Pharmaceutical use of trisaccharide structured compounds in pulmonary fibrosis

Abstract

This invention provides the use of a trisaccharide compound with a core structure of GlcN(1→4)IdoA(1→4)GlcNS in the preparation of a medicament for treating or preventing pulmonary fibrosis. This medicament can improve pulmonary inflammatory responses, reduce the degree of damage to lung tissue structure, improve or inhibit or delay the progression of pulmonary fibrosis, and ultimately improve and enhance pulmonary ventilation function in patients with pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a trisaccharide compound in the preparation of drugs for the treatment or prevention of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis, also known as interstitial pulmonary fibrosis or interstitial lung disease, is a serious lung disease characterized by thickening and hardening of lung tissue, leading to scarring and progressive loss of lung function. Its causes are diverse, including occupational and environmental exposures, medications and treatments, autoimmune diseases, infections, genetic factors, and lifestyle factors. Idiopathic pulmonary fibrosis (IPF) is the most common type, primarily affecting middle-aged and elderly men, and its pathological feature is common interstitial pneumonia. Symptoms of pulmonary fibrosis include dry cough, exertional dyspnea, and fatigue; in later stages, cyanosis and thickening of the fingertips may occur. The prognosis for IPF is poor, with a median survival of only 3-5 years after diagnosis. Currently, there is no specific treatment for pulmonary fibrosis; treatment mainly involves supportive therapies such as antifibrotic drugs, smoking cessation, pulmonary rehabilitation, and oxygen therapy. Some patients may require lung transplantation. There is currently no effective treatment for pulmonary fibrosis in clinical practice. Summary of the Invention

[0003] This invention provides the use of a trisaccharide compound of formula A, or a salt thereof, or a solvate thereof, in the preparation of a medicament for the treatment or prevention of pulmonary fibrosis, wherein the three monosaccharides of the trisaccharide compound of formula A, or a salt thereof, are represented from left to right by B, C, and D, respectively:

[0004]

[0005] in:

[0006] R1, R2, R3, and R4 may be the same or different, and are independently selected from H or -SO3H.

[0007] R5 is a -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3H.

[0008] The salt is a salt formed with a monovalent cation, a divalent cation, and / or a trivalent cation.

[0009] The monovalent cation is, for example, selected from Na. + K + Li + NH4 + etc.; the divalent cation is selected, for example, from Ca... 2+ Cu 2+ Zn 2+ Fe 2+ Mg 2+ Mn 2+etc.; the trivalent cations are selected, for example, from Al 3+ Fe 3+ Etc. Those skilled in the art will understand that when the compound forms a salt, the corresponding group in formula A is its anionic group, such as -COO. - 、 -SO3 - .

[0010] The compound of formula A is a compound with a single optical activity, namely Glc(1→4)IdoA(1→4)GlcNS, where the D sugar terminal group is α or β configuration.

[0011] In some embodiments of the present invention, in formula A, R1, R2, R3 and R4 are the same, all being H.

[0012] In some embodiments of the present invention, in formula A, R5 is -C1-3 alkyl, -C2-4 alkylene-NH2, or -C2-4 alkylene-NHSO3H. Preferably, R5 is methyl, ethyl, -C2H4NH2, -C2H4NHSO3H, -C3H6NH2, or -C3H6NHSO3H.

[0013] In some embodiments of the present invention, the salt is a salt formed with a monovalent cation, wherein the monovalent cation is selected from Na. + K + Li + NH4 + Preferably, the monovalent cation is selected from Na. + K + .

[0014] In some embodiments of the present invention, the salt is a salt formed with a divalent cation selected from Ca. 2+ Cu 2+ Zn 2+ Fe 2+ Mg 2+ Mn 2+ Preferably, the divalent cation is Ca. 2+ .

[0015] In some embodiments of the present invention, in formula A, R1, R2, R3, and R4 are the same, all being H; R5 is -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3H, and the salt is a salt formed with a monovalent cation, a divalent cation, and / or a trivalent cation. Preferably, R5 is -C1-3 alkyl, -C2-4 alkylene-NH2, or -C2-4 alkylene-NHSO3H; more preferably, R5 is methyl, ethyl, -C2H4NH2, -C2H4NHSO3H, -C3H6NH2, or -C3H6NHSO3H. Preferably, the salt is a salt formed with a monovalent cation selected from Na. + K + Li + NH4 + Preferably, the monovalent cation is selected from Na. + K + Alternatively, the salt is a salt formed with a divalent cation selected from Ca. 2+ Cu 2+ Zn 2+ Fe 2+ Mg 2+ Mn 2+ Preferably, the divalent cation is Ca. 2+ .

[0016] In one embodiment of the present invention, the structural formula of compound A is as follows, and is referred to as CV010 in the present invention.

[0017]

[0018] In one embodiment of the present invention, the sodium salt structure of compound A is as follows, referred to as CV016 in the present invention.

[0019]

[0020] According to the present invention, the compound of formula A is derived via a fully protected trisaccharide intermediate of formula E. The compound is obtained by sequentially removing a hydroxyl protecting group, O-sulfonation, optional azide reduction reaction, and finally N-sulfonation. Those skilled in the art will understand that after obtaining compound A, the corresponding salt can be obtained by salting it with a cation, for example, by exchanging compound A with a cation exchange resin to prepare its corresponding monovalent, divalent, or trivalent salt.

[0021] In the fully protected trisaccharide intermediate E, Rx can be an azide group, R 21 R 31 R 41 R 51 and R 61They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; preferably R. 21 R 31 and R 51 Same, R 41 and R 61 Same or different, and both are different from R 21 R 31 and R 51 Further optimization of R 21 R 31 and R 51 They are the same, both are benzyl, R 41 and R 61 Whether the groups are the same or different, they are independently selected from acetyl and benzoyl groups.

[0022] Preferably, in the reaction, all hydroxyl groups to be sulfonated are deprotected in one step, and after the hydroxyl O-sulfonation, all hydroxyl groups not to be sulfonated are deprotected in one step.

[0023] In a specific embodiment of the present invention, the present invention uses the aforementioned synthesis method to synthesize CV010 and CV016:R 21 R 31 R 41 R 51 and R 61 The definitions are the same as before, and the synthesis route is as follows:

[0024]

[0025] In one specific embodiment of the preparation of CV010 and CV016, R 21 R 31 and R 51 All are benzyl, R 41 For acetyl, R 61 The radical is benzoyl, and Y is selected from H and Na. + K + Li + NH4 + H or Na are preferred. + .

[0026] The fully protected trisaccharide intermediate E can be derived from the monosaccharide intermediate F. and disaccharide receptor 4 It is obtained through a glycosylation reaction.

[0027] Rx can be an azide group, R 21 R 31 R 41 R 51 and R 61They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; X is a leaving group suitable for reacting with other acceptors to form bonds between glycosides.

[0028] Preferably, X is a hydroxyl group, a thioalkyl group, a thioaryl group, a halogen, a trichloroiminoacetyl group, a phosphate ester, or a tert-butyldiphenylsilyloxy group.

[0029] Preferred R 21 R 31 and R 51 Same, R 41 and R 61 Same or different, and both are different from R 21 R 31 and R 51 Further optimization of R 21 R 31 and R 51 They are the same, both are benzyl, R 41 and R 61 Whether the groups are the same or different, they are independently selected from acetyl and benzoyl groups.

[0030] According to the present invention, the glycosylation reaction temperature is -80°C to -10°C. The reaction can be carried out under strong acid conditions, such as trifluoromethanesulfonic acid, TBSOTf, TMSOTf, etc.

[0031] In one embodiment of the present invention, the fully protected trisaccharide intermediate 1 is obtained by glycosylation of a monosaccharide intermediate 3 and a disaccharide acceptor 4, as shown in the following reaction formula:

[0032]

[0033] The disaccharide intermediate 4 can be obtained from the monosaccharide intermediate 6 and the monosaccharide intermediate 7, as shown in the following reaction formula:

[0034]

[0035] The monosaccharides 6, 7, and 4 disaccharide receptor used in the above preparation method can be prepared according to synthetic methods known in the art, such as: Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116; Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. ChemMedChem, 2014, 9, 1071–1080. The definitions of the functional groups in monosaccharides 6, 7, and 4 are the same as those of the corresponding functional groups described above.

[0036] Therefore, the present invention also provides the intermediates in the above-described synthesis method and their preparation methods.

[0037] A monosaccharide intermediate 3 has the following structure: Among them, R is preferred. 21 and R 31 All are benzyl, R 41 It is chloroacetyl, acetyl, benzoyl, or pivaloyl. In one embodiment of the invention, the monosaccharide intermediate 3 has R... 21 and R 31 All are benzyl, R 41 It is an acetyl group, which can be either α or β configuration, named 3-1, and has the following structural formula:

[0038] The reaction formula for its preparation method is as follows:

[0039]

[0040] A disaccharide intermediate 4 has the following structure: Among them, R is preferred. 21 and R 51 It is benzyl, R 41 and R 61 The same or different, selected from chloroacetyl, acetyl, benzoyl, or pivaloyl. In one embodiment of the invention, the R of the disaccharide intermediate 4... 21 and R 51 It is benzyl, R 41 For acetyl, R 61 It is benzoyl, named 4-1, and has the following structural formula:

[0041] Disaccharide intermediate 4-1 can be prepared using synthetic methods known in the art, such as Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. ChemMedChem, 2014, 9, 1071–1080.

[0042] A fully protected trisaccharide intermediate 1 has the following structural formula:

[0043]

[0044] Where R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl. In one specific embodiment of the invention, R 21 R 31 and R 51 All are benzyl, R 41 For acetyl, R 61 It is benzoyl.

[0045] The present invention also provides intermediates I, II and III.

[0046] The structure of compound I is as follows:

[0047] Among them, R 21 R 31 and R 51 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl.

[0048] The structure of compound II is as follows:

[0049] Among them, R 21 R 31 and R 51 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl, with Y selected from H and Na. + K + Li + NH4 + Preferably, Y is H or Na. + .

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

[0051] Where Y is selected from H and Na + K + Li + NH4 + Preferably, Y is H or Na. + .

[0052] Intermediates I, II, and III are generated from trisaccharide intermediate 1 through sequential dehydroxylation, sulfonation, and azide reduction, as shown in the following reaction formulas:

[0053]

[0054] The dehydroxyl protecting group, sulfonation, and azide reduction can all be carried out using reaction methods and conditions known in the art. In one embodiment of the invention, intermediate 1, under alkaline conditions, simultaneously removes R. 41 R 61 Intermediate I is obtained by reacting methyl ester with SO3·NMe3. In one embodiment of the invention, intermediate I is subjected to SO3·NMe3 to obtain O-sulfonated intermediate II. In one embodiment of the invention, intermediate II is subjected to catalytic hydrogenation to remove benzyl and Cbz groups, while azide is reduced to generate an amino group, to obtain intermediate III. In one embodiment of the invention, the amino group in compound III is sulfonated under SO3·Py to obtain compound CV010, which is then ion-exchanged with a sodium-type ion exchange resin to obtain compound CV016. The sodium ion exchange resin can be a resin known in the art, including but not limited to AmberliteIR120 Na + Dowex-50-WX4-Na + wait.

[0055] The compound of formula A of the present invention, or its salt or solvate thereof, can be prepared by referring to the method in Chinese patent application CN116854752A.

[0056] The inventors of this application constructed a mouse pulmonary fibrosis model using bleomycin. The study found that the compound of formula A of this invention, or its salt or its solvate, can improve the pulmonary inflammatory response in mice with pulmonary fibrosis, reduce the degree of damage to the lung tissue structure, improve or inhibit or delay the degree of pulmonary fibrosis, and ultimately improve and enhance the pulmonary ventilation function of the model mice.

[0057] Therefore, the compound of formula A of the present invention, or its salt or solvate, can improve the pulmonary inflammatory response, reduce the degree of damage to the lung tissue structure, improve or inhibit or delay the degree of pulmonary fibrosis, and ultimately improve and enhance the pulmonary ventilation function of patients with pulmonary fibrosis when treating or preventing pulmonary fibrosis.

[0058] Pulmonary fibrosis can be caused by various factors, including but not limited to: (1) Occupational and environmental factors, such as long-term inhalation of dust such as asbestos, coal, metal dust, smoke and toxic and harmful gases. Silicosis and pneumoconiosis are typical occupational lung diseases caused by occupational and environmental factors. (2) Smoking. (3) Drugs and radiation damage, such as antitumor drugs such as bleomycin, cyclophosphamide, mitomycin, and basil, antiarrhythmic drugs such as amiodarone and tocainide, and psychotropic drugs such as cocaine, can all cause pulmonary fibrosis. In addition, immunotherapy drugs such as PD-1 / PD-L1, tocilizumab, and targeted therapy drugs such as gefitinib can also cause pulmonary fibrosis. In addition, the inflammatory reaction caused by damage to the lung tissue in the radiation area after chest radiotherapy in patients with malignant tumors is also called radiation pneumonia. Generally, chronic radiation pneumonia manifests as pulmonary fibrosis, and symptoms appear 6-12 months after radiotherapy. (4) Connective tissue disease, which is an autoimmune disease that affects multiple systems and organs. Connective tissue diseases affecting the lungs can cause pulmonary fibrosis, and pulmonary fibrosis may also be the first manifestation of connective tissue diseases. Common connective tissue diseases that can affect the respiratory system include systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, dermatomyositis, and mixed connective tissue disease. (5) Pathogenic microbial infection, recurrent infections caused by viruses, bacteria, fungi, parasites, etc. The pulmonary fibrosis mentioned also includes idiopathic pulmonary fibrosis.

[0059] In one embodiment of the present invention, the pulmonary fibrosis is drug-induced pulmonary fibrosis.

[0060] The compound of formula A in this invention has a clear and highly effective therapeutic effect on pulmonary fibrosis, but has almost no anticoagulant activity, high stability, and no platelet factor 4 binding activity. Therefore, the risk of bleeding is extremely low while using it for therapeutic purposes. In addition, the structure of compound A is well-defined, which is beneficial for preparation and quality control.

[0061] The present invention also provides a pharmaceutical composition for treating or preventing pulmonary fibrosis, comprising a compound of formula A of the present invention, or a salt thereof, or a solvate thereof, as an active ingredient, optionally further comprising one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, etc.

[0062] According to the present invention, the amount of compound A or its salt or solvate thereof in the pharmaceutical composition (calculated as compound A) is 0.1-1000 mg, preferably 1-500 mg, and more preferably 5-100 mg.

[0063] The mass percentage of compound A or its salt or solvate (calculated as compound A) in the pharmaceutical composition is 0.01%-95%, and may be 0.1%-10% or 10%-90% depending on the dosage form, preferably 20%-80%, and more preferably 30%-70%.

[0064] The routes of administration of the pharmaceutical composition include, but are not limited to: oral; sublingual; sublingual; transdermal; pulmonary; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, and intravenous; and by implantation of a reservoir or infusion device.

[0065] The dosage form of the pharmaceutical composition may be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; or an injectable dosage form, such as an injection solution, powder for injection, etc., administered via intravenous, intraperitoneal, subcutaneous, or intramuscular routes. All dosage forms used are well known to those skilled in the art of pharmaceutical science. For example, the pharmaceutical composition may be an injection solution, and the concentration of compound A or its salt or solvation in the injection solution, calculated based on compound A, may be 1-15 mg / ml, such as 5 mg / ml, 10 mg / ml, 12.5 mg / ml, etc.

[0066] According to the present invention, the dosage of compound A or its salt or solvate (calculated as compound A) will depend on the recipient's age, health and weight, the type of concurrent drugs, the frequency of treatment, the route of administration, etc. The drug may be administered in a single daily dose, once daily, once every two days, once every three days, once every four days, or the total daily dose may be administered in two, three, or four separate doses per day. The dosage of compound A or its salt or solvate (calculated as compound A) is 0.01-100 mg / kg / day, preferably 0.1-10 mg / kg / day, for example 0.5 mg / kg / day, 1 mg / kg / day, 2 mg / kg / day, 5 mg / kg / day, etc.

[0067] The pharmaceutical composition may be administered in combination with other therapeutic agents or supportive therapies, or formulated as a combination drug, or may further contain other therapeutic agents. These other therapeutic agents are other medications for treating pulmonary fibrosis.

[0068] The present invention also provides the use of a compound of formula A or a salt thereof or a solvate thereof in combination with other medicaments for treating pulmonary fibrosis in the preparation of medicaments for treating pulmonary fibrosis.

[0069] The present invention also provides the use of a compound of formula A or a salt thereof or a solvate thereof in the preparation of a medicament for the treatment of pulmonary fibrosis in combination with other medicaments for the treatment of pulmonary fibrosis.

[0070] Other medications for treating pulmonary fibrosis include, but are not limited to: pirfenidone, nintedanib, phosphoramide tablets, azathioprine tablets, mycophenolate mofetil capsules, cyclosporine soft capsules, omeprazole enteric-coated tablets, lansoprazole enteric-coated capsules, pantoprazole sodium enteric-coated capsules, etc.

[0071] The present invention also provides a method for treating or preventing pulmonary fibrosis, 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. Attached Figure Description

[0072] Figure 1 In a mouse model of pulmonary fibrosis, the results of vital capacity (FVC) measurements in the control group, BLM group, and BLM+CV016 group were as follows.

[0073] Figure 2 In a mouse model of pulmonary fibrosis, the lung wet weight to body weight ratio of mice in the control group, BLM group, and BLM+CV016 group are statistically analyzed.

[0074] Figure 3 In a mouse model of pulmonary fibrosis, the results of HE staining and Masson staining of lung tissue from mice in the control group, BLM group, and BLM+CV016 group were compared. Detailed Implementation

[0075] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0076] The following describes the synthesis of compounds CV010 and CV016 according to the method described in the examples of Chinese patent application CN116854752A.

[0077] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0078] Ac: Acetyl; AgOTf: Silver trifluoromethanesulfonate; Bn: Benzyl; Bz: Benzoyl; ClAc: Monochloroacetyl; CSA: Camphorsulfonic acid; Cbz: Benzyloxycarbonyl; CsF: Cesium fluoride; DBU: 1,8-Diazabicycloundec-7-ene; DCM: Dichloromethane; DDQ: 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone; DMF: N,N-Dimethylformamide; PMB: p-Methoxybenzyl; TfOH: Trifluoromethanesulfonic acid; TBSOTf: Tert-butyldimethylsilyltrifluoromethanesulfonate; TEMPO: 2,2,6,6-Tetramethylpiperidine oxide; TMSOTf: Trimethylsilyltrifluoromethanesulfonate; Tol: Toluene.

[0079] Example 1: Preparation of trisaccharide intermediate III-1

[0080] 1. Preparation method of monosaccharide intermediate 3-1

[0081] Intermediate 9 was obtained by reacting glucosamine hydrochloride 8 with benzyloxyformyl chloride; 1,6-cyclic intermediate 10 was obtained through a two-step reaction; catalytic hydrogenation and azo transfer reaction were used to obtain a common intermediate 11 with an azido group protected at the 2-position; the hydroxyl groups at the 3- and 4-positions were fully benzylated under the action of benzyl bromide and sodium hydride to obtain intermediate 12; 1,6-cyclic intermediate 13 was obtained under the action of acetic anhydride and TBSOTf; the terminal acetyl group was removed under the action of benzylamine to obtain intermediate 14; finally, trichloroacetylimine ester donor 3-1 was obtained under the action of potassium carbonate and trichloroacetonitrile.

[0082]

[0083] The reaction conditions and yields for each step are as follows: a) Cb2Cl, NaOH, H2O, 59%; b) 1) TsCl, Py, MS, 2) EtOH, DBU, NaI, two-step yield 49%; c) 1) 4atm H2, Pd / C, MeOH, 2) TfN3, CuSO4, Et3N, MeOH, two-step yield 82%; d) BnBr, NaH, DMF, 0℃, 85%; e) Ac2O, TBSOTf, 94%; f) BnNH2, DCM, 89%; g) Cl3CCN, K2CO3, DCM, rt, 98%.

[0084] 2. Preparation method of fully protected trisaccharide intermediate 1-1

[0085] The synthesized monosaccharide intermediate 3-1, in its mixed configuration, can be used directly in the next reaction without separation. Trichloroacetylimine ester is a relatively common glycosyl donor, typically reacting under mild conditions with high yields. Monosaccharide intermediate 3-1 is glycosylated and coupled with disaccharide intermediate 4-1 synthesized according to known literature methods, yielding a fully protected trisaccharide 1-1 in the presence of trifluoromethanesulfonic acid.

[0086]

[0087] Monosaccharide intermediate 3-1 (2.83 g, 502.24 mmol) and glycosyl acceptor disaccharide intermediate 4-1 (2.74 g, 324.23 mmol) were dissolved in redistilled DCM and then added to a solution containing pre-activated DCM. In a reaction flask containing molecular sieves, the mixture was continuously stirred at room temperature for 30 min to equilibrate the reaction. After the reaction system temperature was lowered to -20℃, trifluoromethanesulfonic acid (26.53 μL, 0.33 mmol) was slowly added dropwise. The reaction was monitored by TLC. After the reaction was complete, the molecular sieves were removed by silica gel filtration. The resulting filtrate was concentrated and purified by direct silica gel column chromatography (PE / EA = 5:1) to obtain the fully protected trisaccharide intermediate 1-1 (3.42 g, 87%).

[0088] 1H NMR(400MHz,CDCl3)δ8.08(d,J=7.6Hz,2H),7.41–7.25(m,22H),7.21–7.18(m,6H),5.46(d,J=3.1Hz,1H),5.14(t,J=3.6Hz,1H),5.02(d,J=2.2Hz,2H),4.88(s,1H),4.85(d,J=3.0Hz,2H),4.82(d,J=5.3Hz,2H),4.77(d,J=4.2Hz,1H),4.74(d,J=3.8Hz,1H),4.64(d,J=3.8Hz,1H),4.58(d,J=10.4Hz,1H),4.54(d,J=10.9Hz,1H),4.32(d,J=2.7Hz,1H),4.30(d,J=2.2Hz,3H),4.24(d,J=3.6Hz,1H),4.21(d,J=3.9Hz,1H),4.14(t,J=4.3Hz,1H),4.02(t,J=4.0Hz,1H),3.99–3.90(m,2H),3.73(dt,J=9.9Hz,3.2Hz,1H),3.63(d,J=3.8Hz,1H),3.61(d,J=2.8 1H),3.58(s,3H),3.47(t,J=9.4Hz,1H),3.31(s,3H),3.21(dd,J=10.2,3.5Hz,1H).

[0089] 13 C NMR(100MHz,CDCl3)δ170.75,170.52,169.35,165.42,155.81,138.23,137.69,137.58,137.42,136.24,133.37,130.01,129.38,128.74,128.52,128.49,128.44,128.39,128.24,128.17,128.13,128.06,127.96,127.88,127.76,127.35,99.32,98.87,98.50,80.05,79.12,77.29,75.62,75.28,74.98,74.90,74.56,74.44,73.32,70.07,69.33,69.12,68.92,67.00,63.62,62.45,62.33,55.29,54.56,52.10,20.87.

[0090] HRMS[M+Na] + m / z 1275.46443(calcd for C67 H 72 N4NaO 20 ,1275.4638).

[0091] 3. Preparation method of trisaccharide intermediate III-1

[0092] The fully protected trisaccharide 1-1 was subjected to the combined action of LiOH, H2O2 and NaOH to simultaneously remove Ac, Bz and methyl ester to obtain trihydroxy compound I-1; heating under the action of SO3·NMe3 gave the intermediate compound II-1 after O-sulfonation; benzyl and Cbz were removed by catalytic hydrogenation, and the azide was reduced to generate an amino group to obtain diamino compound III-1.

[0093]

[0094] The fully protected trisaccharide compound 1-1 (342.62 mg, 0.26 mmol) was dissolved in 5.00 mL of tetrahydrofuran. Then, 6.23 mL of 1.25 N LiOH solution and 13.13 mL of 30% H₂O₂ solution were added dropwise at room temperature. After stirring for 12 hours, 14.34 mL of methanol and 7.82 mL of 6 N NaOH solution were added, and stirring continued for at least 12 hours. After the reaction was confirmed to be complete by TLC, the pH was adjusted to 2 with 4 N hydrochloric acid under ice-water bath conditions. The reaction solution was then extracted three times with DCM and concentrated under reduced pressure, followed by silica gel column chromatography (DCM:MeOH = 15:1) to obtain compound I-1 (258.0 mg, 93%). High-performance liquid chromatography analysis of intermediate I-1 revealed only a single peak, indicating its high purity. Under argon protection, intermediate I-1 (258.02 mg, 0.24 mmol) and SO3·NMe3 (897.62 mg, 5.33 mmol) were dissolved in 3 mL of anhydrous DMF. The reaction system was heated to 65 °C and stirred continuously for at least 12 h. The reaction solution was taken and the degree of reaction was monitored by high performance liquid chromatography. The reaction was compared with the peak time of compound I-1. When the newly generated peak had a shorter retention time and was a single peak, the reaction was considered complete. After heating was stopped, the reaction system was allowed to rise naturally to room temperature. The reaction solution was concentrated and purified by Sephadex LH-20 gel column chromatography to obtain intermediate II-1 (304.13 mg, 93%). Intermediate II-1 (304.13 mg, 0.22 mmol) was dissolved in 3.00 mL of a mixed solvent of methanol, tert-butanol and water (v / v / v = 2:1:1), and palladium on carbon (50.00 mg) was added. The mixture was stirred for 24 h under a hydrogen pressure of 4 atm. After removing the palladium on carbon by filtration with filter paper, the reaction solution was concentrated to obtain intermediate III-1 (184.93 mg, 98%).

[0095] 1H NMR(400MHz,D2O)δ5.33(d,J=3.7Hz,1H),5.15(s,1H),4.88(d,J=3.7Hz,1H),4.79(d,J=1.8Hz,1 H),4.32–4.25(m,3H),4.23(d,J=3.1Hz,2H),4.13(d,J=2.1Hz,1H),4.11–4.06(m,2H),3.95(d,J =5.6Hz,1H),3.85(t,J=9.8Hz,2H),3.77(t,J=9.9Hz,1H),3.69(d,J=9.5Hz,1H),3.65(s,1H),3. 47(t,J=9.7Hz,1H),3.35(s,3H),3.26(dd,J=10.7Hz,3.6Hz,1H),3.22(dd,J=10.5Hz,3.7Hz,1H).

[0096] 13 C NMR(100MHz,D2O)δ175.15,98.88,96.32,91.44,76.78,73.00,70.50,70.26, 69.39,69.19,68.94,68.67,67.32,66.61,66.16,62.96,55.32,54.22,54.03.

[0097] HRMS[M–H] - m / z 769.0596 (calcd for C 19 H 33 N2O 24 S3,769.0586).

[0098] Example 2: Preparation of compound CV010

[0099] Intermediate III-1 (35 mg, 0.045 mmol) was dissolved in 0.50 mL of water, and the pH was adjusted to 9-10 with 4N NaOH solution and maintained. Then, sulfur trioxide pyridine complex (216 mg, 1.366 mmol) was added in portions. The reaction was confirmed to be complete by TLC. The pH of the neutralized reaction solution was approximately 7-8. The reaction solution was then concentrated and purified by Sephadex G-25 gel column chromatography to obtain compound CV010 (39 mg, 94%).

[0100]

[0101] 1H NMR(400MHz,D2O)δ5.37(d,J=3.6Hz,1H),5.18(d,J=3.0Hz,1H),4.97(d,J=3.6Hz,1H),4.78 (d,J=2.8Hz,1H),4.33-4.26(m,4H),4.21–4.14(m,2H),4.06(t,J=3.2Hz,1H),3.92(td,J=8. 0Hz,7.5Hz,3.5Hz,2H),3.70(t,J=9.5Hz,1H),3.64(d,J=10.1Hz,1H),3.59(d,J=10.1Hz,1H ),3.52(t,J=9.5Hz,1H),3.37(s,3H),3.35(s,1H),3.22(ddd,J=13.1Hz,10.1Hz,3.6Hz,2H).

[0102] 13 C NMR(100MHz,D2O)δ175.37,99.29,98.32,96.95,76.88,75.94,75.90,70.98, 70.05,69.87,69.24,69.15,68.94,68.56,66.87,66.49,57.92,57.76,55.43.

[0103] HRMS[M–H] - m / z 928.9717 (calcd for C 19 H 33 N2O 30 S5,928.9722).

[0104] Example 3 Preparation of compound CV016

[0105] Compound CV010 (39.4 mg, 0.042 mmol) was treated with Dowex-50-WX4-Na + The column exchanged the sodium salt, and the sugar-containing component was collected to concentrate the solvent, yielding compound CV016 (44 mg, 98%).

[0106]

[0107] 1H NMR(400MHz,D2O)δ5.35(d,J=3.6Hz,1H),5.16(d,J=3.0Hz,1H),4.95(d,J=3.6Hz,1H),4.75(d,J =2.8Hz,1H),4.27(dq,J=13.0Hz,3.8Hz,2.9Hz,4H),4.19–4.12(m,2H),4.04(t,J=3.2Hz,1H),3.9 0(td,J=8.0Hz,7.5Hz,3.5Hz,2H),3.68(t,J=9.5Hz,1H),3.61(d,J=10.1Hz,1H),3.56(d,J=10.1H z,1H),3.50(t,J=9.5Hz,1H),3.35(s,3H),3.33(s,1H),3.20(ddd,J=13.1Hz,10.1Hz,3.6Hz,2H).

[0108] 13 C NMR(100MHz,D2O)δ174.36,99.23,98.22,96.93,76.84,75.93,75.89,70.96, 70.01,69.84,69.22,69.13,68.91,68.51,66.84,66.46,57.89,57.73,55.40.

[0109] HRMS[M-6Na+5H] - m / z 928.9754 (calcd for C 19 H 33 N2O 30 S5,928.9722).

[0110] Example 4: Therapeutic effect of CV016 in a mouse model of pulmonary fibrosis

[0111] 1.1 Construction of a mouse pulmonary fibrosis model

[0112] Experimental animals: Healthy male C57 BL / 6J mice, 4–6 weeks old.

[0113] Experimental materials: Bleomycin (BLM), tribromoethanol, CV016, PBS solution, 1ml syringe, cold light lamp, intravenous catheter (22G), gavage needle, endotracheal intubation platform.

[0114] Reagent preparation:

[0115] (1) BLM solution: Dissolve in PBS to prepare a solution concentration of 35 mg / mL.

[0116] (2) CV016: Dissolve in PBS to prepare a solution with a concentration of 4 mg / mL.

[0117] Experimental methods:

[0118] (1) The mice were divided into three groups of 6 mice each: blank group, BLM group and BLM+CV016 group.

[0119] (2) No processing is done on the blank group.

[0120] (3) Mice in the BLM group and BLM+CV016 group were anesthetized. The fully anesthetized mice were fixed on the operating table with their upper teeth suspended. The mice's tongues were pushed to the left side and held with gauze, which was then inserted to the root of the tongue. The root of the tongue was lifted to expose the glottis, which could be seen as an inverted glottis.

[0121] (4) Insert the endotracheal intubation needle, quickly insert the needle when the mouse's glottis opens during breathing, inject bleomycin, and then quickly pull out the guide needle inside the intubation tube.

[0122] (5) Remove the mouse and repeatedly invert it to make bleomycin evenly distributed in the lungs.

[0123] (6) Feed normally for seven days.

[0124] (7) Starting from day 8, administer once a day until day 21; administer 100 μL of BLM+CV016 subcutaneously per animal, and administer the corresponding dose of PBS subcutaneously to the blank group and BLM group.

[0125] (8) Treat mice on day 22.

[0126] 1.2 Mouse lung function test

[0127] (1) Tracheal separation: After anesthetizing the mice, they were fixed on the operating table, and the skin of the neck was cut along the middle of the mouse's neck to separate the trachea;

[0128] (2) Cut the trachea at the 3rd and 4th ring bones, insert the endotracheal tube to a depth of 5-10 mm, and ligate the trachea with surgical sutures.

[0129] (3) Then place the mouse on the support plate of the body tracing box and connect the endotracheal tube to the airway of the body tracing box.

[0130] (4) Data collection is performed after the body scanning box is turned off.

[0131] Experimental results are as follows Figure 1As shown, compared with the blank group, the FVC (forced vital capacity) of the BLM group was significantly reduced; compared with the BLM group, the FVC of the BLM+CV016 group was significantly increased, approaching the level of the blank group. The results indicate that CV016 can effectively increase the forced vital capacity (FVC) of the model group mice, reduce respiratory resistance, and improve restrictive ventilatory dysfunction caused by pulmonary fibrosis.

[0132] 1.3 Lung wet weight / body weight ratio in each group of mice

[0133] Mice were weighed before sacrifice, and the right lung lobes of each group of mice were collected, washed with PBS, dried, and then weighed. The experimental results are as follows: Figure 2 As shown, compared with the blank group, the lung wet weight / body weight ratio of mice in the BLM group was significantly increased; compared with the BLM group, the lung wet weight / body weight ratio of the BLM+CV016 group was significantly decreased, indicating that the lung wet weight / body weight ratio of mice gradually improved after CV016 treatment.

[0134] 1.4 HE and Masson staining procedures and results

[0135] Mice were sacrificed on day 22. After perfusion of the heart with PBS containing 4% paraformaldehyde, the brain and spinal cord were taken for frozen sections, stained with hematoxylin and eosin (HE) and Masson stain.

[0136] HE staining:

[0137] (1) Place the prepared paraffin slices in a 60℃ oven and bake for 60 minutes;

[0138] (2) Dewaxing and hydration: The dewaxed sections are placed 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);

[0139] (3) Staining cell nuclei: Place the slides in hematoxylin staining solution, stain for 15 minutes, and then rinse with distilled water for 5 minutes;

[0140] (4) Staining the cytoplasm: Place the slide in eosin staining solution, and after 5 minutes, rinse with distilled water for 3 seconds;

[0141] (5) Dehydration, clearing, and mounting: 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 cleared sections have dried, they are mounted with neutral resin.

[0142] (6) Observe under a microscope and take pictures.

[0143] Masson staining:

[0144] (1) Dewax and hydrate the paraffin sections according to the HE staining procedure;

[0145] (2) Stain the nucleus with hematoxylin staining solution for 5-10 minutes;

[0146] (3) Differentiate with 1% hydrochloric acid alcohol for 5-15 seconds, then rinse thoroughly with water;

[0147] (4) Add 0.1-1% lithium carbonate for 5 minutes to increase the blueing effect, then wash with water;

[0148] (5) Use Masson's Ponceau Acid Flavescent Solution for 5-10 minutes;

[0149] (6) Wash with 2% glacial acetic acid aqueous solution for 1 min;

[0150] (7) Differentiate with 1% phosphomolybdic acid aqueous solution for 3-5 min, then wash with 2% glacial acetic acid aqueous solution for 1 min;

[0151] (8) Without rinsing with water, directly stain with aniline blue solution or 1% light green solution for 1-2 minutes; then wash with 0.2% glacial acetic acid solution for 1 minute;

[0152] (9) Dehydrate with 5% ethanol for 2-3 seconds, dehydrate with anhydrous ethanol 3 times for 5 seconds each time, clear with xylene 3 times for 1 minute each time, seal with neutral resin, observe under a microscope and take pictures.

[0153] Experimental results are as follows Figure 3 As shown, HE staining revealed that the lung tissue of mice in the blank control group exhibited normal alveolar structure and morphology, with no obvious inflammatory cell infiltration or fibrosis. In contrast, mice in the BLM group showed extensive inflammatory cell infiltration in the alveolar cavities, severely disordered lung tissue structure, significant alveolar structural damage, alveolar cavity shrinkage or even partial closure, exhibiting a state of consolidation, and a significant increase in fibrotic areas, displaying obvious pathological damage characteristics. In comparison, mice in the BLM+CV016 group showed relatively normal alveolar morphology, well-filled alveolar cavities, and significantly reduced inflammatory cell infiltration, indicating that CV016 effectively alleviated BLM-induced pulmonary fibrosis.

[0154] Further Masson staining results ( Figure 3The results showed that the blank control group had low collagen fiber content in lung tissue, with no obvious blue staining areas. In contrast, the BLM group exhibited extensive collagen fiber proliferation in the lung tissue, displaying widespread blue staining areas, especially in the alveolar walls. The thickening of collagen fibers led to significant alveolar wall thickening, indicating severe fibrosis. In the BLM+CV016 group, the blue staining area was significantly reduced, the degree of collagen fiber proliferation was significantly decreased, and alveolar wall thickness was also restored, indicating that CV016 intervention effectively alleviated lung fibrosis and reduced excessive collagen fiber deposition.

[0155] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a trisaccharide compound of formula A, or a salt thereof, or a solvate thereof, in the preparation of a medicament for the treatment or prevention of pulmonary fibrosis: in, R1, R2, R3, and R4 may be the same or different, and are independently selected from H or -SO3H. R5 is a -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; 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 use as described in claim 1, characterized in that, In equation A, R1, R2, R3, and R4 are the same, all being H.

3. The use as described in claim 1 or 2, characterized in that, In formula A, R5 is -C1-3 alkyl, -C2-4 alkylene-NH2, or -C2-4 alkylene-NHSO3H; Preferably, R5 is methyl, ethyl, -C2H4NH2, -C2H4NHSO3H, -C3H6NH2 or -C3H6NHSO3H.

4. The use as described in any one of claims 1-3, characterized in that, The salt is a salt formed with a monovalent cation, which is selected from Na. + K + Li + NH4 + ; Alternatively, the salt is a salt formed with a divalent cation selected from Ca. 2+ Cu 2+ Zn 2+ Fe 2+ Mg 2+ Mn 2+ .

5. The use as described in claim 1, characterized in that, The compound or its salts are selected from compounds with the following structures:

6. The use as described in any one of claims 1-5, characterized in that, The treatment or prevention of pulmonary fibrosis includes improving the inflammatory response in the lungs, or reducing the degree of damage to the lung tissue structure, or improving, inhibiting or delaying the progression of pulmonary fibrosis.

7. The use as described in any one of claims 1-6, characterized in that, The pulmonary fibrosis mentioned is drug-induced pulmonary fibrosis.

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