Heparin trisaccharide compounds and pharmaceutical uses thereof

Abstract

The present application provides a heparin trisaccharide compound and a preparation method thereof, the heparin trisaccharide compound has a single optical activity, a core structure is Glc (1→4) IdoA (1→4) GlcNS, can treat or prevent sepsis, reduce mortality, protect organs of sepsis patients, reduce cytokines in the body of patients, but almost no anticoagulant activity, and resistance to HPA hydrolysis, no platelet factor 4 binding activity, so that the bleeding risk is extremely low while treating. The structure of the compound of formula A is clear, and the preparation and quality control are beneficial.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, specifically relating to heparin trisaccharide compounds, their preparation methods, and their activity in treating sepsis and pharmaceutical applications. Background Technology

[0002] Currently, sepsis is clinically defined as a life-threatening organ dysfunction caused by a disordered response of the body to infection (e.g., caused by various pathogens). In the past, the clinical definition of sepsis also included "systemic inflammatory response syndrome," "multiple organ dysfunction syndrome" caused by infection, trauma, burns, etc.

[0003] Sepsis is primarily caused by a systemic inflammatory imbalance leading to progressive endothelial dysfunction, leukocyte and platelet activation, and disruption of the anticoagulant / procoagulant system, often ultimately resulting in multiple organ dysfunction syndrome (MODS). Excessive systemic inflammatory response, vascular endothelial cell damage, and abnormally activated coagulation responses are the most prominent pathophysiological features of sepsis. The latter can lead to a hypercoagulable state, resulting in the formation of numerous microthrombi in the microcirculation, thus exacerbating the inflammatory response. Inflammation and coagulation are mutually causal and mutually reinforcing, ultimately leading to disseminated intravascular coagulation (DIC), multiple organ dysfunction syndrome (MODS), and even multiple organ dysfunction failure (MOF). Sepsis accompanied by circulatory and cellular metabolic disturbances, also known as septic shock, is a subtype of sepsis, with persistent hypotension as its main clinical manifestation and an in-hospital mortality rate exceeding 40%.

[0004] Sepsis is a leading cause of death among non-cardiac patients in intensive care units, characterized by its severe nature and high mortality rate.

[0005] Due to the high incidence and highly complex mechanisms of sepsis, there are currently no specific drugs for treatment, and drug therapy is often ineffective. Comprehensive treatment usually requires a combination of multiple medications and equipment, including: controlling the source of infection, incision and drainage of abscesses, removal of infected and necrotic tissue, and in severe cases, the use of anticoagulants, vasoactive drugs, and hormones, as well as ICU oxygen therapy, ventilation, and liver and kidney function support. Sepsis and septic shock have become significant challenges in critical care medicine, making the development of antiseptic drugs an urgent priority. Summary of the Invention

[0006] This invention provides a heparin trisaccharide compound with valuable pharmacological properties, particularly reducing the mortality rate of sepsis, inhibiting organ damage during the development of sepsis, and protecting vascular endothelial cells in sepsis.

[0007] The present invention further provides a method for preparing the heparin trisaccharide compound.

[0008] A heparin trisaccharide compound having the structure of formula A (the three monosaccharides from left to right are represented by B, C, and D):

[0009]

[0010] in:

[0011] R1 may be the same or different, and can be independently selected from -OSO3Y or -NHSO3Y.

[0012] R2, R3, R4, R5, and R6 may be the same or different, and are independently selected from H or -SO3Y.

[0013] R7 is -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3Y.

[0014] Each Y may be the same or different, and is independently selected from H or a monovalent cation, wherein the monovalent cation is selected from Na. + K + Li + NH4 + wait.

[0015] The compound of formula A is a compound with a single optical activity, namely Glc(1→4)IdoA(1→4)GlcNS, where the D sugar terminal group is α or β configuration.

[0016] Preferably, R1 is the same, which is -NHSO3Y.

[0017] Preferably, R1 is different, where R1 is -OSO3Y for B sugar and -NHSO3Y for D sugar.

[0018] Preferably, R2, R3, and R5 are the same and are H, while R4 and R6 are -SO3Y.

[0019] Preferably, R7 is a -C1-3 alkyl, -C2-4 alkylene-NH2, or -C2-4 alkylene-NHSO3Y. In some embodiments of the present invention, R7 is methyl, ethyl, -C2H4NH2, -C2H4NHSO3Y, -C3H6NH2, or -C3H6NHSO3Y.

[0020] Preferably, each Y is identical and is selected from H and Na. + K+ Li + NH4 + .

[0021] Preferably, each Y is the same or different, and is independently selected from Na. + K + Li + NH4 + In one embodiment of the invention, each Y is identical and selected from Na. + K + Li + NH4 + .

[0022] Those skilled in the art will understand that any one or two or more Ys are selected from Na. + K + Li + NH4 + Compound A with a monovalent cation is essentially a salt of compound 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 - .

[0023] In one embodiment of the present invention, Y is H, R1 = NHSO3H, R2 = R3 = R5 = H, R4 = R6 = SO3H, R7 = Me, and the D-sugar terminal methyl ester is in the α configuration. The structural formula of the compound is as follows, and it is referred to as CV010 in the present invention.

[0024]

[0025] In one embodiment of the present invention, Y is Na. + R1 = NHSO3Na, R2 = R3 = R5 = H, R4 = R6 = SO3Na, R7 = Me, and the D-sugar terminal methyl ester is in the α configuration. The structural formula of the compound is as follows, and it is referred to as CV016 in this invention.

[0026]

[0027] In one embodiment of the present invention, Y is H, R1 of B sugar is OSO3H, R1 of D sugar is NHSO3H, R2 is R3 is R5 is H, R4 is R6 is SO3H, R7 is Me, and the terminal methyl ester of D sugar is α configuration. The structural formula of the compound is as follows, and it is referred to as CV012 in the present invention.

[0028]

[0029] In one embodiment of the present invention, Y is Na.+ The B sugar has R1 = OSO3Na, the D sugar has R1 = NHSO3Na, R2 = R3 = R5 = H, R4 = R6 = SO3Na, R7 = Me, and the D sugar terminal methyl ester is in the α configuration. The structural formula of the compound is as follows, and it is referred to as CV018 in this invention.

[0030]

[0031] The present invention also provides a method for preparing compound A.

[0032] According to the present invention, the compound of formula A is derived via a fully protected trisaccharide intermediate of formula E. [E] is obtained by sequentially undergoing dehydroxyl protecting group removal, O-sulfonation, optional azide reduction reaction, and finally N-sulfonation.

[0033] In the fully protected trisaccharide intermediate E, Rx can be an azide group or an OR group. 11 R 11 R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; preferably R. 21 R 31 and R 51 Same, R 11 R 41 and R 61 Same or different, and both are different from R 21 R 31 and R 51 Further optimization of R 21 R 31 and R 51 They are the same, both are benzyl, R 11 R 41 and R 61 Whether the groups are the same or different, they are independently selected from acetyl and benzoyl groups.

[0034] Preferably, in the reaction, all hydroxyl groups to be sulfonated are deprotected in one step, and after the hydroxyl O-sulfonation, all hydroxyl groups not to be sulfonated are deprotected in one step.

[0035] In one embodiment of the present invention, the reaction method described above is used to synthesize compound A of formula R2=R3=R5=H, R4 and R6 being -SO3Y, and R1 being -NHSO3Y. The synthesis method uses fully protected trisaccharide intermediate 1 as a starting material, sequentially undergoing dehydroxylation, O-sulfonation, and azide reduction, followed by N-sulfonation. In a specific embodiment of the present invention, the synthesis method described above is used to synthesize CV010 and CV016:

[0036]

[0037] In another embodiment of the present invention, the present invention uses the aforementioned synthetic reaction method to synthesize compound A of formula R2=R3=R5=H, R4 and R6 being -SO3Y, R1 of the β-sugar being OSO3Y, and R1 of the D-sugar being NHSO3Y. The synthetic method uses fully protected trisaccharide intermediate 2 as a starting material, successively undergoing dehydroxylation, O-sulfonation, and finally N-sulfonation. In a specific embodiment of the present invention, the present invention uses the aforementioned synthetic method to synthesize CV012 and CV018:

[0038]

[0039] Where R 11 R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; Y is defined as described above. In one specific embodiment for the preparation of CV010 and CV016, R 21 R 31 and R 51 All are benzyl, R 41 For acetyl, R 61 It is benzoyl, and Y is H or Na. + In one specific embodiment of the preparation of CV012 and CV018, R 11 and R on D sugar 41 All are acetyl groups, R 21 R 31 and R 51 All are benzyl groups, with R on the β-glucose. 41 and R 61 It is benzoyl, and Y is H or Na. + .

[0040] The fully protected trisaccharide intermediate E can be derived from the monosaccharide intermediate F. [F] and disaccharide receptor 4 It is obtained through a glycosylation reaction.

[0041] Rx can be an azide group or OR 11 R 11 R 21 R 31 R 41 R 51 and R 61 They can be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; X is a leaving group suitable for reacting with other acceptors to form bonds between glycosides.

[0042] Preferably, X is a hydroxyl group, a thioalkyl group, a thioaryl group, a halogen, a trichloroimine acetyl group, a phosphate ester, or a tert-butyldiphenylsilyloxy group.

[0043] Preferred R 21 R 31 and R 51 Same, R 11 R 41 and R 61 Same or different, and both are different from R 21 R 31 and R 51 Further optimization of R 21 R 31 and R 51 They are the same, both are benzyl, R 11 R 41 and R 61 Whether the groups are the same or different, they are independently selected from acetyl and benzoyl groups.

[0044] According to the present invention, the glycosylation reaction temperature is -80°C to -10°C. The reaction can be carried out under strong acid conditions, such as trifluoromethanesulfonic acid, TBSOTf, TMSOTf, etc.

[0045] Unrestricted by any particular theory, the inventors discovered that an acyl group is formed at position 6 of the monosaccharide intermediate F, which is beneficial for increasing the proportion of the α configuration in the product during the synthesis of the fully protected trisaccharide E, and even obtaining a trisaccharide with the full α configuration; furthermore, the acyl group is acid-stable and will not be removed during the glycosylation reaction, ensuring a high yield of the fully protected trisaccharide E.

[0046] In one embodiment of the present invention, the fully protected trisaccharide intermediate 1 is obtained by glycosylation of a monosaccharide intermediate 3 and a disaccharide acceptor 4, as shown in the following reaction formula:

[0047]

[0048] In another embodiment of the present invention, the fully protected trisaccharide intermediate 2 is obtained by a glycosylation reaction between the monosaccharide intermediate 5 and the disaccharide acceptor 4, as shown in the following reaction formula:

[0049]

[0050] The disaccharide intermediate 4 can be obtained from the monosaccharide intermediate 6 and the monosaccharide intermediate 7, as shown in the following reaction formula:

[0051]

[0052] The monosaccharides 6, 7, and 4 disaccharide receptor used in the above preparation method can be prepared according to synthetic methods known in the art, such as: 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. The definitions of the functional groups in monosaccharides 6, 7, and 4 are the same as those of the corresponding functional groups described above.

[0053] Therefore, the present invention also provides the intermediates in the above synthesis method and their preparation methods.

[0054] A monosaccharide intermediate 3 has the following structure: Among them, R is preferred. 21 and R 31 All are benzyl, R 41 It is chloroacetyl, acetyl, benzoyl, or pivaloyl. In one embodiment of the invention, the monosaccharide intermediate 3 has R... 21 and R 31 All are benzyl, R 41 It is an acetyl group, which can be either α or β configuration, named 3-1, and has the following structural formula:

[0055] The reaction formula for its preparation method is as follows:

[0056]

[0057] A monosaccharide intermediate 5 has the following structure: Among them, R is preferred. 21 and R 31 All are benzyl, R 11 and R 41 The same or different, selected from chloroacetyl, acetyl, benzoyl, or pivaloyl. In one embodiment of the invention, the monosaccharide intermediate 5 has R...21 and R 31 All are benzyl, R 41 It is benzoyl, R 11 It is an acetyl group, which can be either α or β configuration, named 5-1, and has the following structural formula:

[0058] The reaction formula for its preparation method is as follows:

[0059]

[0060] A disaccharide intermediate 4 has the following structure: Among them, R is preferred. 21 and R 51 It is benzyl, R 41 and R 61 The same or different, selected from chloroacetyl, acetyl, benzoyl, or pivaloyl. In one embodiment of the invention, the R of the disaccharide intermediate 4... 21 and R 51 It is benzyl, R 41 For acetyl, R 61 It is benzoyl, named 4-1, and has the following structural formula:

[0061] Disaccharide intermediate 4-1 can be prepared using synthetic methods known in the art, such as Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. ChemMedChem, 2014, 9, 1071–1080.

[0062] A fully protected trisaccharide intermediate 1 has the following structural formula:

[0063]

[0064] Where R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl. In one specific embodiment of the invention, R 21 R 31 and R 51 All are benzyl, R 41 For acetyl, R 61 It is benzoyl.

[0065] A fully protected trisaccharide intermediate 2 has the following structural formula:

[0066]

[0067] Where R 11 R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl. In one specific embodiment of the invention, R 11 It is an acetyl group, the R on the β sugar. 41 It is benzoyl, R on the D sugar 41 For acetyl, R 21 R 31 and R 51 All are benzyl, R 61 It is benzoyl.

[0068] The present invention also provides intermediates I, II and III.

[0069] The structure of compound I is as follows:

[0070] Among them, R 21 R 31 and R 51 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl.

[0071] The structure of compound II is as follows:

[0072] Among them, R 21 R 31 and R 51 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl. Y is defined the same as in formula A, and preferably Y is H or Na. + .

[0073] The structure of compound III is as follows:

[0074] Where Y is defined the same as in Equation A, and preferably Y is H or Na. + .

[0075] Intermediates I, II, and III are generated from trisaccharide intermediate 1 through sequential dehydroxylation, sulfonation, and azide reduction, as shown in the following reaction formulas:

[0076]

[0077] The dehydroxyl protecting group, sulfonation, and azide reduction can all be carried out using reaction methods and conditions known in the art. In one embodiment of the invention, intermediate 1, under alkaline conditions, simultaneously removes R. 41 R 61 Intermediate I is obtained by reacting methyl ester with SO3·NMe3. In one embodiment of the invention, intermediate I is subjected to SO3·NMe3 to obtain O-sulfonated intermediate II. In one embodiment of the invention, intermediate II is subjected to catalytic hydrogenation to remove benzyl and Cbz groups, while reducing azide to generate an amino group, to obtain intermediate III. In one embodiment of the invention, the amino group in compound III is sulfonated under the action of SO3·Py to obtain compound CV010, which is then ion-exchanged with a sodium-type ion exchange resin to obtain compound CV016. The sodium ion exchange resin can be a resin known in the art, including but not limited to AmberliteIR120Na. + Dowex-50-WX4-Na + wait.

[0078] The present invention also provides intermediates IV, V and VI.

[0079] The structure of compound IV is as follows:

[0080] Where R 21 R 31 and R 51 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl.

[0081] The structure of compound V is as follows:

[0082] Where R 21 R 31 and R 51 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl. Y is defined the same as in formula A, and preferably Y is H or Na. + .

[0083] The structure of compound VI is as follows:

[0084] Where Y is defined the same as in equation A, and preferably Y is H. + Or Na + .

[0085] Intermediates IV, V, and VI are generated from trisaccharide intermediate 2 by sequentially removing the hydroxyl protecting group and sulfonating, as shown in the following reaction formulas:

[0086]

[0087] The dehydroxyl protecting group removal and sulfonation can both be carried out using reaction methods and conditions known in the art. In one embodiment of the present invention, intermediate 2, under alkaline conditions, simultaneously removes R. 11 R 41 R 61 Intermediate IV is obtained by reacting methyl ester with SO3·NMe3. In one embodiment of the invention, intermediate IV is subjected to SO3·NMe3 to obtain O-sulfonated intermediate V. In one embodiment of the invention, intermediate V is subjected to catalytic hydrogenation to remove the benzyl group and Cbz, yielding intermediate VI. In one embodiment of the invention, the amino group in compound VI is sulfonated under SO3·Py to obtain compound CV012, which is then ion-exchanged with a sodium-type ion exchange resin to obtain compound CV018. The sodium ion exchange resin may be a resin known in the art, including but not limited to Amberlite IR120Na. + Dowex-50-WX4-Na + wait.

[0088] The 2016 international guidelines for the management of sepsis and septic shock use the sequential organ failure assessment (SOFA) score as the standard for assessing organ dysfunction in the diagnosis of sepsis. The guidelines emphasize early identification and appropriate treatment to improve prognosis. Sepsis can be diagnosed when the SOFA score of infected or suspected infected patients increases by ≥2 points from baseline.

[0089] SOFA rating scale

[0090]

[0091]

[0092] Some studies suggest that early microcirculatory thrombosis in sepsis may have a protective effect on the body: ① It can confine pathogens within microthrombi, hindering their movement within blood vessels; ② Microthrombi may act as 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 Intravascular thrombosis can stimulate the aggregation of local antimicrobial peptides, facilitating pathogen clearance. Therefore, this intravascular thrombosis is also known as immune thrombosis, which is beneficial for sepsis patients. If inappropriate anticoagulation therapy is given in the early stages of sepsis, leading to the dissolution of intravascular thrombi, it will disrupt the local immune defense mechanism, allowing pathogens to spread through the bloodstream. Because compound A has weak anticoagulant activity and a very low risk of bleeding, it can be used to treat sepsis as soon as the patient is diagnosed, without waiting for a significant hypercoagulable state, which is beneficial for early treatment of sepsis and improves prognosis.

[0093] The present invention also provides a pharmaceutical composition comprising a compound of formula A or a solvate thereof as an active ingredient, optionally further comprising one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, etc.

[0094] In one embodiment of the present invention, the pharmaceutical composition is used to treat or prevent sepsis or septic shock, protect the organs of patients with sepsis, protect the vascular endothelial cells of patients with sepsis, and / or reduce TNF-α, IL-1β and / or IL-6 in patients with sepsis.

[0095] According to the present invention, the sepsis includes, but is not limited to, infections or inflammation caused by various pathogens, including, but not limited to, viruses, bacteria (e.g., Gram-negative bacteria such as Pseudomonas aeruginosa), fungi, rickettsiae, spirochetes, chlamydiae, etc.

[0096] According to the present invention, the organs include, but are not limited to, the heart, lungs, spleen, liver, and / or kidneys. The protection of the organs (or viscera) includes, but is not limited to, mitigating organ tissue damage and protecting or maintaining organ function.

[0097] According to the present invention, the levels of TNF-α, IL-1β and / or IL-6 in vivo include TNF-α, IL-1β and / or IL-6 in blood, tissues and / or organs.

[0098] The amount of compound A in the pharmaceutical composition (calculated as compound A) is 0.1-1000 mg, preferably 1-500 mg, and more preferably 5-100 mg.

[0099] The compound of formula A (calculated as compound A) in the pharmaceutical composition accounts for 0.01%-95% of the mass percentage of the pharmaceutical composition. Depending on the dosage form, it may be 0.1%-10%, 0.3-5%, or 10%-90%, preferably 20%-80%, and more preferably 30%-70%.

[0100] The dosage form of the pharmaceutical composition may be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; or an injectable dosage form, such as an injection solution, powder for injection, etc., administered via intravenous, intraperitoneal, subcutaneous, or intramuscular routes. All dosage forms used are well known to those skilled in the art of pharmaceutical science. For example, the pharmaceutical composition may be an injection solution, and the concentration of compound A in the injection solution may be 1-15 mg / ml, such as 5 mg / ml, 10 mg / ml, 12.5 mg / ml, etc.

[0101] The routes of administration of the pharmaceutical composition include, but are not limited to: oral; sublingual; sublingual; transdermal; pulmonary; rectal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, and intravenous; and by implantation of a reservoir or infusion device.

[0102] The dosage of compound A (calculated as compound A) will depend on the recipient's age, health, and weight, the type of concomitant medication, treatment frequency, route of administration, etc. The medication can be administered as a single daily dose, once daily, once every two days, once every three days, once every four days, or the total daily dose can be administered in two, three, or four separate doses per day. The dosage of compound A (calculated as compound 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.

[0103] The pharmaceutical composition can be administered in combination with other therapeutic agents or formulated as a combination drug. The other therapeutic agents may be other drugs for treating sepsis, depending on the type of disease and symptom.

[0104] Other medications for treating sepsis include, but are not limited to: antibiotics, blood volume expanders, vasoactive drugs, glucocorticoids, blood products, blood glucose control drugs, and anticoagulants.

[0105] This invention provides the use of compound A in the preparation of a medicament for treating or preventing sepsis or septic shock. The medicament can reduce the mortality rate of patients with sepsis.

[0106] This invention provides the use of compound A in the preparation of a medicament for protecting organs of patients with sepsis.

[0107] This invention provides the use of compound A in the preparation of a medicament for protecting vascular endothelial cells in patients with sepsis.

[0108] This invention provides the use of compound A in the preparation of a medicament for reducing TNF-α, IL-1β and / or IL-6 in patients with sepsis.

[0109] The present invention provides the use of compound A in combination with other drugs for treating sepsis in the preparation of a drug for treating or preventing sepsis or septic shock.

[0110] This invention provides the use of compound A in combination with other drugs for treating sepsis in the preparation of a drug for protecting the organs of patients with sepsis.

[0111] This invention provides the use of compound A in combination with other drugs for treating sepsis in the preparation of a drug for protecting vascular endothelial cells in patients with sepsis.

[0112] This invention provides the use of compound A in combination with other drugs for treating sepsis in the preparation of a drug that reduces TNF-α, IL-1β and / or IL-6 in patients with sepsis.

[0113] This invention provides the use of compound A in the preparation of other drugs for the treatment or prevention of sepsis or septic shock.

[0114] This invention provides the use of compound A in the preparation of a medicament for the protection of organs in patients with sepsis in combination with other medicaments for the treatment of sepsis.

[0115] This invention provides the use of compound A in the preparation of other drugs for treating sepsis and in combination with drugs for protecting vascular endothelial cells in patients with sepsis.

[0116] This invention provides the use of compound A in the preparation of drugs that reduce TNF-α, IL-1β and / or IL-6 in sepsis patients in combination with other drugs for treating sepsis.

[0117] The present invention provides a method for treating or preventing sepsis or septic shock, protecting the organs of sepsis patients, protecting the vascular endothelial cells of sepsis patients, and / or reducing TNF-α, 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, or a pharmaceutical composition containing a compound of formula A or a solvate thereof, is administered to a patient in need.

[0118] Compound A of this invention exhibits a clear and highly effective therapeutic and preventative effect against sepsis, but possesses almost no anticoagulant activity and is resistant to HPA hydrolysis, meaning it is highly stable and lacks platelet factor 4 binding activity. Therefore, it carries an extremely low risk of bleeding while being used for therapeutic purposes. The well-defined structure of compound A facilitates its preparation and quality control. Attached Figure Description

[0119] Figure 1 The effects of CV016 and Suramin on survival in a mouse LPS sepsis model

[0120] Figure 2 HE staining results of organ damage in septic mice using CV016 and Suramin

[0121] Figure 3 Effects of CV016 and Suramin on serum cytokine levels in septic mice

[0122] Figure 4 The role of CV016 in the survival rate of a mouse model of Pseudomonas aeruginosa (PA) infection.

[0123] Figure 5 HE staining results of CV016 on organ damage in mice infected with Pseudomonas aeruginosa

[0124] Figure 6 Results of bacterial count in the lungs of mice infected with Pseudomonas aeruginosa using CV016 Detailed Implementation

[0125] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0126] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0127] 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-butyldimethylsilyltrifluoromethanesulfonate; TEMPO: 2,2,6,6-Tetramethylpiperidine oxide; TMSOTf: Trimethylsilyltrifluoromethanesulfonate; Tol: Toluene.

[0128] Example 1: Preparation of trisaccharide intermediate III-1

[0129] 1. Preparation method of monosaccharide intermediate 3-1

[0130] Intermediate 9 was obtained by reacting glucosamine hydrochloride 8 with benzyloxyformyl chloride; 1,6-cyclic intermediate 10 was obtained through a two-step reaction; catalytic hydrogenation and azo transfer reaction were used to obtain a common intermediate 11 with an azido group protected at the 2-position; the hydroxyl groups at the 3- and 4-positions were fully benzylated under the action of benzyl bromide and sodium hydride to obtain intermediate 12; 1,6-cyclic intermediate 13 was obtained under the action of acetic anhydride and TBSOTf; the terminal acetyl group was removed under the action of benzylamine to obtain intermediate 14; finally, trichloroacetylimine ester donor 3-1 was obtained under the action of potassium carbonate and trichloroacetonitrile.

[0131]

[0132] The reaction conditions and yields for each step are as follows: a) Cb2Cl, NaOH, H2O, 59%; b) 1) TsCl, Py, MS, 2) EtOH, DBU, NaI, two-step yield 49%; c) 1) 4atm H2, Pd / C, MeOH, 2) TfN3, CuSO4, Et3N, MeOH, two-step yield 82%; d) BnBr, NaH, DMF, 0℃, 85%; e) Ac2O, TBSOTf, 94%; f) BnNH2, DCM, 89%; g) Cl3CCN, K2CO3, DCM, rt, 98%.

[0133] 2. Preparation method of fully protected trisaccharide intermediate 1-1

[0134] The synthesized monosaccharide intermediate 3-1, in its mixed configuration, can be used directly in the next reaction without separation. Trichloroacetylimine ester is a relatively common glycosyl donor, typically reacting under mild conditions with high yields. Monosaccharide intermediate 3-1 is glycosylated with disaccharide intermediate 4-1 synthesized according to known literature methods, yielding a fully protected trisaccharide 1-1 in the presence of trifluoromethanesulfonic acid.

[0135]

[0136] Monosaccharide intermediate 3-1 (2.83 g, 502.24 mmol) and glycosyl acceptor disaccharide intermediate 4-1 (2.74 g, 324.23 mmol) were dissolved in redistilled DCM and then added to a solution containing pre-activated DCM. In a reaction flask containing molecular sieves, the mixture was continuously stirred at room temperature for 30 min to equilibrate the reaction. After the reaction system temperature was lowered to -20℃, trifluoromethanesulfonic acid (26.53 μL, 0.33 mmol) was slowly added dropwise. The reaction was monitored by TLC. After the reaction was complete, the molecular sieves were removed by silica gel filtration. The resulting filtrate was concentrated and purified by direct silica gel column chromatography (PE / EA = 5:1) to obtain the fully protected trisaccharide intermediate 1-1 (3.42 g, 87%).

[0137] 1H NMR(400MHz,CDCl3)δ8.08(d,J=7.6Hz,2H),7.41–7.25(m,22H),7.21–7.18(m,6H),5.46(d,J=3.1Hz,1H),5.14(t,J=3.6Hz,1H),5.02(d,J=2.2Hz,2H),4.88(s,1H),4.85(d, J=3.0Hz,2H),4.82(d,J=5.3Hz,2H),4.77(d,J=4.2Hz,1H),4.74(d,J=3.8Hz,1H),4.64(d,J=3.8Hz,1H),4.58(d,J=10.4Hz,1H),4.54(d,J=10.9Hz,1H),4.32(d,J=2.7Hz,1H),4.30 (d,J=2.2Hz,3H),4.24(d,J=3.6Hz,1H),4.21(d,J=3.9Hz,1H),4.14(t,J=4.3Hz,1H),4.02(t,J=4.0Hz,1H),3.99–3.90(m,2H),3.73(dt,J=9.9Hz,3.2Hz,1H),3.63(d,J=3.8Hz,1H), 3.61(d,J=2.8 1H),3.58(s,3H),3.47(t,J=9.4Hz,1H),3.31(s,3H),3.21(dd,J=10.2,3.5Hz,1H).

[0138] 13 C NMR(100MHz,CDCl3)δ170.75,170.52,169.35,165.42,155.81,138.23,137.69, 137.58,137.42,136.24,133.37,130.01,129.38,128.74,128.52,128.49,128.44,128.39,128.24,128.17,128.13,128.06,127.96,127.88,127.76,127.35,99.32,98.87,98.50,80.05,79.12,77.29, 75.62,75.28,74.98,74.90,74.56,74.44,73.32,70.07,69.33,69.12,68.92,67.00,63.62,62.45,62.33,55.29,54.56,52.10,20.87.

[0139] HRMS[M+Na] +m / z 1275.46443 (calcd for C 67 H 72 N4NaO 20 ,1275.4638).

[0140] 3. Preparation method of trisaccharide intermediate III-1

[0141] The fully protected trisaccharide 1-1 was subjected to the combined action of LiOH, H2O2 and NaOH to simultaneously remove Ac, Bz and methyl ester to obtain trihydroxy compound I-1; heating under the action of SO3·NMe3 gave the intermediate compound II-1 after O-sulfonation; benzyl and Cbz were removed by catalytic hydrogenation, and the azide was reduced to generate an amino group to obtain diamino compound III-1.

[0142]

[0143] The fully protected trisaccharide compound 1-1 (342.62 mg, 0.26 mmol) was dissolved in 5.00 mL of tetrahydrofuran. Then, 6.23 mL of 1.25 N LiOH solution and 13.13 mL of 30% H₂O₂ solution were added dropwise at room temperature. After stirring for 12 hours, 14.34 mL of methanol and 7.82 mL of 6 N NaOH solution were added, and stirring continued for at least 12 hours. After the reaction was confirmed to be complete by TLC, the pH was adjusted to 2 with 4 N hydrochloric acid under ice-water bath conditions. The reaction solution was then extracted three times with DCM and concentrated under reduced pressure, followed by silica gel column chromatography (DCM:MeOH = 15:1) to obtain compound I-1 (258.0 mg, 93%). High-performance liquid chromatography analysis of intermediate I-1 revealed only a single peak, indicating its high purity. Under argon protection, intermediate I-1 (258.02 mg, 0.24 mmol) and SO3·NMe3 (897.62 mg, 5.33 mmol) were dissolved in 3 mL of anhydrous DMF. The reaction system was heated to 65 °C and stirred continuously for at least 12 h. The reaction solution was taken and the degree of reaction was monitored by high performance liquid chromatography. The reaction was compared with the peak time of compound I-1. When the newly generated peak had a shorter retention time and was a single peak, the reaction was considered complete. After heating was stopped, the reaction system was allowed to rise naturally to room temperature. The reaction solution was concentrated and purified by Sephadex LH-20 gel column chromatography to obtain intermediate II-1 (304.13 mg, 93%). Intermediate II-1 (304.13 mg, 0.22 mmol) was dissolved in 3.00 mL of a mixed solvent of methanol, tert-butanol and water (v / v / v = 2:1:1), and palladium on carbon (50.00 mg) was added. The mixture was stirred for 24 h under a hydrogen pressure of 4 atm. After removing the palladium on carbon by filtration with filter paper, the reaction solution was concentrated to obtain intermediate III-1 (184.93 mg, 98%).

[0144] 1 H NMR(400MHz,D2O)δ5.33(d,J=3.7Hz,1H),5.15(s,1H),4.88(d,J=3.7Hz,1H),4.79(d,J =1.8Hz,1H),4.32–4.25(m,3H),4.23(d,J=3.1Hz,2H),4.13(d,J=2.1Hz,1H),4.11–4.06 (m,2H),3.95(d,J=5.6Hz,1H),3.85(t,J=9.8Hz,2H),3.77(t,J=9.9Hz,1H),3.69(d,J= 9.5Hz,1H),3.65(s,1H),3.47(t,J=9.7Hz,1H),3.35(s,3H),3.26(dd,J=10.7Hz,3.6Hz, 1H), 3.22 (dd, J=10.5Hz, 3.7Hz, 1H).

[0145] 13 C NMR(100MHz,D2O)δ175.15,98.88,96.32,91.44,76.78,73.00,70.50,70.26,69.39, 69.19,68.94,68.67,67.32,66.61,66.16,62.96,55.32,54.22,54.03.

[0146] HRMS[M–H] - m / z 769.0596 (calcd for C 19 H 33 N2O 24 S3,769.0586).

[0147] Example 2: Preparation of compound CV010

[0148] Intermediate III-1 (35 mg, 0.045 mmol) was dissolved in 0.50 mL of water, and the pH was adjusted to 9-10 with 4N NaOH solution and maintained. Then, sulfur trioxide pyridine complex (216 mg, 1.366 mmol) was added in portions. The reaction was confirmed to be complete by TLC. The pH of the neutralized reaction solution was approximately 7-8. The reaction solution was then concentrated and purified by Sephadex G-25 gel column chromatography to obtain compound CV010 (39 mg, 94%).

[0149]

[0150] 1H NMR (400MHz, D2O) δ5.37(d,J=3.6Hz,1H),5.18(d,J=3.0Hz,1H),4.97(d,J=3.6 Hz,1H),4.78(d,J=2.8Hz,1H),4.33-4.26(m,4H),4.21–4.14(m,2H),4.06(t,J=3.2Hz,1H), 3.92(td,J=8.0Hz,7.5Hz,3.5Hz,2H),3.70(t,J=9.5Hz,1H),3.64(d,J=10.1Hz,1H),3.59(d, J=10.1Hz,1H),3.52(t,J=9.5Hz,1H),3.37(s,3H),3.35(s,1H),3.22(ddd,J=13.1Hz,10.1Hz,3.6Hz,2H).

[0151] 13 C NMR(100MHz,D2O)δ175.37,99.29,98.32,96.95,76.88,75.94,75.90,70.98,70.05, 69.87,69.24,69.15,68.94,68.56,66.87,66.49,57.92,57.76,55.43.

[0152] HRMS[M–H] - m / z 928.9717 (calcd for C 19 H 33 N2O 30 S5,928.9722).

[0153] Example 3 Preparation of compound CV016

[0154] Compound CV010 (39.4 mg, 0.042 mmol) was treated with Dowex-50-WX4-Na + The column exchanged the sodium salt, and the sugar-containing component was collected to concentrate the solvent, yielding compound CV016 (44 mg, 98%).

[0155]

[0156] 1H NMR (400MHz, D2O) δ5.35(d,J=3.6Hz,1H),5.16(d,J=3.0Hz,1H),4.95(d,J=3.6 Hz,1H),4.75(d,J=2.8Hz,1H),4.27(dq,J=13.0Hz,3.8Hz,2.9Hz,4H),4.19–4.12(m,2H),4 .04(t,J=3.2Hz,1H),3.90(td,J=8.0Hz,7.5Hz,3.5Hz,2H),3.68(t,J=9.5Hz,1H),3.61(d,J =10.1Hz,1H),3.56(d,J=10.1Hz,1H),3.50(t,J=9.5Hz,1H),3.35(s,3H),3.33(s,1H),3.20(ddd,J=13.1Hz,10.1Hz,3.6Hz,2H).

[0157] 13 C NMR(100MHz,D2O)δ174.36,99.23,98.22,96.93,76.84,75.93,75.89,70.96,70.01, 69.84,69.22,69.13,68.91,68.51,66.84,66.46,57.89,57.73,55.40.

[0158] HRMS[M-6Na+5H] - m / z 928.9754 (calcd for C 19 H 33 N2O 30 S5,928.9722).

[0159] Example 4: Preparation of trisaccharide intermediate VI-1

[0160] 1. Preparation method of monosaccharide intermediate 5-1

[0161] Starting with pentoxyglucose, it reacts with acetic anhydride to generate a fully acetylated glucose intermediate 16; intermediate 16 reacts with p-toluene in boron trifluoride ether solution to generate a glucosinolate compound 17; under alkaline conditions of MeOH / MeONa, the acetyl group is removed, followed by reaction with the benzylide reagent benzaldehyde dimethyl acetal to give intermediate 18; intermediate 18 is refluxed with di-tert-butyltin oxide in anhydrous methanol, and then the 3-hydroxyl group is selectively protected with 4-methoxybenzyl chloride to give intermediate 19; intermediate 1... 9 was dissolved in pyridine, and acetic anhydride was added dropwise to give compound 20, which is acetylated at the 2-position of glucose; then, the PMB protecting group was removed by DDQ catalysis to give compound 21; under ice bath and strongly alkaline conditions, benzyl bromide was added dropwise to give compound 22; compound 22 was heated and co-refluxed with glacial acetic acid to give compound 23, which has naked hydroxyl groups at the 4 and 6 positions of glucose; under light-protected conditions, benzoyl chloride was added dropwise to selectively protect the 6-position hydroxyl group of glucose with a Bz group to give compound 24; under neutral silver oxide conditions, benzyl bromide was added dropwise to give the final monosaccharide fragment 5-1.

[0162]

[0163] a) Ac₂O, pydine, 89%; b) TolSH, BF₃·Et₂O, DCM, 86%; c) 1) MeONa, MeOH, DCM; 2) Benzaldehyde Dimethylacetal, CSA, DMF, two-step yield 83%; d) 1) Bu₂SnO, MeOH, reflux; 2) PMBCl, C S F, DMF, 90℃, two-step yield 75%; e) Et3N, Ac2O, DMAP, 0℃, 46%; f) DDQ, DCM, H2O, 78%; g) Ag2O, BnBr, DCM, 60%; h) Ac2O, 92℃, 98%; i) BzCl, Et3N, THF, 0℃, 69%; j) Ag2O, BnBr, 60%.

[0164] 2. Preparation method of fully protected trisaccharide intermediate 2-1

[0165] The synthesized monosaccharide intermediate 5-1, in its mixed configuration, can be used directly in the next reaction without separation. Monosaccharide intermediate 5-1 is glycosylated and coupled with disaccharide intermediate 4-1 synthesized according to known literature methods, yielding a fully protected trisaccharide 2-1 under the action of trifluoromethanesulfonic anhydride.

[0166]

[0167] Take a 50mL two-necked bottle, add the magnetic ferrule connecting the double-row tube device, turn on the oil pump, heat the bottle for 1 minute, turn off the oil pump, remove the magnetic ferrule, and weigh... Add 2.5 g of molecular sieve (powder) to the two-necked flask, reconnect the double-row tubing, turn on the oil pump, and heat the flask for about 10 minutes, until the molecular sieve softens and slides freely down the flask wall to the bottom without sticking to the wall. Turn off the oil pump, place the magnetic stir bar in the two-necked flask, turn the oil pump back on, heat the flask for 2 minutes, then place it on a magnetic stirrer, turn on argon gas, and set the flow rate to 5 L / min. Perform a gas exchange operation to ensure the two-necked flask is under argon protection. Turn off the oil pump, adjust the argon flow rate to 1 L / min and maintain it until the reaction is complete. After the two-necked flask cools to room temperature, weigh out 500 mg (0.82 mmol) of thioglycolic acid donor 5-1 and 134 mg (0.66 mmol) respectively, dissolve them in 5 mL of redistilled dichloromethane, and equilibrate with the molecular sieve at room temperature for 30 minutes. The reaction was placed at -78°C, and activated by dropwise addition of trifluoromethanesulfonic anhydride (98 μL, 0.58 mmol). TLC was monitored immediately. After the complete disappearance of the thioglycolic acid donor 5-1, glucosamine acceptor 4-1 (692 mg, 0.82 mmol) was dissolved in 1.5 mL of redistilled dichloromethane and slowly added dropwise to the reaction system. The reaction was allowed to proceed until 0°C. After the acceptor reaction was complete, TLC was monitored, and the reaction was quenched with triethylamine. The mixture was filtered through a molecular sieve, and the filtrate was concentrated. Column chromatography (PE / EA = 5:1 → 3:1 → 2:1) yielded the fully protected trisaccharide compound 2-1 (740 mg, 68%).

[0168] 1 H NMR (400MHz, CDCl3) δ8.08-7.98(m,4H),7.54-7.34(m,7H),7.30-7.21(m,17H),7.16-7.09(m,5H),7.05(dd,J=7.0,2.2Hz,2H),5.50( dd,J=12.3,6.1Hz,2H),5.21(dd,J=9.1,5.2Hz,1H),5.03(dd,J=8.5,4.6Hz,2H),4.84-4.73(m,3H),4.64(s,3H),4.57(dd,J=10.8,3.0 Hz,4H),4.44(dd,J=12.3,3.5Hz,1H),4.38(d,J=12.4Hz,1H),4.31(dd,J=12.3,3.2Hz,1H), 4.23(d,J=12.4Hz,2H),4.17-4.07(m,3H),3.95(dd,J=16.6,6.7Hz,2H),3.71-3.62(m,4H), 3.57(dd,J=19.8,10.2Hz,2H),3.39(dd,J=10.2,3.5Hz,1H),3.27(s,3H),2.10(s,3H),1.88(s,3H).

[0169] 13 C NMR (100MHz, CDCl3) δ170.89,169.92,169.61,166.13,165.61,155.83,138.43, 137.84,137.66,137.54,136.34,133.18,130.12,129.89,129.66,129.21,128.53,128.51,128.49, 128.47,128.44,128.39,128.37,128.34,128.24,128.20,128.17,127.95,127.91,127.79,127.54, 127.46,127.34,98.86,98.63,98.37,78.43,77.42,77.30,77.15,77.10,76.78,76.49,76.35,76.20,75.45,74.45,74.34,73.46,73.16,72.62,71.06,70.94,69.73,68.93,66.91,63.05,62.16,55.27, 54.38,52.28,21.08,20.89.

[0170] HRMS[M+Na] + m / z found 1354.4832(cacld for C 74 H 77 NNaO 22 ,1354.4829).

[0171] 3. Preparation method of trisaccharide intermediate VI-1

[0172] The fully protected trisaccharide 2-1 was subjected to the combined action of LiOH, H2O2 and NaOH to simultaneously remove Ac, Bz and methyl ester to obtain tetrahydroxy compound IV-1; heating under the action of SO3·NMe3 gave the O-sulfonated intermediate compound V-1; and the benzyl group and Cbz were removed by catalytic hydrogenation to obtain amino compound VI-1.

[0173]

[0174] The fully protected trisaccharide 2-1 (100 mg, 0.075 mmol) was dissolved in 6.9 mL of THF and placed on a magnetic stirrer. 1.8 mL of 1.25 N LiOH solution and 3.8 mL of 30% H₂O₂ solution were added dropwise to the system, and the reaction was allowed to proceed at room temperature for at least 12 h. Then, 4.2 mL of MeOH and 2.3 mL of 4 N NaOH solution were added dropwise, and the reaction was continued for another 12 h. The reaction was monitored by TLC; the CMC spot disappeared, and a new spot formed below it with a noticeable tailing. The pH was adjusted to 2–3 by adding 6 N hydrochloric acid at 0 °C, followed by extraction with DCM, drying with anhydrous sodium sulfate, filtering to remove sodium sulfate hydrate, and concentrating under reduced pressure. Rapid column chromatography (DCM / MeOH = 20:1 → 15:1) was performed to obtain the tetrahydroxy naked intermediate IV-1 (66 mg, 88%, colorless solid). Compound IV-1 (100 mg, 0.098 mmol) and SO3·NMe3 (542 mg, 4.0 mmol) were weighed and placed in a reaction flask. The air in the flask was displaced and protected with argon. 5 mL of anhydrous DMF was injected into the flask, and the reaction was carried out at 55 °C. The reaction status was continuously monitored by HPLC. The starting material peak gradually decreased, and the product peak gradually increased until the starting material peak disappeared completely and the product peak was a single peak. The reaction was then stopped, and the DMF was evaporated to dryness. The crude product was dissolved in methanol and loaded onto a Sephadex LH-20 gel column using a 1:1 DCM / MeOH eluent. The column was then subjected to anisaldehyde staining, and the colored fraction was collected and evaporated to dryness to obtain intermediate V-1 (119 mg, 84%, pale yellow solid). Intermediate V-1 was detected by HPLC. The selected HPLC method showed only a single peak at a wavelength of 210 nm, and the peak elution time was consistent with that of the product peak during the reaction. Intermediate V-1 (200 mg, 0.14 mmol) was dissolved in 2 mL of methanol, 1 mL of tert-butanol and 1 mL of water. Pd / C (30 mg) was added to the reaction solution and the mixture was placed in a hydrogen generator and stirred at a hydrogen pressure of 4 atm for 48 h. After the reaction was complete, the mixture was filtered through diatomaceous earth and the filtrate was concentrated to obtain intermediate VI-1 (131 mg, 99%).

[0175] 1 1H NMR (400MHz, D2O) showed a signal peak in the aromatic region without hydrogen. 1H NMR(400MHz,D2O)δ5.13(d,J=3.8Hz,1H),5.03(s,1H),4.85(d,J=3.5Hz,1H),4.27(d,J=9.5Hz,1H),4.24-4.15(m,5H), 4.13-3.94(m,4H),3.85(dd,J=16.3,6.2Hz,3H),3.65(d,J=9.4Hz,1H),3.53(dd,J=6.0,3.6Hz,1H),3.48(d,J=9.5Hz,1H),3.30(d,J=2.4Hz,3H).

[0176] 13 C NMR(100MHz,D2O)δ174.93,99.44,95.89,95.13,81.73,76.29,74.81,72.44,69.95, 69.71,68.92,68.64,68.53,67.50,66.47,66.29,66.01,55.30,55.03,54.05.

[0177] Example 5: Preparation of compound CV012

[0178] Intermediate VI-1 (60 mg, 0.071 mmol) was dissolved in 1.5 mL of water and placed on a magnetic stirrer. The pH was adjusted to 9–10 by adding 2N NaOH solution dropwise. SO3·Py (226 mg, 1.42 mmol) was added to the reaction solution in six portions, one batch every half hour, with each batch containing 38 mg. After each addition of SO3·Py, 2N NaOH solution was added dropwise to adjust the pH to 9–10 and maintained within this pH range until all SO3·Py was added. The reaction was then allowed to proceed at room temperature for 6 hours. After the reaction was complete, the pH was adjusted to neutral with hydrochloric acid. The reaction solution was concentrated, and the crude product was dissolved in water and loaded onto a Sephadex G-25 gel column using water as the eluent. The concentrated compound CV012 (62 mg, 94%) was obtained.

[0179] 1 H NMR(400MHz,D2O)δ5.21(d,J=3.8Hz,1H),5.14(s,1H),4.97(d,J=3.6Hz,1H),4.38(d,J=9.5Hz,1H), 4.35-4.19(m,6H),4.05-3.89(m,3H),3.72-3.58(m,5H),3.37(s,3H),3.21(dd,J=10.1,3.6Hz,1H).

[0180] 13C NMR(100MHz,D2O)δ175.89,99.43,98.57,95.35,81.87,77.43,74.56,72.39,69.99, 69.92,69.79,68.53,68.31,67.53,66.92,66.33,65.29,57.84,55.48.

[0181]

[0182] Example 6 Preparation of compound CV018

[0183] Compound CV012 (66 mg, 0.071 mmol) was eluent with water (Dowex-50-WX4-Na). + The column was exchanged for sodium salt, anisaldehyde was used for color development, and the colored fraction was collected to obtain the target compound CV018 (74 mg, 99%).

[0184] 1 H NMR(400MHz,D2O)δ5.18(d,J=3.8Hz,1H),5.10(s,1H),4.93(d,J=3.6Hz,1H),4.35(d,J=9.5Hz,1H), 4.30-4.16(m,6H),3.98-3.84(m,3H),3.69-3.53(m,5H),3.33(s,3H),3.19(dd,J=10.1,3.6Hz,1H).

[0185] 13 C NMR(100MHz,D2O)δ174.91,99.26,98.18,95.04,81.75,77.23,74.28,72.08,69.95, 69.85,69.70,68.50,68.17,67.46,66.83,66.25,65.24,57.78,55.37.

[0186]

[0187] The following experiments were conducted using CV016 and CV018 prepared in Examples 3 and 6.

[0188] Example 7: Therapeutic effects of CV016 and Suramin in a mouse sepsis model

[0189] Survival rate experiment of CV016 and Suramin against mouse sepsis

[0190] Fifteen male C57BL / 6J mice, 6-8 weeks old and weighing 20-25g, were randomly divided into three groups of five each. The survival rate of mice with septicemia was tested using CV016 and Suramine according to the table below. Each group of mice was intraperitoneally injected with 40mg / kg LPS. 30 minutes later, mice in group ② were subcutaneously injected with CV016 (40mg / kg), and mice in group ③ were subcutaneously injected with Suramine (40mg / kg). The mice were observed and their survival rate was recorded daily.

[0191]

[0192] Experimental results are as follows Figure 1 As shown, all mice in the LPS sepsis model group died within 72 hours, with a survival rate of 0%. During the observation period of 80 hours or even longer, the survival rate of the LPS+CV016 (40 mg / kg) group was 80%, while the survival rate of the LPS+Suramin (40 mg / kg) group was only 60% when using the same concentration of Suramin (40 mg / kg). The experimental results indicate that CV016 can effectively improve the survival rate of sepsis-affected mice, and the positive control Suramin can also improve the survival rate of sepsis-affected mice. The anti-septic effect of CV016 is better than that of Suramin.

[0193] CV016 can improve organ damage in a mouse model of sepsis.

[0194] Twenty male C57BL / 6J mice, 6-8 weeks old and weighing 20-25g, were randomly divided into four groups of five mice each. Organ damage experiments involving CV016 and Suramin in septic mice were conducted according to the table below. Except for the control group, all mice in each group received an intraperitoneal injection of 40mg / kg LPS. Thirty minutes later, mice in group ③ received a subcutaneous injection of CV016 (40mg / kg), and mice in group ④ received a subcutaneous injection of Suramin (40mg / kg). Twenty-four hours later, the mice were dissected, and organs such as the lungs, kidneys, spleen, and liver were harvested. HE staining was used to further investigate organ damage following CV016 administration.

[0195] Paraffin embedding: Isolated organs such as lungs, kidneys, spleens, and livers were fixed in 4% paraformaldehyde for 24 hours. Each group of isolated organs was placed in an embedding cassette and dehydrated using a dehydrator. The dehydration program was as follows: immersion in 70% ethanol, 80% ethanol, 85% ethanol, 90% ethanol, and 95% ethanol for 1 hour each, followed by immersion in anhydrous ethanol, anhydrous ethanol, and anhydrous ethanol for 45 minutes each. The embedding cassettes were then removed and placed in a fume hood. The organs were then immersed in ethanol / xylene (v / v = 1:1) for 30 minutes, xylene for 30 minutes, xylene / paraffin (v / v = 1:1) at 90°C for 1 hour, paraffin at 200-300°C for 1 hour, and paraffin at 200-300°C for 14 hours. After the paraffin immersion was complete, the organs were removed and embedded using a tissue embedding machine.

[0196] HE staining: First, cut the paraffin-embedded tissue block into 3μm sections using a microtome. Preheat a vacuum drying oven to 65℃ and bake the sections for 3-4 hours. Dewax the slides by immersing them in xylene for 15 minutes, repeating this process three times. After dewaxing, immerse the sections sequentially in anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes each, and finally in distilled water for 5 minutes. Next, stain the slides in hematoxylin for 6 minutes, then in distilled water for 30 seconds, 75% ethanol for 30 seconds, and 95% ethanol for 3 minutes. Finally, stain the slides in eosin for 3 minutes, then in 95% ethanol for 1 minute, 95% ethanol for 1 minute, anhydrous ethanol for 1 minute, and xylene for 1 minute. Mount the slides with neutral resin and allow them to stand at room temperature for at least 24 hours before microscopic observation and processing.

[0197]

[0198] Since patients with severe sepsis may experience multiple organ failure, the mice were dissected 24 hours after a sepsis mouse model was established using LPS. Organs such as the lungs, kidneys, spleen, and liver were harvested, and HE staining was used to further investigate the organ damage after CV016 administration.

[0199] The results are as follows Figure 2As shown, the Control group exhibited normal lung tissue structure under an optical microscope, while the LPS-induced septic mice showed severe pulmonary congestion, inflammatory cell infiltration, alveolar wall thickening, and interstitial edema. The LPS+CV016 group showed reduced pulmonary congestion, and the alveolar wall thickening and interstitial edema were significantly better than in the LPS group, indicating that CV016 can effectively improve the lung damage in septic mice, and its effect is superior to that of the Suramine group. The Control group showed normal kidney tissue structure, while the mice in the LPS group showed significant kidney damage, such as renal tubular structure destruction or dilation, vacuolar degeneration, and brush border loss. These pathological changes were significantly improved in the LPS+CV016 group, and the effect was superior to that of the Suramine group. The Control group showed normal liver tissue structure; while the LPS-induced septic model mice showed significant liver damage, such as congestion, swelling, and increased leukocytes in the hepatic sinusoids. These pathological changes were significantly improved in the LPS+CV122 group, and the effect was superior to that of the Suramine group. The control group showed normal spleen tissue structure; while the LPS sepsis model mice showed obvious spleen damage, with unclear boundaries between the blue and red pulp and fusion. The blue pulp was mostly composed of immune cells, and a significant reduction and death of immune cells could be seen. These pathological changes were significantly improved in the LPS+CV122 group, and the effect was better than that in the Suramin group.

[0200] CV016 reduces cytokine levels in LPS sepsis model mice.

[0201] Twenty male C57BL / 6J mice were randomly divided into four groups of five each, as shown in the table below: Group ① mice were injected with 0.9% NaCl (100 μL / mouse), and mice ②-④ were injected intraperitoneally with LPS (20 mg / kg, 100 μL / mouse). 30 min later, group ③ mice were injected with 40 mg / kg CV016, and group ④ mice were injected with 40 mg / kg Suramin. Three h later, blood was collected from the mice by enucleation into 1.5 mL centrifuge tubes. The collected whole blood was incubated at 37°C for 1 h, then at 4°C overnight, and finally centrifuged (3000g, 20 min, 4°C) to separate the serum.

[0202] Serum cytokine levels were detected using a mouse TNF-α, IL-6, and IL-1β ELISA kit. 100 μL of TNF-α, IL-6, and IL-1β capture antibody solution was added to each well of a 96-well plate and incubated overnight at 4°C. The plate was washed three times with Wash Buffer, blocked with 200 μL / well Assay Diluent A, and incubated at 37°C for 1 h, followed by three washes. 100 μL / well of TNF-α, IL-6, and IL-1β standards and samples were added, and the plate was incubated at 37°C for 2 h, followed by three washes. 100 μL of diluted TNF-α, IL-6, and IL-1β antibody solution was added, and the plate was incubated at 37°C for 1 h, followed by three washes. 100 μL of Avidin-HRP solution was added, and the plate was incubated at room temperature for 30 min, followed by three washes. 100 μL of TMB was added, and the plate was incubated at room temperature in the dark for 30 min. The reaction was terminated by adding 100 μL of stop solution. Positive wells should change from blue to yellow. The absorbance at 450 nm was read using an ELISA reader within 15 minutes, and the cytokine levels were calculated based on the standard curve.

[0203]

[0204] The results are as follows Figure 3 As shown: In the control group (normal mice), the levels of TNF-α, IL-6, and IL-1β in serum were very low. However, after LPS stimulation, the levels of TNF-α, IL-6, and IL-1β in mice increased sharply in a short period of time, consistent with the characteristics of a cytokine storm. The LPS+CV016 group significantly inhibited the level of TNF-α compared with the LPS group (***P<0.001). The LPS+Suramin group also significantly inhibited the level of TNF-α (**P<0.01), but the inhibitory effect was not as strong as that of CV016. The same phenomenon was observed in serum IL-6 and IL-1β. CV016 could also significantly inhibit the increase of IL-6 and IL-1β induced by LPS, and the effect was stronger than that of Suramn.

[0205] Example 8: CV016 treatment of Pseudomonas aeruginosa (PA) infection in mice.

[0206] Male C57BL / 6J mice, 6-8 weeks old, weighing 20-25g, were raised at the Nankai University Experimental Animal Center under specific pathogen-free, humidity- and temperature-controlled conditions.

[0207] The experiment was divided into two groups: ① *Pseudomonas aeruginosa* model group, and ② *Pseudomonas aeruginosa* treatment group. Five mice were in each group. Mice were established as a *Pseudomonas aeruginosa* infection model via intraperitoneal injection, with an infectious dose of 1 × 10⁻⁶. 6CFU / mouse. Model group: 0.9% NaCl was injected into each mouse 30 min and 20 h after infection, with a volume of 100 μL. Treatment group: CV016 (50 mg / kg) was administered to each mouse 30 min and 20 h after infection via subcutaneous injection in the back, dissolved in 0.9% NaCl, with a volume of 100 μL per mouse.

[0208] 1. Survival rate experiment

[0209] Mice were observed and their survival rate was recorded hourly. The experimental results are as follows: Figure 4 As shown, the survival rate of the Pseudomonas aeruginosa model group within 72 hours was 40%, while the survival rate of the CV016 treatment group within 72 hours was 80%, an increase of 40% in survival rate.

[0210] 2. HE staining and lung colony count

[0211] Mice were euthanized by cervical dislocation 72 hours after the first injection and dissected. Heart, liver, spleen, lung and kidney tissues were collected and further investigated for organ damage and lung colony counting after CV016 administration using HE staining (method as in Example 7).

[0212] (1) HE staining results

[0213] The results are as follows Figure 5 As shown, the Control group exhibited normal lung tissue structure under an optical microscope, while the PA model mice showed severe pulmonary congestion, inflammatory cell infiltration, alveolar wall thickening, and interstitial edema. The PA+CV016 group showed reduced pulmonary congestion, and the alveolar wall thickening and interstitial edema were significantly better than those in the PA group, indicating that CV016 can effectively improve the lung damage in septic mice. The Control group showed normal liver tissue structure, while the PA model septic mice showed obvious liver damage, such as congestion, swelling, and increased leukocytes in the hepatic sinusoids. These pathological changes were significantly improved in the PA+CV016 group. The Control group showed normal spleen tissue structure, while the PA model septic mice showed obvious spleen damage, with unclear boundaries between the blue and red pulp and signs of fusion. The blue pulp mainly contains immune cells, and a significant reduction in the number and death of immune cells was observed. These pathological changes were significantly improved in the PA+CV016 group. The control group showed normal kidney tissue structure, while the mice in the PA group exhibited obvious kidney damage, such as renal tubular structure destruction or dilation, vacuolar degeneration, and brush border loss. These pathological changes were significantly improved in the PA+CV016 group. No abnormalities were found in the cardiac tissue sections of any group.

[0214] (2) Methods and results of lung colony counting

[0215] Lung tissue was ground and counted using a dropper; the results are as follows: Figure 6 As shown, the number of bacterial colonies in the lungs of the CV016 treatment group was significantly lower than that of the PA group, indicating that CV016 protected lung tissue and reduced bacterial invasion of the lungs.

[0216] Example 9: Determination of the anticoagulant activity of CV016 and CV018

[0217] 1. Experimental Principle

[0218] Heparin first reacts with excess ATIII to form a complex (AT-Hep.), theoretically assuming that all heparin is used to form the AT-Hep. complex, which inhibits thrombin activity. The AT-Hep. complex then binds to excess FXa, and the remaining free FXa can hydrolyze the substrate S. 2765 The pNA product was developed at 405 nm, and its potency was calculated using the BS2000 software with the method of parallel lines of the reaction.

[0219] Heparin + AT III → [AT - Hep.]

[0220] [AT-Hep.]+[FXa(excess)]→[FXa-AT-Hep.]+[residual FXa]

[0221] [residual FXa]+Substrate→Peptide+pNA

[0222] 2. Testing Methods

[0223] The activity test requires ATIII, FXa, and the chromogenic substrate S. 2765 All are commercially available from Beijing Adhoc International Biotechnology Co., Ltd., including: human antithrombin (ATIII), AG00-0132; bovine activated factor X (FXa), AG00-0121; and FXa chromogenic substrate S. 2765 , AG00-0102-10.

[0224] Prepare the corresponding solutions according to the table below:

[0225]

[0226] Preparation of low molecular weight heparin sodium standards / samples: Dilute the standards or samples to 1.0 IU / ml (the starting dose can be adjusted appropriately within the pharmacopoeia range), and then dilute to five concentration gradients: [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 the samples according to the order of operations in the table below, and then read the OD value at 450 nm using an ELISA reader. Calculate the potency using the 4.4 method of the stoichiometric reaction parallel line with BS2000 software.

[0227] Enzyme activity assay procedure sequence

[0228]

[0229] 3. As determined:

[0230] The standard low molecular weight heparin sodium (H0185000, low molecular weight heparin analytical biological standard, purchased from Beijing Adhoc International Biotechnology Co., Ltd.) has a potency of 100 IU / mg.

[0231] The potency PT of CV016 was measured to be 0.00084499 IU / mg, and the potency PT of CV018 was measured to be 0.00003235 IU / mg.

[0232] The higher the potency, the better the anticoagulant activity. As can be seen from the above experiments, CV016 and CV018 have virtually no anticoagulant activity.

[0233] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A heparin trisaccharide compound having the structure of formula A, wherein the three monosaccharides from left to right are represented by B, C, and D: A， in: The R1 for both β- and D-glucoses is -OSO3Y, or the R1 for β-glucose is -OSO3Y and the R1 for D-glucose is -NHSO3Y, or the R1 for β-glucose is -NHSO3Y and the R1 for D-glucose is -OSO3Y. R7 is -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3Y. Each Y may be the same or different, and is independently selected from H or a monovalent cation, wherein the monovalent cation is selected from Na. + K + Li + NH4 + ; R2, R3, and R5 are the same, which is H; R4 and R6 are -SO3Y.

2. The compound according to claim 1, characterized in that, R7 is -C1-3 alkyl, -C2-4 alkylene-NH2, or -C2-4 alkylene-NHSO3Y.

3. The compound according to claim 1 or 2, characterized in that, Each Y is identical and is selected from H and Na. + K + Li + NH4 + .

4. The compound according to claim 1 or 2, characterized in that, Each Y may be the same or different, and is independently selected from Na. + K + Li + NH4 + .

5. The compound according to claim 1 or 2, characterized in that, Each Y is identical and is selected from Na. + K + Li + NH4 + .

6. The compound of claim 1, wherein the compound is selected from compounds with the following structures: ,or .

7. A pharmaceutical composition comprising the compound according to any one of claims 1-6.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is used to treat or prevent sepsis or septic shock, protect the organs of patients with sepsis, protect the vascular endothelial cells of patients with sepsis, and / or reduce TNF-α, IL-1β and / or IL-6 in patients with sepsis.

9. The pharmaceutical composition according to claim 8, characterized in that, The organs mentioned are selected from the heart, lungs, spleen, liver and / or kidneys.

10. The pharmaceutical composition according to any one of claims 7-9, characterized in that, It further includes other therapeutic agents selected from sepsis treatment agents.

11. The use of heparin trisaccharide compounds in the preparation of pharmaceuticals, said pharmaceuticals being used to treat or prevent sepsis or septic shock, to protect organs of patients with sepsis, to protect vascular endothelial cells of patients with sepsis, and / or to reduce TNF-α, IL-1β and / or IL-6 in patients with sepsis; The heparin trisaccharide compound has the structure of formula A, and the three monosaccharides from left to right are represented by B, C, and D, respectively: A， in: R1 may be the same or different, and can be independently selected from -OSO3Y or -NHSO3Y. R2, R3, and R5 are the same, which is H; R4 and R6 are -SO3Y. R7 is -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3Y. Each Y may be the same or different, and is independently selected from H or a monovalent cation, wherein the monovalent cation is selected from Na. + K + Li + NH4 + .

12. The application as described in claim 11, characterized in that, R7 is -C1-3 alkyl, -C2-4 alkylene-NH2, or -C2-4 alkylene-NHSO3Y.

13. The application as described in claim 11 or 12, characterized in that, Each Y is identical and is selected from H and Na. + K + Li + NH4 + .

14. The application as described in claim 11 or 12, characterized in that, Each Y is identical and is selected from Na. + K + Li + NH4 + .

15. The application as described in claim 11, wherein the heparin trisaccharide compound is selected from the following structural compounds: , , ,or .

16. The application as described in claim 11, 12, or 15, wherein the organ is selected from the heart, lungs, spleen, liver, and / or kidneys.

17. Compound VI ,in, Each Y may be the same or different, and is independently selected from H or a monovalent cation, wherein the monovalent cation is selected from Na. + K + Li + NH4 + .

18. Compound VI as claimed in claim 17, characterized in that, Each Y is identical, selected from H or Na. + .

19. A method for preparing the compound of formula A according to any one of claims 1-6, characterized in that, Through the fully protected trisaccharide intermediate E The compound, after undergoing the process of dehydroxyl protecting groups, O - Sulfonation, optional azide reduction reaction, and finally... N - Sulfonation yields the following: Rx is an azide group or OR 11 R 11 R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl.

20. The preparation method according to claim 19, characterized in that, R 21 R 31 and R 51 Same, R 11 R 41 and R 61 Same or different, and both are different from R 21 R 31 and R 51 .

21. The preparation method according to claim 19, characterized in that, R 21 R 31 and R 51 They are the same, both are benzyl, R 11 R 41 and R 61 Whether the groups are the same or different, they are independently selected from acetyl and benzoyl groups.

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