Sulfonated trisaccharide as well as preparation method and treatment application thereof

Abstract

The present invention provides a sulfonated trisaccharide having pharmaceutical activity and capable of treating pulmonary arterial hypertension, inflammatory bowel disease and multiple sclerosis, and a preparation method thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compounds, and particularly relates to a sulfonated trisaccharide, a preparation method thereof, and application thereof in preparing medicines. Background Art

[0002] Oligosaccharides are monosaccharides (usually 2-10) connected by glycosidic bonds to form straight or branched chains. They have various forms and activities in the body. For example, fondaparinux sodium is a synthetic pentasaccharide, which is used as an anticoagulant in clinical practice. Due to the diversity of oligosaccharide structures, diversity of activities, and complexity of synthesis, the development of oligosaccharide drugs is a relatively difficult field in the field of drug research and development.

[0003] On the other hand, there is a lack of effective therapeutic drugs in clinical use for some diseases with unknown causes and pathogenesis, such as multiple sclerosis, pulmonary hypertension and inflammatory bowel disease.

[0004] Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. It is currently believed that the immune system is involved in its occurrence and development. The characteristic pathological changes of MS are multiple demyelinating plaques in the white matter of the central nervous system, mostly located around the lateral ventricles, accompanied by reactive gliosis, and may also have axonal damage. MS often involves the brain, spinal cord white matter, subcortical structures, brainstem, cerebellum and optic nerves, etc., which can cause patients to have paresthesias, limb weakness, loss of muscle coordination, decreased vision, and even paralysis. The etiology and pathogenesis of MS are still unclear, and it is believed that it may be related to multiple factors such as viral infection (common viruses may include human herpes virus type 6, Epstein-Barr virus, measles virus, human T-lymphotropic virus type I, etc.), autoimmune response, genetics and environment.

[0005] Pulmonary hypertension (PH) is a pathophysiological state caused by a variety of known or unknown reasons, characterized by abnormally elevated pulmonary artery pressure. The hemodynamic diagnostic criteria are: mean pulmonary artery pressure (mPAP) ≥ 25 mmHg measured by right cardiac catheterization at sea level and in resting state. Clinically, pulmonary hypertension is divided into five categories: ① arterial pulmonary hypertension (PAH): idiopathic, hereditary, drug- and toxin-induced pulmonary hypertension, disease-related pulmonary hypertension (including connective tissue disease-related PAH, portal hypertension-related PAH, HIV-related PAH, etc.), PAH with pulmonary vein or capillary involvement, and persistent pulmonary hypertension of the newborn; ② pulmonary hypertension caused by left heart disease: left heart disease includes left ventricular systolic or diastolic dysfunction, valvular heart disease, congenital heart disease, etc.; ③ pulmonary hypertension caused by hypoxia and / or lung disease: such as chronic obstructive pulmonary disease (COPD), interstitial lung disease, sleep apnea, long-term residence in plateau environment, etc.; ④ chronic thromboembolic pulmonary hypertension and other pulmonary artery obstructive diseases: such as intravascular tumors, arteritis, etc.; ⑤ pulmonary hypertension caused by unknown and / or multiple factors: such as blood system diseases (chronic hemolytic anemia, myelodysplastic syndrome, etc.), systemic diseases (sarcoidosis, etc.), and metabolic diseases. A variety of factors contribute to the pathogenesis of pulmonary hypertension, including the proliferation of lung cells, which can affect vascular remodeling (i.e., hyperplasia). For example, pulmonary vascular remodeling occurs mainly through the proliferation of arterial endothelial cells and smooth muscle cells in patients with pulmonary hypertension. Further, it has been found that pulmonary pulmonary hypertension may be caused by excessive proliferation of pulmonary arterial smooth cells and pulmonary endothelial cells. Further, the characteristics of late pulmonary hypertension can be muscled tissue of distal pulmonary arterioles, concentric intimal thickening and obstruction of vascular lumen by proliferating endothelial cells. The main symptoms of pulmonary hypertension are dyspnea or shortness of breath, and other symptoms include fatigue, dizziness, fainting, peripheral edema (swelling of feet, legs or ankles), bluish lips and skin, chest pain, angina, dizziness during exercise, non-productive cough, rapid heartbeat and palpitations, etc. Early diagnosis of patients with pulmonary hypertension is difficult, treatment is more difficult, and the prognosis is poor. Current therapies cannot cure pulmonary hypertension, and they do not directly treat the potential vascular remodeling (vascular remodeling) and vascular muscularization observed in many patients with pulmonary hypertension. Therefore, there is a high, unmet need for effective therapies for treating pulmonary hypertension.

[0006] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease with unclear etiology, including ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a chronic nonspecific ulcerative colitis. The lesions mainly involve the colon mucosa and submucosa, and the range often starts from the distal colon, can develop retrogradely to the proximal part, and even involve the entire colon, rectum and terminal ileum, with a continuous distribution; the main clinical manifestations are diarrhea, abdominal pain and mucus, pus and blood in the stool; the severity of the disease varies, often recurring, and lasting for months, years or longer. In clinical practice, ulcerative colitis has many different names according to the gross anatomical location of the lesion. When the inflammation is limited to the rectum, it is called proctitis; when the inflammation involves the rectum and sigmoid colon, it is called proctosigmoiditis; when the inflammation occurs in the colon, it is called colitis; when the inflammation retrogradely invades the terminal ileum, it is called retrograde ileitis. Crohn's disease is a chronic granulomatous inflammation. The lesions can affect various parts of the gastrointestinal tract, mainly the terminal ileum and its adjacent colon, and are often distributed in a segmental and asymmetric manner. The main clinical manifestations are abdominal pain, diarrhea, fistulas, anal lesions, and systemic symptoms of varying degrees. The etiology and pathogenesis of inflammatory bowel disease have not yet been fully clarified and may be related to multiple factors. It is currently assumed that it is caused by a combination of factors such as genetic susceptibility, imbalance between probiotics and pathogenic bacteria in the gastrointestinal tract, impaired intestinal epithelial cell integrity, and host immune dysfunction. Due to the unclear exact cause and complex pathogenesis, the treatment of inflammatory bowel disease has always been a major problem that has plagued the clinical practice of gastroenterology. Summary of the invention

[0007] The present invention provides a sulfonated trisaccharide, which has valuable pharmacological properties and can be used as a medicine. The present invention also provides a preparation method of the sulfonated trisaccharide.

[0008] A sulfonated trisaccharide or a salt thereof, which has a structure of formula A (the three monosaccharides from left to right are represented by B, C, and D respectively):

[0009]

[0010] in:

[0011] R 2 , R 3 , R 4 , R 5 and R 6 The same or different, independently selected from H or -SO 3 H,

[0012] R 7 -C1-5 alkyl, -C1-5 alkylene-NH2 , or -C1-5 alkylene-NHSO 3 H,

[0013] The salts are salts formed with monovalent cations, divalent cations and / or trivalent cations.

[0014] The monovalent cation is selected from Na + , K + , Li + , NH 4 + etc.; the divalent cation is, for example, selected from Ca 2+ , Cu 2+ , Zn 2+ , Fe 2+ ,Mg 2+ , Mn 2+ etc.; the trivalent cation is selected from Al 3+ , Fe 3+ Those skilled in the art will understand that when the compound is salted, the corresponding group in formula A is its anionic group, such as -COO - 、 -SO 3 - .

[0015] In some embodiments of the present invention, in Formula A, R 2 , R 3 , R 4 , R 5 and R 6 Same, both -SO 3 H.

[0016] In some embodiments of the present invention, in Formula A, R 2 , R 3 , R 4 , R 5 and R 6 Same, both are H.

[0017] In some embodiments of the present invention, in Formula A, R 2 , R 3 and R 5 Same as H, R 4 and R 6 Same as -SO 3 H.

[0018] In some embodiments of the present invention, in Formula A, R 2 , R 4 and R 6 Same as -SO 3 H, R 3 and R5 The same is H.

[0019] In some embodiments of the present invention, in Formula A, R 2 , R 4 , R 5 and R 6 Same as -SO 3 H, R 3 For H.

[0020] In some embodiments of the present invention, in Formula A, R 7 -C1-3 alkyl, -C2-4 alkylene-NH 2 、-C2-4 alkylene-NHSO 3 H.

[0021] In some embodiments of the present invention, in Formula A, R 7 Methyl, ethyl, -C 2 H 4 NH 2 , -C 2 H 4 NHSO 3 H, -C 3 H 6 NH 2 or -C 3 H 6 NHSO 3 H.

[0022] In some embodiments of the invention, the D sugar terminal group is in the alpha configuration.

[0023] In some embodiments of the invention, the D sugar terminal group is in the β configuration.

[0024] In some embodiments of the present invention, the salt is a salt formed with a monovalent cation, preferably the monovalent cation is selected from Na + , K + .

[0025] In some embodiments of the present invention, the salt is a salt formed with a divalent cation, preferably the divalent cation is Ca 2+ .

[0026] In some embodiments of the present invention, in Formula A, R 2 , R 3 and R 5 Same, H; R 4 and R 6 Same as -SO 3 H; R 7 -C1-5 alkyl, -C1-5 alkylene-NH 2、-C1-5 alkylene-NHSO 3 H, the salt is a salt formed with a monovalent cation, a divalent cation and / or a trivalent cation. Preferably, R 7 -C1-3 alkyl, -C2-4 alkylene-NH 2 、-C2-4 alkylene-NHSO 3 H, more preferably, R 7 Methyl, ethyl, -C 2 H 4 NH 2 , -C 2 H 4 NHSO 3 H, -C 3 H 6 NH 2 or -C 3 H 6 NHSO 3 H. Preferably, the salt is a salt formed with a monovalent cation, preferably the monovalent cation is selected from Na + , K + Alternatively, the salt is a salt formed with a divalent cation, preferably the divalent cation is Ca 2+ Preferably, the terminal group of the D sugar is in the α configuration.

[0027] In one embodiment of the present invention, in Formula A, R 2 =R 3 =R 5 =H, R 4 =R 6 =SO 3 H, R 7 =Me, and the terminal methyl ester of D sugar is in α configuration, the structural formula of the compound is as follows, and TSC is used in the present invention:

[0028] The sodium salt structural formula of the compound is as follows, which is referred to as TSC19 in the present invention:

[0029]

[0030] The present invention also provides a method for preparing the compound of formula A or a salt thereof.

[0031] According to the present invention, the compound of formula A is prepared by fully protecting the trisaccharide intermediate of formula E [E], successively removed the hydroxyl protecting group, O-sulfonated, and finally N-sulfonated. Those skilled in the art will appreciate that after obtaining the compound of formula A, the corresponding salt of the compound can be obtained by salifying with a cation, that is, the compound of formula A or its salt can be prepared. Methods known in the art can be used, such as exchanging the compound of formula A with a cationic resin to prepare its corresponding monovalent salt, divalent salt or trivalent salt, etc.

[0032] In the fully protected trisaccharide intermediate E, R 11 , R 21 , R 31 , R 41 , R 51 and R 61 The radicals may be the same or different and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl and p-methoxybenzyl.

[0033] Preferably, in the reaction, all hydroxyl groups to be sulfonated are deprotected at one time, and after the hydroxyl groups are O-sulfonated, all hydroxyl groups not to be sulfonated are deprotected at one time.

[0034] In one embodiment of the present invention, the present invention adopts the synthetic reaction method to synthesize TSC and TSC19, using the fully protected trisaccharide intermediate 2 as the raw material, sequentially undergoing dehydroxylation, O-sulfonation, dehydroxylation, and finally N-sulfonation. 11 , R 21 , R 31 , R 41 , R 51 and R 61 The definition of the corresponding group is the same as that of the above-mentioned group, preferably R 11 , R 31 , R 51 and R on D sugar 21 Same, R on B sugar 21 and R on D sugar 41 Same, R 61 and R on B sugar 41 The same; further preferably R 11 , R 31 , R 51 and R on D sugar 21 Same, both are benzyl, R on B sugar 21 and R on D sugar 41 Same, both are acetyl, R 61 and R on B sugar 41 The same, are benzoyl.

[0035]

[0036] The fully protected trisaccharide intermediate 2 can be prepared from the monosaccharide intermediate F [F] and disaccharide receptors Glycosylation reaction occurs to obtain:

[0037]

[0038] R 11 , R 21 , R 31 , R 41 , R 51 and R 61 can be the same or different, independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, p-methoxybenzyl; X is a leaving group suitable for reacting with other acceptors to form a bond between glycosides. Preferably, X is hydroxyl, alkylthio, arylthio, halogen, trichloroacetimidyl, phosphate, tert-butyldiphenylsilyloxy.

[0039] 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, and the like.

[0040] The disaccharide intermediate 4 can be obtained through the monosaccharide intermediate 6 and the monosaccharide intermediate 7, and the reaction formula is as follows:

[0041]

[0042] Monosaccharide 6, monosaccharide 7 and disaccharide receptor 4 used in the above preparation method can be prepared according to the 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. The definitions of the groups in monosaccharide 6, monosaccharide 7 and disaccharide receptor 4 are the same as those of the corresponding groups mentioned above.

[0043] Therefore, the present invention also provides each intermediate in the above-mentioned synthesis method and a preparation method thereof.

[0044] A monosaccharide intermediate F has the following structure: Among them, R 31 is benzyl, R 11 , R21 and R 41 are the same or different and are selected from benzyl, chloroacetyl, acetyl, benzoyl or pivaloyl. In one embodiment of the present invention, R 11 and R 31 All are benzyl, R 41 is benzoyl, R 21 It is an acetyl group, in β configuration, named F-1, with the structural formula

[0045] In one embodiment of the present invention, the reaction formula of the preparation method of F-1 is as follows:

[0046]

[0047] A disaccharide intermediate 4 has the following structure: Among them, R 21 and R 51 is benzyl, R 41 and R 61 are the same or different and are selected from chloroacetyl, acetyl, benzoyl or pivaloyl. In one embodiment of the present invention, R 21 and R 51 is benzyl, R 41 is acetyl, R 61 It is a benzoyl group, named 4-1, with the structural formula

[0048] The disaccharide intermediate 4-1 can be prepared by a synthetic method known in the art, such as Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. Chem Med Chem, 2014, 9, 1071-1080.

[0049] The present invention also provides intermediates IV, V and VI.

[0050] The structure of compound IV is as follows:

[0051] Where R 21 , R 11 , R 31 and R 51 may be the same or different, independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, p-methoxybenzyl, preferably R 21 , R 11 , R 31 and R 51 All are benzyl.

[0052] The structure of the compound V is as follows:

[0053] Where R 21 , R 11 , R 31 and R 51 may be the same or different, independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, p-methoxybenzyl, preferably R 21 , R 11 , R 31 and R 51 All are benzyl; Y is H, Na + , K + , Li + , NH 4 + , preferably Y is H or Na + .

[0054] The structure of the compound VI is as follows:

[0055] Where Y is H, Na + , K + , Li + , NH 4 + , preferably Y is H + Or Na + .

[0056] The intermediates IV, V and VI are respectively produced by removing the hydroxyl protecting group and sulfonating the trisaccharide intermediate 2 in sequence, and the reaction formula is as follows:

[0057]

[0058] The removal of the hydroxyl protecting group and the sulfonation can be carried out by reaction methods and conditions known in the art. In one embodiment of the present invention, intermediate 2 is simultaneously subjected to the removal of R in the presence of a base. 41 , R 61 , R on B sugar 21 and methyl ester to obtain intermediate IV. In one embodiment of the present invention, intermediate IV is SO 3 ·NMe 3 In one embodiment of the present invention, the intermediate V is catalytically hydrogenated to obtain the intermediate V after O-sulfonation. 11 , R 31 , R 51 , R on D sugar 21 and Cbz to obtain intermediate VI. In one embodiment of the present invention, in SO 3The amino group in the compound of formula VI is sulfonated under the action of Py to obtain compound TSC, and ion exchange is performed with a sodium ion exchange resin to obtain compound TSC19. The sodium ion exchange resin can be a resin known in the art, including but not limited to Amberlite IR120 Na + 、Dowex-50-WX4-Na + wait.

[0059] The inventors of the present application have found that the compound of formula A of the present invention or its salt has the activity of treating multiple sclerosis, which can improve the behavior of EAE model mice, inhibit demyelination and inflammation of the nervous system. In addition, the compound of the present invention can also inhibit the weight loss of DSS-induced IBD model mice, improve the stool characteristics and blood in the stool of model mice; histological examination further confirmed that the compound of the present invention can improve the intestinal inflammation of DSS-induced IBD model mice, and repair or maintain the epithelial integrity of the intestinal mucosa. The compound of the present invention can also reduce cardiac hypertrophy in SU5416 and hypoxia-induced pulmonary hypertension models and hypoxia-induced pulmonary hypertension models, and improve cardiac function; histological analysis further revealed that in the model, the compound of the present invention has a significant effect on reducing vascular reconstruction and vascular muscularization.

[0060] Therefore, the present invention also provides a pharmaceutical composition, which comprises the compound of formula A of the present invention or its salt or solvate thereof as an active ingredient, and optionally further comprises 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 regulators, preservatives, lubricants, disintegrants, etc.

[0061] In some embodiments of the invention, the pharmaceutical composition is used to treat pulmonary hypertension, inflammatory bowel disease or multiple sclerosis.

[0062] The pharmaceutical composition can be administered in combination with other therapeutic agents or supportive therapies, or prepared as a combined drug, or the pharmaceutical composition can further contain other therapeutic agents. The other therapeutic agents can be other drugs for treating pulmonary hypertension, inflammatory bowel disease or multiple sclerosis, etc., depending on the type of disease or condition being treated.

[0063] The present invention also provides use of the compound of formula A or its salt or solvate in preparing a drug for treating pulmonary hypertension, inflammatory bowel disease or multiple sclerosis.

[0064] The present invention also provides the use of the compound of formula A or its salt or solvate in combination with other drugs for treating pulmonary hypertension, inflammatory bowel disease or multiple sclerosis in the preparation of drugs for treating pulmonary hypertension, inflammatory bowel disease or multiple sclerosis.

[0065] The present invention also provides the use of the compound of formula A or its salt or solvate in the preparation of a drug for treating pulmonary hypertension, inflammatory bowel disease or multiple sclerosis in combination with other drugs for treating pulmonary hypertension, inflammatory bowel disease or multiple sclerosis.

[0066] The present invention also provides a method for treating a disease, characterized in that a therapeutically effective amount of a compound of formula A or a salt thereof or a solvate thereof, or a pharmaceutical composition containing a compound of formula A or a salt thereof or a solvate thereof, is administered to a patient in need thereof. In one embodiment of the present invention, the disease is pulmonary hypertension. In one embodiment of the present invention, the disease is inflammatory bowel disease. In one embodiment of the present invention, the disease is multiple sclerosis.

[0067] According to the present invention, the amount of the compound of formula A or its salt or solvate 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.

[0068] The mass percentage of the compound of formula A or its salt or solvate thereof (calculated as the compound of formula A) in the pharmaceutical composition is 0.01%-95%. 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%.

[0069] 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 or its salt or solvate in the injection can be 1-15 mg / ml, such as 5 mg / ml, 10 mg / ml, 12.5 mg / ml, etc., based on the compound of formula A.

[0070] 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.

[0071] According to the present invention, the dosage of the compound of formula A or its salt or its solvate (in terms of 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 or its salt or its solvate (in terms of 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.

[0072] According to the present invention, in certain aspects, the multiple sclerosis is relapsing-remitting (RR), secondary-progressive (SP), primary-progressive (PP) or progressive-relapsing (PR).

[0073] In some aspects, the treatment of multiple sclerosis is to delay the time of clinical symptoms of the patient. In some aspects, the treatment of multiple sclerosis is to alleviate or relieve or improve the clinical symptoms and signs of the patient, including but not limited to paresthesia, optic neuritis, limb weakness, movement disorders, tremor of the limbs, unsteady standing, ataxia, limb rigidity and spasm, dysarthria, pain, etc. In some aspects, the treatment of multiple sclerosis is to improve or inhibit nerve demyelination. In some aspects, the treatment of multiple sclerosis is to improve or inhibit inflammation of the central nervous system (including but not limited to the brain and spinal cord).

[0074] Other drugs for the treatment of multiple sclerosis include but are not limited to: fampridine, beta-interferon, teriflunomide, sphingosine-1-phosphate (S1P) receptor modulators (such as siponimod, fingolimod, ozanimod, etc.), ofatumumab, natalizumab, mitoxantrone, dimethyl fumarate, etc.

[0075] According to the present invention, in certain aspects, the treatment of pulmonary hypertension refers to treating, preventing or reducing the progression rate and / or severity of pulmonary hypertension (e.g., treating, preventing or reducing the progression rate and / or severity of one or more complications of pulmonary hypertension).

[0076] In some embodiments of the present invention, the pulmonary hypertension is arterial pulmonary hypertension, including idiopathic, hereditary, drug- and toxin-induced pulmonary hypertension, disease-related pulmonary hypertension (including connective tissue disease-related PAH, portal hypertension-related PAH, HIV-related PAH, etc.), PAH with pulmonary vein or capillary involvement, and persistent pulmonary hypertension of the newborn.

[0077] In some embodiments of the present invention, the pulmonary hypertension is pulmonary hypertension caused by left heart disease: left heart disease includes left ventricular systolic or diastolic dysfunction, valvular heart disease, congenital heart disease, etc.

[0078] In some embodiments of the present invention, the pulmonary hypertension is pulmonary hypertension caused by hypoxia and / or lung disease: for example, chronic obstructive pulmonary disease (COPD), interstitial lung disease, sleep apnea, long-term residence in a plateau environment, etc.

[0079] In some embodiments of the present invention, the pulmonary hypertension is chronic thromboembolic pulmonary hypertension and pulmonary hypertension caused by other pulmonary artery obstructive diseases: for example, pulmonary hypertension caused by intravascular tumors, arteritis, etc.

[0080] In some embodiments of the present invention, the pulmonary hypertension is caused by unknown and / or multiple factors: for example, blood system diseases (chronic hemolytic anemia, myelodysplastic syndrome, etc.), systemic diseases (sarcoidosis, etc.), and metabolic diseases.

[0081] According to the present invention, in certain aspects of the invention, the progression rate and / or severity of pulmonary hypertension is treated, prevented or reduced (e.g., the progression rate and / or severity of one or more complications of pulmonary hypertension is treated, prevented or reduced), wherein the patient has a resting pulmonary artery pressure (PAP) of at least 25 mm Hg (e.g., 25, 30, 35, 40, 45 or 50 mm Hg).

[0082] In some embodiments, the treating, preventing, or reducing the rate of progression and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension) is to adjust one or more hemodynamic parameters of a patient with pulmonary hypertension to a more normal level (e.g., normal compared to healthy people of similar age and gender).

[0083] In some embodiments, the adjustment of the hemodynamic parameters to a more normal level refers to lowering PAP, for example, lowering the patient's PAP by at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 12 mmHg, at least 15 mmHg, at least 20 mmHg, at least 25 mmHg, etc.

[0084] In some embodiments, the adjustment of the hemodynamic parameters to more normal levels refers to a reduction in pulmonary vascular resistance (PVR).

[0085] In some embodiments, adjusting the hemodynamic parameter to a more normal level refers to increasing the pulmonary capillary wedge pressure (PCWP).

[0086] In some embodiments, adjusting the hemodynamic parameter to a more normal level refers to increasing left ventricular end-diastolic pressure (LVEDP).

[0087] In some embodiments, adjusting the hemodynamic parameter to a more normal level refers to increasing the pulmonary acceleration time (PAT).

[0088] In some embodiments, adjusting the hemodynamic parameters to a more normal level refers to increasing the ratio of pulmonary artery acceleration time (PAT) / pulmonary ejection time (PET).

[0089] In some embodiments, the treatment, prevention or reduction of the progression rate and / or severity of pulmonary hypertension (e.g., the progression rate and / or severity of one or more complications of pulmonary hypertension) is the progression rate and / or severity of cell proliferation in the pulmonary artery of a patient with pulmonary hypertension. In some embodiments, the progression rate and / or severity of cell proliferation in the pulmonary artery is the progression rate and / or severity of smooth muscle and / or endothelial cell proliferation in the pulmonary artery. In some embodiments, the progression rate and / or severity of cell proliferation in the pulmonary artery is the progression rate and / or severity of angiogenesis in the pulmonary artery.

[0090] In some embodiments, the treating, preventing, or reducing the rate of progression and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension) is increasing physical activity in a patient with pulmonary hypertension.

[0091] The improvement of the physical activity can use any suitable measure of exercise capacity. For example, the exercise capacity in the 6-minute walk test (6MWT), which measures how far a subject can walk in 6 minutes, i.e., the 6-minute walk distance (6MWD), is often used to assess the severity and disease progression of pulmonary hypertension. The Borg dyspnea index (BDI) is a numerical scale for assessing the dyspnea (discomfort breathing) felt. It measures the degree of dyspnea, for example, after completing the 6MWT, where a BDI of 0 indicates no shortness of breath, and a BDI of 10 indicates maximum shortness of breath.

[0092] In some embodiments, increasing the physical activity of a patient with pulmonary hypertension is increasing their 6MWD by at least 10 meters, at least 15 meters, at least 20 meters, at least 30 meters, at least 40 meters, at least 50 meters, at least 60 meters, at least 70 meters, at least 80 meters, at least 90 meters, at least 100 meters, etc.

[0093] In some embodiments, increasing physical activity in a patient with pulmonary hypertension is by lowering their BDI by at least 0.5 index points, at least 1 index point, at least 1.5 index points, at least 2 index points, at least 2.5 index points, at least 3 index points, at least 3.5 index points, at least 4 index points, at least 4.5 index points, at least 5 index points, at least 5.5 index points, at least 6 index points, at least 6.5 index points, at least 7 index points, at least 7.5 index points, at least 8 index points, at least 8.5 index points, at least 9 index points, at least 9.5 index points, etc.

[0094] In some embodiments, the treating, preventing, or reducing the rate of progression and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension) is treating, preventing, or reducing the rate of progression and / or severity of dyspnea in patients with pulmonary hypertension.

[0095] In some embodiments, the treating, preventing, or reducing the rate of progression and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension) is treating, preventing, or reducing the rate of progression and / or severity of chest pain in a patient with pulmonary hypertension.

[0096] In some embodiments, the treating, preventing, or reducing the rate of progression and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension) is treating, preventing, or reducing the rate of progression and / or severity of fatigue in patients with pulmonary hypertension.

[0097] In some embodiments, the treating, preventing, or reducing the progression rate and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the progression rate and / or severity of one or more complications of pulmonary hypertension) is treating, preventing, or reducing the progression rate and / or severity of pulmonary fibrosis in patients with pulmonary hypertension.

[0098] In some embodiments, the treating, preventing, or reducing the rate of progression and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension) is treating, preventing, or reducing the rate of progression and / or severity of pulmonary vascular remodeling in patients with pulmonary hypertension.

[0099] In some embodiments, the treatment, prevention or reduction of the progression rate and / or severity of pulmonary hypertension (e.g., the treatment, prevention or reduction of the progression rate and / or severity of one or more complications of pulmonary hypertension) is the treatment, prevention or reduction of the progression rate and / or severity of right ventricular hypertrophy in patients with pulmonary hypertension. In some embodiments, the treatment, prevention or reduction of the progression rate and / or severity of right ventricular hypertrophy in patients with pulmonary hypertension is to reduce their right ventricular free wall thickness (RV free wall thickness, RVFWT).

[0100] In some embodiments, the treatment, prevention, or reduction of the progression rate and / or severity of pulmonary hypertension (e.g., the treatment, prevention, or reduction of the progression rate and / or severity of one or more complications of pulmonary hypertension) is to improve right ventricular systolic function in patients with pulmonary hypertension. In some embodiments, the improvement of right ventricular systolic function is to increase tricuspid annular plane systolic excursion (TAPSE).

[0101] According to the present invention, pulmonary hypertension at the treatment baseline can be mild, moderate or severe, as measured, for example, by the World Health Organization (WHO) functional category, which is a measure of disease severity in patients with pulmonary hypertension. The WHO functional system is divided into four functional levels: Class I: pulmonary hypertension that does not lead to physical activity limitation, ordinary physical activity does not cause excessive dyspnea or fatigue, chest pain or near syncope; Class II: pulmonary hypertension that leads to slight limitation of physical activity, the patient is comfortable at rest, ordinary physical activity causes excessive dyspnea or fatigue, chest pain or near syncope; Class III: pulmonary hypertension that leads to significant limitation of physical activity, the patient is comfortable at rest, less than ordinary activity causes excessive dyspnea or fatigue, chest pain or near syncope; Class IV: pulmonary hypertension that leads to the inability to perform any physical activity without symptoms, the patient shows signs of right heart failure, dyspnea and / or fatigue may exist even at rest, and any physical activity increases discomfort.

[0102] In certain aspects, patients in whom the present invention treats, prevents, or reduces the rate of progression and / or severity of pulmonary hypertension (e.g., treats, prevents, or reduces the rate of progression and / or severity of one or more complications of pulmonary hypertension) have grade I, II, III, or IV pulmonary hypertension as recognized by the WHO.

[0103] In some embodiments, the patient is a patient with stage I pulmonary hypertension recognized by the WHO. In some embodiments, the treatment, prevention or reduction of the progression rate and / or severity of pulmonary hypertension (e.g., treatment, prevention or reduction of the progression rate and / or severity of one or more complications of pulmonary hypertension) is to prevent or delay the patient from progressing from stage I pulmonary hypertension to stage II pulmonary hypertension.

[0104] In some embodiments, the patient is a patient with grade II pulmonary hypertension recognized by the WHO. In some embodiments, the patient is prevented or delayed from progressing from grade II pulmonary hypertension to grade III pulmonary hypertension. In some embodiments, the patient is promoted or increased from grade II pulmonary hypertension to grade I pulmonary hypertension.

[0105] In some embodiments, the patient is a patient with grade III pulmonary hypertension recognized by the WHO. In some embodiments, the patient is prevented or delayed from progressing from grade III pulmonary hypertension to grade IV pulmonary hypertension. In some embodiments, the patient is promoted or increased from grade III pulmonary hypertension to grade II pulmonary hypertension. In some embodiments, the patient is promoted or increased from grade III pulmonary hypertension to grade I pulmonary hypertension.

[0106] In some embodiments, the patient is a patient with grade IV pulmonary hypertension recognized by the WHO. In some embodiments, it is to promote or increase the patient's retreat from grade IV pulmonary hypertension to grade III pulmonary hypertension. In some embodiments, it is to promote or increase the patient's retreat from grade IV pulmonary hypertension to grade II pulmonary hypertension. In some embodiments, it is to promote or increase the patient's retreat from grade IV pulmonary hypertension to grade I pulmonary hypertension.

[0107] According to the present invention, other drugs or supportive therapies for treating pulmonary hypertension include, but are not limited to: prostacyclin and its derivatives (e.g., epoprostenol, treprostinil and iloprost); prostacyclin receptor agonists (e.g., celecoxib); endothelin receptor antagonists (e.g., thelin, ambrisentan, macitentan and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem and nifedipine; anticoagulants (e.g., warfarin); diuretics; oxygen therapy; atrial septotomy; pulmonary artery thromboendarterectomy; phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil); activators of soluble guanylate cyclase (e.g., cinaciguat and riociguat); ASK-1 inhibitors (e.g., CIIA; SCH79797; GS-4997; MSC2032964A; 3H-naphtho[1,2,3-de]quinoline-2,7-dione, NQDI-1; 2-thio-thiazolidine, 5-bromo-3-(4-oxo-2-thio-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one); NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2.2-Difluoropropionamide; C28 imidazole (CDDO-Im); 2-cyano-3,12-dioxooleanolic acid-1,9-diene-28-oic acid (CDDO); 3-acetyloleanolic acid; 3-trifluoroacetyloleanolic acid; 28-methyl-3-acetyloleanolic acid; 28-methyl-3-trifluoroacetyloleanolic acid; 28-methoxyoleanolic acid; SZC014; SCZ015; SZC017; polyethylene glycol derivatives of oleanolic acid Biological; 3-O-(β-D-pyranosyl) oleanolic acid; 3-O-[β-D-pyranosyl-(1-->3)-β-D-pyranosyl] oleanolic acid; 3-O-[β-D-pyranosyl-(1-->2)-β-D-pyranosyl] oleanolic acid; 3-O-[β-D-pyranosyl-(1-->3)-β-D-pyranosyl] oleanolic acid 28-O-β-D-pyranosyl ester; 3-O-[β- 28-O-β-D-glucopyranosyl] oleanolic acid ester; 3-O-[α-L-rhamnosyl-(1-->3)-β-D-pyranoglucopyranosyl] oleanolic acid; 3-O-[α-L-rhamnosyl-(1-->3)-β-D-pyranoglucopyranosyl] oleanolic acid ester; 28-O-β-D-glucopyranosyl] oleanolic acid ester; 28-O-β-D-glucopyranosyl- Fruit acid; 3-O-β-D-pyranosyl-glucopyranosyl (1→3)-β-D-pyranosyl-glucopyranosyl (CS1); 3-O-β-D-pyranosyl-glucopyranosyl (1→3)-β-D-pyranosyl-glucopyranosyl (CS2); 3,11-dioxoolean-12-ene-28-acid methyl ester (DIOXOL); ZCVI4-2; 3-dehydroxy-1,2,5-diazo[3',4':2,3]oleanolic acid benzyl ester); lung and / or heart transplantation.

[0108] According to the present invention, in some aspects, the inflammatory bowel disease is ulcerative colitis. In clinical practice, ulcerative colitis is also referred to as proctitis, proctosigmoiditis, backwash ileitis, colitis, etc., depending on the different gross anatomical sites involved in the disease. In some aspects, the inflammatory bowel disease is Crohn's disease.

[0109] According to the present invention, the effects of treating or preventing inflammatory bowel disease include but are not limited to relieving or inhibiting diarrhea, relieving or inhibiting abdominal pain, improving stool characteristics, improving blood in stool, restoring body weight or inhibiting weight loss, relieving or inhibiting mucosal inflammation, relieving or inhibiting intestinal edema, repairing intestinal mucosal epithelium, and / or maintaining the integrity of intestinal mucosal epithelium, etc.

[0110] The improvement of inflammatory bowel disease symptoms can be evaluated by any suitable measurement, such as the disease activity index (DAI) score, which is based on a comprehensive score of weight loss, stool characteristics, and fecal occult blood.

[0111] In some embodiments of the present invention, the improvement of inflammatory bowel disease symptoms is to reduce the DAI score by at least 0.5 index points, at least 1 index point, at least 1.5 index points, at least 2 index points, at least 2.5 index points, at least 3 index points, at least 3.5 index points, at least 4 index points, at least 4.5 index points, at least 5 index points, at least 5.5 index points, at least 6 index points, at least 6.5 index points, at least 7 index points, at least 7.5 index points, at least 8 index points, at least 8.5 index points, at least 9 index points, at least 9.5 index points, etc.

[0112] In some embodiments of the invention, weight loss is inhibited by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to treatment baseline.

[0113] In some embodiments of the present invention, the body weight decreases by at most 20%, at most 15%, at most 10%, at most 5%, or even no weight loss relative to the body weight before the disease occurs.

[0114] Other drugs for the treatment of inflammatory bowel disease include, but are not limited to: aminosalicylic acid preparations, including, but not limited to, salicylic acid sulfazopyridine, olsalazide, balsalazide, mesalazine, and para-aminosalicylic acid. Glucocorticoids, including, but not limited to, prednisone, prednisolone, hydrocortisone, dexamethasone, and budesonide. Immunosuppressants and immunomodulators, including, but not limited to, azathioprine, 6-mercaptopurine, methotrexate, cyclophosphamide, cyclosporin A, tacrolimus, mycophenolate mofetil, infliximab, and the like. Antibiotics, including, but not limited to, metronidazole, ornidazole, tinidazole, tobramycin, vancomycin, ciprofloxacin, and the like.

[0115] According to the present invention, in certain aspects, when the compounds of the present invention are used to treat or prevent inflammatory bowel disease, the compounds of the present invention can also be prepared as nutritional products or health products. According to the present invention, "nutritional products" refer to all products with nutritional and / or physiological effects; this includes in particular food supplements, foods, dietary products, etc. These products can be administered in particular by oral, nasogastric, etc. "Health products" refer to all products that have a beneficial effect on health in prevention or treatment, whether the effect is physiological or pharmacological, especially drugs and pharmaceutical products. These products can be administered in particular by oral, gastric, nasogastric, etc.

[0116] The compound of formula A of the present invention has a clear therapeutic effect, but has almost no anticoagulant activity, and is resistant to HPA hydrolysis, that is, it has high stability and does not have the binding activity of platelet factor 4. Therefore, the risk of bleeding is extremely low while the therapeutic use is used. The compound of formula A has a clear structure, which is conducive to preparation and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 The hydrogen spectrum and carbon spectrum of compound 2, the upper figure is the hydrogen spectrum and the lower figure is the carbon spectrum

[0118] Figure 2 The hydrogen spectrum and carbon spectrum of compound TSC19, the upper figure is the hydrogen spectrum and the lower figure is the carbon spectrum

[0119] Figure 3 Statistical graph of the behavioral scores of mice in the control group (normal), model group (EAE) and TSC19 treatment group over time in the EAE model mouse experiment

[0120] Figure 4 Statistical graph of the weight changes over time in the control group (normal), model group (EAE) and TSC19 treatment group mice in the EAE model mouse experiment

[0121] Figure 5 In the EAE model mouse experiment, the statistical chart of the detection results of pro-inflammatory factors (TNF-α and IFN-γ) and anti-inflammatory factors (IL-4) in the brain, spinal cord and spleen of mice in the control group (normal), model group (EAE) and TSC19 treatment group

[0122] Figure 6 In the EAE model mouse experiment, HE staining results of the spinal cord of the control group (control), model group (EAE) and TSC19 treatment group mice

[0123] Figure 7 HE staining results of brain tissue of mice in the control group (control), model group (EAE) and TSC19 treatment group in the EAE model mouse experiment

[0124] Figure 8 Results of LFB staining of spinal cord tissues of mice in the control group (control), model group (EAE) and TSC19 treatment group in the EAE model mouse experiment

[0125] Fig. 9 In the HY+SU pulmonary hypertension model, the cardiac ultrasound results of the control group (control), model group (HY+SU) and drug-treated group (TSC19), where A is the cardiac ultrasound result of tricuspid annular plane systolic excursion (TAPSE); B is the cardiac ultrasound results of pulmonary artery acceleration time (PAT) and pulmonary ejection time (PET); C is a bar chart of quantitative analysis of right ventricular free wall thickness (RVFWT), tricuspid annular plane systolic excursion (TAPSE) and pulmonary artery acceleration time (PAT) / pulmonary ejection time (PET)

[0126] Fig.10 In the HY pulmonary hypertension model, the cardiac ultrasound results of the control group (control), model group (HY) and drug-treated group (TSC19), where A is the cardiac ultrasound result of tricuspid annular plane systolic excursion (TAPSE); B is the cardiac ultrasound results of pulmonary artery acceleration time (PAT) and pulmonary ejection time (PET); C is a bar chart of quantitative analysis of right ventricular free wall thickness (RVFWT), tricuspid annular plane systolic excursion (TAPSE) and pulmonary artery acceleration time (PAT) / pulmonary ejection time (PET)

[0127] Fig.11 The results of the therapeutic effect of TSC19 on lung tissue in the pulmonary hypertension model, where A is the HE staining photos of lung tissues of the control group (control), model group (HY+SU, HY) and drug group (TSC19) in the HY+SU model, as well as the quantitative analysis of pulmonary vascular wall thickness; B is the HE staining photos of lung tissues of the control group, model group and drug group in the HY model, as well as the quantitative analysis of pulmonary vascular wall thickness

[0128] Fig.12 The results of TSC19's protective effect on the heart in the pulmonary hypertension model, where A is the HE and Masson staining photos of the heart tissues of the control group (control), model group (HY+SU, HY) and drug-treated group (TSC19) in the HY+SU model; B is the HE and Masson staining photos of the heart tissues of the control group, model group and drug-treated group in the HY model

[0129] Fig.13 Inhibitory effect of TSC19 on weight loss in mice in the DSS-induced mouse inflammatory bowel disease model. Left: Time-dependent weight change. Right: Statistical graph of weight loss in each group of mice on day 8. In the figure: *P<0.05, **P<0.01

[0130] Fig.14 Statistical graph of DAI score changes in each group of mice in the DSS-induced mouse inflammatory bowel disease model. Left: DAI time change graph. Right: DAI score statistics of each group of mice on the 8th day. In the figure, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0131] Fig.15 HE staining of colon tissues of mice in each group in the DSS-induced mouse inflammatory bowel disease model. The scale bar is 100 μm.

[0132] Fig.16 Statistical graphs of colon weight, colon length, and spleen weight of each group of mice in the DSS-induced mouse inflammatory bowel disease model. Left: colon weight, middle: colon length, right: spleen weight; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 DETAILED DESCRIPTION

[0133] 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.

[0134] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0135] 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.

[0136] Example 1 Preparation of fully protected trisaccharide intermediate 2

[0137] 1. Preparation of monosaccharide intermediate F-1

[0138] Compound 17 in MeOH / K 2 CO 3The acetyl group is removed under alkaline conditions, and then a benzylidene reagent is added and adjusted to acidity with p-toluenesulfonic acid monohydrate to obtain intermediate 18. Intermediate 18 is refluxed with dibutyltin oxide in anhydrous methanol, and then cesium fluoride and 4-methoxybenzyl chloride are added to selectively protect the 3-hydroxyl group to obtain intermediate 19; compound 19 reacts with benzyl bromide under neutral silver oxide conditions to obtain compound 20. Compound 20 removes the PMB protecting group under the action of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to obtain compound 21; using dichloromethane as a solvent, triethylamine and acetic anhydride are added dropwise, and DMAP is used as a catalyst to obtain compound 22. Compound 22 is dissolved in dichloromethane, and trifluoroacetic acid is added to remove the benzylidene to obtain compound 23. Under ice bath conditions, an equal amount of benzoyl chloride is added to selectively protect the 6-position, and the intermediate is purified. Under silver oxide conditions, the benzyl group is moved to the 4-position to obtain the monosaccharide compound F-1. The reaction process is as follows:

[0139]

[0140] a)1)K 2 CO 3 , MeOH, 25℃-rt; 2) Benzaldehyde Dimethylacetal, Toluene-4-sulfonic acid, DMF, two-step yield 78%;

[0141] b)1)Bu 2 SnO, MeOH, reflux; 2) PMBCl, CsF, DMF, 90℃, two-step yield 67%; c) Ag 2 O,BnBr,DCM,88%; d)DDQ,DCM,H 2 O, 71%; e) Et 3 N,Ac 2 O, DMAP, DCM, 90%; f) TFA, DCM, 79%; g) 1) BzCl, Et 3 N,THF,0℃-rt; 2)Ag 2 O, BnBr, DCM, two-step yield 72%.

[0142]

[0143] The specific steps are as follows: 10g of compound 17 (30.21mmol) was dissolved in methanol, 12.51g of potassium carbonate (90.63mmol) was added in batches, and the mixture was heated to 25°C. A new spot was generated by TLC monitoring, and the crude product was directly used for the next step. The obtained solid was dissolved in DMF, placed on a stirrer, 9.06ml of benzaldehyde dimethyl acetal (60.42mmol) was added, and the pH was adjusted to 2-3 with p-toluenesulfonic acid monohydrate. The reaction was allowed to proceed overnight. The next day, the raw material on the plate disappeared, and the reaction was stopped. The reaction was quenched with triethylamine. After the solvent was drained, it was extracted with DCM and saturated NaCl system, the organic phase was collected, dried, and separated by column chromatography to obtain a white solid 18 (5.92g, two-step reaction yield 78%).

[0144] 1 H NMR (400 MHz, CDCl 3 )δ7.49-7.42(m,4H),7.36(m,3H),7.14(m,2H),5.50(s,1H),4.54(d,J=9.7Hz,1H),4.35 (dd,J=10.4,3.9Hz,1H),3.83-3.73(m,2H),3.49-3.38(m,3H),2.92(s,1H),2.36(s,3H).

[0145] 13 C NMR (100 MHz, CDCl 3 )δ138.95,137.02,133.78,130.01,129.44,128.48,127.44,126.43,102.02,88.80,80.35,74.65,72.62,70.62,68.70,29.81,21.30.

[0146]

[0147] 10 g of compound 18 (26.73 mmol) and 9.95 g of dibutyltin oxide (40.107 mmol) were dissolved in 30 mL of anhydrous methanol, placed on a magnetic stirrer, and refluxed at 60 ° C for 6 h. The reaction system gradually changed from a suspension to a clear liquid. After the reaction liquid was slowly cooled to room temperature, it was concentrated under reduced pressure. After removing the anhydrous methanol, the reactant was directly dissolved in 20 mL of dry DMF without purification. 7.22 mL of p-methoxybenzyl chloride (53.47 mmol) and 4.87 g of cesium fluoride (32.086 mmol) was added to the reaction system, and it was refluxed and stirred at 90 ° C overnight. After the reaction of the raw material disappeared completely under TLC monitoring, the reaction solution was slowly cooled to room temperature, and then concentrated under reduced pressure using an oil pump to remove the reaction solvent DMF. The obtained oil was redissolved with EA, and then the reaction solution was extracted with a saturated NaCl solution. The forward extraction was three times and the reverse extraction was two times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the hydrated sodium sulfate, and the organic phase was concentrated under reduced pressure using a water pump. Column chromatography was used to separate and obtain compound 19 (9.906 g, 67%).

[0148] 1 H NMR (400 MHz, CDCl 3 )δ7.49(m,2H),7.43(m,2H),7.40-7.36(m,3H),7.28(m,2H),7.13(m,2H),6.87-6.83(m,2H),5.56(s,1H),4.88(d,J=11.1Hz,1H),4.71(d, J=11.1Hz,1H),4.56(d,J=9.6Hz,1H),4.38(dd,J=10.4,4.9Hz,1H),3.79(m,4H),3.64(m,2H),3.51-3.44(m,2H),2.55(s,1H),2.35(s,3H).

[0149] 13 C NMR (100 MHz, CDCl 3 )δ159.52,138.87,137.35,133.97,130.39,129.94,129.14,128.39,127.33,126.13,1 14.02,101.35,88.61,81.32,81.28,77.36,74.59,72.14,70.86,68.76,55.38,21.30.

[0150]

[0151] 9.90 g of compound 19 (26.68 mmol) was dissolved in redistilled dichloromethane, 18.51 g of silver oxide (80.04 mmol) was quickly weighed in batches and added to the reaction system. The system was cooled to 0°C in an ice bath, and 9.63 ml of benzyl bromide (80.04 mmol) was added dropwise. After the reaction of the raw material disappeared completely as monitored by TLC, the silver oxide was filtered out, the organic phase was concentrated, and the separation was performed by column chromatography to obtain compound 20 (10.82 g, yield 88%).

[0152] 1 H NMR (400 MHz, CDCl 3 )δ7.55-7.30(m,14H),7.12(m,2H),6.82(m,2H),5.59(s,1H),4.86(m,3H),4.71(t,J=10.0Hz,2H),4 .39(dd,J=10.6,5.0Hz,1H),3.80(d,J=6.0Hz,5H),3.67(t,J=9.4Hz,1H),3.46(m,2H),2.35(s,3H).

[0153] 13 C NMR (100 MHz, CDCl 3 )δ159.44,138.30,137.44,133.17,130.59,129.95,129.89,129.25,129.09,128.52,128.38,128.29,127.96, 126.13,113.93,101.24,88.60,82.81,81.62,80.49,77.36,75.92,75.09,70.35,68.85,55.38,29.82,21.26.

[0154]

[0155] 4.43 g of compound 20 (9.62 mmol) was dissolved in 30 mL of a mixed solution of dichloromethane and water in a volume ratio of 10:1, 3.27 g of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (14.44 mmol) was weighed and added to the reaction bottle. The reaction was carried out for 3 h at room temperature. After the raw material spot completely disappeared as monitored by TLC, the red precipitate generated in the reaction solution was filtered out, and DCM was added to the reaction system. The mixture was extracted with DCM and saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain compound 21 (2.34 g, 71%).

[0156] 1 H NMR (400 MHz, CDCl 3)δ7.49-7.34(m,12H),7.15(m,2H),5.52(s,1H),4.95(d,J=10.9Hz,1H),4.82(d,J=10.9Hz,1H),4.69(d,J=9.7Hz,1H),4.36(dd,J= 10.5, 4.9Hz, 1H), 3.90 (t, J = 8.8Hz, 1H), 3.77 (t, J = 10.1Hz, 1H), 3.50 (d, J = 9.3Hz, 1H), 3.46–3.39 (m, 2H), 2.50 (s, 1H), 2.37 (s, 3H).

[0157] 13 C NMR (100 MHz, CDCl 3 )δ138.32,138.22,137.09,133.01,132.52,129.95,129.92,129.87,129.38,129.32,129.11,128.76,128.67,1 28.45,128.34,128.27,128.15,126.40,101.90,88.33,80.87,80.41,77.36,75.56,75.53,70.15,68.77,21.27.

[0158]

[0159] 2.34g of compound 21 (6.86mmol) was dissolved in dichloromethane, ice bath was added and the reaction bottle was fixed on the stirrer, 2.85ml of triethylamine (20.58mmol), 1.93ml of acetic anhydride (20.58mmol) and catalytic amount of dimethylaminopyridine were added, and the ice bath was removed after the addition was completed. After 3h, the plate was tapped, the raw material disappeared and a slightly less polar product was generated, and the reaction was stopped. The product was spin-dried and purified by column chromatography to obtain a white solid 22 (2.3g, 90%).

[0160] 1 H NMR (400 MHz, CDCl 3 )δ7.47-7.42(m,4H),7.36-7.32(m,8H),7.15(m,2H),5.47(s,1H),5.39(t,J=9.2Hz,1H),4.93(d,J=11.0Hz,1H),4.77(d,J=9.7H z,1H),4.64(d,J=10.9Hz,1H),4.37(dd,J=10.5,4.8Hz,1H),3.79(d,J=10.2Hz,1H),3.61-3.51(m,3H),2.37(s,3H),1.96(s,3H).

[0161] 13 C NMR (100 MHz, CDCl 3 )δ169.87,138.52,137.79,137.04,133.12,130.02,129.18,128.60,128.36,128.27,128. 07,126.27,101.47,88.86,79.32,78.71,77.36,75.32,74.45,70.45,68.75,21.32,21.04.

[0162]

[0163] 2.3 g (6.00 mmol) of compound 22 was dissolved in dichloromethane solution, and 9.8 ml of trifluoroacetic acid (60.00 mmol) was slowly added. The reaction was stopped when the white solid disappeared and the reaction solution became clear. Sodium bicarbonate was adjusted to neutral, and then extracted with EA and saturated NaCl system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and dried by spin drying, and purified by column chromatography to obtain a white solid 23 (1.41 g, 79%).

[0164] 1 H NMR (400 MHz, CDCl 3 )δ7.45-7.41(m,2H),7.36-7.28(m,5H),7.13(m,2H),5.03(t,J=9.2Hz,1H),4 .89(d,J=11.0Hz,1H),4.68(d,J=9.8Hz,1H),4.59(d,J=11.0Hz,1H),3.89(dd ,J=12.0,3.2Hz,1H),3.78(dd,J=12.0,4.8Hz,1H),3.59(t,J=9.6Hz,1H),3.4 5(t,J=9.3Hz,1H),3.39-3.33(m,1H),2.66(s,2H),2.34(s,3H),1.99(s,3H).

[0165] 13 C NMR (100 MHz, CDCl 3 )δ172.15,138.31,137.87,132.78,129.97,129.26,128.56,128.10,1 28.02,87.82,79.70,79.37,78.62,75.25,69.92,62.48,21.24,21.08.

[0166]

[0167] 1.41g of compound 23 (4.78mmol) was dissolved in dry tetrahydrofuran, 0.73ml of triethylamine (5.26mmol) was added under ice bath condition, and then 0.61ml of benzoyl chloride (5.26mmol) was added dropwise. After a new spot was generated by TLC monitoring, an acidic resin was added to neutralize the triethylamine, and the mixture was filtered and dried to obtain a crude product of Bz at the 6th position, which was directly used in the next step. The intermediate was dissolved in redistilled dichloromethane, 3.3g of silver oxide (14.34mmol) was weighed in batches and added to the reaction system, the system was cooled to 0°C by ice bath, and 1.7ml of benzyl bromide (14.34mmol) was added dropwise. After the reaction of the raw materials disappeared completely by TLC monitoring, the silver oxide was filtered off, the organic phase was concentrated, and the compound F-1 (1.68g, two-step yield 72%) was obtained.

[0168] 1 H NMR (400 MHz, CDCl 3 )δ8.07-8.01(m,2H),7.62(d,J=7.4Hz,1H),7.46(dd,J=7.8,6.6Hz,5H),7.34-7.25(m,7H),7.22( dd,J=8.2,6.2Hz,2H),6.94(dd,J=8.2,6.1Hz,2H),5.35(d,J=9.2Hz,1H),4.90(d,J=11.0Hz,1H), 4.65(dd,J=16.7,5.9Hz,2H),4.55(dd,J=11.3,3.1Hz,3H),4.46(dd,J=12.0,5.4Hz,1H),3.72(dd d,J=9.6,5.3,2.0Hz,1H),3.60(d,J=9.5Hz,1H),3.45(d,J=9.4Hz,1H),2.27(s,3H),1.87(s,3H).

[0169] 13 C NMR (100 MHz, CDCl 3 )δ169.79,166.09,138.09,137.80,137.18,133.17,129.95,129.84,129.70,128.94,128.58,128.48,128.44,128 .33,128.13,128.09,127.91,87.47,78.74,77.41,77.09,76.91,76.77,76.26,74.76,74.59,63.50,21.18,21.02.

[0170] 2. Preparation method of fully protected trisaccharide intermediate 2

[0171] The synthesized monosaccharide intermediate F-1 is glycosylated and coupled with the disaccharide intermediate 4-1 synthesized according to a known literature method to generate a fully protected trisaccharide 2 under the action of trimethylsilyl.

[0172]

[0173] Preparation before the reaction: Use a microwave oven to bake the molecular sieve to remove water, and put the reaction bottle and magnet into an oven to dry.

[0174] Take out the two bottles after half an hour of baking, put the magnet in your pocket, and quickly add the baked Molecular sieve (2.5001g), connect the double-row tube, turn on the oil pump, and connect the bottle firmly with strong suction force to ensure the airtightness of the device. Then use a torch to bake the reaction bottle, and keep shaking the molecular sieve and knocking the reaction bottle until the molecular sieve becomes soft, smooth, and non-sticky. Close the oil pump valve, put in the magnet, plug the stopper, open the oil pump valve, and continue baking with a torch for 10s. Fix the reaction bottle on the iron frame, open the argon valve, and ventilate it three times. After the ventilation is completed, turn off the oil pump, and the preparation before the reaction is completed. Dissolve 100mg donor F-1 (0.20mmol) and 173mg acceptor 4-1 (0.20mmol) in redistilled dichloromethane, slowly inject into the reaction bottle with a syringe, turn on stirring, and balance for 40min. Pour anhydrous ethanol and liquid nitrogen into a Dewar flask and adjust to -40°C to cool the reaction flask. After 15 minutes, add 138 mg N-iodosuccinimide (0.60 mmol), add a catalytic amount of trifluoromethanesulfonic acid trimethylsilyl grease with a microinjector, and continue cooling. After 1 hour, the spot plate reaction is complete, and triethylamine is added to quench, and the argon gas is turned off for post-treatment. Use a funnel equipped with a section of silica gel for suction filtration, rinse with ethyl acetate several times, concentrate, and purify by column chromatography to obtain a fully protected trisaccharide compound 2 (183 mg, 69%).

[0175] 1H NMR (400MHz, CDCl3) δ8.06-7.94(m,4H),7.59-7.33(m,7H),7.29(m,6H),7.25-7.19(m,11H),7.17-7.08(m,5H),7.08-7 .03(m,2H),5.56-5.44(m,2H),5.21(t,J=5.3Hz,1H),5.04(m,2H),4.85-4.71(m,3H),4.64(s,3H),4.60-4.51(m,4H),4. 44(dd,J=12.2,3.5Hz,1H),4.38(d,J=12.4Hz,1H),4.31(dd,J=12.3,3.6Hz,1H),4.23(m,2H),4.18-4.06(m,3H),4.02- 3.89(m,2H),3.72-3.62(m,4H),3.62-3.52(m,2H),3.38(dd,J=10.1,3.5Hz,1H),3.26(s,3H),2.10(s,3H),1.89(s,3H).

[0176] 13 C NMR (100MHz, CDCl3) δ170.95,169.98,169.67,166.19,165.67,155.89,138.49,137.90,137.72,137.60,136. 40,133.25,130.18,129.96,129.72,129.27,128.58,128.53,128.43,128.30,128.26,128.23,128.01,127.97 ,127.85,127.60,127.52,127.40,98.92,98.70,98.43,78.49,77.36,76.56,76.41,76.26,75.52,74.51,74.41,73.52,73.22,72.69,71.12,70.99,69.80,68.99,66.98,63.11,62.22,55.33,54.44,52.34,21.14,20.95.

[0177] Example 2 Preparation of Compound TSC19

[0178]

[0179] Deprotection

[0180] 90 mg of fully protected trisaccharide 2 was dissolved in an appropriate amount of tetrahydrofuran, and 1 mL of 1.25 N LiOH solution and 2.1 mL of 30% H2 O 2 Solution, fix the reaction bottle on the stirrer, after 12h, add 2.2mL anhydrous methanol and 1.2mL 4N NaOH solution, continue the reaction for 12h, spot the plate to find a thin and long product spot, adjust the pH of the reaction solution to 5-6 with 6N HCl, extract with DCM and saturated NaCl solution system, collect the organic phase, dry with anhydrous sodium sulfate, filter, and spin dry. Column chromatography separation (DCM: MeOH = 15: 1 → 10: 1) to obtain the deprotected compound (40mg, 58%).

[0181] Oxysulfonation

[0182] Before the reaction, place the 25 mL flask and magnet to be used in an oven and bake for 30 min. Keep the flask and magnet dry. Weigh 220 mg SO according to 20 equivalents per equivalent of hydroxyl. 3 ·NMe 3 (1.58mmol) was added into the bottle and the stopper was plugged tightly. 20mg of the deprotected trisaccharide compound (0.015mmol) was dissolved in as little anhydrous DMF as possible, and the solution was added dropwise into the reaction bottle with a syringe. The reaction was stirred continuously for 36h at 60°C and the solvent was dried up. The oil was redissolved with methanol and column chromatography was performed using a Sephadex LH-20 gel column. A mixed solution of dichloromethane and methanol in a volume ratio of 1:1 was used as the eluent to collect the sugar-containing components. After the solvent was dried up, methanol was used as the eluent to pass through a Dowex-50-WX4-Na+ column to exchange it into a sodium salt. The sugar-containing components were collected and concentrated to obtain the oxysulfonated compound (24mg, 87%).

[0183] Hydrogenation and nitrogen sulfonation

[0184] 24 mg of the oxysulfonated compound (0.017 mmol) was dissolved in 3 mL of methanol and an equal volume of H 2 O, then weigh 20 mg of palladium carbon and place it in a reaction bottle. After reacting in a 4 atm hydrogen atmosphere for 24 hours, use an organic phase filter to filter out the palladium carbon. The solution is colorless. The filtrate is dried by spin drying. NMR shows no peak in the aromatic region, and the next step can be continued. The hydrogenated product is dissolved in 4 mL H 2 O, add 2N NaOH solution to adjust the pH of the reaction solution to 9-10, weigh SO 3 Py (53 mg, 0.33 mmol) was divided into 4 equal portions and added to the reaction solution at intervals of 30 min each time. After the addition was completed, the reaction solution was stirred for 4 h at room temperature. During the entire addition and reaction period, the pH of the reaction solution was constantly monitored to maintain it between 9 and 10. After the reaction time was reached, the pH was neutralized to 7-8 with hydrochloric acid, and then Sephadex G-25 gel column chromatography was performed to obtain H 2O was used as the eluent to collect the sugar-containing components, and H 2 O was used as the eluent for Dowex-50-WX4-Na+ column exchange to exchange it into sodium salt, the sugar-containing components were collected, and the solvent was concentrated to obtain the final compound TSC19 (15 mg, two-step yield was 84%).

[0185] 1 H NMR (400 MHz, D 2 O)δ5.26(d,J=3.8Hz,1H),5.17(s,1H),5.01(d,J=3.5Hz,1H),4.43(t,J=9.7Hz,1H),4.33(m,3H),4.27(s,1H),4.21-4.18(m,1H),4. 16-4.12(m,1H),3.99(m,3H),3.82-3.75(m,1H),3.73(d,J=9.6Hz,1H),3.71-3.62(m,3H),3.41(s,3H),3.27(dd,J=10.2,3.6Hz,1H).

[0186] 13 C NMR (100MHz, D 2 O) δ174.96,99.21,98.13,94.97,81.69,77.15,74.21,72.02,69.89,69.79,69.64,68.45,68.13,67.40,66.76,66.20,65.18,57.73,55.32.

[0187] The following experiments were performed using the prepared TSC19.

[0188] Example 3 The therapeutic effect of TSC19 in multiple sclerosis models

[0189] 1.1 Construction of EAE (Experimental Autoimmune Encephalomyelitis, EAE) model in mice:

[0190] Preparation of MOG (myelin oligodendrocyte glycoprotein) solution: dissolve MOG35-55 polypeptide powder in PBS in a clean bench to a concentration of 2 mg / mL; preparation of CFA (complete Freund's adjuvant) solution: add 7 mg of Mycobacterium tuberculosis to complete Freund's adjuvant to a concentration of 8 mg / mL; mix and emulsify MOG and CFA solutions: take two 10 mL syringes, mix MOG solution and CFA solution in a 1:1 volume ratio to fully emulsify them. At this time, the MOG concentration in the syringe is 1 mg / mL; the CFA concentration is 4 mg / mL.

[0191] Ten C57BL / 6 mice were injected intraperitoneally with chloral hydrate to fully anesthetize the mice. After the mice were fully anesthetized, four points on both sides of the spine of each mouse were subcutaneously injected with an emulsion of MOG and CFA, totaling 0.2 mL, and about 50 μL at each site.

[0192] PTX (pertussis toxin) injection: 200 ng of PTX was injected intraperitoneally to each mouse, and 48 hours later, 200 ng of PTX was injected intraperitoneally again.

[0193] The body weight and behavioral scores (Kono 5-point method) of mice in the control group (normal mice, given an equal volume of solvent), model group and treatment group were recorded: after modeling, the body weight of mice was measured and recorded daily, and clinical scores were performed daily for 36 days.

[0194] Scoring criteria: 0 points: normal; 0.5 points: only the tip of the tail of the mouse droops; 1 point: tail weakness or staggering gait; 1.5 points: tail weakness with staggering gait; 2 points: incomplete paralysis of both hind limbs or complete paralysis of one hind limb; 2.5 points: complete paralysis of one hind limb with incomplete paralysis of the other hind limb; 3 points: complete paralysis of both hind limbs; 3.5 points: paralysis of both hind limbs with weakness of one forelimb; 4 points: quadriplegia; 5 points: death.

[0195] The main pathological features of EAE mice are inflammation accompanied by demyelination, which manifests itself in the loss of muscle coordination, movement disorders, paralysis, etc. After TSC19 treatment, movement improved, weight was restored, and clinical scores showed a downward trend. Figure 3 As shown in the figure, PBS was subcutaneously administered to the model group on day 8 of immune induction, and TSC19 was administered to the treatment group for 26 consecutive days. The mice in the model group began to show clinical symptoms on the 12th day of modeling, and the mice in the TSC19 treatment group showed clinical symptoms on the 14th day after modeling. The clinical score of the mice in the model group reached a maximum of 4.24 points on the 19th day, and then gradually eased, and the score gradually decreased. On the 25th day, it dropped to 3.08 and then gradually worsened. After treatment, the score of the mice in the TSC19 treatment group reached a maximum of 2.48 on the 20th day, and began to gradually ease on the 21st day, and dropped to 0.98 on the 31st day. Throughout the process, the scores of the mice in the model group were higher than those in the TSC19 treatment group, and the difference between the two groups of data was statistically significant (P<0.0001). TSC19 can delay the onset of EAE symptoms and effectively alleviate the clinical symptoms of EAE mice.

[0196] The experimental results of the weight of mice in each group are as follows Figure 4As shown in the figure, the weight of normal mice fluctuated slightly for 36 days but showed an upward trend. The weight of mice in the model group and the TSC19 treatment group decreased significantly on the first day after immunization, with the normal group increasing by 100.5%, the model group by 97.0%, and the TSC19 treatment group by 97.5%. Starting from the second day, the weight of the model group and the treatment group increased until the seventh day. During this period, there was no significant difference between the weight of the two groups of mice and the normal group. The weight of mice in the model group reached the highest value of 107.8% on the seventh day of immune induction. From the eighth day, the mice in the model group showed clinical symptoms and their weight also decreased. As the disease progressed, the weight of mice in the model group continued to decrease, and on the 25th day of immune induction, it dropped to the lowest value of 85.8%, which was 24.5% different from normal mice. For the treatment group, after TSC19 treatment started on the 8th day, the weight of the mice increased, reaching a maximum of 107.4% on the 10th day. Thereafter, the weight of the mice in this group decreased slightly, and on the 16th day the weight of the mice in this group dropped to the lowest, which was 101.5% of the initial body weight, a difference of 4.3% from the weight of the mice in the normal group. Afterwards, the weight of the mice in this group slowly stabilized.

[0197] 1.2ELISA cytokine detection

[0198] Grind the brain, spinal cord and spleen of each group of mice and add samples to the standard wells, test sample wells and blank wells according to the instructions. Take a suitable sealing film to cover the enzyme-labeled coated plate, place it in a 37°C constant temperature incubator and incubate for 60 minutes; take out the coated plate, carefully peel off the sealing film, and carefully pour out all the liquid; fill each well with diluted washing solution, let it stand for 30 seconds, pour out the washing solution, invert the coated plate and pat it dry with toilet paper, and repeat 5 times; then add the color developer, shake it gently to mix, wrap it with tin foil, and place it in a 37°C constant temperature incubator to develop color for 15 minutes away from light; after the color development is completed, add 50 μL of stop solution to each well to stop the reaction, and the liquid turns from blue to yellow; measure the absorbance (OD value) at 450nm within 15 minutes, and adjust it to zero with a blank well; calculation: use the standard concentration and OD value as the horizontal and vertical coordinates, respectively, draw a standard curve, and obtain the linear regression equation of the standard curve. Substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to obtain the actual concentration of the sample.

[0199] In the early stages of multiple sclerosis, activated microglia and macrophages can release pro-inflammatory factors (such as IL-6, IL-23 and TNF-α) and chemokines (such as CCL-2, CCL-5 and CCL-22), which further activate and recruit inflammatory cells such as macrophages and T lymphocytes to attack the CNS, aggravating nerve demyelination. Figure 5 As shown, TSC19 could significantly reduce the levels of pro-inflammatory factors (TNF-α and IFN-γ) and increase the levels of anti-inflammatory factors (IL-4) in the brain, spinal cord and spleen.

[0200] 1.3 Histological observation

[0201] The mice were killed on the 36th day after immune induction, and the animal hearts were perfused with PBS containing 4% paraformaldehyde. The brain and spinal cord were then frozen and sectioned for histological observation using HE and LFB staining, respectively.

[0202] Hematoxylin-eosin (H&E) staining:

[0203] 1) Place the prepared paraffin sections in a 60°C oven for 60 minutes; 2) Dewaxing and hydrating: Place the dewaxing sections in xylene I (10 minutes) → xylene II (10 minutes) → anhydrous ethanol I (3 minutes) → anhydrous ethanol II (3 minutes) → 95% ethanol (3 minutes) → 85% ethanol (2 minutes) → 75% ethanol (2 minutes) → distilled water (3 minutes); 3) Stain the nucleus: Place the sections in hematoxylin staining solution, stain for 15 minutes, and then rinse with distilled water for 5 minutes; 4) Stain the cytoplasm: Place the sections in eosin staining solution, and after 5 minutes, rinse with distilled water for 3 seconds; 5) Dehydration, transparency and sealing: 70% ethanol (30 seconds) → 80% ethanol (30 seconds) → microscopic examination → 95% ethanol (2 minutes) → anhydrous ethanol I (2 minutes) → anhydrous ethanol II (2 minutes) → xylene I (3 minutes) → xylene II (5 minutes). After the transparent slices are dried, they are sealed with neutral gum; 6) Observe under a microscope and take pictures.

[0204] HE staining results Figure 6 and Figure 7 As shown, the spinal cord of normal mice was intact and the medulla was tight, and there was no obvious microglial aggregation in the brain and spinal cord. The model group had more inflammatory cell infiltration in the brain and spinal cord, disordered nerve fiber arrangement, and vacuolar changes. The inflammatory cell infiltration in the brain and spinal cord of mice in the TSC19 treatment group was significantly reduced, and the nerve fibers were neatly arranged, indicating that TSC19 can significantly reduce the inflammatory response in the spinal cord of EAE mice.

[0205] Laboratoire blue (LFB) myelin staining:

[0206] 1) Dewax the prepared paraffin sections with xylene and hydrate with ethanol gradient; 2) Preheat the fast blue dye solution in a 60°C water bath for 30 minutes, then immerse the sections in the fast blue dye solution at 37°C overnight; 3) Take out the sections and rinse them in 0.05% lithium carbonate solution; 4) Take out the sections and put them in 70% ethanol for differentiation, then rinse them with tap water for 1 minute; 5) Soak the sections in lithium carbonate differentiation solution for a few seconds again, then take them out immediately, rinse them with running water, examine them under a microscope, and observe the color separation effect; 6) If the color separation is complete, continue to rinse with running water for 5 minutes; 7) Dehydrate the sections with alcohol, make them transparent with xylene, and seal them with neutral gum; 8) Observe under a microscope and take pictures.

[0207] The results of spinal cord demyelination in each group were observed by LFB staining. Figure 8 As shown in the figure, the myelin sheaths of the spinal cord of normal mice are neatly arranged, with clear texture, high myelin density, and no demyelination. The spinal cords of mice in the model group showed demyelination, such as loose myelin structure, disintegration, and reduced density. The spinal cord myelin sheaths of the TSC19 treatment group were intact after administration, the vacuolar changes in the spinal cord were significantly reduced, and the demyelinating changes were significantly alleviated. This shows that TSC19 can reduce demyelination in the spinal cord tissue of EAE mice.

[0208] Example 4 TSC19 therapeutic experiment in pulmonary hypertension mouse model

[0209] 1.1 Establishment of mouse pulmonary hypertension model

[0210] HY+SU hypoxia model: 24 8-week-old male SPF wild-type WT (C57BL / 6) mice (weight 20-25 g) were randomly divided into two groups according to the principle of weight balance, namely the control group (8 mice) and the experimental group (16 mice). SU5416 was dissolved in the solvent DMSO, and the experimental group mice were given SU5416 (20 mg / kg) intraperitoneal injection once a week. After being placed in a hypoxia box for 21 days of continuous hypoxia (oxygen partial pressure 10%), the experimental group was randomly divided into two groups according to the principle of weight balance, namely HY+SU (subcutaneous injection of PBS with the same volume as the treatment group) and TSC19 group (TSC19 20 mg / kg / day, subcutaneous injection method). Each mouse was subcutaneously injected with TSC19 at a dose of 20 mg / kg every day for 1 week. The mice in the control group were raised in a standard sterile breeding environment for 4 weeks, and the same dose of DMSO was intraperitoneally injected at the same time point in the first 3 weeks, and the same dose of PBS was subcutaneously injected at the same time point in the last week.

[0211] HY hypoxia model: 24 8-week-old male SPF wild-type WT (C57BL / 6) mice (weight 20-25 grams). They were randomly divided into two groups according to the principle of weight balance, namely the Control group (8 mice) and the experimental group (16 mice). The mice in the Control group were raised in a standard sterile breeding environment, without treatment for the first three weeks, and subcutaneously injected with the same dose of PBS at the same time point in the last week. The mice in the experimental group were placed in a hypoxia box for continuous hypoxia (oxygen partial pressure 10%) for 21 days, and then randomly divided into two groups according to the principle of weight balance, namely the HY group (subcutaneously injected with the same volume of PBS as the treatment group) and the TSC19 group (TSC1920mg / kg / day, subcutaneous injection method). Each mouse was subcutaneously injected with TSC19 at a dose of 20mg / kg every day for 1 week.

[0212] 1.2 Experimental Results

[0213] 1.2.1 TSC19 can effectively alleviate right ventricular function damage and improve hemodynamic parameters in both HY+SU and HY models

[0214] Right ventricular free wall thickness (RVFWT) is related to right ventricular hypertrophy, tricuspid annular plane systolic excursion (TAPSE) is a parameter of right ventricular systolic function, and pulmonary acceleration time (PAT) and pulmonary acceleration time (PAT) / pulmonary ejection time (PET) are good parameters reflecting right ventricular afterload.

[0215] The above indicators of mice were detected using an ultra-high-resolution small animal ultrasound imaging system (High-Resolution In Vivo Imaging System).

[0216] The experimental results are as follows Fig. 9 and 10 As shown, the thickness of the right ventricular free wall in the HY+SU and HY-induced mouse models was significantly thickened, the tricuspid annular plane systolic excursion was reduced, and the pulmonary artery acceleration time (PAT) / pulmonary ejection time (PET) ratio was reduced. TSC19 treatment can effectively improve right ventricular hypertrophy, right ventricular function damage and hemodynamic parameters.

[0217] 1.2.2 TSC19 can effectively improve the cardiopulmonary tissue damage in both HY+SU and HY models

[0218] After one week of administration according to the model in 1.1, the mice were killed, and the lung tissue and heart of the mice were quickly removed and rinsed with normal saline to obtain bloodless lung tissue and heart. The right upper lobe of the lung was separated and immersed in 4% formalin. After fixation at room temperature for 24 hours, the lung tissue was dehydrated, embedded and sliced. Subsequently, the lung tissue was stained with HE, and the heart tissue was stained with HE and Masson.

[0219] Lung test results Fig.11 As shown in the lung HE results, the pulmonary vascular wall of the HY+SU group or the HY group was significantly thickened and the lumen became narrower compared with the Control group, indicating that the pulmonary vascular remodeling occurred to a certain extent. The pulmonary vascular wall of the TSC19 group was thinned to a certain extent, and the lumen diameter was enlarged, indicating that the compound can effectively improve the pulmonary vascular remodeling phenomenon in mice with pulmonary hypertension.

[0220] The results of the heart test were as follows Fig.12As shown in the figure, from the heart HE and Masson results, it can be seen that the myocardial cells in the myocardial tissue of the mice in the Control group are neatly arranged, uniform in size, with normal intercellular matrix and no overall damage, while the myocardial cells of the mice in the HY+SU group or HY group are uneven in size and irregularly arranged, with aggravated cardiac fibrosis and obvious myocardial fiber rupture, indicating that the heart tissue has been damaged to a certain extent. After treatment, the TSC19 group had a certain degree of improvement, indicating that the compound has a good improvement effect on the hearts of the two pulmonary hypertension model mice.

[0221] Example 5 TSC19 therapeutic experiment in DSS-induced mouse inflammatory bowel disease model

[0222] 1.1 Establishment of mouse IBD model

[0223] Reagents: Solution preparation: 3% DSS stock solution, weigh 9 g of DSS, add 300 mL of sterile drinking water, stir to completely dissolve, dispense into the animal's drinking water bottle, and change it every two days.

[0224] Animals: 25 SPF C57 mice weighing 16-18g, 6-8 weeks old, male mice. The ambient temperature was 18 to 22°C, and the ambient humidity was 50 to 60%. They were kept in an SPF room, 5 mice per cage, and the bedding and feed were changed in time, and free food and water were guaranteed.

[0225] Methods: 25 mice were randomly divided into a control group (5 mice) and an experimental group (20 mice) according to the principle of weight balance. The mice in the control group drank normal drinking water; the mice in the experimental group drank 3% DSS aqueous solution, and 3% DSS was replaced every two days. The body weight and mouse status were recorded. When the body weight dropped by 10% or blood in the stool appeared, that is, starting from the 4th day, the experimental group was randomly divided into a DSS group (5 mice) and a DSS+TSC19 group (5 mice) according to the principle of weight and clinical score balance, and the mice without disease were discarded. DSS+TSC19 was subcutaneously administered, and 3% DSS was replaced with a 1% DSS maintenance dose to maintain the symptoms of inflammatory bowel disease. When the body weight no longer dropped significantly and blood in the stool appeared after administration, the observation was terminated.

[0226] Grouping: 20 mice were divided into 4 groups. The specific grouping and positive drug administration were as follows:

[0227] mark Number of mice DSS induction amount (%) DSS maintenance amount (%) drug Control 5 0 0 / DSS 5 3 1 / DSS+TSC19 5 3 1 0.4mg / piece

[0228] Observation indicators: Observe the general conditions of mice such as eating, activity, and hair every day, weigh the animals every day, observe the characteristics of the mice's feces and fecal occult blood, bleeding, and evaluate the severity of colitis. The disease activity is scored according to the principles in the following table. The disease activity index (DAI) score = weight loss score + stool characteristics score + fecal occult blood score, and the disease activity index score of each mouse is calculated.

[0229]

[0230]

[0231] Post-processing steps:

[0232] 1. Weigh the mice in each group;

[0233] 2. Dissect and separate the spleen, take photos for comparison and measure the weight of the spleen in each group;

[0234] 3. Separate the colon and cecum by separating them from the small intestine at the ileocecal region and the anus at the distal end of the rectum. At this point, take a representative mouse from each group or each group and take a gross picture of the intestine from the cecum to the rectum;

[0235] 4. Measure the length of the colon. Colitis can increase edema and shorten the length of the entire colon.

[0236] 5. Then, the colon was separated from the cecum (ileocecal junction) and rapidly flushed with cold PBS using a 5-10 ml syringe (18-G, 3-in. feeding needle) to remove feces and blood; after flushing with PBS, the colon was weighed.

[0237] 6. Prepare colon tissue specimens for histological examination.

[0238] 1.2 Experimental Results

[0239] 1.2.1TSC19 can effectively alleviate the weight loss of DSS IBD model mice

[0240] The experimental results are as follows Fig.13 As shown in the figure, the weight of mice in the Control group gradually increased over time; the weight of mice in the DSS group began to drop sharply from the 4th day, and the weight loss exceeded 20% on the 8th day, which was considered as death (the mice were euthanized); compared with the DDS group, the weight of mice in the drug-added group began to slowly decrease from the 4th day until the 6th day, and the overall weight was higher than that of the DSS group. From the 7th day, the weight of mice in the DSS+TSC19 group showed an upward trend. Comparison of the weight of mice in each group on the 8th day ( Fig.13 The right figure shows that TSC19 can effectively alleviate the weight loss of mice after DSS stimulation.

[0241] The disease activity index score of each mouse was calculated according to the DAI score = weight loss score + stool characteristics score + stool occult blood score. The experimental results are shown in Fig.14 As shown in the figure, the DAI score is proportional to the severity of the disease. The DAI score of the DSS group was the highest, and the DSS+TSC19 group was close to the Control group, indicating that TSC19 can effectively alleviate the symptoms of colitis. The DAI scores of mice in each group on the 8th day showed ( Fig.14 Right), TSC19 can effectively alleviate the aggravation of the disease in mice after DSS stimulation.

[0242] 1.2.2TSC19 can effectively improve pathological damage in DSS IBD model mice

[0243] The damage was further observed by HE staining of colon tissue. Fig.15 As shown, the colon mucosal epithelium of mice in the Control group was intact, the crypt structure was intact and regularly arranged, and there was no inflammatory cell infiltration and ulcers; the colon mucosa of the DSS model group was destroyed, the crypts were reduced, and there was inflammatory cell infiltration and ulcers; compared with the DSS group, the colon mucosal destruction in the DSS+TSC19 group was significantly improved, and the crypt structure was more complete.

[0244] 1.2.3TSC19 can effectively improve the degree of colon inflammation in DSS IBD model mice

[0245] In addition to the three indicators of weight loss, DAI score and colon tissue HE staining that can reflect the disease condition, the length and weight of the colon and the weight of the spleen can also be used as evaluation criteria for IBD inflammation. Colitis will increase tissue edema and shorten the length of the entire colon. Therefore, the colon length of mice with colitis should be shorter than that of normal mice in the control group. At the same time, the colon mass should also be larger than that of normal mice in the control group. In addition, spleen enlargement is also one of the signs of inflammation. We dissected the mice at the end of the model investigation, weighed the colon weight and spleen weight of each group of mice, and measured the colon length of each group of mice. The results are as follows: Fig.16 As shown in the figure, the colon weight, colon length and spleen weight of the TSC19-administered group were reduced, increased and decreased to varying degrees compared with the DSS group, indicating that TSC19 can effectively alleviate colon inflammation.

[0246] 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. A sulfonated trisaccharide or a salt thereof, which has a structure of formula A, wherein the three monosaccharides from left to right are represented by B, C, and D respectively: in: R2, R3, R4, R5 and R6 are the same or different and are independently selected from H or -SO3H; R7 is -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3H; the salt is a salt formed with a monovalent cation, a divalent cation and / or a trivalent cation; Preferably, the monovalent cation is selected from Na + , K + , Li + , NH4 + ; The divalent cation is selected from Ca 2+ , Cu 2+ , Zn 2+ , Fe 2+ ,Mg 2+ , Mn 2+ ; The trivalent cation is selected from Al 3+ , Fe 3+ .

2. The sulfonated trisaccharide or its salt according to claim 1, characterized in that In formula A, R2, R3, R4, R5 and R6 are the same and are -SO3H; or, R2, R3, R4, R5 and R6 are the same and are H; or, R2, R3 and R5 are the same and are H, and R4 and R6 are the same and are -SO3H; or, R2, R4 and R6 are the same and are -SO3H, and R3 and R5 are the same and are H; or, R2, R4, R5 and R6 are the same and are -SO3H, and R3 is H.

3. The sulfonated trisaccharide or its salt according to claim 1 or 2, characterized in that In formula A, R7 is -C1-3 alkyl, -C2-4 alkylene-NH2, -C2-4 alkylene-NHSO3H; Preferably, in formula A, R7 is methyl, ethyl, -C2H4NH2, -C2H4NHSO3H, -C3H6NH2 or -C3H6NHSO3H.

4. The sulfonated trisaccharide or its salt according to any one of claims 1 to 3, characterized in that The D sugar terminal group is in the α configuration; alternatively, the D sugar terminal group is in the β configuration.

5. The sulfonated trisaccharide or its salt according to claim 1, characterized in that In formula A, R2, R3 and R5 are the same, which is H; R4 and R6 are the same, which is -SO3H; R7 is -C1-5 alkyl, -C1-5 alkylene-NH2, -C1-5 alkylene-NHSO3H, and the salt is a salt formed with a monovalent cation, a divalent cation and / or a trivalent cation; Preferably, R7 is -C1-3 alkyl, -C2-4 alkylene-NH2, -C2-4 alkylene-NHSO3H, more preferably, R7 is methyl, ethyl, -C2H4NH2, -C2H4NHSO3H, -C3H6NH2 or -C3H6NHSO3H; Preferably, the salt is a salt formed with a monovalent cation, preferably the monovalent cation is selected from Na + , K + Alternatively, the salt is a salt formed with a divalent cation, preferably the divalent cation is Ca 2+ ; And / or, preferably, the terminal group of the D sugar is in the alpha configuration.

6. The sulfonated trisaccharide or its salt according to claim 1, selected from the following compounds:

7. Use of the sulfonated trisaccharide or its salt according to any one of claims 1 to 6 in the preparation of a medicament for treating multiple sclerosis, inflammatory bowel disease or pulmonary hypertension; Preferably, the multiple sclerosis is relapsing-remitting, secondary progressive, primary progressive or progressive relapsing; Preferably, the pulmonary hypertension is arterial pulmonary hypertension, pulmonary hypertension caused by left heart disease, pulmonary hypertension caused by hypoxia and / or lung disease, pulmonary hypertension caused by chronic thromboembolic pulmonary hypertension and other pulmonary artery obstructive diseases, or pulmonary hypertension caused by unknown and / or multiple factors; Preferably, the inflammatory bowel disease is ulcerative colitis; Preferably, the inflammatory bowel disease is Crohn's disease.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the sulfonated trisaccharide or its salt according to any one of claims 1 to 6; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

9. An intermediate having the following structure: Where Y is H, Na + , K + , Li + , NH4 + , preferably Y is H + Or Na + .

10. A method for preparing the sulfonated trisaccharide or its salt according to any one of claims 1 to 6, characterized in that: Using the fully protected trisaccharide intermediate 2 as the raw material, it is sequentially subjected to dehydroxylation, O-sulfonation, dehydroxylation, and finally N-sulfonation to obtain: In the fully protected trisaccharide intermediate 2, R 11 , R 21 , R 31 , R 41 , R 51 and R 61 may be the same or different, and independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, p-methoxybenzyl; preferably, wherein R 11 , R 31 , R 51 and R on D sugar 21 Same, R on B sugar 21 and R on D sugar 41 Same, R 61 and R on B sugar 41 The same; further preferably R 11 , R 31 , R 51 and R on D sugar 21 Same, both are benzyl, R on B sugar 21 and R on D sugar 41 Same, both are acetyl, R 61 and R on B sugar 41 The same, both are benzoyl; Y is H, Na + , K + , Li + , NH4 + , preferably Y is H or Na + .

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