Application of heparin pentasaccharide compound in preparing medicine for treating viral hemorrhagic fever

Abstract

The present invention provides an application of a heparin pentasaccharide compound in the preparation of a drug for treating viral hemorrhagic fever. The compound can reduce the mortality rate of viral hemorrhagic fever, improve the pathophysiological changes of viral hemorrhagic fever, and inhibit organ damage during the development of the disease.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compound medicine, and in particular relates to the application of a heparin pentasaccharide compound in the preparation of a medicine for treating viral hemorrhagic fever. Background Art

[0002] Since the 1930s, more than a dozen viral diseases with fever, hemorrhage and shock as the main clinical features have been discovered in the world. Through the joint efforts of virologists, clinicians, epidemiologists and pathophysiologists, it was found that although these different hemorrhagic diseases have different pathogens, they are a group of diseases in terms of pathogenesis. Therefore, "viral hemorrhagic fever" was established as a general term for a group of diseases and is widely used.

[0003] This group of diseases can be classified according to different etiologies. From the perspective of etiology, at least 13 pathogens of viral hemorrhagic fever have been established: Togaviridae (its Alphavirus genus causes Chikungunya hemorrhagic fever, Flavivirus genus causes yellow fever, Omsk hemorrhagic fever, dengue hemorrhagic fever, and Kyosanur Forest disease); Bunyaviridae (Nairovirus genus causes Crimean-Congo hemorrhagic fever, Hantavirus genus causes epidemic hemorrhagic fever, Phlebovirus genus causes Rif Valley fever); Arenaviridae Arenavirus genus (cause Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever); Filovirus genus of Filoviridae (cause Ebola hemorrhagic fever and Marburg hemorrhagic fever), etc.

[0004] The common clinical features of viral hemorrhagic fever are early fever, systemic signs and symptoms similar to those of influenza and malaria, followed by bleeding, including subcutaneous bleeding (such as bleeding spots, congestion, oozing at puncture sites, epistaxis, gingival bleeding, hemorrhagic conjunctivitis), visceral bleeding (such as hematemesis, melena, hematuria, vaginal bleeding, etc.), and the degree of bleeding varies from disease to disease. Severe viral hemorrhagic fever can affect the cardiovascular system, leading to reversible or irreversible shock syndrome, and other complications will soon appear, such as dehydration, renal failure, liver failure, hemolysis, jaundice, acidosis, central nervous system involvement, and secondary bacterial infection. On the other hand, viral infection may also directly cause organ damage, such as pitting necrosis of organs such as the liver, kidneys, heart, and spleen, and inflammatory cell infiltration. Local pathological changes and systemic pathophysiological changes will occur in parallel and promote each other during the development of the disease. Therefore, the mortality rate of viral hemorrhagic fever varies from disease to disease. For example, the mortality rate of Chikungunya hemorrhagic fever is very low, the mortality rate of yellow fever is less than 5%, the mortality rate of epidemic hemorrhagic fever is less than 10%, the mortality rate of Crimean-Congo hemorrhagic fever is 10-50%, and the mortality rate of Ebola hemorrhagic fever can be as high as over 80%.

[0005] As a group of diseases, viral hemorrhagic fevers have different pathogens and epidemiological characteristics. Some are transmitted by insects (mosquitoes, ticks, sand flies, midges), some are transmitted by rodents, and some have unclear transmission routes. Although the incidence rate is far lower than that of influenza, malaria, etc., their epidemic potential, relatively high mortality rate and extremely difficult prevention and treatment still pose special problems to public health.

[0006] At present, humans still lack effective therapeutic drugs for viral pathogens, so the treatment of viral hemorrhagic fever is mainly symptomatic and comprehensive, including antipyretic, fluid replacement, hemostasis, application of glucocorticoids, treatment of shock, etc. However, due to the bleeding tendency of patients, drugs such as aspirin that can cause bleeding cannot be used for antipyretic.

[0007] In terms of prevention, various types of vaccines are one of the commonly used means, including inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, etc., which can provide certain protection. Taking Crimean-Congo hemorrhagic fever (CCHF) as an example, CCHFV inactivated vaccines and live attenuated vaccines have been developed (cell-derived inactivated vaccines can avoid causing autoimmune and allergic reactions in the vaccine recipients, and have fewer side effects, but the spatial conformation of the virus's structural proteins is often destroyed during the inactivation process, which may affect its immunological characteristics and weaken its ability to stimulate the body's specific immune response; compared with inactivated vaccines, live attenuated vaccines have stronger immunity themselves, but have potential pathogenic risks), CCHFV subunit vaccines (although research on CCHFV subunit vaccines has made progress, it has also encountered some bottleneck problems, such as how to increase the expression of CCHFV antigen proteins, especially GP, how to effectively secrete and correctly modify recombinant antigen proteins, how to purify and maintain the activity of antigen proteins, and how to enhance their ability to stimulate the body's immune response, etc. If these problems can be effectively solved This issue will greatly promote the further development of CCHFV subunit vaccines), CCHFV nucleic acid vaccine (although CCHFV nucleic acid vaccine research has shown good application prospects, the drawback is that the vaccine mainly expresses endogenous antigen proteins, simply activates the major histocompatibility complex class I antigen presentation processing pathway and induces specific cellular immune responses mainly based on CTL. At the same time, due to the defects of the injection method, DNA vaccines often cannot effectively enter cells and are prematurely degraded by nucleases in the body), CCHFV virus live vector vaccine (although live vector vaccines can effectively stimulate the body to produce specific immune responses and protect animals from viral attacks, their biggest drawback is that the pre-existing antiviral vector antibodies in the body of the vaccinated individual will affect the immune effect of the live vector vaccine), CCHFV virus-like particle vaccine (virus-like particle vaccine (virus-like VLPs, as a candidate vaccine for genetic engineering, have been widely used in the development of other viral vaccines. However, their main drawback is that as an exogenous recombinant protein, they cannot continuously stimulate the body's immune response, and their ability to stimulate the body's cellular immune response is not strong. In addition, the most critical issue in the development of VLP vaccines is how to express and purify VLPs in large quantities, so choosing a suitable expression system is particularly important).

[0008] There is still a need to develop drugs that can treat viral hemorrhagic fever, especially those that can improve patients' bleeding tendency and symptoms, thereby improving the progression of the disease, preventing shock caused by cardiovascular involvement and a series of subsequent pathophysiological changes, and reducing mortality. Summary of the invention

[0009] The invention provides a heparin pentasaccharide compound, which can treat viral hemorrhagic fever, avoid various organ damages in the development process of hemorrhagic fever disease, and reduce mortality.

[0010] The structure of the heparin pentasaccharide compound is as follows: Structure A (the five monosaccharides from left to right are represented by D, E, F, G, and H, respectively):

[0011]

[0012] Wherein each Y is the same or different and is independently selected from H, Na + , K + , Li + , NH4 + Monovalent cations.

[0013] Preferably, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cations.

[0014] Preferably, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

[0015] In one embodiment of the present invention, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

[0016] It will be understood by those skilled in the art that any one or two or more Y is selected from Na + , K + , Li + , NH4 + The compound of formula A with a monovalent cation is essentially a salt of the compound of formula A with Y being H. When Y is a monovalent cation, the corresponding group in formula A is its anionic group, such as -COO - 、-SO3 - .

[0017] The compound of formula A is a compound having a single optical activity, namely, GlcNS6S(1→4)GlcA3Sβ(1→4)GlcNS3S6S(1→4)IdoA2S(1→4)GlcNS6S, and the H sugar terminal methyl ester is in an α configuration.

[0018] In one embodiment of the present invention, Y is H, and the structural formula of the compound is as follows, which is represented by CV001 in the present invention.

[0019]

[0020] In one embodiment of the present invention, Y is Na + The structural formula of the compound is as follows, which is referred to as CV122 in the present invention, and is essentially the sodium salt of CV001.

[0021]

[0022] In one embodiment of the present invention, Y is K + The structural formula of the compound is as follows, which is referred to as CV123 in the present invention, and is essentially the potassium salt of CV001.

[0023]

[0024] Since the compound of formula A (eg CV122) has weak anticoagulant activity and has an extremely low risk of causing bleeding, it can be used in the early stages of the disease when treating viral hemorrhagic fever.

[0025] The present invention provides use of a compound of formula A or a solvate thereof in preparing a medicament for treating viral hemorrhagic fever.

[0026] According to the present invention, the viral hemorrhagic fever is a disease caused by viral infection and characterized by fever, bleeding and shock, including but not limited to Chikungunya hemorrhagic fever, yellow fever, Omsk hemorrhagic fever, dengue hemorrhagic fever, Kyosanur Forest Sickness, Crimean-Congo hemorrhagic fever, epidemic hemorrhagic fever, Rif Valley Fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Ebola hemorrhagic fever, and Marburg hemorrhagic fever.

[0027] The inventors of the present invention found through research using a viral hemorrhagic fever animal model that the compound can reduce the mortality rate of viral hemorrhagic fever animal model mice, and observed that the compound has a protective effect on the organs of viral hemorrhagic fever animal model mice, reducing the organ tissue structure and function damage that occurs during the development of the disease.

[0028] The present invention provides the use of a compound of formula A or a solvate thereof in the preparation of a medicament for protecting organs of patients with viral hemorrhagic fever. In some embodiments of the present invention, the organs include but are not limited to spleen, heart, lungs, liver and / or kidneys. The protective organs (or organs) include but are not limited to reducing tissue damage of the organs, for example, reducing necrosis of cells in the organs, reducing bleeding in the organs, reducing infiltration of inflammatory cells in the organs, thereby protecting or maintaining organ function.

[0029] The present invention provides use of a compound of formula A or a solvate thereof in preparing a drug for protecting the vascular endothelium or cells thereof of patients with viral hemorrhagic fever.

[0030] The present invention provides use of a compound of formula A or a solvate thereof in combination with other drugs for treating viral hemorrhagic fever in the preparation of a drug for treating viral hemorrhagic fever.

[0031] The present invention provides use of a compound of formula A or a solvate thereof in preparing a drug for treating viral hemorrhagic fever in combination with other drugs for treating viral hemorrhagic fever.

[0032] In one embodiment of the present invention, the compound of formula A is CV001.

[0033] In one embodiment of the present invention, the compound of formula A is CV122.

[0034] In one embodiment of the present invention, the compound of formula A is CV123.

[0035] The compound of formula A has a clear structure, is easy to prepare and control quality, does not bind to other proteins, has a long half-life in vivo, and has a low half-life in the two-compartment model. 1 / 2 For 17 hours.

[0036] The present invention further provides a pharmaceutical composition, characterized in that it comprises the compound of formula A or a solvate thereof as an active ingredient. The pharmaceutical composition is used for treating viral hemorrhagic fever, protecting organs of patients with viral hemorrhagic fever, and protecting vascular endothelium or cells thereof of patients with viral hemorrhagic fever.

[0037] The pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0038] The pharmaceutically acceptable carrier is various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH regulators, preservatives, lubricants, disintegrants, etc.

[0039] Examples of the diluent include lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol, etc. Examples of the binder include starch, gelatin, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, etc. Examples of the antioxidant include vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, etc. Examples of the pH adjuster include hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, etc. Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, metacresol, benzalkonium chloride, etc. Examples of the lubricant include magnesium stearate, micropowder silica gel, talc, etc. Examples of the disintegrant include starch, methyl cellulose, xanthan gum, cross-linked sodium carboxymethyl cellulose, etc.

[0040] The amount of the compound of formula A contained in the pharmaceutical composition (calculated as the compound of formula A) is 0.1-1000 mg, preferably 1-500 mg, and more preferably 5-100 mg.

[0041] The mass percentage of the compound of formula A (calculated as the compound of formula A) in the pharmaceutical composition is 0.01%-95% of the pharmaceutical composition. Depending on the dosage form, it can be, for example, 0.1%-10%, 0.3-5%, or 10%-90%, preferably 20%-80%, and more preferably 30%-70%.

[0042] The dosage form of the pharmaceutical composition can be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; it can also be an injectable dosage form, such as an injection, a powder injection, etc., which is injected intravenously, intraperitoneally, subcutaneously or intramuscularly. All dosage forms used are well known to ordinary technicians in the pharmaceutical field. For example, the pharmaceutical composition can be an injection, and the concentration of the compound of formula A in the injection can be 1-15 mg / ml, such as 5 mg / ml, 10 mg / ml, 12.5 mg / ml, etc.

[0043] Routes of administration of the pharmaceutical composition include, but are not limited to: oral; buccal; sublingual; transdermal; pulmonary; rectal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous; by implanted reservoir or reservoir.

[0044] The dosage of the compound of formula A (calculated as the compound of formula A) will depend on the age, health and weight of the recipient, the type of the combined drug, the frequency of treatment, the route of administration, etc. The drug can be administered in a single daily dose, once a day, once every two days, once every three days, once every four days, or the total daily dose is administered in divided doses twice, three times or four times a day. The dosage of the compound of formula A (calculated as the compound of formula A) is 0.01-100 mg / kg / day, preferably 0.1-10 mg / kg / day, for example 0.5 mg / kg / day, 1 mg / kg / day, 2 mg / kg / day, 5 mg / kg / day, etc.

[0045] The pharmaceutical composition can be used in combination with other drugs for treating viral hemorrhagic fever, protecting organs of patients with viral hemorrhagic fever, and protecting vascular endothelium or cells thereof of patients with viral hemorrhagic fever.

[0046] The pharmaceutical composition may further contain a second therapeutic agent, which is another drug for treating viral hemorrhagic fever, protecting organs of patients with viral hemorrhagic fever, or protecting the vascular endothelium or cells thereof of patients with viral hemorrhagic fever.

[0047] The present invention provides a method for treating viral hemorrhagic fever, characterized in that a therapeutically effective amount of a compound of formula A or a solvate thereof is administered to a patient in need thereof.

[0048] The present invention provides a method for protecting organs of patients with viral hemorrhagic fever, characterized in that a therapeutically effective amount of a compound of formula A or a solvate thereof is administered to a patient in need thereof.

[0049] The present invention provides a method for protecting the vascular endothelium or cells thereof of patients with viral hemorrhagic fever, characterized in that a therapeutically effective amount of a compound of formula A or a solvate thereof is administered to a patient in need.

[0050] The administration routes of the compound of formula A, or its solvate, include, but are not limited to: oral; buccal; sublingual; transdermal; pulmonary; rectal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial; by implanted reservoir or reservoir.

[0051] The method further comprises administering to the patient in need thereof other drugs for treating viral hemorrhagic fever.

[0052] Other drugs for treating viral hemorrhagic fever include, but are not limited to: antiviral agents (such as ribavirin, favipiravir, chloroquine, etc.), monoclonal antibodies, blood volume expanders, vasoactive drugs, glucocorticoids, blood products (such as serum from recovering patients), anticoagulants, etc.

[0053] The compound of formula A can be synthesized using a heparin pentasaccharide synthesis method known in the art, such as a 4+1 synthesis strategy, or a 3+2 synthesis strategy, etc.

[0054] In one embodiment of the present invention, a 3+2 synthesis route is used, and a trisaccharide donor leaving group different from the prior art, a different hydroxyl protecting group strategy, and a different monosaccharide module synthesis method are selected to fully protect the pentasaccharide intermediate 1, which is sequentially subjected to dehydroxylation and carboxyl protecting group, sulfonation and azide reduction, and finally sulfonation to obtain a compound of formula A:

[0055]

[0056] The dehydroxylation and carboxyl protecting group, sulfonation and azide reduction can all be carried out using reaction methods and conditions known in the art.

[0057] In the intermediate 1, R1, R2 and R3 may be the same or different and independently selected from chloroacetyl, acetyl, benzoyl and pivaloyl; the definition of Y in the compounds of formula II and formula III is as described above. In one embodiment of the present invention, R1 and R2 are both acetyl, R3 is benzoyl, and Y is H. In one embodiment of the present invention, R1 and R2 are both acetyl, R3 is benzoyl, and Y is Na + In a specific embodiment of the present invention, R1 and R2 are both acetyl, R3 is benzoyl, and Y is K + .

[0058] The fully protected pentasaccharide intermediate 1 can be obtained by glycosylation reaction of the trisaccharide intermediate 2 and the disaccharide receptor 3, and the reaction formula is as follows:

[0059]

[0060] The glycosylation reaction can be carried out by methods known in the art. In some embodiments of the present invention, the reaction temperature is -10°C to -25°C. The reaction can be carried out under strong acid conditions, such as trifluoromethanesulfonic acid, TBSOTf, TMSOTf, etc.

[0061] The trisaccharide intermediate 2 can be coupled with a monosaccharide intermediate 7 and a disaccharide intermediate 8 through a (1→4) glycosidic bond to obtain a trisaccharide intermediate 4 with a specific stereo configuration, and the 1,6 ring opening of the trisaccharide intermediate 4 obtains a trisaccharide intermediate 5, and then obtains a trisaccharide intermediate 2, and the reaction formula is as follows. Each of the reaction steps can be carried out using methods known in the art. In some embodiments of the present invention, the reaction of the monosaccharide intermediate 7 and the disaccharide intermediate 8 to synthesize the trisaccharide intermediate 4 is carried out under acidic conditions in an organic solvent, and the reaction temperature is -10°C to -25°C. The organic solvent can be selected from benzene, toluene, dichloromethane, etc. The acidic conditions can be formed by adding acidic substances such as TfOH, TMSOTf or TBSOTf. In some embodiments of the present invention, 1,6 ring-opening of trisaccharide intermediate 4 is carried out in the corresponding acid anhydride (e.g., chloroacetic anhydride, acetic anhydride, benzoic anhydride, pivalic anhydride) to obtain trisaccharide intermediate 5, and TBSOTf, TMSOTf, Et2OBF3 or Et3SiOTf is added, and the reaction temperature is -20°C to 0°C.

[0062]

[0063] Disaccharide intermediate 8 can be obtained by glycosylation of monosaccharides 10 and 11 and separation of the β-configuration product. The reaction formula is as follows:

[0064] In some embodiments of the present invention, the Koenigs-Knorr glycosylation reaction for generating the disaccharide intermediate 9 can be carried out using an Ag2CO3 / AgOTf reaction system or an Ag2O / TMSOTf reaction system.

[0065] The monosaccharide 10 can be prepared by using glucose as a raw material through a reaction comprising the following steps: 1) converting the hydroxyl group at position 1 of glucose into a glucosinolate; 2) protecting the hydroxyl groups at positions 4 and 6 of glucose, protecting the hydroxyl group at position 3 with a temporary protecting group, and then protecting the hydroxyl group at position 2 with a benzyl group, and then removing the temporary protecting group and replacing the hydroxyl group at position 3 with R2; 3) deprotecting the hydroxyl groups at positions 4 and 6 of glucose and reacting them, respectively oxidizing and methylating the hydroxyl group at position 6, and protecting the hydroxyl group at position 4 with a chloroacetyl group; 4) converting the glucosinolate at position 1 into bromine to obtain the monosaccharide intermediate 10.

[0066] Monosaccharide 11, monosaccharide 7, and disaccharide receptor 3 used in the above preparation method can be prepared according to a synthesis method known in the art, for example: Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116; Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. ChemMedChem, 2014, 9, 1071–1080.

[0067] The definitions of R1, R2 and R3 in each intermediate compound in the above preparation method are the same as the definitions of the corresponding groups in Intermediate 1; X is a leaving group suitable for reacting with other acceptors to form a bond between glycosides, and preferably X is hydroxyl, alkylthio, arylthio, halogen, trichloroacetylimido, phosphate, or tert-butyldiphenylsilyloxy. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Survival of mice after administration of CV122 in a viral hemorrhagic fever mouse model

[0069] Figure 2 HE staining results of spleen and liver tissues of mice after administration of CV122 in viral hemorrhagic fever mouse model DETAILED DESCRIPTION

[0070] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0071] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0072] Ac: acetyl; AgOTf: silver trifluoromethanesulfonate; Bn: benzyl; Bz: benzoyl; ClAc: monochloroacetyl; CSA: camphorsulfonic acid; Cbz: benzyloxycarbonyl; CsF: cesium fluoride; DBU: 1,8-diazabicycloundec-7-ene; DCM: dichloromethane; DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMF: N,N-dimethylformamide; PMB: p-methoxybenzyl; TfOH: trifluoromethanesulfonic acid; TBSOTf: tert-butyldimethylsilyl trifluoromethanesulfonate; TEMPO: 2,2,6,6-tetramethylpiperidinoxide; TMSOTf: trimethylsilyl trifluoromethanesulfonate; Tol: toluene.

[0073] Example 1 Synthesis of Pentasaccharide Intermediate III

[0074] 1. Preparation method of monosaccharide intermediate 10-1

[0075] Using glucose 12 as the starting material, monosaccharide intermediate 13 was obtained by full acetylation; in BF3 . Under the action of Et2O, it reacts with p-toluene thiophenol to obtain monosaccharide intermediate 14; under the action of catalytic amount of sodium methoxide, all acetyl groups are removed, and then the 4,6-dihydroxyl group is protected with benzylide to obtain 2,3-dihydroxy naked monosaccharide intermediate 15; under the action of dibutyltin oxide, PMB protecting group is selectively used as temporary protecting group at the 3-position to obtain monosaccharide intermediate 16; the 2-position hydroxyl group is benzyl etherified to obtain monosaccharide intermediate 17; under the action of DDQ, the temporary protecting group PMB is oxidized and removed to obtain intermediate 18, which is then acetylated to obtain monosaccharide intermediate 19; the benzylide protecting group is removed by heating under acetic acid conditions to obtain 4,6-dihydroxy monosaccharide intermediate 20; TEMPO oxidation and methyl esterification to obtain monosaccharide intermediate 21; the 4-position hydroxyl group is protected with chloroacetyl to obtain monosaccharide intermediate 22; finally, iodine bromide is used to treat the β-thioglycoside to convert it into α-configuration monosaccharide intermediate 10-1.

[0076]

[0077] The reaction conditions and yields of each step are as follows: a) Ac2O, HClO4, 77%; b) TolSH, BF3·Et2O, DCM, 86%; c) 1) MeONa, MeOH, DCM; 2) Benzaldehyde Dimethylacetal, CSA, DMF, two-step total yield 83%; d) 1) Bu2SnO, MeOH, reflux; 2) PMBCl, CsF, DMF, 90°C, two-step total yield 75%; e) BnBr, NaH, DMF, 92%; f) DDQ, DCM, H2O, 78%; g) Ac2O, Et3N, DCM, 98%; h) 80% AcOH, 90°C, 76%; i) 1) 2,2,6,6-Tetramethylpiperidinooxy (TEMPO), Iodobenzene diacetate, DCM, H2O; 2) MeI, KHCO3, DMF, two-step total yield 57%; j) ClAc2O, Py, DCM, 76%; k) IBr, DCM, 76%.

[0078] 2. Preparation method of disaccharide intermediate 9-1

[0079] Monosaccharide intermediates 10-1 and 11-1 reacted overnight in the presence of insoluble silver carbonate and a catalytic amount of silver trifluoromethanesulfonate to generate disaccharide intermediate 9-1. NMR identification showed that the α / β ratio of the product was approximately 1:1.

[0080]

[0081] Add to the two bottles Molecular sieves (1g, powder), vacuum the flask, pass argon protection and cool to room temperature. Dissolve the monosaccharide intermediate 10-1 (300mg, 0.63mmol) and the monosaccharide intermediate 11-1 (172mg, 0.75mmol) in 10mL dry dichloromethane, add them to the reaction flask with a syringe, stir at room temperature for half an hour, add Ag2CO3 (260mg, 0.94mmol) and catalytic amount of AgOTf (16mg, 0.063mmol) to the reaction flask, protect from light, react overnight, TLC detection donor 10-1 completely disappears, generate a main substance, add diatomaceous earth to filter, the filtrate is concentrated under reduced pressure and directly purified by column chromatography (petroleum ether / ethyl acetate = 2:1) to obtain α / β configuration mixed disaccharide monosaccharide intermediate 9-1. For the synthesis method of monosaccharide intermediate 11-1, please refer to Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharidefondaparinux Sodium.Org.Chem.Front.,2019,6,3116.

[0082] HRMS [M+Na] + m / z 650.1327 (calculated: C 26 H 30 ClN3NaO 13 ,650.1365).

[0083] 3. Preparation method of disaccharide intermediate 8-1

[0084] The chloroacetyl group at the 4-position of the disaccharide intermediate 9-1 is removed, and the α-configuration product is separated to obtain a pure β-configuration disaccharide intermediate 8-1.

[0085]

[0086] The above disaccharide intermediate 9-1 was dissolved in 15 mL of a mixed solvent of chloroform and methanol (V / V=1:1), thiourea (192 mg, 2.52 mmol) was added and heated to 60°C, and the plate was monitored. When the raw material disappeared completely and three single spots were generated, the heating was stopped, and the mixture was cooled to room temperature. An appropriate amount of silica gel was added and spin-dried, and the mixture was directly separated by column chromatography (petroleum ether / ethyl acetate=1:1) to obtain a colorless foamy disaccharide intermediate 8-1 (583 mg, two-step yield 42%).

[0087] 1H NMR (400MHz, CDCl3) δ7.38–7.23(m,6H),5.49(s,1H),5.28(s,1H),5.11(t,J=9.3Hz,1H), 4.92(d,J=11.8Hz,1H),4.69(dd,J=12.9,9.8Hz,2H),4.58(d,J=5.3Hz,1H),4.01(d,J=7.6 Hz,1H),3.94(d,J=9.8Hz,1H),3.87(t,J=9.6Hz,1H),3.81(s,3H),3.80–3.73(m,1H),3.6 3(s,1H),3.51(dd,J=9.4,7.7Hz,1H),3.34(s,1H),3.21(s,1H),2.11(s,3H),2.01(s,3H). 13 C NMR (101MHz, CDCl3) δ171.07,169.27,169.21,138.06,128.41,127.98,127.78,103.50,100.1 7,78.28,76.22,75.18,74.63,74.22,74.03,70.61,70.07,64.88,58.73,52.88,21.03,20.94.

[0088] HRMS [M+Na] + m / z 574.1642 (calculated: C 24 H 29 N3NaO 12 ,574.1649).

[0089] 4. Preparation method of trisaccharide intermediate 2-1-1

[0090] The monosaccharide intermediate 7-1 is glycosylated with the disaccharide intermediate 8-1 to obtain the stereospecific trisaccharide intermediate 4-1; acetylation is performed to obtain the 1,6-ring-opened trisaccharide intermediate 5-1; the terminal acetyl group is removed under the action of benzylamine to obtain the trisaccharide intermediate 6-1, and then trichloroacetimidation is added to obtain the trisaccharide intermediate 2-1-1.

[0091]

[0092] Preparation method of trisaccharide intermediate 4-1: Add Molecular sieve (1g, powder), vacuum baking bottle, pass argon protection and cool to room temperature. Disaccharide intermediate 8-1 (430mg, 0.78mmol) and monosaccharide intermediate 7-1 (578mg, 1.0mmol) were dissolved in 10mL dry toluene, added to the reaction bottle with a syringe, stirred at room temperature for half an hour, cooled to -20℃, TfOH (8.9μL, 0.1mmol) was added dropwise, and stirred at -20℃ for 1.5h. After TLC monitoring, the reaction of disaccharide intermediate 8-1 was completed, excess triethylamine was added to quench the reaction, and the molecular sieve was filtered out with a small piece of silica gel. The filtrate was concentrated under reduced pressure, and the crude product was directly separated by column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain trisaccharide intermediate 4-1 (651mg, 87%).

[0093] 1 H NMR (400MHz, CDCl3) δ7.30(ddd,J=23.3,15.2,7.2Hz,16H),5.47(s,1H),5.32–5.13(m,2H),4.99(d,J=3.4Hz,1H),4.90(d,J=11.9Hz ,1H),4.81(d,J=13.4Hz,3H),4.69(t,J=9.2Hz,2H),4.59(d,J=5.6Hz,1H),4.54(d,J=10.9Hz,1H),4.28(d,J=11.3Hz,1H),4.19(dd,J =12.2,2.4Hz,1H),4.11–3.98(m,2H),3.94(d,J=9.5Hz,1H),3.86(t,J=9.6Hz,1H),3.82–3.72(m,4H),3.69(d,J=10.0Hz,1H),3.63(s ,1H),3.54(d,J=9.6Hz,1H),3.49(d,J=9.4Hz,1H),3.34(dd,J=10.3,3.4Hz,1H),3.20(s,1H),2.09(s,3H),2.01(s,3H),1.98(s,3H). 13C NMR (101MHz, CDCl3) δ170.55,169.75,169.12,167.49,137.94,137.62,13 7.53,128.52,128.40,128.13,128.11,128.02,127.98,127.91,127.78,10 3.45,100.20,99.11,79.83,78.24,77.18,76.35,75.56,74.99,74.93,74. 45,73.95,73.66,70.41,70.21,64.93,63.79,62.00,20.95,20.94,20.80.

[0094] HRMS[M+K] + m / z 999.3036 (calculated: C 46 H 52 KN6O 17 ,999.3026).

[0095] Preparation method of trisaccharide intermediate 5-1: dissolve trisaccharide intermediate 4-1 (370 mg, 0.39 mmol) in 10 mL acetic anhydride, protect with nitrogen, cool to 0°C in an ice bath, add TBSOTf (38 μL, 0.16 mmol) dropwise, maintain the reaction at low temperature for 20 min, monitor the reaction of the raw material by TLC, and quench the reaction by adding triethylamine. The reaction solution is concentrated under reduced pressure, and the crude product is directly purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain a colorless oily product trisaccharide intermediate 5-1 (409 mg, 100%).

[0096] 1 H NMR (400MHz, CDCl3) δ7.87–7.03(m,15H),6.41–3.17(m,28H),2.23–1.89(m,15H). 13C NMR (101MHz, CDCl3) δ170.54,170.03,169.98,169.59,168.58,167.58,137.54,137.50 ,137.42,128.52,128.45,128.15,128.11,128.07,128.04,128.01,127.92,127.88,103 .34,99.07,89.91,79.83,78.96,77.30,77.20,75.94,75.61,74.99,74.94,73.67,70.97,70.33,69.97,63.79,61.91,61.01,60.37,52.83,29.69,20.99,20.86,20.79,20.64.

[0097] HRMS [M+Na] + m / z 1085.3638 (calculated: C 50 H 58 NaN6O 20 ,1085.3604).

[0098] Preparation method of trisaccharide intermediate 2-1-1: dissolve trisaccharide intermediate 5-1 (100 mg, 0.094 mmol) in 5 mL of THF, add benzylamine (51 μL, 0.47 mmol) and react at room temperature. After TLC monitoring, transfer the reaction solution to a separatory funnel, wash away the remaining benzylamine with 1N hydrochloric acid, re-extract the aqueous phase with ethyl acetate twice, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude product that is directly purified by column chromatography to obtain trisaccharide intermediate 6-1. Dissolve the above trisaccharide intermediate 6-1 in 5 mL of dichloromethane, add anhydrous potassium carbonate (19 mg, 0.14 mmol), and add trichloroacetonitrile (14 μL, 0.14 mmol) dropwise at room temperature. After the addition, the reaction solution was stirred at room temperature for 2 hours. TLC monitored that the raw materials were basically reacted. Potassium carbonate was filtered off with a small piece of silica gel. The filtrate was concentrated under reduced pressure. The crude product was directly purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain a colorless syrup trisaccharide intermediate 2-1-1 (79 mg, two-step yield 72%). Two configurations, α configuration and β configuration, can be separated by silica gel column chromatography. The following are the NMR data of the two configurations.

[0099] The mixed-configuration intermediate 2-1-1 can be directly used in the next reaction to obtain the fully protected compound 1-1 of the α configuration, or the α configuration and β configuration obtained by separating the intermediate 2-1-1 can be used in the next step to obtain the fully protected compound 1-1 of the α configuration. That is, no matter which configuration or mixed-configuration intermediate 2-1-1 is used in the subsequent synthesis, it will not affect the subsequent reaction and yield.

[0100] NMR of β-configuration product: 1 H NMR (400MHz, CDCl3) δ8.81(s,1H),7.49–7.09(m,17H),6.42(s,1H),5.53(t,J=9.7Hz,1H),5.21(t,J=9.1H z,1H),4.94(s,1H),4.82(d,J=9.9Hz,3H),4.69(d,J=11.5Hz,1H),4.62–4.51(m,2H),4.46(dd,J=15.5,10. 2Hz,2H),4.30(d,J=12.0Hz,1H),4.23–4.02(m,4H),3.98(t,J=9.0Hz,1H),3.87(d,J=9.8Hz,3H),3.77(s,3 H),3.64(d,J=9.1Hz,2H),3.53(t,J=9.1Hz,1H),3.44–3.24(m,2H),2.05(s,7H),2.02(s,3H),1.93(s,3H). 13 C NMR (101MHz, CDCl3) δ170.55,169.92,169.76,169.60,167.56,160.50,137. 53,137.50,137.33,128.54,128.46,128.17,128.05,127.94,127.89,103.3 3,99.11,94.09,79.83,78.82,77.19,76.01,75.62,74.99,73.68,71.31,70.33,69.84,63.79,61.91,60.95,60.66,52.84,20.89,20.85,20.81,20.64.

[0101] HRMS [M+Na] + m / z 1186.2578 (calculated: C 50 H 56 Cl3NaN7O 19 ,1186.2594).

[0102] NMR of the product with α configuration: 1H NMR (400MHz, CDCCl3) δ8.77(s,1H),7.40–7.28(m,10H),7.27–7.18(m,5H),5.68(d,J=8.4Hz,1H),5.19(t,J=9.4Hz,1H),5.10(t,J= 9.7Hz,1H),4.94(d,J=3.5Hz,1H),4.82(m,2H),4.80(s,1H),4.69(d,J=11.9Hz,1H),4.53(m,3H),4.42(d,J=7.7Hz,1H),4.29(dd,J =12.3,2.1Hz,1H),4.16(d,J=11.7,1H),4.14(s,1H),3.94(t,J=9.3Hz,1H),3.88–3.80(m,3H),3.77(s,3H),3.73(t,J=8.8,1H),3. 68–3.60(m,2H),3.54(t,J=9.3Hz,1H),3.3(d,J=9.8,1H),3.29(t,J=9.8,1H),2.06(s,3H),2.06(s,3H),2.02(s,3H),1.92(s,3H). 13 C NMR (101MHz, CDCl3) δ170.58,169.94,169.78,169.62,167.57,160.50,137.49, 137.32,128.56,128.47,128.19,127.95,103.35,99.13,94.09,79.83,78.82,77 .26,77.17,76.03,75.64,75.01,74.97,73.68,71.31,70.32,69.83,63.77,61.90,60.95,60.65,31.94,29.72,29.68,29.38,22.71,20.91,20.87,20.83,20.66.

[0103] 5. Preparation method of fully protected pentasaccharide intermediate 1-1

[0104] The trisaccharide trichloroacetimidate donor trisaccharide compound 2-1-1 and disaccharide intermediate 3-1 are catalyzed by trifluoromethanesulfonic acid to generate a fully protected pentasaccharide intermediate 1-1.

[0105]

[0106] Add to the two bottles Molecular sieves (1g, powder), vacuum baking bottle, pass argon protection and cool to room temperature. Dissolve the trisaccharide intermediate 2-1-1 (110mg, 0.095mmol) and disaccharide intermediate 3-1 (95.7mg, 0.11mmol) in 10mL dry dichloromethane, add to the reaction bottle with a syringe, stir at room temperature for half an hour, cool to -20°C, add TfOH (2μL, 0.019mmol) dropwise, monitor the trisaccharide intermediate 2-1-1 after complete reaction by TLC, add triethylamine to quench the reaction, filter the molecular sieve with diatomaceous earth, concentrate the filtrate under reduced pressure, and purify the crude product by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain a colorless oily pentasaccharide intermediate 1-1 (133mg, 76%).

[0107] 1 H NMR (400MHz, CDCl3) δ8.14(d,J=7.6Hz,2H),7.58(t,J=7.3Hz,1H),7.47(t,J=7.6Hz,2H),7 .42–7.07(m,30H),5.60(d,J=5.3Hz,1H),5.37(t,J=9.9Hz,1H),5.30–5.13(m,2H),5.15–4. 46(m,18H),4.42(d,J=7.7Hz,1H),4.31(t,J=12.1Hz,2H),4.25–4.01(m,6H),4.01–3.60(m, 1H),3.54(q,J=9.5Hz,2H),3.40–3.14(m,6H),2.10(s,3H),2.07–1.96(m,9H),1.92(s,3H). 13C NMR (101MHz, CDCl3) δ170.95,170.55,170.01,169.88,169.64,167.62,165.42,155.88,138.47,137.55,137.51,137.33,137.28,136 .31,133.51,130.04,129.11,128.71,128.56,128.52,128.37,128.25,128.18,128.04,127.95,127.92,127.67,127.37,103.47,99. 11,98.84,98.21,97.48,79.82,78.55,77.35,76.64,76.48,76.42,75.62,75.01,74.92,74.74,74.69,74.33,73.55,72.88,71.78,71.10,70.32,69.65,69.47,68.88,66.91,63.76,62.13,61.91,61.08,60.71,60.40,55.27,54.40,52.83,52.43,20.93,20.85,20.63.

[0108] HRMS [M+Na] + m / z 1868.6488 (calculated: C 93 H 103 NaN7O 33 ,1868.6494).

[0109] 6. Preparation method of pentasaccharide intermediate III-1

[0110] The fully protected pentasaccharide 1-1 is subjected to the combined action of LiOH, H2O2 and NaOH to simultaneously remove Ac, Bz and methyl ester to obtain the hexahydroxy compound I; heated under the action of SO3·NEt3 to obtain the O-sulfonated intermediate compound II-1; the benzyl and Cbz are removed by catalytic hydrogenation reaction, and the azide is reduced to form the amino group to obtain the triamino compound III-1.

[0111]

[0112] Preparation method of pentasaccharide intermediate III-1: After the fully protected pentasaccharide intermediate 1-1 (100 mg, 0.054 mmol) was dissolved in 4.9 mL THF, 1.3 mL 1.25 N LiOH solution and 2.7 mL 30% H2O2 solution were added dropwise in sequence. After reacting at room temperature for 12 hours, 3 mL methanol and 1.6 mL 4 N NaOH solution were added. The reaction continued at room temperature for 12 hours. The TLC color development generated a long strip with a tail and no spots on the top. The pH was adjusted to 2 with 6 N hydrochloric acid under ice bath, and the reaction solution was transferred to a separatory funnel and extracted with dichloromethane three times. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The pentasaccharide intermediate I (73 mg, 90%) was obtained by rapid column chromatography separation (dichloromethane / methane = 15:1). Only one single peak was found by HPLC purity detection, proving that its purity was very high. Pentasaccharide intermediate I (100 mg, 0.067 mmol) and SO3·NEt3 (1.1 g, 6.0 mmol) were dissolved in 10 mL of anhydrous DMF and reacted at 65°C for 24 hours. The reaction was monitored by HPLC. When a single peak was generated, the reaction was stopped and the DMF was dried by spin drying. The oil was dissolved in methanol and directly passed through a Sephadex LH-20 gel column. A mixed solution of dichloromethane and methanol (V / V=1:1) was used as an eluent to collect the sugar-containing components. After the solvent was dried by spin drying, methanol was used as an eluent to pass through a Dowex-50-WX4-Na + The column was exchanged into sodium salt, and the sugar-containing components were collected and concentrated to obtain a light yellow solid pentasaccharide intermediate II-1 (136 mg, 95%). The purity of the compound was tested again by HPLC, and it was found that the peak time was consistent with the product in the reaction solution. The pentasaccharide intermediate II-1 (692 mg, 0.32 mmol) was dissolved in 2 mL of methanol, 1 mL of tert-butanol and 1 mL of water were added, and Pd / C (40 mg) was added. Stir at room temperature for 2 days under a hydrogen pressure of 4 atm, filter with diatomaceous earth, and spin dry the filtrate to obtain a gray-green solid pentasaccharide intermediate III-1 (488 mg, 100%). NMR showed that there was no hydrogen in the aromatic region.

[0113] 1 H NMR (400MHz, D2O) δ5.67(d,J=3.8Hz,1H),5.51(d,J=3.7Hz,1H),5.29(s,1H),5.05(d,J=3.5Hz,1H),4.92(s,1H),4.63(dd,J=20.4,9. 8Hz,3H),4.53(d,J=10.2Hz,1H),4.36(dd,J=30.1,11.2Hz,7H),4.26–3.69(m,16H),3.62(dd,J=13.4,6.0Hz,2H),3.55–3.36(m,6H). 13C NMR(101MHz,D2O)δ174.87,174.29,101.21,99.10,96.00,94.88,91.35,83.58,76.95,76.52,74.96,74.06,73.19,72.42 ,72.15,71.12,70.63,69.44,69.32,69.02,68.80,68.48,67.43,66.68,66.02,65.68,63.31,55.51,54.27,53.92,53.38.

[0114] HRMS[M-2Na] + m / z 738.4654 (calculated: C 31 H 47 N3Na6O 43 S6 2- ,738.4647).

[0115] Example 2 Preparation of Compound CV001

[0116] Preparation method of CV001: Take the pentasaccharide intermediate III-1 (200 mg, 0.13 mmol) prepared in Example 1 and dissolve it in 2 mL of water, add 2N NaOH solution to adjust the pH to between 9 and 10 (and continuously add 2N NaOH to maintain the pH between 9 and 10 in the following reaction), add SO3·Py (110 mg, 0.7 mmol), add another batch after half an hour, repeat six times, then stir at room temperature for 4 hours and neutralize with hydrochloric acid to pH 7-8, concentrate and pass directly through Sephadex G-25 gel column with water as eluent, collect sugar-containing components, and concentrate to obtain the target compound CV001 (198 mg, 96%).

[0117]

[0118] 1 H NMR(400MHz,D2O)δ5.55(d,J=3.5Hz,1H),5.37(d,J=3.5Hz,1H),5.13(d,J=3.1Hz,1H),5.02(d,J=3.1Hz,1H),4.89(d,J=3.6Hz,1H),4.64(s,3 H),4.48(t,J=7.3Hz,1H),4.36(s,1H),4.35–3.98(m,11H),3.82-3.57( m,8H),3.47(d,J=9.4Hz,1H),3.43-3.22(m,5H),3.15(d,J=5.4Hz,1H). 13C NMR(101MHz,D2O)δ176.25,175.38,103.82,101.28,99.44,99.30,98.29,82.34,78.43,77.65,76.78,76.09,75.98,75 .46,74.99,73.34,71.46,70.87,69.95,69.57,69.45,69.09,68.87,67.36,66.87,66.13,58.65,57.89,56.23,56.35.

[0119] HRMS [M–3H] 3- m / z 527.9675 (calculated: C 31 H 53 N3O 52 S9,527.9616)

[0120] Example 3 Synthesis of Compound CV122

[0121] Synthesis method 1: Compound CV001 is subjected to sodium exchange to obtain sodium salt CV122.

[0122]

[0123] After CV001 was dissolved in water, it was filtered through Dowex-50-WX4-Na + The column was exchanged to the sodium salt, the sugar-containing fractions were collected, and the solvent was concentrated to obtain CV122 as a white solid.

[0124] Synthesis method 2: Take the pentasaccharide intermediate III-1 (200 mg, 0.13 mmol) prepared in Example 1 and dissolve it in 2 mL of water. Add 2N NaOH solution to adjust the pH to between 9 and 10 (and continue to add 2N NaOH to maintain the pH between 9 and 10 in the following reaction). Add SO3·Py (110 mg, 0.7 mmol), add another batch after half an hour, repeat six times, and then stir at room temperature for 4 hours and neutralize with hydrochloric acid to pH 7-8. After concentration, use water as eluent to directly pass through Sephadex G-25 gel column, collect sugar-containing components, concentrate, and pass through Dowex-50-WX4-Na with water. + The column was exchanged into sodium salt, the sugar-containing fractions were collected, and the solvent was concentrated to give CV122 (235 mg, 98%) as a white solid.

[0125] 1H NMR(400MHz,D2O)δ5.45(d,J=3.3Hz,1H),5.33(d,J=3.3Hz,1H),5.21(d,J=2.9Hz, 1H),5.08(d,J=2.9Hz,1H),4.99(d,J=3.4Hz,1H),4.72(s,3H),4.53(t,J=7.8Hz,1 H),4.44(s,1H),4.41–4.08(m,11H),3.92(dd,J=19.1,9.2Hz,4H),3.79–3.60(m,4 H),3.57(d,J=9.6Hz,1H),3.47–3.31(m,4H),3.30–3.22(m,1H),3.22–3.11(m,1H). 13 C NMR(101MHz,D2O)δ174.87,174.29,101.74,99.38,98.57,98.30,97.31,80.65,76.97,76.45,76.17,76.13,75.78,75 .35,74.69,72.12,70.73,70.59,69.82,69.24,69.11,68.53,68.50,66.64,66.27,65.88,57.92,57.79,56.74,55.50.

[0126] HRMS [M–3Na] 3- m / z 586.5803 (calculated: C 31 H 45 N3Na8O 52 S9 3- ,586.5801).

[0127] Example 4 Synthesis of Compound CV123

[0128] Synthesis method 1: Potassium salt CV123 can be obtained by potassium exchange of compound CV001.

[0129]

[0130] Dissolve CV001 in water and filter with Dowex-50-WX4-K + The column was exchanged into potassium salt, the sugar-containing fractions were collected, and the solvent was concentrated to obtain CV123 as a white solid.

[0131] Synthesis method 2: Take the pentasaccharide intermediate III-1 (137 mg, 0.09 mmol) prepared in Example 1 and dissolve it in 2 mL of water. Add 2N NaOH solution to adjust the pH to between 9 and 10 (and continue to add 2N NaOH to maintain the pH between 9 and 10 in the following reaction). Add SO3·Py (99 mg, 0.63 mmol), add another batch after half an hour, repeat six times, and then stir at room temperature for 4 hours and neutralize with hydrochloric acid to pH 7-8. After concentration, use water as eluent to directly pass through Sephadex G-25 gel column, collect sugar-containing components, concentrate, and pass through Dowex-50-WX4-K with water. + The column was exchanged into potassium salt, the sugar-containing fractions were collected, and the solvent was concentrated to give CV123 (175 mg, 97%) as a white solid.

[0132] 1 H NMR(400MHz,D2O)δ5.48(d,J=3.4Hz,1H),5.35(d,J=3.4Hz,1H),5.24(d,J=2.8H z,1H),5.09(d,J=2.7Hz,1H),4.93(d,J=3.6Hz,1H),4.75(m,3H),4.55(t,J=7.6 Hz,1H),4.49(s,1H),4.40–4.09(m,11H),4.23-3.84(m,4H),3.74–3.62(m,4H), 3.55(d,J=9.4Hz,1H),3.49–3.34(m,4H),3.32–3.24(m,2H),3.22–3.11(m,2H). 13 C NMR(101MHz,D2O)δ175.35,174.32,101.85,99.68,98.54,98.26,97.43,81.77,77.23,76.69,76.13,76.10,75.98,75 .35,74.57,73.42,70.64,70.56,69.96,69.37,69.26,68.36,68.22,66.57,66.46,65.79,57.65,57.34,56.74,55.26.

[0133] In the following examples, CV122 is used as an example to illustrate the therapeutic activity of the compound of formula A. CV122 is prepared by the synthesis method 2 of Example 3.

[0134] Example 5 The therapeutic effect of CV122 on viral hemorrhagic fever

[0135] Interferon α / β receptor (IFNAR - / -) gene knockout mice were infected with Crimean-Congo hemorrhagic fever virus (CCHFV) to construct an animal model of viral hemorrhagic fever.

[0136] The experimental mice were divided into two groups: (1) Control group (administered with 0.9% NaCl): 5 male mice (7-8 weeks IFNAR - / - ), (2) Treatment group (CV122 administration): 5 male mice (7-8 weeks IFNAR - / - ).

[0137] Virus infection of mice: intraperitoneal injection of CCHFV, 5000 TCID per mouse 50 The mice in the treatment group were subcutaneously administered CV122 at 0.4 mg / mouse at 48h, 72h, 96h, and 120h after infection, and CV122 at 0.58 mg / mouse at 144h and 168h, respectively, dissolved in 0.9% NaCl, 100 μL per mouse. The mice in the control group were injected with the same dose of normal saline, 100 μL per mouse. The clinical symptoms and survival rate of the mice were monitored daily. The survival rate of mice is shown in Figure 1 .from Figure 1 It can be seen that within 15 days after being infected with the virus, the mice in the control group gradually died. The first dead mouse appeared on the 5th day, two dead mice appeared on the 6th day, and one dead mouse appeared on the 7th day. In the end, a total of four mice died, with a mortality rate of 80%. The only dead mouse in the CV122 treatment group appeared on the 7th day after virus infection during the entire observation period, with a mortality rate of 20%.

[0138] After the experiment, the surviving mice were killed and their livers and spleens were taken for tissue preparation and HE staining for pathological examination.

[0139] The HE staining results of the tissues are shown in Figure 2 .from Figure 2 It can be seen that the spleen of the mice in the control group was hemorrhagic and edematous; the boundary between the white and medulla disappeared, the spleen corpuscle structure collapsed, the lymphocytes were necrotic, and the number was significantly reduced; the macrophages and mononuclear lymphocytes in the medulla proliferated; the hepatocytes were occasionally seen in the liver tissue with sheet-like coagulative necrosis, glycogen depletion, homogeneous cytoplasm, red staining, and nuclear condensation; accompanied by obvious inflammatory cell infiltration. In the mice in the treatment group, the congestion and edema of the spleen were slightly alleviated; the boundary between the white and medulla was blurred, the spleen corpuscles became smaller or even disappeared, the number of lymphocyte necrosis was significantly reduced; the macrophages and mononuclear lymphocytes in the medulla proliferated, and the overall degree of lesions was slightly lighter than that of the control group; the degree of liver lesions was not significantly different from that of the control group.

[0140] Example 6 Anticoagulant activity of CV122

[0141] 1. Experimental Principle

[0142] Heparin first forms a complex (AT-Hep.) with excess ATIII. Theoretically, it is believed that all heparin forms an AT-Hep. complex that inhibits thrombin activity. The AT-Hep. complex then combines with excess FXa, and the remaining free FXa can hydrolyze the substrate S 2765 The pNA product was visualized at 405 nm, and the titer was calculated using the bs2000 software using the quantitative response parallel line 4.4 method.

[0143] Heparin+AT III → [AT-Hep.]

[0144] [AT-Hep.]+[FXa(excess)]→[FXa-AT-Hep.]+[residual FXa]

[0145] [residual FXa]+Substrate→Peptide+pNA

[0146] 2. Test methods

[0147] Activity assay requires ATIII, FXa, and chromogenic substrate S 2765 All of them are commercially available from Beijing Adhoc International Biotechnology Co., Ltd., including human antithrombin (ATIII), AG00-0132; bovine activated factor X (FXa), AG00-0121; FXa factor chromogenic substrate S 2765 , AG00-0102-10.

[0148] Prepare the corresponding solutions according to the following table:

[0149]

[0150] Preparation of low molecular weight heparin sodium standards / samples: The standards or samples are diluted to 1.0 IU / ml (the starting dose can be adjusted appropriately within the pharmacopoeia range), and then diluted to five concentration gradients of AE [A] 0.1600 (or 0.2 / 0.18) IU / ml×0.75=[B] 0.1200 IU / ml×0.75=[C] 0.0900 IU / ml×0.75=[D] 0.0675 IU / ml×0.75=[E] 0.0506 IU / ml. Add samples according to the operation sequence in the table below, then read the OD value at 450nm with a microplate reader, and calculate the potency using the bs2000 software using the quantitative response parallel line 4.4 method.

[0151] Enzyme activity assay operation sequence

[0152]

[0153] 3. After determination:

[0154] The titer of CV122 was PT = 119.96 IU / mg, and the confidence limit of the titer was FL = 4.6284%;

[0155] The potency of fondaparinux sodium (provided by Tianjin Hongri Pharmaceutical Co., Ltd.) was measured to be PT = 923.01 IU / mg, and the confidence limit of the potency was FL = 6.014%.

[0156] The greater the titer, the better the anticoagulant activity. From the above experiments, it can be seen that the anticoagulant activity of CV122 is lower than that of fondaparinux sodium.

[0157] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a compound of formula A in the preparation of a drug for treating viral hemorrhagic fever, wherein the structure of the compound of formula A is as follows: Wherein each Y is the same or different and is independently selected from H, or Na + , K + NH4 + Monovalent cation.

2. The use as claimed in claim 1, wherein each Y is the same and is selected from H, or Na + , K + NH4 + Monovalent cation.

3. The use as claimed in claim 1, wherein each Y is the same or different and is independently selected from Na + , K + , NH4 + .

4. The use as claimed in claim 1, wherein each Y is the same and is selected from Na + , K + , NH4 + .

5. The use according to claim 1, wherein the compound of formula A has the following structural formula:

6. Use of a compound of formula A in the preparation of a drug for protecting organs of patients with viral hemorrhagic fever, wherein the structure of the compound of formula A is as follows: Wherein each Y is the same or different and is independently selected from H, or Na + , K + NH4 + Monovalent cation.

7. The use as claimed in claim 6, wherein each Y is the same and is selected from H, or Na + , K + NH4 + Monovalent cation.

8. The use as claimed in claim 6, wherein each Y is the same or different and is independently selected from Na + , K + , NH4 + .

9. The use as claimed in claim 6, wherein each Y is the same and is selected from Na + , K + , NH4 + .

10. The use according to claim 6, wherein the compound of formula A has the following structural formula:

11. The use according to any one of claims 6 to 10, wherein the organ is spleen and / or liver.

12. Use of a compound of formula A in the preparation of a drug for use in combination with other drugs for treating viral hemorrhagic fever; the drug is used to treat viral hemorrhagic fever and / or protect organs of patients with viral hemorrhagic fever; the other drugs for treating viral hemorrhagic fever are selected from: antiviral agents, monoclonal antibodies, blood volume expanders, vasoactive drugs, glucocorticoids, blood products, anticoagulants; the structure of the compound of formula A is as follows: Wherein each Y is the same or different and is independently selected from H, or Na + , K + NH4 + Monovalent cation.

13. The use according to claim 12, wherein each Y is the same and is selected from H, or Na + , K + NH4 + Monovalent cation.

14. The use according to claim 12, wherein each Y is the same or different and is independently selected from Na + , K + , NH4 + .

15. The use according to claim 12, wherein each Y is the same and is selected from Na + , K + , NH4 + .

16. The use according to claim 12, wherein the compound of formula A has the following structural formula:

17. The use according to any one of claims 12 to 16, characterized in that: The antiviral agent is selected from ribavirin, favipiravir and chloroquine.

18. The use according to any one of claims 12 to 16, characterized in that: The organ is spleen and / or liver.

19. The use according to any one of claims 1 to 10 or 12 to 16, characterized in that: The viral hemorrhagic fevers are Chikungunya hemorrhagic fever, yellow fever, Omsk hemorrhagic fever, dengue hemorrhagic fever, Kyosanur Forest Sickness, Crimean-Congo hemorrhagic fever, epidemic hemorrhagic fever, Rif Valley Fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Ebola hemorrhagic fever, and Marburg hemorrhagic fever.

20. The use according to any one of claims 1 to 10 or 12 to 16, characterized in that: The viral hemorrhagic fever is Crimean-Congo hemorrhagic fever.

21. The use according to claim 11, characterized in that The viral hemorrhagic fevers are Chikungunya hemorrhagic fever, yellow fever, Omsk hemorrhagic fever, dengue hemorrhagic fever, Kyosanur Forest Sickness, Crimean-Congo hemorrhagic fever, epidemic hemorrhagic fever, Rif Valley Fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Ebola hemorrhagic fever, and Marburg hemorrhagic fever.

22. The use according to claim 11, characterized in that The viral hemorrhagic fever is Crimean-Congo hemorrhagic fever.

23. The use according to claim 17, characterized in that The viral hemorrhagic fevers are Chikungunya hemorrhagic fever, yellow fever, Omsk hemorrhagic fever, dengue hemorrhagic fever, Kyosanur Forest Sickness, Crimean-Congo hemorrhagic fever, epidemic hemorrhagic fever, Rif Valley Fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Ebola hemorrhagic fever, and Marburg hemorrhagic fever.

24. The use according to claim 17, characterized in that The viral hemorrhagic fever is Crimean-Congo hemorrhagic fever.

25. The use according to claim 18, characterized in that The viral hemorrhagic fevers are Chikungunya hemorrhagic fever, yellow fever, Omsk hemorrhagic fever, dengue hemorrhagic fever, Kyosanur Forest Sickness, Crimean-Congo hemorrhagic fever, epidemic hemorrhagic fever, Rif Valley Fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Ebola hemorrhagic fever, and Marburg hemorrhagic fever.

26. The use according to claim 18, characterized in that The viral hemorrhagic fever is Crimean-Congo hemorrhagic fever.

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