Application of heparin pentasaccharide compounds in the preparation of sepsis drugs

Abstract

The present invention provides a heparin pentasaccharide, which can be used for the treatment of sepsis or septic shock, reduce the mortality rate of sepsis, inhibit organ damage in the development of sepsis, protect vascular endothelial cells in sepsis, and reduce the serum TNF-α, IL-1β and / or IL-6 content of sepsis patients. The heparin pentasaccharide has a weak anticoagulant activity and a very low risk of causing bleeding. When treating sepsis, it can be used when it is determined that the patient has sepsis, without waiting until the patient has an obvious hypercoagulable state, which is conducive to the early treatment of sepsis and improves its prognosis.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compound manufacturing, and in particular relates to application of a heparin pentasaccharide compound in preparing a drug for treating sepsis. Background Art

[0002] At present, the clinical definition of sepsis is life-threatening organ dysfunction caused by the disorder of the body's response to infection. Sepsis is mainly caused by the imbalance of systemic inflammatory disorders, which leads to progressive endothelial dysfunction, leukocyte and platelet activation, and anticoagulant / procoagulant system disorders, and finally often causes multiple organ dysfunction syndrome (MODS). Systemic excessive inflammatory response, vascular endothelial cell damage, and abnormally activated coagulation reaction are the main pathophysiological characteristics of sepsis. The latter can lead to a hypercoagulable state in the body and the formation of a large number of microthrombi in the microcirculation, thereby aggravating the body's inflammatory response. Inflammation and coagulation are mutually causal and mutually reinforcing, eventually leading to disseminated intravascular coagulation (DIC) and multiple organ dysfunction syndrome (MODS), and even multiple organ dysfunction failure (MOF). Sepsis with circulatory and cellular metabolic disorders, also known as septic shock, is a subtype of sepsis. Its main clinical manifestation is persistent hypotension, and its in-hospital mortality rate exceeds 40%. In the past, the clinical definition of sepsis also used "systemic inflammatory response syndrome", "systemic inflammatory response syndrome", "multiple organ dysfunction syndrome" or "multiple organ dysfunction syndrome" caused by infection, trauma, burns, etc.

[0003] Sepsis is the leading cause of death in non-cardiac patients in intensive care units, with the characteristics of severe illness and high mortality rate. Sepsis is also one of the characteristics of severe cases of Corona Virus Disease 2019 (COVID-19). There are more than 18 million cases of severe sepsis worldwide each year, and about 14,000 people die from its complications every day, and the rate is increasing by 1.5% to 8.0% per year. A 2018 study on sepsis mortality used data from my country's National Mortality Surveillance System (NMSS). According to the ICD codes of the causes of death of residents in the 2015 NMSS database, the deaths related to various infections were identified. It was found that among the 1,937,299 deaths reported in the NMSS database in 2015, sepsis-related deaths accounted for 12.6%, and the standardized mortality rate calculated based on census data was 66.7 (95% CI 66.4-67.0) cases / 100,000. Based on this estimate, the number of deaths from sepsis in the country that year was about 1 million.

[0004] Due to the high incidence and complex mechanism of sepsis, there is currently no specific clinical treatment drug, and the drug treatment effect is poor. It usually requires a combination of multiple drugs and devices for comprehensive treatment, including: controlling the source of infection, incision and drainage of lesions and abscesses, and clearing infected necrotic tissues. In severe cases, anticoagulants, vascular activation drugs, hormone drugs, and ICU ward oxygen therapy ventilation, liver and kidney function support and other device-assisted means are required. The patient mortality rate is as high as 30% to 70%. Sepsis and septic shock have become an important problem facing critical care medicine. The development of drugs for the treatment of sepsis is imminent.

[0005] In 2001, Eli Lilly launched drotrecogin α, the world's only innovative drug for the treatment of severe sepsis. However, due to uncertain efficacy and the failure of subsequent large-scale clinical control studies to reconfirm the efficacy of rhAPC, it was withdrawn from the market in 2011.

[0006] Statistics show that the ICU clinical drug "Xuebijing" can only reduce the mortality rate of patients with severe pneumonia by 8.8%, and its efficacy advantage is not obvious. Summary of the invention

[0007] The invention provides a heparin pentasaccharide compound, which can reduce the mortality rate of sepsis, inhibit organ damage in the development of sepsis, and protect vascular endothelial cells in sepsis.

[0008] 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):

[0009]

[0010] Wherein each Y is the same or different and is independently selected from H, Na + , K + , Li + , NH 4 + Monovalent cations.

[0011] Preferably, each Y is the same and is selected from H, Na + , K + , Li + , NH 4 + Monovalent cations.

[0012] Preferably, each Y is the same or different and is independently selected from Na + , K + , Li + , NH 4 + .

[0013] In one embodiment of the present invention, each Y is the same and is selected from Na + , K + , Li + , NH 4 + .

[0014] It will be understood by those skilled in the art that any one or two or more Y is selected from Na + , K + , Li + , NH 4 + 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 - 、-SO 3 - .

[0015] 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.

[0016] 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.

[0017]

[0018] 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.

[0019]

[0020] 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.

[0021]

[0022] The 2016 International Guidelines for the Management of Sepsis and Septic Shock revised the concept and diagnosis of sepsis, using the Sequential (sepsis-related) Organ Failure Assessment (SOFA) as the standard for evaluating organ dysfunction for the diagnosis of sepsis, and emphasized that early identification and appropriate treatment are necessary to improve prognosis. Sepsis can be diagnosed when the SOFA score of an infected or suspected infected patient increases by ≥2 points compared to the baseline.

[0023] SOFA Rating Table

[0024]

[0025]

[0026] Some studies have shown that microcirculatory thrombosis in the early stage of sepsis may have a protective effect on the body: ① pathogens can be enclosed in microthrombi, hindering their movement in blood vessels; ② microthrombi may become a protective barrier, limiting the spread of pathogens through the bloodstream; ③ fibrin, fibrinogen and fibrin degradation products can recruit and activate neutrophils and macrophages, enhancing local cellular immune responses; ④ intramicrovascular thrombi can stimulate the aggregation of local antimicrobial peptides, which is beneficial to the removal of pathogens. Therefore, this type of intramicrovascular thrombosis is also called immune thrombosis, which is beneficial to patients with sepsis. If inappropriate anticoagulant treatment is given in the early stage of sepsis, the microvascular thrombus will dissolve, which will destroy the local immune defense mechanism and cause pathogens to spread with the bloodstream. Since the compound of formula A (such as CV122) has weak anticoagulant activity and a very low risk of causing bleeding, it can be used when it is determined that the patient is sepsis, without waiting until the patient is in a state of obvious hypercoagulability, which is beneficial to the early treatment of sepsis and improve its prognosis.

[0027] The present invention provides the use of a compound of formula A in preparing a drug for treating sepsis or septic shock. The drug can reduce the mortality rate of sepsis patients.

[0028] The present invention provides the use of a compound of formula A in the preparation of a medicament for protecting organs of patients with sepsis. In some embodiments of the present invention, the organs include but are not limited to lungs, liver and / or kidneys. The protection of organs (or organs) includes but is not limited to reducing tissue damage of organs, protecting or maintaining organ function.

[0029] The present invention provides use of a compound of formula A in preparing a medicament for protecting vascular endothelial cells of sepsis patients.

[0030] The present invention provides the use of a compound of formula A in preparing a drug for reducing TNF-α in sepsis patients.

[0031] The present invention provides use of a compound of formula A in preparing a drug for reducing IL-1β and / or IL-6 in sepsis patients.

[0032] The present invention provides use of a compound of formula A in combination with other drugs for treating sepsis in preparing a drug for treating sepsis or septic shock.

[0033] The present invention provides use of a compound of formula A in combination with other drugs for treating sepsis in the preparation of a drug for protecting organs of sepsis patients.

[0034] The present invention provides use of a compound of formula A in combination with other drugs for treating sepsis in the preparation of a drug for protecting vascular endothelial cells of sepsis patients.

[0035] The present invention provides use of a compound of formula A in combination with other drugs for treating sepsis in the preparation of a drug for reducing TNF-α in sepsis patients.

[0036] The present invention provides use of a compound of formula A in combination with other drugs for treating sepsis in the preparation of a drug for reducing IL-1β and / or IL-6 in sepsis patients.

[0037] The present invention provides use of the compound of formula A in preparing a drug for treating sepsis or septic shock in combination with other drugs for treating sepsis.

[0038] The present invention provides use of the compound of formula A in preparing a drug for protecting organs of sepsis patients in combination with other drugs for treating sepsis.

[0039] The present invention provides use of a compound of formula A in preparing a drug for protecting vascular endothelial cells of sepsis patients in combination with other drugs for treating sepsis.

[0040] The present invention provides use of a compound of formula A in preparing a drug for reducing TNF-α in sepsis patients in combination with other drugs for treating sepsis.

[0041] The present invention provides use of the compound of formula A in the preparation of a drug for treating sepsis in combination with other drugs for treating sepsis to reduce IL-1β and / or IL-6 in sepsis patients.

[0042] According to the present invention, the content of TNF-α, IL-1β and / or IL-6 in the body includes TNF-α, IL-1β and / or IL-6 in the blood, tissues and / or organs.

[0043] In one embodiment of the present invention, the compound of formula A is CV001.

[0044] In one embodiment of the present invention, the compound of formula A is CV122.

[0045] In one embodiment of the present invention, the compound of formula A is CV123.

[0046] 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.

[0047] The present invention further provides a pharmaceutical composition, characterized in that it comprises a compound of formula A, or a solvate thereof as an active ingredient. The pharmaceutical composition is used to treat sepsis or septic shock, protect organs of sepsis patients, protect vascular endothelial cells of sepsis patients, and / or reduce TNF-α, IL-1β and / or IL-6 in sepsis patients.

[0048] The pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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%.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] The pharmaceutical composition can be used in combination with other drugs for treating sepsis.

[0057] The pharmaceutical composition may further contain a second therapeutic agent, which is another drug for treating sepsis.

[0058] The present invention provides a method for treating sepsis or septic shock, 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.

[0059] The present invention provides a method for protecting organs of patients with sepsis, 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.

[0060] The present invention provides a method for protecting vascular endothelial cells of patients with sepsis, 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.

[0061] The present invention provides a method for reducing TNF-α in sepsis patients, characterized in that a therapeutically effective amount of a compound of formula A or a solvate thereof is administered to a patient in need.

[0062] The present invention provides a method for reducing IL-1β and / or IL-6 in sepsis patients, characterized in that a therapeutically effective amount of a compound of formula A or a solvate thereof is administered to a patient in need.

[0063] 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.

[0064] The method further comprises administering to the patient in need thereof an additional drug for treating sepsis.

[0065] Other drugs for treating sepsis include, but are not limited to, antibiotics, blood volume expanders, vasoactive drugs, glucocorticoids, blood products, blood sugar control drugs, anticoagulants, etc.

[0066] 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.

[0067] 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:

[0068]

[0069] The dehydroxylation and carboxyl protecting group, sulfonation and azide reduction can all be carried out using reaction methods and conditions known in the art.

[0070] Intermediate 1 R 1 , R 2 and R 3 can be the same or different, and independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl; the definition of Y in the compounds of formula II and formula III is as described above. In a specific embodiment of the present invention, R 1 and R 2 All are acetyl, R 3 is benzoyl, and Y is H. In a specific embodiment of the present invention, R 1 and R 2 All are acetyl, R 3 is benzoyl, Y is Na + In one embodiment of the present invention, R 1 and R 2 All are acetyl, R 3 is benzoyl, Y is K + .

[0071] 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:

[0072]

[0073] 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.

[0074] 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 stereo-specific trisaccharide intermediate 4, and the 1,6 ring opening of the trisaccharide intermediate 4 obtains a trisaccharide intermediate 5, and then obtains the trisaccharide intermediate 2, and the reaction formula is as follows. Each reaction step can be carried out by 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, the 1,6 ring opening of the trisaccharide intermediate 4 is carried out in the corresponding acid anhydride (such as chloroacetic anhydride, acetic anhydride, benzoic anhydride, pivalic anhydride) to obtain the trisaccharide intermediate 5, and TBSOTf, TMSOTf, Et 2 OBF 3 or Et 3 SiOTf, etc., the reaction temperature is -20℃~0℃.

[0075]

[0076] 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:

[0077]

[0078] In some embodiments of the present invention, Ag can be used 2 CO 3 / AgOTf reaction system or Ag 2 The O / TMSOTf reaction system was used to carry out the Koenigs-Knorr glycosylation reaction to generate the disaccharide intermediate 9.

[0079] The monosaccharide 10 can be prepared by using glucose as a raw material through the following steps: 1) converting the hydroxyl group at position 1 of glucose into 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 it with R 2 Protect the hydroxyl group at position 3; 3) deprotect the hydroxyl groups at positions 4 and 6 of glucose and then react, oxidize and methylate the hydroxyl group at position 6, and protect the hydroxyl group at position 4 with a chloroacetyl group; 4) convert the glucosinolate at position 1 to bromine to obtain the monosaccharide intermediate 10.

[0080] 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. Chem Med Chem, 2014, 9, 1071–1080.

[0081] In the above preparation method, R 1 , R 2 and R 3 The definitions of are the same as those of the corresponding groups in Intermediate 1; X is a leaving group suitable for reacting with other acceptors to form a bond between glycosides, preferably X is hydroxyl, alkylthio, arylthio, halogen, trichloroacetylimido, phosphate, or tert-butyldiphenylsilyloxy. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 Survival of mice after CV122 administration in the LPS-stimulated sepsis model

[0083] Figure 2 Results of determination of TNF-α content in mouse serum after administration of CV122 in LPS-stimulated sepsis model

[0084] Figure 3 HE staining results of mouse lung tissue after CV122 administration in LPS-stimulated sepsis model

[0085] Figure 4 HE staining results of mouse kidney tissue after CV122 administration in LPS-stimulated sepsis model

[0086] Figure 4-1 HE staining results of mouse liver tissue after CV122 administration in LPS-stimulated sepsis model

[0087] Figure 4-2 Measurement results of TNF-α, IL-1β, and IL-6 in lung, kidney, and liver tissue homogenates of mice after CV122 administration in the LPS-stimulated sepsis model

[0088] Figure 5 The protective effect of CV122 on human umbilical vein endothelial cells treated with LPS

[0089] Figure 6Survival of mice after administration of fondaparinux in the LPS-stimulated sepsis model

[0090] Figure 7 Survival of mice after administration of low molecular weight heparin in the LPS-stimulated sepsis model

[0091] Figure 8 Survival rate of mice after administration of CV122 and Suramin in the LPS-stimulated sepsis model

[0092] Fig. 9 Results of measurement of TNF-α, IL-1β, IL-6, AST, and ALT in mouse serum after administration of CV122 and Suramin in the LPS-stimulated sepsis model

[0093] Fig.10 Cell survival results after CV122 and Suramin administration

[0094] Fig.11 Flow cytometric analysis of the effects of CV122 and Suramin on the expression of ICAM-1 and VCAM-1 in cells induced by LPS and TNF-α DETAILED DESCRIPTION

[0095] 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.

[0096] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0097] 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.

[0098] Example 1 Synthesis of Pentasaccharide Intermediate III

[0099] 1. Preparation method of monosaccharide intermediate 10-1

[0100] Using glucose 12 as the starting material, monosaccharide intermediate 13 was obtained by full acetylation; 3. E 2 O, react 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-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; heating under acetic acid conditions to remove the benzylide protecting group to obtain 4,6-dihydroxy monosaccharide intermediate 20; TEMPO oxidation and methyl esterification to obtain monosaccharide intermediate 21; the 4-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.

[0101]

[0102] The reaction conditions and yields of each step are as follows: a) Ac 2 O,HClO 4 ,77%; b)TolSH,BF 3 ·Et 2 O, DCM, 86%; c) 1) MeONa, MeOH, DCM; 2) Benzaldehyde Dimethylacetal, CSA, DMF, two-step total yield 83%; d) 1) Bu 2 SnO, MeOH, reflux; 2) PMBCl, CsF, DMF, 90°C, two-step total yield 75%; e) BnBr, NaH, DMF, 92%; f) DDQ, DCM, H 2 O, 78%; g)Ac 2 O,Et 3 N, DCM, 98%; h) 80% AcOH, 90°C, 76%; i) 1) 2,2,6,6-Tetramethylpiperidinooxy (TEMPO), Iodobenzene diacetate, DCM, H 2 O; 2)MeI,KHCO 3 ,DMF, two-step total yield 57%; j)ClAc 2 O, Py, DCM, 76%; k) IBr, DCM, 76%.

[0103] 2. Preparation method of disaccharide intermediate 9-1

[0104] 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.

[0105]

[0106] Add to the two bottles Molecular sieve (1g, powder), vacuum the flask, pass argon protection and cool to room temperature. Dissolve the monosaccharide intermediate 10-1 (300mg, 0.63mmol) and 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, and add Ag 2 CO 3 (260 mg, 0.94 mmol) and catalytic amount of AgOTf (16 mg, 0.063 mmol) were added to the reaction bottle, protected from light, and reacted overnight. TLC detected that the donor 10-1 completely disappeared and a main substance was generated. It was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure and directly purified by column chromatography (petroleum ether / ethyl acetate = 2:1) to obtain a disaccharide monosaccharide intermediate 9-1 with a mixed α / β configuration. For the synthesis method of monosaccharide intermediate 11-1, see Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116.

[0107] HRMS [M+Na] + m / z 650.1327 (calculated: C 26 H 30 C1N 3 NaO 13 ,650.1365).

[0108] 3. Preparation method of disaccharide intermediate 8-1

[0109] The chloroacetyl group at position 4 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.

[0110]

[0111] 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%).

[0112] 1 H NMR (400 MHz, CDCl 3 )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=7.6Hz,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),3.63(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 CNMR (101MHz, CDCl 3 )δ171.07,169.27,169.21,138.06,128.41,127.98,127.78,103.50,100.17,78.28, 76.22,75.18,74.63,74.22,74.03,70.61,70.07,64.88,58.73,52.88,21.03,20.94.

[0113] HRMS [M+Na] + m / z 574.1642 (calculated: C 24 H 29 N 3 NaO 12 ,574.1649).

[0114] 4. Preparation method of trisaccharide intermediate 2-1-1

[0115] 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.

[0116]

[0117] 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%).

[0118] 1 H NMR (400 MHz, CDCl 3 )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). 13 C NMR (101 MHz, CDCl3 )δ170.55,169.75,169.12,167.49,137.94,137.62,137.53,128.52, 128.40,128.13,128.11,128.02,127.98,127.91,127.78,103.45,100 .20,99.11,79.83,78.24,77.18,76.35,75.56,74.99,74.93,74.45,7 3.95,73.66,70.41,70.21,64.93,63.79,62.00,20.95,20.94,20.80.

[0119] HRMS[M+K] + m / z 999.3036 (calculated: C 46 H 52 KN 6 O 17 ,999.3026).

[0120] 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%).

[0121] 1 H NMR (400 MHz, CDCl 3 )δ7.87–7.03(m,15H),6.41–3.17(m,28H),2.23–1.89(m,15H). 13 C NMR (101 MHz, CDCl 3)δ170.54,170.03,169.98,169.59,168.58,167.58,137.54,137.50,137.42,128. 52,128.45,128.15,128.11,128.07,128.04,128.01,127.92,127.88,103.34,99.0 7,89.91,79.83,78.96,77.30,77.20,75.94,75.61,74.99,74.94,73.67,70.97,70.33,69.97,63.79,61.91,61.01,60.37,52.83,29.69,20.99,20.86,20.79,20.64.

[0122] HRMS [M+Na] + m / z 1085.3638 (calculated: C 50 H 58 NaN 6 O 20 ,1085.3604).

[0123] 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.

[0124] 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.

[0125] NMR of β-configuration product: 1 H NMR (400 MHz, CDCl 3 )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=9.1Hz,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,3H),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 CNMR (101MHz, CDCl 3 )δ170.55,169.92,169.76,169.60,167.56,160.50,137.53,137.50,13 7.33,128.54,128.46,128.17,128.05,127.94,127.89,103.33,99.11, 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.

[0126] HRMS [M+Na] + m / z 1186.2578 (calculated: C 50 H 56 Cl 3 NaN 7 O 19 ,1186.2594).

[0127] NMR of the product with α configuration: 1 H NMR (400 MHz, CDCCl 3)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 CNMR(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.

[0128] 5. Preparation method of fully protected pentasaccharide intermediate 1-1

[0129] 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.

[0130]

[0131] 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%).

[0132] 1 H NMR (400 MHz, CDCl 3 )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,3 0H),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). 13 C NMR (101 MHz, CDCl 3)δ170.95,170.55,170.01,169.88,169.64,167.62,165.42,155.88,138.47,137.55,137.51,137.33,137.28,136.31,133.51,1 30.04,129.11,128.71,128.56,128.52,128.37,128.25,128.18,128.04,127.95,127.92,127.67,127.37,103.47,99.11,98.84 ,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.

[0133] HRMS [M+Na] + m / z 1868.6488 (calculated: C 93 H 103 NaN 7 O 33 ,1868.6494).

[0134] 6. Preparation method of pentasaccharide intermediate III-1

[0135] The fully protected pentasaccharide 1-1 was reacted with LiOH and H 2 O 2 Under the combined action of NaOH, Ac, Bz and methyl ester are removed simultaneously to obtain hexahydroxy compound I; in SO 3 ·NEt 3 The intermediate compound II-1 after O-sulfonation is obtained by heating under the action of ; the benzyl group and Cbz are removed by catalytic hydrogenation reaction, and the azide is reduced to form an amino group to obtain the triamino compound III-1.

[0136]

[0137] Preparation of pentasaccharide intermediate III-1: The fully protected pentasaccharide intermediate 1-1 (100 mg, 0.054 mmol) was dissolved in 4.9 mL THF, and 1.3 mL 1.25 N LiOH solution and 2.7 mL 30% H 2 O 2Solution, react at room temperature for 12 hours, add 3mL methanol and 1.6mL 4N NaOH solution, continue to react at room temperature for 12 hours, monitor with a spot plate, TLC color will generate a long strip with a tail, and there are no spots on the top. Adjust the pH to 2 with 6N hydrochloric acid in an ice bath, transfer the reaction solution to a separatory funnel and extract it three times with dichloromethane, combine the organic layers, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate by rapid column chromatography (dichloromethane / methane = 15:1) to obtain pentasaccharide intermediate I (73mg, 90%). Only one single peak was found by HPLC purity detection, proving that its purity is very high. Pentasaccharide intermediate I (100mg, 0.067mmol) and SO 3 ·NEt 3 (1.1 g, 6.0 mmol) was dissolved in 10 mL of anhydrous DMF and reacted at 65 °C for 24 hours. The degree of 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 the eluent to collect the sugar-containing components. After the solvent was dried by spin drying, methanol was used as the 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.

[0138] 1 H NMR (400 MHz, D 2 O)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). 13 C NMR (101MHz, D 2O)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.

[0139] HRMS[M-2Na] + m / z 738.4654 (calculated: C 31 H 47 N 3 Na 6 O 43 S 6 2- ,738.4647).

[0140] Example 2 Preparation of Compound CV001

[0141] 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 continue to add 2N NaOH to maintain the pH between 9 and 10 in the following reaction). Add SO 3 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. After concentration, 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%).

[0142]

[0143] 1 H NMR (400 MHz, D 2O)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,3H),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). 13 C NMR (101MHz, D 2 O) 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.

[0144] HRMS[M-3H] 3- m / z 527.9675 (calculated: C 31 H 53 N 3 O 52 S 9 ,527.9616)

[0145] Example 3 Synthesis of Compound CV122

[0146] Synthesis method 1: Compound CV001 is subjected to sodium exchange to obtain sodium salt CV122.

[0147]

[0148] 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.

[0149] 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 SO 3Py (110 mg, 0.7 mmol), add another batch half an hour later, repeat six times, then stir at room temperature for 4 hours and neutralize with hydrochloric acid to pH 7-8, concentrate and directly pass through Sephadex G-25 gel column with water as eluent, collect sugar-containing components, concentrate and pass through Dowex-50-WX4-Na + 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.

[0150] 1 H NMR (400 MHz, D 2 O)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,1H),4.44 (s,1H),4.41–4.08(m,11H),3.92(dd,J=19.1,9.2Hz,4H),3.79–3.60(m,4H),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, D 2 O) 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.

[0151] HRMS [M–3Na] 3- m / z 586.5803 (calculated: C 31 H 45 N 3 Na 8 O 52 S 9 3- ,586.5801).

[0152] Example 4 Synthesis of Compound CV123

[0153] Synthesis method 1: Potassium salt CV123 can be obtained by potassium exchange of compound CV001.

[0154]

[0155] 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.

[0156] 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 SO 3 Py (99 mg, 0.63 mmol), add another batch half an hour later, repeat six times, then stir at room temperature for 4 hours and neutralize with hydrochloric acid to pH 7-8, concentrate and directly pass through Sephadex G-25 gel column with water as eluent, 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.

[0157] 1 H NMR (400 MHz, D 2 O)δ5.48(d,J=3.4Hz,1H),5.35(d,J=3.4Hz,1H),5.24(d,J=2.8Hz,1H),5.0 9(d,J=2.7Hz,1H),4.93(d,J=3.6Hz,1H),4.75(m,3H),4.55(t,J=7.6Hz,1H) ,4.49(s,1H),4.40–4.09(m,11H),4.23-3.84(m,4H),3.74–3.62(m,4H),3.5 5(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, D 2 O) 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.

[0158] 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.

[0159] Example 5 The therapeutic effect of CV122 on sepsis

[0160] LPS (lipopolysaccharide) stimulated sepsis model: C57BL / 6Cnc mice were intraperitoneally injected with LPS (40 mg / kg, 100 μL). The mice died within 48 hours after LPS administration, indicating that the model was successfully established.

[0161] 1. Survival rate of mice in LPS-stimulated sepsis model

[0162] Mice C57BL / 6Cnc; LPS (40 mg / kg, 100 μL) was injected intraperitoneally, and CV122 was injected subcutaneously 30 minutes later. The experimental animals were divided into four groups, with 5 mice in each group, and the drugs were administered as follows:

[0163] ①5 mice, LPS; ②5 mice, LPS+CV122: 20mg / kg; ③5 mice, LPS+CV122: 30mg / kg; ④5 mice, LPS+CV122: 40mg / kg. The death of mice in each group was recorded within 80 hours. The results are as follows Figure 1 shown.

[0164] from Figure 1 It can be seen that among the five mice in the LPS group, two died 25 hours after administration, and all died 30 hours after administration. In the three groups given CV122, the survival rate of mice increased, and there was an obvious dose-effect relationship. In the CV122 20 mg / kg group, one died 25 hours after administration, the second died 40 hours after administration, and the remaining three survived throughout the observation period; in the CV122 30 mg / kg group, only one died 25 hours after administration, and the remaining four survived throughout the observation period; in the CV122 40 mg / kg group, all survived throughout the observation period.

[0165] 2. HE staining of mouse lung, kidney and liver tissues

[0166] Mice C57BL / 6Cnc; LPS (40 mg / kg, 100 μL) was injected intraperitoneally, and CV122 was injected subcutaneously 30 minutes later. The experimental animals were divided into three groups, with 5 mice in each group, and the drugs were administered as follows:

[0167] ①5 mice, PBS; ②5 mice, LPS; ③5 mice, LPS+CV122: 30 mg / kg. 12 hours after LPS injection, the mice were dissected to obtain lung and kidney tissues, fixed with 4% paraformaldehyde, and paraffin sections were obtained according to the experimental procedures. HE staining was performed, and the microscopic examination results were as follows: Figure 3 and 4 shown.

[0168] from Figure 3 HE staining of lung tissue showed that the alveolar structure was intact in the PBS group, and there was no edema and thickening of the alveolar wall, and no inflammatory cell infiltration; in the LPS group, the alveolar wall was thickened with inflammatory cell infiltration, and the pulmonary capillaries were significantly dilated with edema of the pulmonary interstitial tissue. The alveolar edema and inflammatory cell infiltration in the CV122 group were significantly reduced compared with the LPS group.

[0169] from Figure 4 HE staining of renal tissue showed that: no obvious damage was found in the PBS group, and the size of the tissue gap was normal; the LPS group showed obvious renal damage phenomena such as tubular structure destruction or expansion and vacuole degeneration; compared with the LPS group, the renal damage in the CV122 group was effectively improved.

[0170] The inventors further conducted the same histological study on the liver of mice, and the results were as follows: Figure 4-1 As shown in the figure, in the PBS group, the size of the liver tissue cells of mice was normal, and there was no pathological change. In the LPS group, the liver sinusoidal congestion, congestion and hepatocyte turbidity in the liver tissue were aggravated, and there was a little punctate necrosis of hepatocytes. Compared with the LPS group, the size of the liver tissue cells in the CV122 group was relatively normal, and the congestion, congestion, hepatocyte turbidity and hepatocyte necrosis were reduced, and the liver damage was effectively improved.

[0171] The inventors also homogenized the lung tissue, kidney tissue and liver tissue of mice, and measured the content of TNF-α, IL-1β and IL-6 in the homogenates of the three organ tissues (grinded in PBS at 0.1 g tissue / mL, centrifuged and the supernatant was taken, and the ELISA kits for mouse TNF-ɑ, IL-6 and IL-1β were used for detection, respectively, according to the operating instructions of the kits). The results are as follows: Figure 4-2 As shown. Figure 4-2 It can be seen that the contents of TNF-α, IL-1β, and IL-6 in the three organ tissue homogenates of the LPS group were significantly higher than those of the PBS group, and the contents of TNF-α, IL-1β, and IL-6 in the CV122 group were significantly lower than those in the LPS group, indicating that CV122 can significantly reduce the increase of TNF-α, IL-1β, and IL-6 caused by LPS.

[0172] 3. Protection of vascular endothelial cells

[0173] Experimental method: HUVEC cells in the logarithmic growth phase were plated in a 24-well plate (with cell slides) for 10 6 / well, divided into three groups, namely PBS group, LPS group (LPS 5μg / ml) and LPS+CV122 group (LPS 5μg / ml, CV122 50μM). After 2h of drug stimulation according to the group, the culture medium was discarded and washed twice with PBS, and the cell slides were taken out for examination under atomic force microscope.

[0174] Experimental results: see Figure 5 After LPS stimulated the cells, the glycocalyx structure on the cell surface was destroyed, and the roughness of the cell surface became greater compared with the normal group (PBS). When CV122 was added, the roughness of the cell surface decreased. According to statistical analysis, there was a significant difference in the surface roughness of HUVEC between the CV122 group and the LPS group.

[0175] 4. Determination of serum TNF-α content

[0176] Mice C57BL / 6Cnc; LPS (40 mg / kg, 100 μL) was injected intraperitoneally, and CV122 was injected subcutaneously 30 minutes later. The experimental animals were divided into five groups, with 5 mice in each group, and the drugs were administered as follows:

[0177] ①5 mice, PBS; ②5 mice, LPS; ③5 mice, LPS+CV122: 20 mg / kg; ④5 mice, LPS+CV122: 30 mg / kg; ⑤5 mice, LPS+CV122: 40 mg / kg. 3 hours after LPS injection, the eyeballs of mice in each group were removed to collect blood, and the serum was separated by centrifugation and the TNF-α content in the serum was detected using an ELISA kit. The results are shown in Figure 2 .

[0178] from Figure 2 It can be seen that the TNF-α content in the serum of mice in the LPS group was significantly higher than that in the PBS group, with statistical significance. The TNF-α content in the serum of mice in each CV122-administered group was lower than that in the LPS group, and the TNF-α content in each CV122 group was significantly different from that in the LPS group, and showed a dose-effect relationship.

[0179] 5. Effects of fondaparinux and low molecular weight heparin on LPS-induced sepsis

[0180] The modeling, grouping and administration were carried out in the same manner as in point 1 above, with the dosage of fondaparinux being 30 mg / kg. Figure 6As shown in the figure, 2 mice died in the LPS group 25 hours after administration, 1 mouse died 30 hours after administration, 1 mouse died 70 hours after administration, and all died 90 hours after administration; in the fondaparinux group 1 mouse died 30 hours after administration, 1 mouse died 50 hours after administration, 1 mouse died 70 hours after administration, and 1 mouse died 90 hours after administration. After 80 hours, 2 mice survived, and after 100 hours, only 1 mouse survived. It can be seen that fondaparinux is not as effective as CV122 in controlling the mortality rate of sepsis.

[0181] The same method as in point 1 was used for modeling, grouping and drug administration, with the dosage of low molecular weight heparin (LMWH) being 40 mg / kg. Figure 7 As shown in the figure, all the patients in the LPS group died more than 30 hours after administration; half of the patients in the low molecular weight heparin group died 30 hours after administration, and this mortality rate remained until 70 hours after administration. It can be seen that the control effect of low molecular weight heparin on sepsis mortality is not as good as CV122.

[0182] 6. Comparative study between CV122 and Suramin

[0183] The inventors later conducted further comparative studies on CV122 and Suramin, including studying the effects of the two on the survival rate of septic mice, cytokines and blood biochemical indicators in the LPS sepsis mouse model, and conducted drug cytotoxicity experiments on cells.

[0184] Experimental methods:

[0185] Fifteen male C57BL / 6Cnc mice, 6-8 weeks old, weighing 20-25g, were randomly divided into 3 groups, 5 mice in each group, and the CV122 and Suramin sepsis mouse survival rate experiments were performed according to Table 1.1. Each group of mice was intraperitoneally injected with 40mg / kg LPS. 30 minutes later, mice in group ② were subcutaneously injected with CV122 (40mg / kg), and mice in group ③ were subcutaneously injected with Suramin (40mg / kg). The mice were observed every day and the survival rate was recorded. The results are shown in Figure 8 As shown. Figure 8 It can be seen that 40 mg / kg of CV122 and Suramin can both reduce the mortality of LPS-induced sepsis mice. The survival rate of the CV122 group during the observation period was 100%, and the survival rate of the Suramin group during the observation period was 60%. CV122 has a stronger protective effect on the survival of septic mice than Suramin.

[0186] Table 1.1CV122 and Suramin sepsis mouse survival experiment

[0187]

[0188] ELISA method to detect cytokine levels in the serum of CV122 and Suramin mice

[0189] Twenty male C57BL / 6Cnc mice were randomly divided into four groups, with five mice in each group, as shown in Table 1.2: The mice in group ① were injected with 0.9% NaCl (100 μL / mouse), and the mice in groups ②-④ were intraperitoneally injected with LPS (20 mg / kg, 100 μL / mouse). 30 minutes later, the mice in group ③ were injected with 40 mg / kg CV122, and the mice in group ④ were injected with 40 mg / kg Suramin. 3 hours later, the mice were eyeballed and blood was collected in a 1.5 mL centrifuge tube. The whole blood was placed at 37°C for 1 hour, then placed in a refrigerator at 4°C overnight, and then placed in a centrifuge (3000g, 20min, 4°C) to separate serum. The cytokine levels in serum were detected using mouse ELISA kits for TNF-ɑ, IL-6, and IL-1β, respectively. 100 μL of capture antibody solution of TNF-ɑ, IL-6, and IL-1β was added to a 96-well plate and incubated overnight at 4°C. Wash 3 times with Wash Buffer, add 200μL / well Assay Diluent A to block, incubate at 37℃ for 1h, and wash 3 times. Add 100μL / well TNF-ɑ, IL-6, IL-1β standards and samples, incubate at 37℃ for 2h, wash 3 times, add 100μL diluted TNF-ɑ, IL-6, IL-1β antibody solution, incubate at 37℃ for 1h, wash 3 times, add 100μL Avidin-HRP solution, incubate at room temperature for 30min, wash 3 times, add 100μL TMB, incubate at room temperature in the dark for 30min. Add 100μL stop solution to terminate the reaction. The positive wells should turn from blue to yellow. Read the absorbance at 450nm with an enzyme reader within 15min, and calculate the cytokine level according to the standard curve.

[0190] Table 1.2 Experimental table of cytokine content detection in serum and tissues

[0191]

[0192]

[0193] 20 male C57BL / 6Cnc mice were randomly divided into 4 groups, 5 mice in each group, as shown in Table 1.3: The mice in group ① were injected with 0.9% NaCl (100 μL / mouse), and the mice in groups ②-④ were intraperitoneally injected with LPS (20 mg / kg, 100 μL / mouse). 30 minutes later, the mice in group ③ were subcutaneously injected with 40 mg / kg CV122, and the mice in group ④ were subcutaneously injected with 40 mg / kg Suramin. 24 hours later, the eyeballs of the mice were removed and blood was collected in 1.5 mL centrifuge tubes. The whole blood was placed at 37℃ for 1 hour, then placed in a refrigerator at 4℃ overnight, and then placed in a centrifuge (3000g, 20min, 4℃) to separate the serum.

[0194] AST and ALT kits were used to detect the levels of AST and ALT in serum. In a 96-well plate, 20 μL / well of AST and ALT matrix solution was added to the test wells and control wells, and 5 μL / well of the sample to be tested was added to the test wells at 37°C for 30 minutes. 20 μL / well of 2,4-dinitrophenylhydrazine solution was added to each well, and 5 μL / well of the sample to be tested was added to the control wells at 37°C for 20 minutes. Then 200 μL / well of 0.4 mol / L sodium hydroxide solution was added, and the 96-well plate was gently mixed and the plate was kept at room temperature for 15 minutes at a wavelength of 510 nm. The OD value of each well was measured by an enzyme-labeled instrument. The absolute OD value = the OD value of the test well - the OD value of the control well was used to check the standard curve to obtain the corresponding AST and ALT activity units.

[0195] Table 1.3 Experimental table of biochemical index detection in septic mice

[0196]

[0197] The results are as follows Fig. 9 As shown. Fig. 9 It can be seen that the serum TNF-α, IL-1β, IL-6, AST and ALT of the mice in the LPS group were significantly higher than those in the control group, and the serum TNF-α, IL-1β, IL-6, AST and ALT of the CV122 group and Suramin group were significantly lower than those of the LPS group, and the reduction degree of serum TNF-α, IL-1β, IL-6, AST and ALT in the CV122 group was greater than that in the Suramin group.

[0198] Detection of CV122 and Suramin cytotoxicity using CCK-8 cell proliferation-toxicity assay kit

[0199] Raw and HUVEC cell suspensions (100 μL / well) were added to a 96-well cell culture plate, with approximately 5,000 cells per well, and incubated at 37°C, 5% CO 2Culture in an incubator for 12 hours. Give 10 μL of 50 μM CV122 and Suramin, 100 μM CV122 and Suramin, 200 μM CV122 and Suramin drug stimulation for 24 hours. Add 10 μL CCK-8 solution to each well. The wells with the corresponding amount of cell culture medium and CCK-8 but no cells can be used as blank controls. After 2 hours of protection from light, use an enzyme reader to detect respectively, and use the enzyme reader to measure the absorbance at 450nm to calculate the cell survival rate. The results are as follows Fig.10 shown.

[0200] Depend on Fig.10 It can be seen that the survival rates of HUVEC (human umbilical vein endothelial cells) and RAW (mouse mononuclear macrophages) in the three dose groups of CV122, 50μM, 100μM, and 200μM, were the same as those in the control group, indicating that CV122 had no toxic effect on cells at extremely high concentrations. The cell survival rates of the three dose groups of Suramin, 50μM, 100μM, and 200μM, decreased with increasing doses, among which the cell survival rates of the Suramin 200μM dose group in RAW cells and the Suramin 100μM and 200μM dose groups in HUVEC cells were significantly reduced compared with the control group.

[0201] The study showed that CV122 has a higher activity than Suramin in reducing the inflammatory cytokines induced by LPS, but a lower cytotoxicity than Suramin.

[0202] 7. Research on cell adhesion molecules ICAM-1 and VCAM-1

[0203] ICAM-1 (Intercellular adhesion molecule-1), also known as CD54, is a member of the immunoglobulin superfamily (IGSF) of adhesion molecules. It is an important adhesion molecule that mediates adhesion reactions and is widely distributed on epithelial cells, vascular endothelial cells, fibroblasts, reticular cells, monocytes, macrophages and lymphocytes.

[0204] VCAM-1 (vascular cell adhesion molecule-1), also known as CD106, is a member of the immunoglobulin superfamily and is widely expressed on the surface of activated endothelial cells, smooth muscle cells, bone marrow stromal cells, macrophages, dendritic cells, fibroblasts, etc. Its biological effects include: participating in the regulation of inflammatory response, differentiation, development, immune response of cells and tissues, and participating in lymphocyte homing, recirculation, and tumor spread and metastasis.

[0205] The inventors investigated the effect of CV122 on the expression of ICAM-1 and VCAM-1 by cells.

[0206] HUVEC (human umbilical vein endothelial cell line) and Raw264.7 (mouse mononuclear macrophage) were purchased from the cell bank of Synbio (Shanghai).

[0207] HUVEC cells in the logarithmic growth phase were digested and centrifuged, and then 1×10 6 The cells were plated at a density of 100 / well in a six-well cell culture plate and continued to be cultured overnight to allow the cells to fully adhere to the wall. A total of 4 groups of cells were grouped as follows: ①PBS control group, ②TNF-α group, ③TNF-α+CV122, ④TNF-α+Surammin. After the cells adhered to the wall, fresh complete medium was replaced, and CV122 (50μM) was added to the dishes of group ③ for co-incubation for 3h, and Surammin (50μM) was added to the dishes of group ④ for co-incubation for 3h, and then TNF-α (20ng / mL) was added to groups ②, ③, and ④ for stimulation for 1h. Finally, the culture medium was discarded, and the cells were washed three times with PBS before the subsequent flow cytometry test of cell adhesion molecules was performed.

[0208] Take the Raw264.7 in the logarithmic growth period according to 1×10 6 The cells were plated at a density of 100 / well in a six-well cell culture plate and continued to be cultured overnight to allow the cells to fully adhere to the wall. A total of 4 dishes of cells were grouped as follows: ①PBS control group, ②LPS group, ③LPS+CV122, ④LPS+Surammin. After the cells adhered to the wall, fresh complete medium was replaced, and CV122 (50μM) was added to the dish of group ③ for co-incubation for 3h, and Surammin (50μM) was added to the dish of group ④ for co-incubation for 3h, and then LPS (5μg / mL) was added to groups ②, ③, and ④ for stimulation for 1h. Finally, the culture medium was discarded, and the cells were washed three times with PBS before the subsequent flow cytometry test of cell adhesion molecules was performed.

[0209] Method for detecting cell adhesion molecules by flow cytometry:

[0210] (1). Take the six-well cell culture plate after the above treatment, discard the cell supernatant with a pipette, and rinse with PBS + 0.5% FBS. (2). Collect the cells in each well with 10mL PBS + 0.5% FBS into a 15mL centrifuge tube, centrifuge at 1000rpm for 7min, and wash twice. (3). Discard the supernatant and invert the tube on absorbent paper. (4). Preparation of antibody mixture:

[0211] ①FITC Anti-Mouse CD54 Antibody (Elabscience, 5μL / sample), AF647 Anti-MouseCD106 Antibody (Elabscience, 5μL / sample), PBS+0.5% FBS plus 100μL / sample; ②FITC Anti-HumanCD54 Antibody (Elabscience, 5μL / sample), AF647 Anti-Human CD106 Antibody (Elabscience, 5μL / sample), PBS+0.5% FBS plus 100μL / sample.

[0212] (5). Add antibody mixture ① to each sample of Raw264.7, and add antibody mixture ② to each sample of HUVEC, and blow evenly with a gun. Incubate at room temperature for 30 minutes, away from light. (6). Add 10mL PBS + 0.5% FBS to each tube, centrifuge at 1000rpm for 7min. (7). Discard the supernatant and invert the tube on absorbent paper. (8). Add 500ul PBS + 0.5% FBS to resuspend the cells, and filter into the flow tube with a 200-mesh copper mesh, wrap the flow tube with tin foil, and detect on a flow cytometer.

[0213] The experimental results are as follows Fig.11 shown. Fig.11 Middle A: Raw264.7 cells stimulated with LPS and HUVEC cells stimulated with TNF-ɑ were highly activated, and the release of adhesion molecule ICAM-1 increased. Pretreatment with CV122 and positive control Suramin significantly inhibited the release of two cell adhesion molecules ICAM-1, thereby weakening the adhesion of Raw264.7 cells stimulated with LPS and the adhesion of HUVEC stimulated with TNF-ɑ, and CV122 had a better inhibitory effect than Suramin. Fig.11 Middle B: Raw264.7 cells stimulated with LPS and HUVEC cells stimulated with TNF-ɑ were highly activated, and the release of adhesion molecule VCAM-1 increased. Pretreatment with CV122 and positive control Suramin significantly inhibited the release of two cell adhesion molecules VCAM-1, thereby weakening the adhesion of Raw264.7 cells stimulated with LPS and the adhesion of HUVEC stimulated with TNF-ɑ, and CV122 had a better inhibitory effect than Suramin.

[0214] The inventors believe that the increase in the release of adhesion molecules ICAM-1 and VCAM-1 is due to the degradation of endothelial cell glycocalyx induced by LPS and TNF-ɑ, and CV122 and Suramin significantly inhibit the release of adhesion molecules, thus proving that CV122 and Suramin have a good protective effect on endothelial glycocalyx, and the protective effect of CV122 is significantly better than that of Suramin. Similarly, CV122 also has a good protective effect on the glycocalyx of RAW cells.

[0215] Example 6 Anticoagulant activity of CV122

[0216] 1. Experimental Principle

[0217] 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.

[0218] Heparin+AT III → [AT-Hep.]

[0219] [AT-Hep.]+[FXa(excess)]→[FXa-AT-Hep.]+[residual FXa]

[0220] [residual FXa]+Substrate→Peptide+pNA

[0221] 2. Test methods

[0222] 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.

[0223] Prepare the corresponding solutions according to the following table:

[0224]

[0225] 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.

[0226] Enzyme activity assay operation sequence

[0227]

[0228] 3. After determination:

[0229] The titer of CV122 was PT = 119.96 IU / mg, and the confidence limit of the titer was FL = 4.6284%;

[0230] 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%.

[0231] 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.

[0232] 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 sepsis or septic shock, 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, Na + , K + , Li + , NH4 + Monovalent cation.

2. The use according to claim 1, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

3. The use according to claim 1, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

4. The use according to claim 3, characterized in that In formula A, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

5. The use according to claim 1, characterized in that The structural formula of the compound of formula A is as follows:

6. Use of a compound of formula A in the preparation of a drug for protecting organs of patients with sepsis, 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, Na + , K + , Li + , NH4 + Monovalent cation.

7. The use according to claim 6, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

8. The use according to claim 6, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

9. The use according to claim 8, characterized in that In formula A, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

10. The use according to claim 6, characterized in that The structural formula of the compound of formula A is as follows:

11. The use according to any one of claims 6 to 10, characterized in that: The organs are lungs, liver and / or kidneys.

12. Use of a compound of formula A in the preparation of a drug for protecting vascular endothelial cells in patients with sepsis, 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, Na + , K + , Li + , NH4 + Monovalent cation.

13. The use according to claim 12, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

14. The use according to claim 12, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

15. The use according to claim 14, characterized in that In formula A, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

16. The use according to claim 12, characterized in that The structural formula of the compound of formula A is as follows:

17. Use of a compound of formula A in the preparation of a drug for reducing TNF-α, IL-1β and / or IL-6 in sepsis patients, 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, Na + , K + , Li + , NH4 + Monovalent cation.

18. The use according to claim 17, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

19. The use according to claim 17, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

20. The use according to claim 17, characterized in that In formula A, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

21. The use according to claim 17, characterized in that The structural formula of the compound of formula A is as follows:

22. A pharmaceutical composition for treating sepsis or septic shock, protecting organs of sepsis patients, protecting vascular endothelial cells of sepsis patients, and / or reducing TNF-α, IL-1β and / or IL-6 in sepsis patients, characterized in that: Contains a compound of formula A as an active ingredient, and 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, Na + , K + , Li + , NH4 + Monovalent cation.

23. The pharmaceutical composition according to claim 22, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

24. The pharmaceutical composition according to claim 22, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

25. The pharmaceutical composition according to claim 24, characterized in that In formula A, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

26. The pharmaceutical composition according to claim 22, characterized in that The structural formula of the compound of formula A is as follows:

27. The pharmaceutical composition according to any one of claims 22 to 26, characterized in that: It further contains other drugs for treating sepsis, and the other drugs for treating sepsis are selected from: antibiotics, blood volume expanders, vasoactive drugs, glucocorticoids, blood products, blood sugar control drugs, and anticoagulants.

28. The pharmaceutical composition according to any one of claims 22 to 26, characterized in that The mass percentage of the compound of formula A in the pharmaceutical composition is 0.01%-95%.

29. The pharmaceutical composition according to claim 28, characterized in that The mass percentage of the compound of formula A in the pharmaceutical composition is 10%-90%.

30. The pharmaceutical composition according to claim 28, characterized in that The mass percentage of the compound of formula A in the pharmaceutical composition is 20%-80%.

31. The pharmaceutical composition according to claim 28, characterized in that The mass percentage of the compound of formula A in the pharmaceutical composition is 30%-70%.

32. Use of a compound of formula A in combination with other drugs for treating sepsis in the preparation of a drug, wherein the drug is used to treat sepsis or septic shock, protect organs of sepsis patients, protect vascular endothelial cells of sepsis patients, and / or reduce TNF-α, IL-1β and / or IL-6 in sepsis patients; the other drugs for treating sepsis are selected from: antibiotics, blood volume expanders, vasoactive drugs, glucocorticoids, blood products, blood sugar control drugs, 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, Na + , K + , Li + , NH4 + Monovalent cation.

33. The use according to claim 32, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

34. The use according to claim 32, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

35. The use according to claim 34, characterized in that In formula A, each Y is the same and is selected from Na + , K + , Li + , NH4 + .

36. The use according to claim 32, characterized in that The structural formula of the compound of formula A is as follows:

37. Use of a compound of formula A in the preparation of a drug for use in combination with other drugs for treating sepsis; the drug is used to treat sepsis or septic shock, protect organs of sepsis patients, protect vascular endothelial cells of sepsis patients, and / or reduce TNF-α, IL-1β and / or IL-6 in sepsis patients; the other drugs for treating sepsis are selected from: antibiotics, blood volume expanders, vasoactive drugs, glucocorticoids, blood products, blood sugar control drugs, 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, Na + , K + , Li + , NH4 + Monovalent cation.

38. The use according to claim 37, characterized in that In formula A, each Y is the same and is selected from H, Na + , K + , Li + , NH4 + Monovalent cation.

39. The use according to claim 37, characterized in that In formula A, each Y is the same or different and is independently selected from Na + , K + , Li + , NH4 + .

40. The use according to claim 39, characterized in that In formula A, each Y is selected from Na + , K + , Li + , NH4 + .

41. The use according to claim 37, characterized in that The structural formula of the compound of formula A is as follows:

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