Use of heparin oligosaccharides for the preparation of a medicament for the treatment of inflammatory bowel disease

Abstract

The present application provides the use of heparin oligosaccharide compounds including trisaccharide compounds, pentasaccharide compounds, fondaparinux in the preparation of drugs for treating or preventing inflammatory bowel disease, which can reduce the colon pathological injury of inflammatory bowel disease mice to different degrees, and effectively reduce the inflammation of the colon.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of heparin oligosaccharide compounds in the preparation of drugs for the treatment or prevention of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease of unknown etiology, including Crohn's disease (CD) and ulcerative colitis (UC). Its pathogenesis involves genetic susceptibility, environmental factors, and abnormal immune responses in the gut microbiota. IBD is protracted and recurrent, and currently remains incurable, imposing a significant burden on patients, families, and society. Despite advancements in various drugs such as monoclonal antibodies, biologics, and novel small molecule drugs, most IBD patients cannot achieve sustained disease remission with existing medications. Furthermore, the pathogenesis of IBD is not fully elucidated, requiring further research to develop novel functionalized oral drug delivery therapies for breakthroughs in IBD treatment. Simultaneously, comprehensive and systematic evaluations of safety and efficacy are essential for its potential clinical application in IBD treatment. Therefore, IBD drug development must overcome these challenges and utilize opportunities at each stage of the drug development process to find ways to overcome these challenges and gradually discover more effective IBD treatments. Summary of the Invention

[0003] The inventors of this application administered heparin-like oligosaccharide compounds selected from the present invention, including trisaccharide compounds, pentasaccharide compounds, and fondaparinux sodium, to a mouse model of inflammatory bowel disease. They found that these compounds could alleviate colonic pathological damage in mice to varying degrees, effectively reduce colonic inflammation, and thus treat inflammatory bowel disease. Based on this, the present invention was completed.

[0004] This invention provides the use of heparin oligosaccharide compounds or their salts or solvates in the preparation of medicaments for the treatment or prevention of inflammatory bowel disease, wherein the heparin oligosaccharide compounds or their salts or solvates are selected from trisaccharide compounds of formula A or their salts or solvates, pentasaccharide compounds of formula M or their salts or solvates, and fondaparinux sodium.

[0005] In one embodiment of the invention, the 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 inflammatory bowel disease.

[0006] In one embodiment of the invention, the invention provides the use of a pentasaccharide compound of formula M, or a salt thereof, or a solvate thereof, in the preparation of a medicament for the treatment or prevention of inflammatory bowel disease.

[0007] In one embodiment of the invention, the invention provides the use of fondaparinux sodium in the preparation of a medicament for the treatment or prevention of inflammatory bowel disease.

[0008] According to the present invention, in the trisaccharide compound of formula A, the three monosaccharides from left to right are represented by B, C, and D, respectively:

[0009]

[0010] in:

[0011] R1, R2, R3, and R4 may be the same or different, and are independently selected from H or -SO3H.

[0012] R5 is a -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3H.

[0013] The salt of Formula A is a salt formed with a monovalent cation, a divalent cation, and / or a trivalent cation.

[0014] 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 - .

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

[0016] In some embodiments of the present invention, in formula A, R1, R2, R3 and R4 are the same, all being H.

[0017] 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.

[0018] 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 + .

[0019] 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+ .

[0020] 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+ .

[0021] 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.

[0022]

[0023] 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.

[0024]

[0025] According to the present invention, in the pentasaccharide compound of formula M, the five monosaccharides from left to right are represented by G, H, J, K, and L, respectively:

[0026]

[0027] The salt of formula M is a salt formed with a monovalent cation, a divalent cation, and / or a trivalent cation.

[0028] 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 cation is selected, for example, from Al 3+ Fe 3+ Etc. Those skilled in the art will understand that when the compound of formula M forms a salt, the corresponding group in formula M is its anionic group, such as -COO. - 、 -SO3 - .

[0029] 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 + .

[0030] 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+ .

[0031] The compound of formula M is a compound with a single optical activity, namely GlcNS6S(1→4)GlcA3Sβ(1→4)GlcNS3S6S(1→4)IdoA2S(1→4)GlcNS6S, and the L-sugar terminal methyl ester is in the α configuration.

[0032] In one embodiment of the present invention, the structural formula of formula M is as follows, which is referred to as CV001 in the present invention.

[0033]

[0034] In one embodiment of the present invention, the sodium salt structure of formula M is as follows, referred to as CV122 in the present invention, which is essentially the sodium salt of CV001.

[0035]

[0036] In one embodiment of the present invention, the potassium salt structure of formula M is as follows, referred to as CV123 in the present invention, which is essentially the potassium salt of CV001.

[0037]

[0038] Preparation method of the compound:

[0039] 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.

[0040] In the fully protected trisaccharide intermediate E, Rx can be an azide group, R 21 R 31 R 41 R 51 and R 61 They may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; preferably R. 21 R 31 and R 51 Same, R 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 51They 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.

[0041] 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.

[0042] 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:

[0043]

[0044] 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. + .

[0045] The fully protected trisaccharide intermediate E can be derived from the monosaccharide intermediate F. and disaccharide receptors It is obtained through a glycosylation reaction.

[0046] Rx can be an azide group, R 21 R 31 R 41 R 51 and R 61 They can be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl, and p-methoxybenzyl; X is a leaving group suitable for reacting with other acceptors to form bonds between glycosides.

[0047] Preferably, X is a hydroxyl group, a thioalkyl group, a thioaryl group, a halogen, a trichloroimine acetyl group, a phosphate ester, or a tert-butyldiphenylsilyloxy group.

[0048] Preferred R 21 R 31 and R 51 Same, R 41 and R 61Same 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.

[0049] 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.

[0050] 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:

[0051]

[0052] The disaccharide intermediate 4 can be obtained from the monosaccharide intermediate 6 and the monosaccharide intermediate 7, as shown in the following reaction formula:

[0053]

[0054] 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.

[0055] Therefore, the present invention also provides the intermediates in the above synthesis method and their preparation methods.

[0056] 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:

[0057] The reaction formula for its preparation method is as follows:

[0058]

[0059] 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:

[0060]

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

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

[0063]

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

[0065] The present invention also provides intermediates I, II and III.

[0066] The structure of compound I is as follows:

[0067] 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.

[0068] The structure of compound II is as follows:

[0069] 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. + .

[0070] The structure of compound III is as follows:

[0071] Where Y is selected from H and Na + K + Li + NH4 + Preferably, Y is H or Na. + .

[0072] 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:

[0073]

[0074] 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 61Intermediate 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.

[0075] The compound of formula A of the present invention, or its salt or solvate thereof, may also be prepared by referring to the method in Chinese patent application CN116854752A.

[0076] The pentasaccharide compound of formula M and its salts in this invention can be prepared by referring to the method in Chinese Invention Patent ZL202111544675.9. Those skilled in the art will know that the synthesized pentasaccharide compound of formula M can be used to prepare the corresponding salts by means of ion exchange resins, etc.; other salts can be prepared by treating the sodium or potassium salts of the prepared pentasaccharide compound of formula M by means of, for example, ion exchange resins.

[0077] The fondaparinux sodium used in this invention is a commercially available drug, and the synthesis methods are all publicly disclosed.

[0078] This invention also provides a pharmaceutical composition for treating or preventing inflammatory bowel disease, comprising a heparin oligosaccharide compound or its salt or solvate as an active ingredient, wherein the heparin oligosaccharide compound or its salt or solvate is selected from compounds of formula A or their salts or solvates, compounds of formula M or their salts or solvates, and fondaparinux sodium. Optionally, the pharmaceutical composition 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: fillers, diluents, antioxidants, pH adjusters, preservatives, etc.

[0079] The pharmaceutical composition may be administered in combination with other drugs for the treatment or prevention of inflammatory bowel disease, or formulated as a combination drug, or may further contain other drugs for the treatment or prevention of inflammatory bowel disease.

[0080] The present invention also provides a method for treating or preventing inflammatory bowel disease, characterized in that a therapeutically effective amount of a heparin oligosaccharide compound or its salt or solvate, or a pharmaceutical composition containing a heparin oligosaccharide compound or its salt or solvate, is administered to a patient in need; wherein the heparin oligosaccharide compound or its salt or solvate is selected from a trisaccharide compound of formula A or its salt or solvate, a pentasaccharide compound of formula M or its salt or solvate, and fondaparinux sodium.

[0081] In one embodiment of the invention, the method for treating or preventing inflammatory bowel disease is characterized by administering to a patient in need a therapeutically effective amount of a compound of formula A or a salt or solvation thereof, or a pharmaceutical composition containing a compound of formula A or a salt or solvation thereof.

[0082] In one embodiment of the invention, the method for treating or preventing inflammatory bowel disease is characterized by administering to a patient in need a therapeutically effective amount of a compound of formula M or a salt or solvation thereof, or a pharmaceutical composition containing a compound of formula M or a salt or solvation thereof.

[0083] In one embodiment of the invention, the method for treating or preventing inflammatory bowel disease is characterized by administering a therapeutically effective amount of fondaparinux sodium, or a pharmaceutical composition containing fondaparinux sodium, to a patient in need.

[0084] Other medications for treating inflammatory bowel disease include, but are not limited to: aminosalicylic acid preparations, including but not limited to: sulfamethoxazole salicylate, olsalazine, balsalazine, 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, cyclosporine A, tacrolimus, mycophenolate mofetil, and infliximab; and antibiotics, including but not limited to metronidazole, ornidazole, tinidazole, tobramycin, vancomycin, and ciprofloxacin.

[0085] According to the invention, in some aspects, the inflammatory bowel disease is ulcerative colitis. In other aspects, the inflammatory bowel disease is Crohn's disease.

[0086] According to the present invention, the effects of the treatment or prevention of inflammatory bowel disease include, but are not limited to, restoring 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.

[0087] In some embodiments of the invention, weight loss is inhibited relative to the treatment baseline 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%.

[0088] In some embodiments of the invention, the body weight decreases by a maximum of 20%, a maximum of 15%, a maximum of 10%, a maximum of 5%, or even no weight loss occurs relative to the body weight before the onset of the disease. Attached Figure Description

[0089] Figure 1 Figure 1. Changes in body weight over time in mice in a DSS-induced mouse model of inflammatory bowel disease.

[0090] Figure 2 HE staining results of colon tissue from different groups of mice in a DSS-induced mouse model of inflammatory bowel disease.

[0091] Figure 3 Statistical graphs of colon weight and colon length in mice of different groups in a DSS-induced mouse inflammatory bowel disease model. Left: Colon weight statistical graph; Right: Colon length statistical graph. Detailed Implementation

[0092] 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.

[0093] The compounds CV010 and CV016 were synthesized according to the method described in the examples of Chinese patent application CN116854752A.

[0094] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0095] 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.

[0096] Example 1: Preparation of trisaccharide intermediate III-1

[0097] 1. Preparation method of monosaccharide intermediate 3-1

[0098] 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.

[0099]

[0100] 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) 4atmH2, 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%.

[0101] 2. Preparation method of fully protected trisaccharide intermediate 1-1

[0102] The synthesized monosaccharide intermediate 3-1, in its mixed configuration, can be used directly in the next reaction without separation. Trichloroacetylimine ester is a relatively common glycosyl donor, typically reacting under mild conditions with high yields. Monosaccharide intermediate 3-1 is glycosylated with disaccharide intermediate 4-1 synthesized according to known literature methods, yielding a fully protected trisaccharide 1-1 in the presence of trifluoromethanesulfonic acid.

[0103]

[0104] 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%).

[0105] 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).

[0106] 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.

[0107] HRMS[M+Na] + m / z 1275.46443(calcd for C67 H 72 N4NaO 20 ,1275.4638).

[0108] 3. Preparation method of trisaccharide intermediate III-1

[0109] 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.

[0110]

[0111] 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%).

[0112] 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).

[0113] 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.

[0114] HRMS[M–H] - m / z 769.0596 (calcd for C 19 H 33 N2O 24 S3,769.0586).

[0115] Example 2: Preparation of compound CV010

[0116] 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%).

[0117]

[0118] 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).

[0119] 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.

[0120] HRMS[M–H] - m / z 928.9717 (calcd for C 19 H 33 N2O 30 S5,928.9722).

[0121] Example 3 Preparation of compound CV016

[0122] 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%).

[0123]

[0124] 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).

[0125] 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.

[0126] HRMS[M-6Na+5H] - m / z 928.9754 (calcd for C 19 H 33 N2O 30 S5,928.9722).

[0127] Example 4: Therapeutic effects of heparin oligosaccharides CV016, CV122 and fondaparinux sodium in a mouse model of inflammatory bowel disease.

[0128] 1.1 Establishment of a mouse model of inflammatory bowel disease

[0129] Reagents: Solution preparation: 3% DSS stock solution, weigh 9g of sodium dextran sulfate (DSS), add 300mL of sterile drinking water, stir to dissolve completely, dispense into animal drinking bottles, and change every two days.

[0130] Animals: 25 male SPF-grade C57 mice, weighing 16-18g, aged 6-8 weeks. The ambient temperature was 18-22℃ and the ambient humidity was 50-60%. The mice were kept in an SPF room, 5 mice per cage. The bedding was changed and supplemented with feed in a timely manner, and free access to food and water was ensured.

[0131] Methods: Twenty-five mice were randomly divided into a Control group (n=5) and an Experimental group (n=20) based on a balanced weight distribution. Mice in the Control group received normal drinking water, while mice in the Experimental group received 3% DSS solution. The 3% DSS was replaced every two days, and mouse weight and condition were recorded. Starting on day 4, when a 10% weight loss or bloody stools occurred, the experimental group was further divided into four groups based on a balanced weight and clinical score: DSS group, CV016 group (DSS+CV016), CV122 group (DSS+CV122), and fondaparinux group (DSS+Fon), with five mice in each group. The CV016 group (20 mg / kg), CV122 group (20 mg / kg), and fondaparinux group (20 mg / kg) received subcutaneous administration of the medication, and the 3% DSS was replaced with a 1% DSS maintenance dose to maintain inflammatory bowel disease symptoms. The study was terminated when the mice no longer experienced significant weight loss after administration.

[0132] Post-processing steps:

[0133] 1. Weigh the mice in each group;

[0134] 2. The colon and cecum were separated from the small intestine and the anus distal to the rectum in the ileocecal region. At this point, gross intestinal photographs were taken from the cecum to the rectum of each group of mice, or one representative mouse from each group.

[0135] 3. Measure the length of the colon; colitis can increase edema and shorten the overall length of the colon.

[0136] 4. Next, separate the colon from the cecum (the junction of the ileocecal junction) and quickly flush with cold PBS using a 5-10 ml syringe (18-G, 3-in). A feeding needle is used to remove feces and blood; after flushing with PBS, weigh the colon.

[0137] 5. Prepare a colon tissue specimen for histological examination.

[0138] 1.2 Experimental Results

[0139] 1.2.1 The compound described above can effectively alleviate weight loss in mice with DSS (Digital Superficial Syndrome) inflammatory bowel disease model.

[0140] The statistical results of mouse weight change are as follows Figure 1As shown, the body weight of mice in the Control group gradually increased over time; the body weight of mice in the model group decreased significantly starting from day 4, and the body weight of mice in the DSS group continued to decrease thereafter, reaching more than 20% by day 8, at which point the mice were considered dead (euthanized); compared with the DDS group, the body weight loss trend of mice in each treatment group slowed down after administration, and the body weight of each treatment group began to increase from day 2 after administration. Among them, the body weight recovery of the DSS+CV016 group was better than that of the DSS+CV122 group, and the body weight recovery of these two groups was better than that of the DSS+Fon group. Therefore, CV016, CV122, and fondaparinux sodium can all effectively alleviate the body weight loss in mice after DSS stimulation.

[0141] 1.2.2 The compound described above can effectively improve the pathological damage in mice with DSS inflammatory bowel disease model.

[0142] Further observation of intestinal damage was performed using HE staining of colon tissue. The HE staining results are as follows: Figure 2 As shown, the colonic 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 or ulceration; the colonic mucosa of the DSS model group was damaged, the crypts were reduced, and there was inflammatory cell infiltration and ulceration; compared with the DSS group, the damage to the colonic mucosa in each treatment group was significantly improved, and the crypt structure was more intact.

[0143] 1.2.3 The compound described above can effectively improve the degree of colonic inflammation in mice with DSS inflammatory bowel disease model.

[0144] Besides weight loss and HE staining of colonic tissue, which reflect disease status, colon length and weight can also serve as evaluation criteria for inflammatory bowel disease. Colitis increases tissue edema and shortens the overall length of the colon. We dissected mice after the model study concluded, weighed the colon of each group, and measured the colon length. The statistical results are as follows: Figure 3 As shown, the colon weight of mice in the DSS group was greater than that of mice in the Control group, while the colon length was shorter. The colon weight of mice in all treatment groups was lower than that of mice in the DSS group, while the colon length was greater. Specifically, the colon weight and colon length of the DSS+CV016 and DSS+CV122 groups showed significant differences compared to the DSS group, and the colon length of the DSS+Fon group showed a significant difference compared to the DSS group. This indicates that CV016, CV122, and fondaparinux sodium can effectively alleviate colonic inflammation.

[0145] 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 heparin oligosaccharide compounds or their salts or solvates in the preparation of medicaments for the treatment or prevention of inflammatory bowel disease, wherein the heparin oligosaccharide compounds or their salts or solvates are selected from trisaccharide compounds of formula A or their salts or solvates, pentasaccharide compounds of formula M or their salts or solvates, and fondaparinux sodium; The trisaccharide compound of formula A is shown in the following formula: in, R1, R2, R3, and R4 may be the same or different, and are independently selected from H or -SO3H. R5 is -C1-5 alkyl, -C1-5 alkylene-NH2, or -C1-5 alkylene-NHSO3H; The pentasaccharide compound of formula M is shown in the following formula: The salt of formula A or the salt of formula M 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 of formula A or the salt of formula M is a salt formed with a monovalent cation selected from Na. + K + Li + NH4 + Preferred from Na + K + Alternatively, the salt of formula A or the salt of formula M is a salt formed with a divalent cation selected from Ca. 2+ Cu 2+ Zn 2+ Fe 2+ Mg 2+ Mn 2+ The preferred form is Ca. 2+ .

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

6. The use as described in claim 1, characterized in that, The compound of formula M or a salt thereof is selected from compounds with the following structures:

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