50 μL of APTT reagent preheated at 37°C and incubate the mixture at 37°C for 3 minutes; transfer the test tube from the incubation well to the test well, add 50 μL of CaCl2 solution preheated at 37°C, and time and record the coagulation time.

[0169] PT test

[0170] Add 45 μL of human coagulation quality control plasma and 5 μL of sample solution, reference solution or solvent to the test tube preheated at 37°C and incubate at 37°C for 2 minutes; transfer the test tube from the incubation well to the test well, add 100 μL of PT reagent preheated at 37°C, and time and record the coagulation time.

[0171] TT test

[0172] Add 90 μL of human coagulation quality control plasma and 10 μL of sample solution, reference solution or solvent to the test tube preheated at 37°C and incubate at 37°C for 2 minutes; transfer the test tube from the incubation well to the test well, add 50 μL of TT reagent preheated at 37°C, and time and record the coagulation time.

[0173] Data processing: The experimental data were input into an Excel document and saved. GraphPad Prism was used to perform a linear fit between the compound concentration and the mean clotting time, and the drug concentration that doubled the clotting time (EC 2.0× ).

[0174] In this example, compounds (I-1'), (I-3'), and (I-4') were used as experimental groups. Two positive control groups were set up, one group was enoxaparin and the other group was compound D-1 (prepared according to Example 9). APTT, PT, and TT tests were performed according to the above methods. Table 2 Drug concentrations that doubled the clotting time

[0175] The results showed (see Table 2): The experimental group compounds could prolong the APTT of human coagulation quality control plasma in a concentration-dependent manner at 2–32 μg / mL, and the concentration of compound (I-1') that doubled the APTT (EC 2.0× ) was 13.7 μg / mL, and the concentration of compound (I-3') that doubled APTT (EC 2.0× ) was 6.4 μg / mL, and the concentration of compound (I-4') that doubled APTT (EC 2.0× ) was 7.4 μg / mL, and its APTT prolongation activity was similar to that of enoxaparin (EC 2.0× In addition, the experimental group compounds had no significant effect on the PT of human quality control plasma at the test concentration (25-200 μg / mL), and had a weak effect on TT. The positive control enoxaparin had a strong APTT and TT prolongation activity, and its EC 2.0× The experimental group compounds can prolong the APTT of human coagulation quality control plasma in a concentration-dependent manner at 2–32 μg / mL, and their APTT prolongation activity is better than that of compound D-1.

[0176] The conclusions show that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention (taking compound (I-1'), compound (I-3'), and compound (I-4') as examples) can selectively act on the intrinsic coagulation pathway (APTT), and their APTT prolongation activity is comparable to that of enoxaparin and significantly superior to that of compound D-1, indicating that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention have more potent anticoagulant activity. In addition, the anticoagulant activity characteristics of the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention are different from those of enoxaparin. The former has no significant effect on the PT of human quality control plasma and has a weak effect on TT, while the latter can strongly inhibit the intrinsic coagulation pathway (APTT) and the common coagulation pathway (TT), further demonstrating that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention have better safety potential.

[0177] Example 12 In vivo antithrombotic activity and bleeding risk study

[0178] General experimental methods:

[0179] (1) Rat inferior vena cava thrombosis model

[0180] Surgery: After anesthetizing SD rats, the abdominal wall was longitudinally incised along the midline, the viscera were removed, the inferior vena cava and its branches were isolated, and a ligature was passed through the inferior margin of the left renal vein of the inferior vena cava. Drug administration and thrombosis induction: Compound solution or vehicle (1 mL / kg) was administered subcutaneously. One hour later, a thrombus-inducing agent (2% rabbit brain powder suspension, 1 mL / kg) was injected into the femoral vein to induce thrombosis. Ten seconds later, the proximal end of the inferior vena cava was ligated along the inferior margin of the left renal vein. 20 minutes later, the distal end of the inferior vena cava was ligated 1.5 cm from the ligated site.

[0181] Plasma sample acquisition: After the experiment, blood was collected from the abdominal aorta of rats using a disposable blood collection needle and a sodium citrate anticoagulant blood collection tube. The blood was mixed and centrifuged (1000 g, 15 min) to obtain plasma samples, which were stored in a -20°C refrigerator and thawed before the experiment. APTT, PT, and TT tests were performed (see Example 11 for the method).

[0182] Obtaining thrombus samples: longitudinally dissect the inferior vena cava, remove the thrombus in the inferior vena cava segment between the two ligatures, place it on weighed weighing paper, and weigh the wet weight of the thrombus; dry it at 60°C for 1 hour and then weigh the dry weight of the thrombus.

[0183] Data processing: Graphpad Prism software was used for statistical analysis. One-way ANOVA (Fisher's LSD Test) was used for significance analysis, and the thrombosis inhibition rate (%) was calculated:

[0184] (2) Mouse tail-cut bleeding model

[0185] Mouse tail bleeding volume: Add 8 ml of pure water to a transparent plastic tube and place it in a 37°C water bath. One hour after the subcutaneous injection of the compound, positive control drug, or vehicle, the mouse is anesthetized and the 5 mm tip of the tail is cut off. The tail is then immersed in water pre-warmed at 37°C. Collect the above blood in 8 ml of pure water, add pure water to 10 ml, and let it stand at room temperature for 1 hour. After the red blood cells are fully lysed, the optical density (OD) at 540 nm (λmax of the ultraviolet absorption of the hemoglobin solution) is measured using a UV spectrophotometer. 540nm ), and the bleeding volume was calculated using a standard curve.

[0186] Bleeding volume-optical density standard curve: Whole blood was collected from three untreated mice. 0, 2.5, 5, 10, 20, 40, 60, and 80 μL of whole blood were injected into 8 mL of pure water, and pure water was added to 10 mL. After standing at room temperature for 1 hour, the OD was measured using a UV spectrophotometer. 540nm .

[0187] Data processing: Graphpad Prism software was used to plot bleeding volume-OD 540nm A standard curve and a plot of drug dose-bleeding volume were drawn, and significance analysis was performed using one-way ANOVA (Fisher's LSD test).

[0188] Experiment a: In vivo antithrombotic activity of compound (I-1') and compound (I-3')

[0189] In this example, Compound (I-1') and Compound (I-3') were used as experimental groups, and vehicle (ie, saline) was used as the control group. The above-mentioned general experimental method was used to explore the in vivo antithrombotic activity in a rat inferior vena cava thrombosis model.

[0190] The results show:

[0191] (1) Referring to Figures 1-3, in a rat inferior vena cava thrombosis model, compound (I-1') at doses of 2.5-10.0 mg / kg subcutaneously injected can dose-dependently reduce thrombus length, thrombus wet weight, and thrombus dry weight. Based on thrombus dry weight, the thrombus inhibition rate of compound (I-1') at a dose of 5.0 mg / kg can reach approximately 66%, and the thrombus inhibition rate at doses of 10.0 mg / kg and above can reach more than 94%. Referring to Figures 4-6, compound (I-1') at doses of 2.5, 5.0, and 10.0 mg / kg subcutaneously injected can dose-dependently prolong rat plasma APTT, and the APTT prolongation activity is consistent with its in vivo antithrombotic activity; it has little effect on the extrinsic (PT) and common coagulation pathways (TT), and has no obvious dose-dependency.

[0192] (2) Referring to Figures 7-9, compound (I-3') dosed at 1.25-10.0 mg / kg subcutaneously reduced thrombus length, wet weight, and dry weight in a dose-dependent manner. Based on dry weight, compound (I-3') achieved an approximately 80% thrombus inhibition rate at a dose of 2.5 mg / kg and over 95% at doses of 5.0 mg / kg and above.

[0193] 10-12 , compound (I-3') can dose-dependently prolong rat plasma APTT upon subcutaneous injection at doses of 1.25, 2.5, 5.0, and 10.0 mg / kg, and the intensity of its APTT prolongation activity is consistent with its in vivo antithrombotic activity; it has little effect on the extrinsic (PT) and common coagulation pathways (TT), and has no obvious dose-dependency.

[0194] The conclusion shows that: in the rat inferior vena cava thrombosis model, the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention (taking compound (I-1') and compound (I-3') as examples) have good antithrombotic activity.

[0195] Experiment b: Comparison of in vivo antithrombotic activity with compound D-1

[0196] In this example, compound (I-1') and compound (I-3') were used as experimental groups, compound D-1 was used as the positive control group, and the solvent (i.e., normal saline) was used as the control group. The above general experimental method was used to explore the in vivo antithrombotic activity in the rat inferior vena cava thrombosis model. Table 3 Antithrombotic activity of compounds Note: n is the number of experimental animals.

[0197] The results showed (see Table 3 and Figures 13-15) that the thrombus inhibition rate of the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention (using compound (I-1') and compound (I-3') as examples) was better than that of the positive control group (Compound D-1) at a dose of 5.0 mg / kg, regardless of whether the thrombus length, thrombus wet weight, or thrombus dry weight were measured. This indicates that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention have better anti-thrombotic activity.

[0198] In addition, referring to Figure 16, the number of animals in the Control group, the positive control group, the compound (I-1') experimental group, and the compound (I-3') experimental group were all 8. By the end of the experiment, 2 animals died in the Control group, 1 animal died in the positive control group, and no animals died in the compound (I-1') experimental group and the compound (I-3') experimental group. This indicates that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention have better safety potential.

[0199] Experiment c: Comparison of antithrombotic activity and bleeding volume at equivalent antithrombotic doses

[0200] In this example, compound (I-3') was used as the experimental group, enoxaparin was used as the positive control group, and vehicle (i.e., saline) was used as the control group. The above general experimental methods were used to investigate the in vivo antithrombotic activity and safety in the rat inferior vena cava thrombosis model and the mouse tail-cut bleeding model.

[0201] Compound (I-3') and enoxaparin were tested in a rat venous thrombosis model to explore their equivalent antithrombotic doses. Comparison revealed that compound (I-3') at a dose of 5.0 mg / kg had comparable antithrombotic activity to enoxaparin (3.6 mg / kg), with thrombosis inhibition rates exceeding 95%. This dose was converted to the mouse dose and then amplified eightfold for bleeding risk studies.

[0202] The results (see Figure 17) show that in a mouse tail-amputation bleeding model, compound (I-3') had no significant effect on bleeding volume or duration at a dose of 56.0 mg / kg, whereas enoxaparin at a dose of 40.32 mg / kg significantly increased bleeding volume, with the bleeding volume being approximately 3.4 times that of the model control group. This demonstrates that the fucosylated chondroitin sulfate oligosaccharide derivatives provided by the present invention (exemplified by compound (I-3')) possess significant antithrombotic activity and a superior safety profile compared to enoxaparin.

[0203] The above description is merely a preferred embodiment, which is intended to be illustrative and non-limiting of the combinations of features necessary to implement the present invention. The titles provided are not intended to limit the various embodiments of the present invention. Terms such as "comprising," "including," and "including" are not intended to be limiting. In addition, unless otherwise indicated, the absence of a numeral modifier includes the plural form, and "or" and "or" mean "and / or." Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0204] All disclosures and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, multiple modifications and variants of the described method and composition of the present invention will be apparent to those skilled in the art. Although the present invention has been described by specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, those multiple variants of the described pattern that are apparent to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A fucosylated chondroitin sulfate oligosaccharide derivative, characterized in that, The fucosylated chondroitin sulfate oligosaccharide derivative described above is a compound having a structure shown in formula (I) or its tautomer, meso form, racemate, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof: Wherein: The aforementioned R1 is selected from H, The aforementioned R2 is selected from Ac, The aforementioned R3 is selected from H, R4 is selected from H, R5 is selected from H, alkali metal, alkaline earth metal; m is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; The wavy line is a covalent linkage site.

2. The fucosylated chondroitin sulfate oligosaccharide derivative according to claim 1, wherein R5 is selected from H, sodium, lithium, potassium, calcium, magnesium.

3. The fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1-2, characterized in that, The fucosylated chondroitin sulfate oligosaccharide derivative is a compound selected from the following structures or its tautomer, mesomer, racemate, enantiomer, diastereomer, or its pharmaceutically acceptable salt:

4. The fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1 to 3, characterized in that m is an integer selected from 1, 2, 3, 4; preferably, m is an integer selected from 1, 2, 3; more preferably, m is 2.

5. The fucosylated chondroitin sulfate oligosaccharide derivative according to claim 4, characterized in that, The fucosylated chondroitin sulfate oligosaccharide derivative described above is a compound selected from the following structures or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or its pharmaceutically acceptable salt:

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1-5.

7. The pharmaceutical composition according to claim 6, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or additive.

8. Use of the fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1-5 or a pharmaceutical composition comprising the fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 6-7 in the preparation of a medicament for preventing and treating thromboembolic diseases, preferably, the thromboembolic diseases are selected from venous thromboembolic diseases, arterial thromboembolic diseases.

9. The application according to claim 8, wherein The venous thromboembolic diseases include but are not limited to pulmonary thromboembolism and deep vein thrombosis; or the arterial thromboembolic diseases include but are not limited to acute coronary syndrome, atrial fibrillation, arterial ischemic attack, stroke.

10. Preparation process of the fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1-5.

11. An intermediate compound for preparing the fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1-5, characterized in that, The intermediate compounds described above are selected from:

12. Preparation process of any intermediate compound according to claim 11.

13. Use of any intermediate compound according to claim 11 in the preparation of the fucosylated chondroitin sulfate oligosaccharide derivative according to any one of claims 1-5.

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