CN115947769B - Sulfonated tetrasaccharide structural compound and pharmaceutical application thereof - Google Patents

Abstract

The present invention relates to a sulfonated tetrasaccharide structure compound and its pharmaceutical application. The present invention provides a heparin tetrasaccharide structure compound and a preparation method thereof. The heparin tetrasaccharide structure compound has a single optical activity, can effectively inhibit the activity of heparanase, has a certain therapeutic potential, and has no cytotoxicity, so that the safety of the drug is guaranteed while the therapeutic use is carried out. Formula I:

Description

Sulfonated tetrasaccharide structure compound and its pharmaceutical application

Technical Field

The invention belongs to the technical field of pharmaceutical compounds, and specifically relates to a sulfonated tetrasaccharide structure compound, a preparation method thereof, and its activity in treating vascular endothelial injury and pharmaceutical application.

Background Art

Heparanase includes two subtypes: heparanase-1 (Hpse-1) and heparanase-2 (Hpse-2). Heparanase-1 is the subtype with biological activity of cleaving heparan sulfate (HS) and is the focus of this study. Therefore, it is simplified and replaced by heparanase (Hpse-2) [105]. Heparanase was first reported by Nakajima's group in 1984 and is the only mammalian endoglucuronidase that can cleave the HS side chain of glycosaminoglycan of HSPG in a unique way.

Under normal physiological conditions, heparanase is expressed at high levels in the placenta and certain blood-borne cells (including platelets, neutrophils, mast cells and lymphocytes), and at lower levels in other human tissues. Under pathological conditions, the expression of heparanase is enhanced in a variety of malignancies, and heparanase is also overexpressed in endothelial and epithelial cells in inflammatory diseases. The enzymatic degradation and remodeling of HS by heparanase alters the integrity and state of tissues and greatly affects various physiological and pathological processes, including tissue repair, cell adhesion, proliferation, survival and differentiation, tumor development and metastasis, angiogenesis, neurite outgrowth, inflammation and autoimmunity.

HS is a highly sulfonated heterogeneous polysaccharide. Heparanase can cleave the glycosidic bond between β-D-glucuronic acid and the subsequent N-(sulfonated)-D-glucosamine, releasing a carbohydrate product with a significant molecular weight that can still bind to protein ligands. Therefore, sulfonated HS derivatives are a class of small molecule heparanase inhibitors with research prospects.

Summary of the invention

The present invention provides a sulfonated tetrasaccharide structure compound, which has valuable pharmacological properties, especially inhibiting the activity of heparanase.

The present invention further provides a method for preparing the sulfonated tetrasaccharide structure compound.

The compound having the structure of Formula I, or its stereoisomers, pharmaceutically acceptable salts or polymorphs, has four monosaccharides represented from left to right by A, B, C, and D respectively:

in:

R ₁ are the same or different and are independently selected from OSO ₃ Y or NHSO ₃ Y;

R ₂ , R ₃ , R ₄ and R ₅ are the same or different and are independently selected from hydrogen or SO ₃ Y;

_R6 is independently selected from hydrogen, an alkyl group of 1 to 4 carbon atoms, an alkoxy group of 1 to 4 carbon atoms, a halogenated alkane group of 1 to 4 carbon atoms or an alkylphenyl group of 1 to 4 carbon atoms;

Y are the same or different and are independently selected from hydrogen or a monovalent cation, wherein the monovalent cation includes but is not limited to Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ , etc.;

Monosaccharide A is selected from sulfonated or unsulfonated glucuronic acid or iduronic acid, R represents a substituent of a hydroxyl group on the sugar ring of monosaccharide A, and R includes a substituent OR ₇ at position 2, a substituent OR ₈ at position 3, and a substituent OR ₉ at position 4;

Indicates that the glycosidic bond configuration is α or β;

Preferably, R ₁ is the same, both are NHSO ₃ Y;

Preferably, R ₂ is different and is selected from hydrogen or SO ₃ Y, R ₂ of the B sugar is selected from SO ₃ Y, and R ₂ of the D sugar is selected from hydrogen;

Preferably, R ₃ is the same, both are SO ₃ Y;

Preferably, R ₄ is selected from hydrogen;

Preferably, R ₅ is selected from SO ₃ Y

Preferably, R ₆ is hydrogen or an alkyl group of 1 to 4 carbon atoms;

Preferably, each Y is the same and is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ .

In one embodiment of the present invention, wherein monosaccharide A is selected from glucuronic acid, which has the structure of formula II:

According to one embodiment of the present invention, exemplary compounds having a structure shown in Formula II include the compounds described in Table 1 and their pharmaceutically acceptable salts or solvates thereof:

Table 1

In one embodiment of the present invention, wherein monosaccharide A is selected from iduronic acid, which has the structure of formula III: R ₇ , R ₈ and R ₉ are the same or different and are independently selected from hydrogen or SO ₃ Y.

According to one embodiment of the present invention, exemplary compounds having a structure shown in Formula III include the compounds set forth in Table 2 and pharmaceutically acceptable salts or solvates thereof:

Table 2

The present invention also provides methods for preparing the compounds of formula I, II and III.

According to the present invention, the formulas I, II and III are prepared by fully protected tetrasaccharide intermediates.

The compound is obtained by sequentially removing the hydroxy protecting group, O-sulfonating, optionally performing an azide reduction reaction, and finally performing an N-sulfonating reaction.

In the fully protected tetrasaccharide compound of formula IV or formula V, R ₂₁ , R ₃₁ , R ₄₁ , R ₅₁ , R ₇₁ , R ₈₁ and R ₉₁ may be the same or different and are independently selected from hydrogen, chloroacetyl, acetyl, benzoyl, pivaloyl, benzyl and p-methoxybenzyl.

According to one embodiment of the present invention, exemplary compounds having a structure shown in Formula IV or Formula V include compounds described in Table 3 and pharmaceutically acceptable salts or solvates thereof:

Table 3

In one embodiment of the present invention, the present invention adopts the reaction method to synthesize a compound of formula I in which sugar A is glucuronic acid, R ₁ =NHSO ₃ Y, sugar B is R ₂ =H or SO ₃ Y, sugar D is R ₂ =H, R ₃ =R ₅ =R ₉ =SO ₃ Y, R ₄ =R ₇ =H, R ₆ =Me, and R ₈ =H or SO ₃ Y. The synthesis method is to prepare a fully protected tetrasaccharide compound of formula IV in which sugar B is R ₂₁ =Ac or Bn, sugar D is R ₂₁ =Bn, R ₃₁ =R ₉₁ =Ac, R ₅₁ =Bz, R ₄₁ =R ₇₁ =Bn, and R ₈₁ =Ac or Bn, sequentially through dehydroxylation, O-sulfonation, optional azide reduction, and finally N-sulfonation to obtain the compound. In a specific embodiment of the present invention, the present invention adopts the method to synthesize the corresponding sodium sulfonated tetrasaccharides CV025, CV026, CV027, CV028, CV029 and CV030 respectively using fully protected tetrasaccharides CV025-1, CV026-1, CV027-1, CV028-1, CV029-1 and CV030-1 as raw materials:

In another embodiment of the present invention, the present invention adopts the reaction method to synthesize the compound of formula I, wherein the A sugar is iduronic acid, R ₁ = NHSO ₃ Y, the B sugar R ₂ = SO ₃ Y, the D sugar R ₂ = H, R ₃ = R ₅ = R ₇ = R ₉ = SO ₃ Y, R ₄ = R ₈ = H, and R ₆ = Me. The synthesis method uses the B sugar R ₂₁ = Ac, the D sugar R ₂₁ = Bn, R ₃₁ = R ₇₁ = Ac, R ₅₁ = R ₉₁ = Bz, R ₄₁ = R ₈₁ = Bn, and the fully protected tetrasaccharide compound of formula IV, and sequentially undergoes dehydroxylation, O-sulfonation, optional azide reduction reaction, and finally N-sulfonation to obtain. In a specific embodiment of the present invention, the present invention adopts the method to synthesize the corresponding sodium sulfonated tetrasaccharide CV031-1 as a raw material:

In one embodiment of the present invention, the fully protected tetrasaccharide intermediate IV is obtained by glycosylation coupling of the disaccharide intermediate 7 and the disaccharide receptor 8, and the reaction formula is as follows:

Wherein X is a leaving group suitable for reacting with other acceptors, including but not limited to trichloroacetimidate, glucosinolate, halogen, etc., and preferably X is selected from trichloroacetimidate.

In one embodiment of the present invention, the fully protected tetrasaccharide intermediate V is obtained by glycosylation coupling of the disaccharide intermediate 9 and the disaccharide receptor 8, and the reaction formula is as follows:

Wherein X is a leaving group suitable for reacting with other acceptors, including but not limited to trichloroacetimidate, glucosinolate, halogen, etc., and preferably X is selected from trichloroacetimidate.

The disaccharide intermediate 7 can be obtained by glycosylation coupling of monosaccharide intermediates 10 and 11 followed by ring opening, and the reaction formula is as follows:

The disaccharide intermediate 9 can be obtained by glycosylation coupling of monosaccharide intermediates 11 and 13 followed by ring opening, and the reaction formula is as follows:

The disaccharide receptor 8 and monosaccharide intermediate 11 used in the above preparation method can be prepared according to a synthetic method known in the art, for example: Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116.

The present invention also provides various intermediates in the above synthesis method and preparation methods thereof.

A monosaccharide intermediate 10 has the following structure: Preferably, R ₇₁ is benzyl, R ₈₁ is benzyl or acetyl, R ₉₁ is acetyl or chloroacetyl, and X is bromine or glucosinolate. In one embodiment of the present invention, R ₇₁ and R ₈₁ of the monosaccharide intermediate 10 are both benzyl, R ₉₁ is acetyl, and X is glucosinolate, which is named 10-1 and has the following structure: In another embodiment of the present invention, R ₇₁ of the monosaccharide intermediate 10 is benzyl, R ₈₁ is acetyl, R ₉₁ is chloroacetyl, and X is bromine, which is named 10-2 and has the following structure:

The preparation method of the monosaccharide intermediates 10-1 and 10-2 is as follows:

A monosaccharide intermediate 11 has the following structure: Preferably, R ₂₁ is an acetyl group or a chloroacetyl group. In one embodiment of the present invention, R ₂₁ of the monosaccharide intermediate 11 is an acetyl group, which is named 11-1 and has the following structure: In another embodiment of the present invention, R ₂₁ of the monosaccharide intermediate 11 is a benzyl group, named 11-2, and its structure is as follows: The preparation of monosaccharide intermediate 11-1 can be prepared according to a synthetic method known in the art, for example: Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116.

A monosaccharide intermediate 13 has the following structure: Preferably, R ₇₁ , R ₈₁ and R ₉₁ are benzyl or benzoyl, and X is bromine or glucosinolate. In one embodiment of the present invention, R ₇₁ and R ₉₁ of the monosaccharide intermediate 13 are both benzoyl, R ₈₁ is benzyl, and X is glucosinolate, which is named 13-1 and has the following structure:

The preparation method of the monosaccharide intermediate 13-1 is as follows:

A disaccharide intermediate 8 has the following structure: In one embodiment of the present invention, R ₂₁ and R ₄₁ of the disaccharide intermediate 8 are both benzyl, R ₃₁ is acetyl, and R ₅₁ is benzoyl, named 8-1, with the following structure: The preparation of monosaccharide intermediate 8-1 can be prepared according to a synthetic method known in the art, for example: Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116.

A disaccharide intermediate 12 has the following structure: Preferably, R ₂₁ and R ₈₁ are acetyl or benzyl, R ₇₁ is benzyl, and R ₉₁ is acetyl or chloroacetyl. In one embodiment of the present invention, R ₂₁ and R ₉₁ of the disaccharide intermediate 12 are both acetyl, and R ₇₁ and R ₈₁ are both benzyl, and the disaccharide intermediate 12 is named 12-1, and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₈₁ and R ₉₁ of the disaccharide intermediate 12 are all acetyl groups, and R ₇₁ is benzyl group, which is named as 12-2 and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₇₁ and R ₈₁ of the disaccharide intermediate 12 are all benzyl groups, and R ₉₁ is all acetyl groups, and the disaccharide intermediate 12 is named 12-3, and has the following structure:

A disaccharide intermediate 7 has the following structure: Preferably, R ₃₁ and R ₉₁ are acetyl groups, R ₇₁ is benzyl group, R ₂₁ and R ₈₁ are benzyl groups or acetyl groups, and X is trichloroacetimidate or glucosinolate. In one embodiment of the present invention, R ₂₁ , R ₃₁ and R ₉₁ of the disaccharide intermediate 7 are all acetyl groups, R ₇₁ and R ₈₁ are all benzyl groups, and X is trichloroacetimidate, which is named 7-1 and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₃₁ , R ₈₁ and R ₉₁ of the disaccharide intermediate 7 are all acetyl groups, R ₇₁ is benzyl, and X is trichloroacetimidate, named as 7-2, with the following structure: In another embodiment of the present invention, R ₃₁ , R ₈₁ and R ₉₁ of the disaccharide intermediate 7 are all acetyl groups, R ₂₁ and R ₇₁ are benzyl groups, and X is trichloroacetimidate, named as 7-3, with the following structure:

The preparation method of the disaccharide intermediate 7-1 is as follows:

The preparation method of the disaccharide intermediate 7-2 is as follows:

The preparation method of the disaccharide intermediate 7-3 is as follows:

A disaccharide intermediate 14 has the following structure: Preferably, R ₂₁ , R ₇₁ and R ₉₁ are acetyl or benzoyl, and R ₈₁ is benzyl. In one embodiment of the present invention, R ₂₁ of the disaccharide intermediate 14 is acetyl, R ₇₁ and R ₉₁ are both benzoyl, and R ₈₁ is benzyl, which is named as 14-1 and has the following structure:

A disaccharide intermediate 9 has the following structure: Preferably, R ₂₁ , R ₃₁ , R ₇₁ and R ₉₁ are acetyl or benzoyl, R ₈₁ is benzyl, and X is trichloroacetimidate or glucosinolate. In one embodiment of the present invention, R ₂₁ and R ₃₁ of the disaccharide intermediate 9 are acetyl, R ₇₁ and R ₉₁ are benzoyl, R ₈₁ is benzyl, and X is trichloroacetimidate, which is named 9-1 and has the following structure:

The preparation method of the disaccharide intermediate 9-1 is as follows:

A fully protected tetrasaccharide IV has the following structure:

It is preferred that R ₃₁ , R ₅₁ and R ₉₁ are acetyl or benzoyl, R ₄₁ and R ₇₁ are benzyl, R ₂₁ and R ₈₁ are acetyl or benzyl, The glycosidic bond configuration is α or β configuration. In one embodiment of the present invention, in the fully protected tetrasaccharide IV, R ₂₁ of the B sugar is an acetyl group, R ₂₁ of the D sugar is a benzyl group, R ₃₁ and R ₉₁ are acetyl groups, R ₄₁ , R ₇₁ and R ₈₁ are benzyl groups, R ₅₁ is a benzoyl group, The glycosidic bond configuration is β configuration, that is, compound CV025-1; in another embodiment of the present invention, in the fully protected tetrasaccharide IV, R ₂₁ of the B sugar is an acetyl group, R ₂₁ of the D sugar is a benzyl group, R ₃₁ and R ₉₁ are acetyl groups, R ₄₁ , R ₇₁ and R ₈₁ are benzyl groups, R ₅₁ is a benzoyl group, The glycosidic bond configuration is α configuration, that is, compound CV026-1; in another embodiment of the present invention, the B sugar R ₂₁ of the fully protected tetrasaccharide IV is an acetyl group, the D sugar R ₂₁ is a benzyl group, R ₃₁ , R ₈₁ and R ₉₁ are acetyl groups, R ₄₁ and R ₇₁ are benzyl groups, R ₅₁ is a benzoyl group, The glycosidic bond configuration is β configuration, that is, compound CV027-1; in another embodiment of the present invention, the B sugar R ₂₁ of the fully protected tetrasaccharide IV is an acetyl group, the D sugar R ₂₁ is a benzyl group, R ₃₁ , R ₈₁ and R ₉₁ are acetyl groups, R ₄₁ and R ₇₁ are benzyl groups, R ₅₁ is a benzoyl group, The glycosidic bond configuration is α configuration, that is, compound CV028-1; in another embodiment of the present invention, R ₂₁ of the B sugar and the D sugar of the fully protected tetrasaccharide IV are both benzyl, R ₃₁ and R ₉₁ are acetyl, R ₄₁ , R ₇₁ and R ₈₁ are benzyl, R ₅₁ is benzoyl, The glycosidic bond configuration is β configuration, that is, compound CV029-1; in another embodiment of the present invention, R ₂₁ of the B sugar and the D sugar of the fully protected tetrasaccharide IV are both benzyl, R ₃₁ and R ₉₁ are acetyl, R ₄₁ , R ₇₁ and R ₈₁ are benzyl, R ₅₁ is benzoyl, The glycosidic bond configuration is α configuration, that is, compound CV030-1.

A fully protected tetrasaccharide V has the following structure:

Preferably, R ₂₁ is acetyl or benzyl, R ₃₁ , R ₅₁ , R ₇₁ and R ₉₁ are acetyl or benzoyl, and R ₄₁ and R ₈₁ are benzyl. In one embodiment of the present invention, the B sugar R ₂₁ of the fully protected tetrasaccharide V is acetyl, the D sugar R ₂₁ is benzyl, R ₃₁ is acetyl, R ₄₁ and R ₈₁ are benzyl, and R ₅₁ , R ₇₁ and R ₉₁ are benzoyl, which is compound CV031-1.

The preparation methods of the fully protected tetrasaccharides CV025-1 to CV030 are similar and the disaccharide acceptors used in the reaction are the same. Different disaccharide trichloroacetimidate donors are glycosylated and coupled with the disaccharide acceptor 8-1 to obtain fully protected tetrasaccharides of corresponding structures. The preparation methods are as follows:

A tetrasaccharide compound 1, Preferably, R ₂₁ and R ₈₁ are benzyl or hydrogen. R ₄₁ and R ₇₁ are benzyl. In one embodiment of the present invention, R ₂₁ of the B sugar is hydrogen, R ₂₁ of the D sugar is benzyl, and R ₄₁ , R ₇₁ and R ₈₁ are all benzyl, named 1-1, with the following structure:

In another embodiment of the present invention, R ₂₁ of the B sugar is hydrogen, R ₂₁ of the D sugar is benzyl, R ₄₁ and R ₇₁ are both benzyl, and R ₈₁ is hydrogen, which is named 1-2 and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₄₁ , R ₇₁ and R ₈₁ are all benzyl groups, named as 1-3, with the following structure:

A tetrasaccharide compound 2, Wherein R ₂₁ , R ₄₁ and R ₇₁ are benzyl groups, R ₂₁₁ and R ₈₁₁ are hydrogen, benzyl or SO ₃ Y, respectively, and Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, R ₂₁ , R ₄₁ , R ₇₁ and R ₈₁₁ are all benzyl groups, R ₂₁₁ is SO ₃ Y, and Y is Na ⁺ , which is named as 2-1, and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₄₁ and R ₇₁ are all benzyl, R ₂₁₁ and R ₈₁₁ are SO ₃ Y, Y is Na ⁺ , and the compound is named 2-2, and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₄₁ , R ₇₁ , R ₂₁₁ and R ₈₁₁ are all benzyl groups, Y is Na ⁺ , and is named as 2-3, with the following structure:

A tetrasaccharide compound 3, Wherein R ₂₁ , R ₄₁ and R ₇₁ are benzyl, R ₂₁₁ and R ₈₁₁ are hydrogen or SO ₃ Y, and Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, R ₂₁ , R ₄₁ , R ₇₁ and R ₈₁₁ are all hydrogen, R ₂₁₁ is SO ₃ Y, and Y is Na ⁺ , which is named 3-1 and has the following structure: In another embodiment of the present invention, R ₂₁ , R ₄₁ and R ₇₁ are all hydrogen, R ₂₁₁ and R ₈₁₁ are SO ₃ Y, Y is Na ⁺ , named as 3-2, with the following structure: In another embodiment of the present invention, R ₂₁ , R ₄₁ , R ₇₁ , R ₂₁₁ and R ₈₁₁ are all hydrogen, Y is Na ⁺ , named as 3-3, with the following structure:

A tetrasaccharide compound II-1, wherein R ₂ and R ₈ are hydrogen or SO ₃ Y, respectively, and Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, R ₂ is SO ₃ Y, R ₈ is hydrogen, and Y is Na ⁺ , The glycosidic bond configuration is β configuration, that is, compound CV025. In another embodiment of the present invention, R ₂ is SO ₃ Y, R ₈ is hydrogen, Y is Na ⁺ , The glycosidic bond configuration is α configuration, that is, compound CV026. In one embodiment of the present invention, R ₂ and R ₈ are SO ₃ Y, Y is Na ⁺ , The glycosidic bond configuration is β configuration, that is, compound CV027. In another embodiment of the present invention, R ₂ and R ₈ are SO ₃ Y, Y is Na ⁺ , The glycosidic bond configuration is α configuration, that is, compound CV028; in another embodiment of the present invention, R ₂ and R ₈ are hydrogen, Y is Na ⁺ , The glycosidic bond configuration is β configuration, that is, compound CV029; in another embodiment of the present invention, R ₂ and R ₈ are hydrogen, Y is Na ⁺ , The glycosidic bond configuration is α configuration, that is, compound CV030.

The preparation method of the tetrasaccharide compounds CV025 and CV026 is as follows:

The preparation method of the tetrasaccharide compounds CV027 and CV028 is as follows:

The preparation method of the tetrasaccharide compounds CV029 and CV030 is as follows:

A tetrasaccharide compound 4, Wherein R ₂₁ , R ₄₁ and R ₈₁ are benzyl groups, and Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, R ₂₁ , R ₄₁ , and R ₈₁ are all benzyl groups, and Y is hydrogen, named as 4-1, and has the following structure:

A tetrasaccharide compound 5, Wherein R ₂₁ , R ₄₁ and R ₈₁ are benzyl groups, and Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, R ₂₁ , R ₄₁ , and R ₈₁ are all benzyl groups, and Y is Na ⁺ , which is named as 5-1 and has the following structure:

A tetrasaccharide compound 6, Wherein R ₂₁ , R ₄₁ and R ₈₁ are hydrogen, and Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, R ₂₁ , R ₄₁ , and R ₈₁ are all hydrogen, and Y is Na ⁺ , named as 6-1, and having the following structure:

A tetrasaccharide compound III-1, Y is selected from hydrogen, Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ . In one embodiment of the present invention, Y is Na ⁺ , that is, compound CV031.

The preparation method of the compound CV031 is as follows:

Unless otherwise specified, the compounds reported in the literature in the above preparation methods can be prepared using reaction methods and conditions known in the art.

The present invention also provides a pharmaceutical composition, which comprises the compound of formula I of the present invention or a 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, adhesives, antioxidants, pH regulators, preservatives, lubricants, disintegrants, etc.

In one embodiment of the present invention, the pharmaceutical composition is used to treat or prevent diseases caused by overexpression of heparanase, such as inflammatory diseases, cancer, sepsis, etc., and its main function is to inhibit the activity of heparanase.

The amount of the compound of formula I contained in the pharmaceutical composition (calculated as the compound of formula I) is 0.1-1000 mg, preferably 1-500 mg, and more preferably 5-100 mg.

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

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 injection dosage form, such as injections, powder injections, etc., which are 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 I in the injection can be 1-15 mg/ml, such as 5 mg/ml, 10 mg/ml, 12.5 mg/ml, etc.

The 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.

The dosage of the compound of formula I (calculated as the compound of formula I) 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 I (calculated as the compound of formula I) 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.

The pharmaceutical composition can be administered in combination with other therapeutic agents or prepared into a combined drug. The other therapeutic agents can be other drugs for treating cancer or sepsis, etc., depending on the type of disease and condition.

The compound of formula I of the present invention has a clear and efficient effect of inhibiting the activity of heparanase and has no cytotoxicity. Therefore, the safety of medication is guaranteed while the compound of formula I has a clear structure, which is conducive to preparation and quality control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 : Inhibition of heparanase activity by compounds CV025, CV026, CV027, CV028, CV029 and CV030 and their IC ₅₀ .

DETAILED DESCRIPTION

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.

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

BF ₃ .Et ₂ O: boron trifluoride ethyl ether; TEMPO: 2,2,6,6-tetramethylpiperidinoxide; PMB: p-methoxybenzyl; DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; TMSOTf: trimethylsilyl trifluoromethanesulfonate; Ac: acetyl; AgOTf: silver trifluoromethanesulfonate; Ag ₂ CO ₃ : silver carbonate; Bn: benzyl; Bz: benzoyl; ClAc: monochloroacetyl; Cbz: benzyloxycarbonyl; DCM: dichloromethane; DMF: N,N-dimethylformamide; PE: petroleum ether; EA: ethyl acetate; MeOH: methanol.

Example 1 Preparation of Monosaccharide Intermediates

1. Preparation method of monosaccharide intermediates 10-1 and 10-2

Using glucose 15 as the starting material, monosaccharide intermediate 16 is obtained by full acetylation; under the action of _{BF3.Et2O ,} it reacts with p-toluene thiophenol to obtain monosaccharide intermediate 17; all acetyl groups are removed under the action of a catalytic amount of sodium methoxide, and then the 4,6-dihydroxyl group is protected with benzylide to obtain the 2,3-dihydroxy-exposed monosaccharide intermediate 18; it is then reacted with benzyl bromide to obtain the 2,3-benzyl-protected intermediate 19; the benzylide-protected group is removed by heating under acetic acid conditions to obtain the 4,6-dihydroxy monosaccharide intermediate 20; TEMPO oxidation and methyl esterification obtain monosaccharide intermediate 21; finally, the 4-hydroxyl group is protected with acetyl to obtain the monosaccharide intermediate 10-1.

Starting from the common intermediate 18, under the action of dibutyltin oxide, the PMB protecting group was selectively used as a temporary protecting group at the 3-position to obtain the monosaccharide intermediate 22; then it reacted with benzyl bromide to obtain the monosaccharide intermediate 23; under the action of DDQ, the temporary protecting group PMB was oxidized and removed to obtain the intermediate 24, which was then acetylated to obtain the monosaccharide intermediate 25; the benzyl protecting group was removed by heating under acetic acid conditions to obtain the 4,6-dihydroxy monosaccharide intermediate 26; TEMPO oxidation and methyl esterification gave the monosaccharide intermediate 27; the 4-position hydroxyl group was protected with a chloroacetyl group to obtain the monosaccharide intermediate 28; finally, the β-thioglycoside was treated with iodine bromide to convert the α-configuration monosaccharide intermediate 10-2.

The reaction conditions and yields of each step are as follows: a) Ac ₂ O, HClO ₄ , 77%; b) TolSH, BF ₃ ·Et ₂ O, DCM, 86%; c) 1) MeONa, MeOH, DCM; 2) Benzaldehyde Dimethylacetal, CSA, DMF, 83% for two steps; d) BnBr, NaH, DMF, 78%; e) 80% AcOH, 90°C, 89%; f) 1) 2,2,6,6-Tetramethylpiperidinooxy (TEMPO), Iodobenzene diacetate, DCM, H ₂ O; 2) MeI, KHCO ₃ , DMF, 57% for two steps; g) Ac ₂ O, Et ₃ N, DCM, 96%; h) 1) Bu ₂ SnO, MeOH, reflux; 2) PMBCl, CSF, DMF, 90°C, 75% for two steps. i) BnBr, NaH, DMF, 92%; j) DDQ, DCM, H ₂ O, 78%; k) Ac ₂ O, Et ₃ N, DCM, 98%; l) 80% AcOH, 90°C, 76%; m) 1) TEMPO, Iodobenzene diacetate, DCM, H ₂ O; 2) Mel, KHCO ₃ , DMF, 57% for two steps; n) ClAc ₂ O, Py, DCM, 76%; o) IBr, DCM, 76%.

NMR data of monosaccharide intermediate 10-1:

¹ H NMR (400MHz, CDCl ₃ ) δ7.49 (d, J＝7.8Hz, 2H), 7.43–7.19 (m, 10H), 7.12 (d, J＝7.8Hz, 2H), 5.11 (t, J＝ 9.7Hz,1H),4.87(d,J＝10.2Hz,1H),4.80(d,J＝11.4Hz,1H),4.68(t,J＝11.0Hz,2H),4.59(d,J＝9.7Hz ,1H),3.88(d,J＝9.9Hz,1H),3.74(s,3H),3.70(t,J＝9.0Hz,1H),3.52(t,J＝9.2Hz,1H),2.34(s ,3H),1.92(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.66,21.19,29.32,52.78,71.06,75.56,76.35,79.79,83.34,87.96,127.83,127.87,128.02,128.49,128.69,129.86,133.3 2,137.82,137.96,138.41,167.69 ,169.60.

NMR data of monosaccharide intermediate 10-2:

¹ H NMR (400MHz, CDCl ₃ ) δ7.40–7.28(m,5H),6.32(d,J＝3.9Hz,1H),5.55(t,J＝9.6Hz,1H),5.18(t,J＝ 12.0Hz,1H),4.66–4.56(m,3H),4.03(s,2H),3.74(s,3H),3.61(dd,J=9.6,3.9Hz,1H),2.03(s,3H). ¹³ C NMR (100MHz, CDCl ₃ )δ20.69,40.32,53.22,70.09,70.89,71.59,72.95,75.84,87.54,128.06,128.52,128.73,136.55,166.39,166.79,169.72.

2. Preparation method of monosaccharide intermediate 13-1

Using commercially purchased monosaccharide intermediate 29 as the starting material, all acetyl groups were removed under the action of a catalytic amount of sodium methoxide to obtain intermediate 30; after TEMPO oxidation and methyl esterification, it reacted with a benzoyl group to obtain monosaccharide intermediate 13-1.

The reaction conditions and yields of each step are as follows: a) MeONa, MeOH, DCM; b) 1) TEMPO, Iodobenzenediacetate, DCM, H ₂ O; 2) MeI, KHCO ₃ , DMF, 61% for two steps; c) BzCl, Py, 81%.

NMR data of monosaccharide intermediate 13-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.12(d,J＝6.8Hz,1H),8.03(d,J＝6.7Hz,2H),7.56–7.33(m,12H),7.12(d,J＝ 7.9Hz,2H),7.01(t,J＝7.7Hz,2H),5.78(s,1H),5.64(d,J＝1.9Hz,1H),5.51(s,1H),5.45(s,1H) ,4.97(d,J＝11.8Hz,1H),4.87(d,J＝11.8Hz,1H),4.20(s,1H),3.76(s,3H),2.31(s,3H). ¹³ C NMR( 100MHz,CDCl ₃ )δ21.10,29.15,52.60,67.11,68.57,68.90,71.78,72.93,86.81,127.72,128.45,128.49,128.57,128.89,129.81,129.85,130.01,130.59,131 .84,133.20,133.75,134.54,137.05,137.76 ,165.30,165.48,168.91.

Example 2 Preparation of disaccharide intermediates

1. Preparation method of disaccharide intermediates 12-1 and 7-1

Monosaccharide intermediates 10-1 and 11-1 underwent glycosylation coupling under the action of NIS and catalytic amount of TMSOTf to obtain disaccharide intermediate 12-1. The mixed configuration of 12-1 was separated by column chromatography to obtain α-configuration intermediate 12-1α and β-configuration intermediate 12-1β. Subsequently, 1,6-ring-opened intermediates 32α and 32β were obtained respectively under the combined action of acetic anhydride and TMSOTf. Intermediate 32 reacted with benzylamine to selectively remove the acetyl group at position 1, and then reacted with trichloroacetonitrile to finally obtain disaccharide intermediates 7-1α and 7-1β. It is worth noting that the trichloroacetimidate esters in 7-1α and 7-1β can be used as glycosyl donors regardless of the mixed configuration or the separated single configuration, that is, the trichloroacetimidate configuration has no specific effect on the subsequent glycosylation reaction. Therefore, based on the consideration of shortening the reaction route and improving the yield, we generally use a mixed configuration of trichloroacetimidate ester donors in the reaction. The separation here is to facilitate the determination of a single configuration structure.

The reaction conditions and yields of each step are as follows: a) NIS, TMSOTf, MS, DCM, -40°C, 2h, 65%; b) Ac ₂ O, TMSOTf, 0°C, 83%; c) 1) BnNH ₂ , THF, rt, overnight; 2) Cl ₃ CCN, K ₂ CO ₃ , DCM, rt, 72% for two steps.

NMR data of disaccharide intermediate 12-1α:

¹ H NMR (400MHz, CDCl ₃ ) δ7.46–7.18 (m, 10H), 5.59 (s, 1H), 5.14 (d, J = 3.5Hz, 1H), 5.09–4.97 (m, 2H), 4.90– 4.71(m,3H),4.70–4.60(m,2H),4.43(d,J＝10.0Hz,1H),4.12(t,J＝9.4Hz,1H),4.00(d,J＝7.6Hz,1H ),3.83–3.74(m,1H),3.69(s,3H),3.64(dd,J＝9.6,3.5Hz,1H),3.50(s,1H),3.05(s,1H),2.12(s, 3H),1.90(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ19.99,21.05,22.11,29.59,29.69,52.84,58.22,64.97,69.51,70.81,74.94,75.34,75.52,78.53,98.81,100.39,127.61 ,127.86,127.94,128.02 ,128.33,128.46,137.99,138.48,168.80,169.51,169.89,178.01.

NMR data of disaccharide intermediate 12-1β:

¹ H NMR (400MHz, CDCl ₃ ) δ7.40–7.19(m,10H),5.49(s,1H),5.26(s,1H),5.20–5.09(m,1H),5.01(d,J＝10.9 Hz,1H),4.83(d,J＝11.6Hz,1H),4.76(d,J＝10.9Hz,1H),4.66(d,J＝11.7Hz,2H ),4.59(d,J＝5.7Hz,1H),4.01(d,J＝7.5Hz,1H),3.92(d,J＝10.0Hz,1H),3.82–3.75(m,1H),3.72(s ,3H),3.69–3.62(m,3H),3.23(s,1H),2.09(s,3H),1.93(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.06,21.02,29.37,52.73,58.95,64.96,70.73,70.77,72.89,73.78,75.18,75.91,81.03,81.13,100.29,102.87,127.7 6,127.84,128.26,128.39 ,128.45,138.11,138.16,167.60,169.24,169.51.

NMR data of disaccharide intermediate 7-1β:

1)

¹ H NMR (400MHz, CDCl ₃ ) δ8.81 (s, 1H), 7.29 (dd, J＝7.5, 5.6Hz, 6H), 7.22 (dd, J＝7.3, 2.2Hz, 4H), 6.42 (d, J＝3.6Hz,1H),5.64–5.51(m,1H),5.05(t,J＝9.6Hz,1H),4.79(d,J＝11.6Hz,3H),4.65(d,J＝11.6Hz, 1H), 4.43–4.30(m,2H),4.20(dd,J＝12.4,4.3Hz,1H),4.08(d,J＝10.1Hz,1H),3.90–3.78(m,2H),3.71(s,3H) ,3.68–3.61(m,2H),3.46(t,J＝8.5Hz,1H),2.21(s,3H),2.02(s,3H),1.92(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.59,20.73,20.85,22.72,29.72,31.95,52.66,60.65,61.27,69.60,71.09,71.38,72.95,75.32,75.37,75.47,77.25,81. 21,81.39,95.08 ,102.73,127.65,127.72,127.80,127.84,128.41,137.67,137.99,160.57,167.49,169.59,170.13.

2)

¹ H NMR (400MHz, CDCl ₃ ) δ8.78 (s, 1H), 7.35–7.27 (m, 7H), 7.26–7.18 (m, 3H), 5.67 (d, J = 8.4Hz, 1H), 5.15– 5.08(m,1H),5.04(t,J＝9.6Hz,1H),4.79–4.70(m,3H),4.63(d,J＝11.6Hz,1H),4.43(dd,J＝12.3 ,2.1Hz,1H),4.32(d,J＝7.8Hz,1H),4.22(dd,J＝12.3,4.6Hz,1H),3.85–3.77(m,2H),3.72(s,4H),3.62 (t,J＝9.2Hz,2H),3.47–3.41(m,1H),2.21(s,3H),2.03(s,3H),1.91(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.59,20.71,20.86,21.09,29.72,52.65,60.43,61.42,63.35,71.08,71.78,72.91,73.81,75.11,75.31,75.48,77.26,81. 16,81.49,96.35 ,102.70,127.72,127.77,127.85,128.40,128.45,137.68,138.86,161.51,168.29,169.58,170.17.

NMR data of disaccharide intermediate 7-1α:

1)

¹ H NMR (400MHz, CDCl ₃ ) δ8.82 (s, 1H), δ7.43–7.28 (m, 10H), 5.71 (d, J = 8.2Hz, 1H), 5.27–5.18 (m, 1H), 5.06(t,J＝8.2Hz,1H),4.88(d,J＝2.9Hz,1H),4.76–4.61(m,4H),4.56(dd,J＝12.5,2.1Hz,1H),4.30–4.21 (m,2H),3.91(t,J＝9.3Hz,1H),3.86(t,J＝8.3Hz,1H),3.79(dd,J＝10.0,8.2Hz,1H),3.76–3.70(m, 1H),3.67(s,3H),3.49(dd,J＝8.5,2.9Hz,1H),2.03(s,3H),2.00(s,3H),1.90(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.67,20.77,28.98,52.67,62.31,63.35,69.93,70.65,73.24,73.92,74.27,74.87,76.47,77.13,78.18,96.32,98.87,1 27.84,127.89,128.24 ,128.26,128.45,128.62,137.78,138.00,161.41,168.39,169.83,170.19.

2)

¹ H NMR (400MHz, CDCl ₃ ) δ8.78 (s, 1H), δ7.37–7.28 (m, 10H), 6.47 (d, J = 3.7Hz, 1H), 5.69–5.55 (m, 1H), 5.08(t,J＝7.9Hz,1H),4.94(d,J＝2.9Hz,1H),4.75–4.62(m,5H),4.52(dd,J＝12.5,2.0Hz ,1H),4.32(d,J＝8.2Hz,1H),4.24(dd,J＝12.5,4.2Hz,1H),3.90(q,J＝8.6,8.0Hz,2H),3.67(s,4H) ,3.51(dd,J＝8.3,2.9Hz,1H),2.04(s,3H),2.03(s,3H),1.93(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.71,21.04,29.69,52.66,60.39,60.97,62.22,69.85,70.84,71.06,71.39,74.22,74.77,76.80,77.97,94.13,98.98,1 27.76,127.83,128.24 ,128.45,128.58,137.77,137.97,160.68,167.78,169.77,170.14.

2. Preparation method of disaccharide intermediates 12-2 and 7-2

Monosaccharide intermediates 10-2 and 11-1 underwent glycosylation coupling in the presence of Ag ₂ CO ₃ and a catalytic amount of AgOTf to obtain disaccharide intermediate 33; after reacting with thiourea to remove the chloroacetyl group, the α and β configurations were separated by column chromatography, and then the 4-position acetyl group was protected under the action of acetic anhydride to obtain the α-configuration intermediate 12-2α and the β-configuration intermediate 12-1β, respectively; subsequently, under the combined action of acetic anhydride and TMSOTf, the 1,6-ring-opened intermediates 34 were obtained, respectively; intermediate 34 reacted with benzylamine to selectively remove the 1-position acetyl group, and then reacted with trichloroacetonitrile to finally obtain the disaccharide intermediates 7-2α and 7-2β. It is worth noting that the trichloroacetimidate esters in 7-2α and 7-3β can be used as glycosyl donors regardless of the mixed configuration or the separated single configuration, that is, the trichloroacetimidate configuration has no specific effect on the subsequent glycosylation reaction. Therefore, based on the consideration of shortening the reaction route and improving the yield, we generally use a mixed configuration of trichloroacetimidate ester donors in the reaction. The separation here is to facilitate the determination of a single configuration structure.

The reaction conditions and yields of each step are as follows: a) Ag ₂ CO ₃ , MS, DCM, rt, 2d, 58%; b) 1) Thiourea, CHCl ₃ , MeOH, 60°C, 83%, 2) Ac ₂ O, Py, 0°C, 65% c) Ac ₂ O, TMSOTf, 0°C, 97%; d) 1) BnNH ₂ , THF, rt, overnight; 2) Cl ₃ CCN, K ₂ CO ₃ , DCM, rt, 72% for two steps

NMR data of disaccharide intermediate 12-2α:

¹ H NMR (400MHz, CDCl ₃ ) δ7.40–7.27 (m, 5H), 5.63–5.54 (m, 2H), 5.15 (d, J = 3.6Hz, 1H), 5.08 (t, J = 9.7Hz, 1H),5.01(s,1H),4.69(d,J＝4.7Hz,3H),4.54(d,J＝10.1Hz,1H),3.99(d,J＝7.7Hz,1H),3.82–3.77( m,1H),3.72(s,3H),3.62(dd,J＝10.0,3.6Hz,1H),3.46(s,1H),3.07(s,1H),2.12(s,3H),2.02(s ,3H),2.00(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.59,20.81,21.02,29.70,52.90,58.27,64.95,68.95,69.70,70.76,70.85,73.15,74.73,75.97,98.45,100.39,127.99,128 .53,137.63,168.40,169.42 ,169.63,169.93.

NMR data of disaccharide intermediate 12-2β:

¹ H NMR (400 MHz, CDCl ₃ )δ7.30(dt,J＝14.3,7.4Hz,5H),5.49(s,1H),5.27(t,J＝1.5Hz,1H),5.24–5.10(m,2H),4.90(d,J＝11.8Hz,1H),4.76–4.66(m,2H),4.60(d,J＝5.7Hz,1H) ,4.03(dd,J＝8.6,6.2Hz,2H),3.78(dd,J＝7.6,5.8Hz,1H),3.73(s,3H),3.67(s,1H),3.58(dd,J＝9.0,7.6Hz,1H),3.22(s,1H),2.10(s,3H),1.99(s,3H) ,1.91(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ169.52,169.21,167.11,137.86,128.42,128.11,127.85,103.18,100.25,78.17,77.36,77.24,77.04,76.72,76.34,74.66,73 .92,72.93,72.59,70.60,69.43,64.94,58.87,52.82,31.92,29.69,29.36,22.69,21.05,20.69,20.54,14.20.

NMR data of disaccharide intermediate 7-2β:

1)

¹ H NMR (400MHz, CDCl ₃ ) δ8.81 (s, 1H), 7.37–7.27 (m, 3H), 7.20 (d, J = 7.1Hz, 2H), 6.42 (d, J = 3.6Hz, 1H) ,5.57(t,J＝9.7Hz,1H),5.19(t,J＝9.6Hz,1H),5.05(t,J＝9.8Hz,1H),4.71(d,J＝11.6Hz,1H),4.57 (d,J＝11.6Hz,1H),4 .46(d,J＝8.0Hz,2H),4.18–4.05(m,2H),3.96(d,J＝9.8Hz,1H),3.89(d,J＝9.5Hz,1H),3.74(s, 3H),3.66(d,J＝14.0Hz,1H),3.44(t,J＝8.7Hz,1H),2.18(s,3H),2.03(s,3H),1.99(s,3H),1.88( s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.46,20.60,20.71,20.79,29.69,52.72,60.71,61.04,69.67,69.74,71.34,72.72,72.98,75.13,75.73,77.24,78.76,94. 09,102.83,127.88 ,127.98,128.45,137.30,160.51,167.05,169.55,169.84,169.92,170.02.

2)

¹ H NMR (400MHz, CDCl ₃ ) δ8.79 (s, 1H), 7.38–7.28 (m, 3H), 7.23 (d, J = 7.2Hz, 2H), 5.69 (d, J = 8.3Hz, 1H) ,5.15(q,J＝10.2Hz,2H),5.03(t,J＝9.5Hz,1H),4.71(d,J＝11.8Hz,1H),4.61–4.47(m,2H),4.44(d, J＝7.8 Hz,1H),4.21–4.05(m,2H),3.95(d,J＝9.9Hz,1H),3.86(t,J＝9.6Hz,1H),3.74(s,4H),3.66(d,J ＝10.2Hz,1H),3.40(t,J＝8.7Hz,1H),2.18(s,3H),2.05(s,3H),1.99(s,3H),1.86(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.45,20.59,20.68,20.79,29.64,52.71,61.20,63.32,69.73,71.87,72.66,72.90,73.73,75.07,75.43,77.26,78.69,96. 30,102.79,127.98 ,128.06,128.47,137.34,160.61,167.56,169.52,169.84,169.95,170.06.

NMR data of disaccharide intermediate 7-2α:

1)

¹ H NMR (400MHz, CDCl ₃ ) δ8.83 (s, 1H), 7.37–7.31 (m, 5H), 6.47 (d, J = 3.7Hz, 1H), 5.66 (dd, J = 10.6, 9.1Hz, 1H),5.36(t,J＝9.3Hz,1H),5.09–5.02(m,2H),4.64–4.47(m,2H),4.33(d,J＝9.6H z,1H),4.22(dd,J＝12.6,3.9Hz,1H),4.15–4.09(m,2H),3.95(t,J＝9.6Hz,1H),3.72(s,3H),3.63–3.55 (m,2H),2.07(s,3H),2.01(s,3H),2.01(s,3H),1.97(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.58,20.74,20.88,21.08,29.71,52.88,61.15,62.11,69.28,69.58,70.27,70.80,71.09,73.82,75.90,76.33,77.26,94. 18,98.13,127.83 ,128.13,128.40,128.69,128.81,137.21,168.07,169.41,169.51,169.82,170.11.

2)

¹ H NMR (400MHz, CDCl ₃ ) δ8.78(s,1H), δ7.36–7.29(m,5H),6.05(s,1H),5.46–5.37(m,2H),5.24(s,1H ),5.07(t,J＝9.8Hz,1H),4.97(d,J＝3.6Hz,1H),4.66(d,J＝12.1Hz,1H),4.54(d,J＝12.1Hz,1H), 4.38–4.26(m,4H),4.23(d,J＝16.8Hz,1H),3.71(s,3H),3.60(dd,J＝10.0,3.7Hz,1H),2.03(s,3H),2.02 (s,3H),2.01(s,6H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.58,20.73,20.83,23.22,29.71,43.75,52.89,62.94,67.68,68.86,69.57,70.15,70.88,73.83,76.01,77.30,88.38,95. 40,127.55,127.86 ,128.72,128.78,168.14,169.83,169.99,170.22,170.71.

3. Preparation method of disaccharide intermediates 12-3 and 7-3

Monosaccharide intermediates 10-1 and 11-2 underwent glycosylation coupling under the action of Ag ₂ CO ₃ and a catalytic amount of AgOTf to obtain disaccharide intermediate 35; after reacting with thiourea to remove the chloroacetyl group, the α-configuration and β-configuration were separated by column chromatography, and then the 4-position acetyl group was protected under the action of acetic anhydride to obtain the α-configuration intermediate 12-3α and the β-configuration intermediate 12-3β, respectively; subsequently, under the combined action of acetic anhydride and TMSOTf, the 1,6-ring-opened intermediates 35 were obtained, respectively; intermediate 34 reacted with benzylamine to selectively remove the acetyl group at the 1-position, and then reacted with trichloroacetonitrile to finally obtain the disaccharide intermediates 7-3α and 7-3β. It is worth noting that the trichloroacetimidate esters in 7-3α and 7-3β can be used as glycosyl donors regardless of the mixed configuration or the separated single configuration, that is, the trichloroacetimidate configuration has no specific effect on the subsequent glycosylation reaction. Therefore, based on the consideration of shortening the reaction route and improving the yield, we generally use a mixed configuration of trichloroacetimidate ester donors in the reaction. The separation here is to facilitate the determination of a single configuration structure.

The reaction conditions and yields of each step are as follows: a) Ag ₂ CO ₃ , MS, DCM, rt, 2d, 63%; b) 1) Thiourea, CHCl ₃ , MeOH, 60°C, 80%, 2) Ac ₂ O, Py, 0°C, 62% c) Ac ₂ O, TMSOTf, 0°C, 95%; d) 1) BnNH ₂ , THF, rt, overnight; 2) Cl ₃ CCN, K ₂ CO ₃ , DCM, rt, 73% for two steps

NMR data of disaccharide intermediate 12-3α:

¹ H NMR (400MHz, CDCl ₃ ) δ7.36–7.24 (m, 17H), 5.58 (s, 1H), 5.05 (t, J = 9.4Hz, 1H), 4.82 (d, J = 11.8Hz, 2H) ,4.74–4.57(m,4H),4.52(d,J＝12.2Hz,2H),4.45(d,J＝10.0Hz,1H),4.11(q,J＝8.0,6.5Hz,2H),3.74( s, 2H), 3.70 (s, 3H), 3.57 (d, J = 9.2Hz, 2H), 3.14 (s, 1H), 1.91 (s, 3H). ¹³ C NMR (100MHz, CDCl ₃ )δ20.64,22.70,29.70,52.84,58.57,65.05,69.52,70.93,72.28,73.67,75.13,75.34,77.25,78.03,78.71,99.12,100.82,127.62,127.64,127 .87,128.15,128.35,128.52,128.66 ,137.10,138.02,138.41,168.77,169.85.

NMR data of disaccharide intermediate 12-3β:

¹ H NMR (400MHz, CDCl ₃ ) δ7.41–7.26 (m, 15H), 5.52 (s, 1H), 5.16 (d, J = 7.3Hz, 1H), 5.01 (d, J = 10.3Hz, 1H) ,4.84(d,J＝11.3Hz,1H),4.67(q,J＝18.4,15.4Hz,6H),4.08(s,1H),3.95(s,1H),3.85(d,J＝9.5Hz, 1H), 3.77 (s, 2H), 3.68 (d, J = 16.8Hz, 5H), 3.21 (s, 1H), 1.94 (s, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ14.14,20.70,22.71,29.38,29.71,31.94,52.81,59.87,65.09,70.82,72.73,72.79,74.34,75.26,75.35,76.63,76.76,77.08,77.39,77.82,8 1.01,81.10,100.80,103.19,127.72,127.79,127.84,127.92,127.97,128.42,128.48,128.51,128.54,137.57,138.05,138.13,167.54,169.5 7.

NMR data of disaccharide intermediate 7-3β:

1)

¹ H NMR (400MHz, CDCl ₃ ) δ8.71 (s, 1H), 7.43 (d, J = 7.3Hz, 2H), 7.39–7.34 (m, 3H), 7.33–7.28 (m, 8H), 7.23 ( d,J＝7.6Hz,2H),5.57(d,J＝8.4Hz,1H),5.18–5.09(m,2H),4.81–4.72(m,4H),4.65(d,J＝11.5Hz,1H ),4.46(t,J＝10.5Hz,2H),4.28(dd,J＝12.2,4.5Hz,1H),3.92–3.86(m,1H),3.81(d,J＝9.9Hz,1H),3.64 (dt,J＝13.9,8.9Hz,2H),3.55–3.47(m,6H),2.03(d,J＝8.8Hz,3H),1.92(s,3H). ¹³ C NMR (101MHz, CDCl ₃ )δ20.61,20.84,29.70,52.63,61.81,65.17,71.14,73.08,73.90,75.40,75.47,76.71,77.03,77.23,77.35,80.78,81.46,81.69,96.41,102.72 ,127.68,127.76,127.81,127.89 ,127.91,128.17,128.30,128.42,128.45,137.68,137.97,138.10,160.99,167.44,169.56,170.30.

2)

¹ H NMR (400MHz, CDCl ₃ ) δ8.77 (s, 1H), 7.26 (t, J = 16.4Hz, 17H), 5.67 (d, J = 8.3Hz, 1H), 5.12 (t, J = 9.6Hz ,1H),5.04(t,J＝9.5Hz,1H),4.84–4.69(m,4H),4.62(d,J＝11.5Hz,1H),4.42(d,J＝11.8Hz,1H) ,4.32(d,J＝7.7Hz,1H),4.25–4.19(m,1H),3.80(d,J＝9.6Hz,2H),3.72(s,4H),3.62(t,J＝9.0Hz, 2H), 3.53 (d, J = 14.6Hz, 1H), 3.44 (t, J = 8.4Hz, 1H), 2.21 (s, 3H), 1.91 (s, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ14.13,20.57,20.69,20.84,22.70,29.71,31.94,52.63,61.44,63.37,71.10,71.82,72.95,73.83,75.10,75.29,75.48,76. 71,77.03,77.23 ,77.35,81.20,81.50,96.35,99.98,102.69,127.70,127.76,127.82,128.38,128.43,137.70,160.67,167.44,169.53,170.13.

NMR data of disaccharide intermediate 7-3α:

α/β configurations cannot be separated, so the spectrum is a mixed one. ¹ H NMR (400MHz, CDCl ₃ )δ8.79(s,1H),7.23(d,J＝21.0Hz,17H),6.06(d,J＝305.0Hz,1H),5.54(s,1H),5.12(s,1H),4.85–4.60(m,5H),4.46(d,J＝22.0Hz,2H),4.29(t,J＝22.6Hz,2H),4.09(d,J＝21.4Hz,2H),3.93(s,1H),3.61(t,J＝19.1Hz,6H),2.00(d,J＝13.9Hz,6H). ¹³ C NMR (101MHz,CDCl ₃ )δ20.76,29.72,52.62,62.45,63.01,65.39,69.94,70.41,70.91,71.21,73.62,74.33,75.34,76.63,76.76,77.07,77.39,79.58,82.64,94.44, 96.60,97.68,97.75,127.08,127.54,127.69,127.80,127.92,128.43,137.57,160.64,168.56,170.00,170.54.

4. Preparation method of disaccharide intermediates 14-1 and 9-1

Monosaccharide intermediates 13-1 and 11-1 undergo glycosylation coupling under the action of NIS and catalytic amount of TMSOTf to obtain disaccharide intermediate 14-1; then, 1,6 ring-opened intermediates 35 are obtained respectively under the joint action of acetic anhydride and TMSOTf; intermediate 35 reacts with benzylamine to selectively remove the acetyl group at position 1, and then reacts with trichloroacetonitrile to finally obtain disaccharide intermediate 9-1. It is worth noting that the trichloroacetimidate in 9-1 can be used as a glycosyl donor regardless of the mixed configuration or the separated single configuration, that is, the trichloroacetimidate configuration has no specific effect on the subsequent glycosylation reaction. Therefore, based on the consideration of shortening the reaction route and improving the yield, we generally use a mixed configuration trichloroacetimidate donor when reacting. The separation here is to facilitate the determination of the single configuration structure.

The reaction conditions and yields of each step are as follows: a) NIS, TMSOTf, MS, DCM, -40°C, 2h, 64%; b) Ac ₂ O, TMSOTf, 0°C, 97%; c) 1) BnNH ₂ , THF, rt, overnight; 2) Cl ₃ CCN, K ₂ CO ₃ , DCM, rt, 72% for two steps.

NMR data of disaccharide intermediate 14-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.04(d,J＝7.5Hz,2H),7.74(d,J＝7.7Hz,2H),7.58(t,J＝7.4Hz,1H),7.47(d ,J＝7.6Hz,2H),7.43–7.32(m,5H),7.31(d,J＝7.3Hz,1H),7.04(t,J＝7.8Hz,2H),5.58(s,1H),5.52 (s,1H),5.46 (s,1H),5.30(d,J＝12.1Hz,2H),5.03(s,1H),4.96(d,J＝12.0Hz,1H),4.86–4.76(m,2H),4.17–4.07( m,1H),4.02(d,J＝7.6Hz,1H),3.86–3.79(m,2H),3.77(s,3H),3.16(s,1H),2.09(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.99,29.57,52.64,58.99,60.43,65.05,66.63,68.40,71.50,72.03,72.28,77.25,95.97,100.50,127.52,127.79,128. 09,128.45,128.48,128.81 ,129.25,129.76,130.01,132.21,133.45,135.85,165.23,165.38,168.10,170.48.

NMR data of disaccharide intermediate 9-1:

1)

¹ H NMR (400MHz, CDCl ₃ ) δ8.78 (s, 1H), 8.06 (d, J = 7.0Hz, 2H), 7.70 (d, J = 7.0Hz, 2H), 7.62 (t, J = 7.5Hz ,1H),7.44(t,J＝7.8Hz,3H),7.40–7.37(m,5H),7.05–7.01(m,2H),5.72(d,J＝8.4Hz,1H),5.43(s, 1H),5.23–5.16(m,2H),5.07(d,J＝2.4Hz,1H),4.96(s,1H),4.83(s,2H),4.45–4.38(m,2H),4.08(s ,1H),4.03(t,J＝9.4Hz,1H),3.73(d,J＝2.8Hz,5H),2.17(s,3H),2.11(s,3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.45,20.99,22.71,29.72,52.42,61.83,64.03,67.41,67.92,68.09,72.33,72.54,73.17,73.52,77.24,96.38,99.64,1 25.47,127.54,128.00 ,128.15,128.56,129.81,130.04,133.05,134.13,137.17,161.43,163.49,166.21,168.72,170.77.

2)

¹ H NMR (400MHz, CDCl ₃ ) δ8.82 (s, 1H), 8.07 (d, J = 3.1Hz, 2H), 7.70 (d, J = 6.7Hz, 2H), 7.63 (t, J = 7.5Hz ,1H),7.45(t,J＝7.8Hz,3H),7.40–7.35(m,5H),7.03(t,J＝7.8Hz,2H),6.46(d,J＝3.6Hz,1H),5.59 (dd,J＝10.6,8.4Hz,1H),5 .46(s,1H),5.24(s,1H),5.12(d,J＝2.5Hz,1H),4.97(s,1H),4.84(d,J＝2.1Hz,2H),4.45–4.26( m,2H),4.08(dd,J＝15.5,8.9Hz,3H),3.73(s,3H),3.61(dd,J＝10.6,3.7Hz,1H),2.19(s,3H),2.12(s ,3H). ¹³ CNMR (100MHz, CDCl ₃ ) δ20.15,20.85,22.71,29.72,45.43,52.39,61.07,61.94,67.53,67.75,68.03,70.98,71.12,72.42,72.52,95.88,99.87,1 27.38,127.92,128.16, 128.50,128.63,129.18,130.04,133.28,133.62,165.54,165.95,169.22,170.14,170.80.

Example 3 Preparation method of fully protected tetrasaccharide compound

1. Preparation of compound CV025-1

Add to the two bottles Molecular sieves (1g, powder), vacuum the flask, pass argon protection and cool to room temperature. Disaccharide donor 7-1β (164.0mg, 0.197mmol) and disaccharide acceptor 8-1 (168mg, 0.21mmol) were dissolved in about 10mL of dry dichloromethane, stirred at room temperature for 30 minutes and then cooled to -20℃, TfOH (4.5μL, 0.025mmol) was added, and then the reaction was slowly restored to room temperature. After 1 hour, TLC monitoring showed that the donor completely disappeared and a major substance was generated. Then triethylamine was added to quench the reaction, and the molecular sieve was filtered with silica gel. The filtrate was concentrated under reduced pressure and directly purified by column chromatography (PE: EA = 3: 1) to obtain a white solid CV025-1 (262mg, 64%).

NMR data of compound CV025-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.06 (d, J = 7.2Hz, 2H), 7.51 (t, J = 7.4Hz, 1H), 7.40 (t, J = 7.6Hz, 2H), 7.24–7.05 (m,25H),5.55(d,J＝5.9Hz,1H),5.37–5.26(m,1H),5.15(d,J＝6.2Hz,1H),5.03(d,J＝3.6Hz,1H) ,5.00–4.85(m,3H),4.82(d,J＝11.7Hz,1H),4.74–4.58(m,7H),4.58–4.49(m,3H),4.33(d,J ＝12.2Hz,1H),4.29–4.19(m,2H),4.12(d,J＝12.1Hz,2H),4.09–3.95(m,4H),3.89(t,J＝9.5Hz,2H),3.73 (d,J＝10.0Hz,1H),3.69–3.51(m,8H),3.51–3.43(m,1H),3.40–3.33(m,1H),3.17(s,3H),3.12(d,J =3.5Hz,1H),2.09(s,3H),2.04(s,3H),1.87(s,3H),1.83(s,3H). ¹³ C NMR (100MHz, CDCl ₃ )δ20.58,20.69,20.79,20.84,52.43,52.60,54.37,55.26,60.42,60.78,61.50,62.06,66.92,68.85,69.24,69.72,71.15,71.31,71.98,72.93, 73.08,74.44,74.70,75.32 ,75.37,75.83,77.29,78.53,81.10,81.43,97.67,98. 17,98.83,102.88,127.64,127.69,127.75,127.91,128.16,128.25,128.33,128.52,128.71,129.07,130.02,133.52,136.28,137.35,137.51, 138.05,138.55,155.87,165.40,167.53,169.57,169.99, 170.16,170.23,171.00.

2. Preparation of compound CV026-1

The synthesis steps are the same as compound CV025-1, that is, the disaccharide donor 7-1α and the disaccharide 8-1 undergo glycosylation coupling.

NMR data of compound CV026-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.15(d,J＝7.6Hz,2H),7.57(t,J＝7.4Hz,1H),7.49(d,J＝7.6Hz,2H),7.37–7.27 (m,16H),7.24–7.15(m,9H),5.63(d,J＝6.1Hz,1H),5.46(t,J＝9.8Hz,1H),5.23(d,J＝6.2Hz,1H) ,5.14(s,1H),5.11–4.98(m,3H),4.95–4.85(m,2H),4.78–4.56(m ,10H),4.48(d,J＝12.0Hz,1H),4.37–4.08(m,7H),3.96(d,J＝27.5Hz,3H),3.86(t,J＝7.7Hz,1H),3.75 (s,4H),3.66(s,3H),3.62(s,1H),3.54(t,J＝9.4Hz,1H),3.49(d,J＝8.0Hz,1H),3.25(s,4H) ,2.13(s,3H),1.99(s,4H),1.93(d,J＝2.8Hz,6H). ¹³ C NMR (100MHz, CDCl ₃ )δ20.74,20.77,20.85,20.98,30.05,30.21,31.45,37.12,37.24,52.35,52.69,54.39,55.27,60.92,62.10,62.49,66.95,68.84,69.67,70.75, 71.44,72.18,73.02,74.23 ,74.61,74.72,77.27,78.06,78.44,97.58,98.17,98.58, 98.84,127.39,127.76,127.82,127.87,127.91,127.93,128.17,128.21,128.34,128.45,128.50,128.58,128.67,129.10,130.03,133.50,136 .30,137.34,137.79,137.94,155.87,165.34,168.48,169.85, 169.99,170.18,170.98.

3. Preparation of compound CV027-1

The synthesis steps are the same as compound CV025-1, that is, the disaccharide donor 7-2β and the disaccharide 8-1 are glycosylated and coupled.

NMR data of compound CV027-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.13(d,J＝7.7Hz,2H),7.58(d,J＝7.5Hz,1H),7.48(t,J＝7.8Hz,2H),7.35–7.27 (m,7H),7.25–7.16(m,13H),5.60(d,J＝5.9Hz,1H),5.40(t,J＝10.2Hz,1H),5.25–5.14(m,2H),5.11– 4.97(m,4H),4.87(d,J＝11.5Hz,1H),4.75–4.44(m,10H),4.31(d,J＝12 .3Hz,1H),4.22–4.03(m,8H),3.93(d,J＝9.8Hz,2H),3.77–3.70(m,6H),3.64(d,J＝10.1Hz,1H),3.54( t,J＝9.7Hz,1H),3.40(t,J＝8.6Hz,1H),3.23(d,J＝15.2Hz,3H),2.15–2.09(m,6H),2.04(d,J＝2.3 Hz,3H),1.98(d,J＝2.6Hz,3H),1.85(d,J＝2.3Hz,3H). ¹³ CNMR(100MHz,CDCl ₃ )δ170.94,170.14,169.99,169.55,167.09,165.39,137.32,137.23,136.28,133.48,130.01,129.09,128.69,128.54,128.49,128.33,128.14,1 28.05,127.96,127.91,127.82,127.65,127.33,102.91,98.83 ,98.20,97.49,78.54,77.35,77.24 ,77.03,76.71,76.14,74.89,74.69,74.35,72.80,72.68,71.20,69.76,69.67,69.25,68.86,66.92,62.10,61.19,60.78,60.39,55.26,52.69, 52.44,31.93,29.70,29.66,29.36 ,22.69,21.04,20.81,20.78,20.67,20.60,20.47,14.20,14.12.

4. Preparation of compound CV028-1

The synthesis steps are the same as compound CV025-1, that is, the disaccharide donor 7-2α and the disaccharide 8-1 undergo glycosylation coupling.

NMR data of compound CV028-1:

¹ H NMR (400 MHz, CDCl ₃ )δ8.14(d,J＝7.2Hz,2H),7.60(d,J＝7.4Hz,1H),7.49(t,J＝7.6Hz,2H),7.40–7.27(m,10H),7.18(m,10H),5.65(d,J＝6.2Hz,1H),5.50–5.40(m,1H),5.3 7(t,J＝9.2Hz,1H),5.23(t,J＝6.4Hz,1H),5.16(d,J＝3.5Hz,1H),5.12–4.96(m,4H),4.88(d,J＝11.5Hz,1H),4.76(d,J＝9.8Hz,1H),4.70(s,2H),4.65–4 .52( m,4H),4.48(d,J＝12.7Hz,1H),4.33(dd,J＝12.3,3.4Hz,1H),4.28(d,J＝9.6Hz,1H),4.26–4.08(m,5H),4.05–3.89(m,3H),3.79(d,J＝10.6Hz,4H),3.72( s,3H),3.63(d,J＝9.5Hz,1H),3.59–3.48(m,2H),3.25(s,3H),3.12(dd,J＝10.6,3.5Hz,1H),2.13(s,3H),2.04(s,3H),2.02(s,3H),1.96(d,J＝2.4Hz, 6H). ¹³ C NMR (100MHz, CDCl ₃ ) δ20.20,20.69,20.83,22.70,29.37,29.71,32.53,52.48,52.87,56.08,60.42,61.69,63.30,66.74,69.34,69.51,70.19,7 2.57,73.85,74.59,77.25,78.41,97.67,97.74,98.11,98.8 4,127.37,127.76,127.91,127.94,128.17,128.30,128.33,128.50,128.63,128.70,129.05,129.99,133.72,136.24,137.29,155.82,165.33, 168.06,169.23,169.86,170.12,171.21.

5. Preparation of Compound CV029-1

The synthesis steps are the same as compound CV025-1, that is, the disaccharide donor 7-3β and the disaccharide 8-1 are glycosylated and coupled.

NMR data of compound CV029-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.13(d,J＝7.5Hz,2H),7.54(t,J＝7.4Hz,1H),7.44(t,J＝7.4Hz,2H),7.32(dd ,J＝17.3,14.0Hz,20H),7.22(d,J＝8.7Hz,10H),5.45(s,1H),5.20–5.12(m,2H),5.08–4.98(m,3H),4.88– 4.73(m,8H),4.64(d,J＝11.5Hz,2H),4.55(d,J＝11.6Hz,1H),4.50–4. 40(m,2H),4.33–4.20(m,4H),4.13(d,J＝5.1Hz,1H),4.04–3.90(m,4H),3.78(t,J＝8.8Hz,2H),3.67 (dd,J＝18.9,10.2Hz,3H),3.55(dd,J＝14.4,8.8Hz,2H),3.46(d,J＝20.6Hz,6H),3.29(s,3H),3.23(d, J=9.8Hz, 1H), 2.03 (s, 3H), 1.96 (s, 3H), 1.91 (s, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ14.13,20.62,20.78,20.85,22.70,29.37,29.67,31.94,52.02,52.55,53.45,54.50,55.27,62.31,62.85,66.97,68.99,69.83,70.42,71.19, 73.24,73.74,74.59,75.22 ,75.45,76.09,77.25,77.94,78.28,78.75,81.45,81.82,98.38,98.48,98.82,99.98,103 .11,127.29,127.43,127.53,127.75,127.80,127.84,127.89,128.03,128.08,128.14,128.18,128.24,128.36,128.43,128.51,128.60,128.6 6,129.34,129.95,133.54,136.24,137.44,137.99,138.15,155.83 ,165.46,167.42,169.47,169.55,170.21,170.75.

6. Preparation of compound CV030-1

The synthesis steps are the same as compound CV025-1, that is, the disaccharide donor 7-3α and the disaccharide 8-1 undergo glycosylation coupling.

NMR data of compound CV030-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.13 (d, J = 7.6Hz, 2H), 7.50–7.26 (m, 19H), 7.25–7.08 (m, 14H), 5.49 (t, J = 4.7Hz, 2H),5.17(d,J＝4.9Hz,1H),5.09(t,J＝8.1Hz,1H),5.00(d,J＝7.7Hz,2H),4.90–4.75(m ,6H),4.73–4.56(m,7H),4.49–4.39(m,2H),4.37–4.20(m,5H),4.04–3.87(m,6H),3.72(d,J＝9.1Hz,2H ),3.64(s,3H),3.54(s,5H),3.30(s,3H),1.97(s,3H),1.96(s,3H). ¹³ C NMR (100MHz, CDCl ₃ )δ20.67,20.76,20.87,22.70,29.37,29.70,31.93,52.00,52.60,54.46,55.28,62.38,62.62,63.31,66.98,68.81,69.32,69.93,70.44,73.72, 73.91,74.17,74.48,74.65 ,75.12,77.25,78.76,79.58,97.81,98.09,98.36,98.83,126.97,127. 34,127.56,127.67,127.81,127.85,127.93,128.05,128.16,128.24,128.29,128.38,128.43,128.51,128.62,129.37,129.95,133.45,136.24 ,137.37,137.56,137.75,137.83,138.36,155.61,165.50,168.56, 169.57,169.87,170.47,170.80.

6. Preparation of compound CV031-1

The synthesis steps are the same as compound CV025-1, that is, the disaccharide donor 7-2α and the disaccharide 8-1 undergo glycosylation coupling.

NMR data of compound CV031-1:

¹ H NMR (400MHz, CDCl ₃ ) δ8.14(d,J＝7.7Hz,2H),8.07(d,J＝7.8Hz,2H),7.72(d,J＝7.7Hz,2H),7.66–7.53 (m,2H),7.51–7.27(m,17H),7.21(dd,J＝17.2,5.7Hz,8H),7.05(t,J＝7.6Hz,2H),5 .64(d,J＝6.3Hz,1H),5.44(s,1H),5.34(t,J＝9.7Hz,1H),5.23(d,J＝6.1Hz,1H),5.19(s,1H) ,5.15(d,J＝3.6Hz,1H),5.09–4.96(m,4H),4.90(d,J＝11.5Hz,1H),4.83( s,2H),4.78(d,J＝9.9Hz,1H),4.70(s,2H),4.66–4.56(m,2H),4.54(d,J＝4.5Hz,1H),4.39(s,2H ),4.33(d,J＝12.3Hz,1H),4.18(d,J＝12.1Hz,1H),4.11(q,J＝4.5,2.6 Hz,3H),4.06–3.91(m,3H),3.87(t,J＝9.4Hz,1H),3.73(s,3H),3.66–3.50(m,5H),3.26(s,3H),3.15 (dd,J=10.6,3.5Hz,1H),2.13(s,3H),2.12(s,3H),2.06(s,3H). ¹³ C NMR (100MHz, CDCl ₃ )δ20.83,20.94,22.70,29.37,29.67,29.71,31.94,52.19,52.41,54.37,55.26,61.28,61.99,63.01,66.91,67.44,68.06,68.18,68.83,70.00, 70.68,71.49,72.40,72.57 ,72.89,73.15,74.55,77.27,77.70,78.41,97.88,98.04,98.85,100.5 6,127.37,127.63,127.75,127.79,127.89,128.17,128.23,128.32,128.50,128.61,128.67,129.09,129.21,129.80,130.02,133.50,133.60, 135.81,137.07,137.36,138.59,156.64,165.19,165.33,166.08, 168.80,170.29,170.72,171.02.

Example 4 Preparation of sulfonated tetrasaccharide compound

1. Preparation of compound CV025

The fully protected tetrasaccharide compound CV025-1 (342.62 mg, 0.26 mmol) was dissolved in 5.00 mL of tetrahydrofuran, and then 6.23 mL of 1.25 N LiOH solution and 13.13 mL of 30% hydrogen peroxide solution were added dropwise at room temperature. After continuous stirring for 12 hours, 14.34 mL of methanol and 7.82 mL of 6 N NaOH solution were added, and stirring was continued for at least 12 hours. After TLC detection, the pH was adjusted to 2 with 4 N hydrochloric acid under ice-water bath conditions, and then the reaction solution was extracted with DCM three times and concentrated under reduced pressure and silica gel column chromatography (DCM: MeOH = 15: 1) was performed to obtain a white solid 1-1β (258.0 mg, 93%). A small amount was taken for high performance liquid chromatography analysis and it was found that there was only one single peak, indicating that its purity was very high. Under argon protection, the above product 1-1β (258.02 mg, 0.24 mmol) and SO ₃ ·NMe ₃ (897.62 mg, 5.33 mmol) were dissolved in 3 mL of anhydrous DMF. The reaction system was heated to 65°C and stirred for at least 12 h. The reaction solution was taken and monitored by HPLC. After stopping the heating, the reaction system was allowed to naturally warm to room temperature. The reaction solution was concentrated and purified by Sephadex LH-20 gel column, and then ion exchanged with Dowex-50-WX4-Na+ to obtain the intermediate 2-1β (304.13 mg, 93%). Compound 2-1β (304.13 mg, 0.22 mmol) was dissolved in 3.00 mL of a mixed solvent consisting of methanol, tert-butanol and water (v/v/v = 2:1:1), palladium carbon (50.00 mg) was added, and the mixture was stirred for 24 h under a hydrogen pressure of 4 atm. The palladium carbon was then filtered through filter paper to remove the palladium carbon, and the reaction solution was concentrated. Dowex-50-WX4-Na+ was used for ion exchange to obtain the intermediate 3-1β (184.93 mg, 98%). Intermediate 3-1β (35.23 mg, 0.045 mmol) was dissolved in 0.50 mL of water, and then the pH was adjusted to 9-10 with 4N NaOH solution, and then sulfur trioxide pyridine complex (216.82 mg, 1.366 mmol) was added in batches. Note that sodium hydroxide solution was continuously added dropwise to maintain the pH when the sulfur trioxide pyridine complex was added. TLC detected that the reaction was complete and the reaction solution was neutralized with 1N hydrochloric acid to a pH of about 9. The reaction solution was then concentrated and separated and purified with a Sephadex G-25 gel column. After concentration, it was exchanged into sodium salt with a Dowex-50-WX4-Na+ column, and the sugar-containing component was collected and the solvent was concentrated to obtain the target product CV025 (41.2 mg, 95%).

NMR data of compound CV025:

¹ H NMR (400MHz, D ₂ O) δ5.40(d,1H),5.17(s,1H),4.93(d,1H),4.78(d,1H),4.52-4.39(m,3H),4.27 -3.87(m,12H),3.73-3.62(m,4H),3.41-3.20(m,6H). ¹³ C NMR(100MHz,D ₂ O)δ45.52,53.25,54.11,55.31,63.19,66.56,67.36,68.75,68.92,69.26,70.84,72.20,72.63,73.20,74.09,74.92,77.21,78.50,81.59,91.13 ,95.87,99.17,101.17, 174.41,176.63.

HRMS[M-2Na] ⁺ m/z 730.8857(calcd for C ₂₅ H ₃₃ N ₂ Na ₉ O ₄₂ S ₇ ^2- ,730.8738).

2. Preparation of compound CV026

The synthesis steps are the same as those of compound CV025-1, namely, deprotection to obtain intermediate 1-1α, O-sulfonation to obtain intermediate 2-1α, catalytic hydrogenation to obtain intermediate 3-1α, and N-sulfonation to obtain the target product CV026.

NMR data of compound CV026:

¹ H NMR (400MHz, D ₂ O) δ5.35 (d, J = 3.8 Hz, 1H), 5.21 (d, J = 4.8 Hz, 1H), 5.10 (s, 1H), 4.86 (d, J = 5.3 Hz,2H),4.27–4.07(m,13H),3.99–3.78(m,8H),3.66–3.51(m,6H),3.30(s,3H),3.26(s,3H),3.12(dd, J=9.9, 3.6Hz, 1H). ¹³ C NMR (100MHz, D ₂ O)δ53.19,54.51,55.39,58.14,62.85,66.58,68.50,68.99,69.40,69.83,70.89,72.60,73.32,74.57,76.45,76.83,77.74,79.39,82.67,95.94 ,98.34,98.88,99.05, 160.88,167.47.

3. Preparation of compound CV027

The synthesis steps are the same as those of compound CV025-1, namely, deprotection to obtain intermediate 1-2β, O-sulfonation to obtain intermediate 2-2β, catalytic hydrogenation to obtain intermediate 3-2β, and N-sulfonation to obtain the target product CV027.

NMR data of compound CV027:

¹ H NMR (400MHz, D ₂ O) δ 5.54 (d, J = 3.5 Hz, 1H), 5.21 ( d, J = 3.9 Hz, 1H), 5.05 ( d, J = 3.6 Hz, 1H), 4.75 ( d,J＝7.8Hz,2H),4.54–4.30(m,10H),4.19(td,J＝7.5,4.0Hz,3H),4.08–3.93(m,4H),3.79(dt,J＝13.2, 9.0Hz, 2H), 3.68 (dd, J＝9.1, 7.3Hz, 2H), 3.51–3.42 (m, 5H), 3.33–3.29 (m, 1H). ¹³ C NMR (100MHz, D ₂ O) δ100.71,99.56,98.28,96.20,80.93,77.15,76.37,76.22,76.13,75.56,73.30,72.53,70.28,70.17,69.81,69.53,68.54,66.73,66.11,57.7 0,56.66,55.43.

4. Preparation of compound CV028

The synthesis steps are the same as those of compound CV025-1, namely, deprotection to obtain intermediate 1-2α, O-sulfonation to obtain intermediate 2-2α, catalytic hydrogenation to obtain intermediate 3-2α, and N-sulfonation to obtain the target product CV028.

NMR data of compound CV028:

¹ H NMR (400MHz, D ₂ O) δ5.38 (d, J = 3.8 Hz, 1H), 5.29 (d, J = 2.0 Hz, 1H), 5.11 (s, 1H), 4.86 (d, J = 3.7 Hz,1H),4.83(d,J＝2.0Hz,1H),4.78(t,J＝3.4Hz,1H),4.64(d,J＝2.8Hz,1H),4.57–4.49(m,3H), ¹³ C NMR(100MHz,D ₂ O)δ53.15,54.19,55.38,62.81,66.57,66.68,67.25,68.67,68.97,69.02,70.57,72.71,73.10,74.15,74.51,74.71,76.39,76.97,81.79,90.99 ,95.03,97.14,99.09, 173.24,173.84.

5. Preparation of compound CV029

The synthesis steps are the same as those of compound CV025-1, namely, deprotection to obtain intermediate 1-3α, O-sulfonation to obtain intermediate 2-3α, catalytic hydrogenation to obtain intermediate 3-3α, and N-sulfonation to obtain the target product CV029.

NMR data of compound CV029:

¹ H NMR (400MHz, D ₂ O) δ5.34 (d, J = 3.5 Hz, 1H), 5.03 (d, J = 3.6 Hz, 1H), 4.86 (d, J = 3.6 Hz, 1H), 4.47 ( d,J＝7.8Hz,1H),4.32(d,J＝9.2Hz,1H),4.28–4.07(m,8H),4.04–3.96(m,3H),3.85–3.77(m,2 H),3.68(d,J＝9.9Hz,1H),3.64–3.59(m,1H),3.56(d,J＝9.2Hz,1H),3.53–3.46(m,1H),3.36–3.29(m ,2H),3.28(d,J＝3.4Hz,1H),3.26(s,3H),3.12(dd,J＝10.4,3.6Hz,1H). ¹³ C NMR (100MHz, D ₂ O) δ55.41,56.59,57.68,65.95,66.67,68.51,69.53,69.78,70.00,70.45,72.72,73.00,74.18,75.94,76.01,76.28,76.91,78. 96,96.71, 97.79,99.48,100.89,174.13,174.32.

6. Preparation of compound CV030

The synthesis steps are the same as those of compound CV025-1, namely, deprotection to obtain intermediate 1-3α, O-sulfonation to obtain intermediate 2-3α, catalytic hydrogenation to obtain intermediate 3-3α, and N-sulfonation to obtain the target product CV030.

NMR data of compound CV030:

¹ H NMR (400MHz, D ₂ O) δ5.32(t,J＝4.2Hz,2H),5.13(s,1H),4.91(d,J＝3.7Hz,1H),4.27–4.00(m,11H ),3.86(dd,J＝10.4,7.8Hz,2H),3.70–3.52(m,5H),3.34(s,1H),3.30(s,3H),3.16(dd,J＝9.2,2.9Hz, 1H). ¹³ C NMR (100MHz, D ₂ O)δ53.88,55.35,56.18,57.69,59.78,66.36,66.77,67.69,69.33,69.94,70.49,71.05,71.46,72.18,72.38,72.84,75.14,76.71,77.33,95.56 ,96.88,98.17,99.31, 167.18,168.86.

7. Preparation of compound CV031

The synthesis steps are the same as those of compound CV031-1, namely, deprotection to obtain intermediate 4-1, O-sulfonation to obtain intermediate 5-1, catalytic hydrogenation to obtain intermediate 6-1, and N-sulfonation to obtain the target product CV031.

NMR data of compound CV031:

¹ H NMR (400MHz, D ₂ O) δ5.34 (d, J = 3.9 Hz, 1H), 5.15 (s, 1H), 5.11 (s, 1H), 5.07 (s, 1H), 4.85 (d, J ＝3.6Hz,1H),4.81(s,1H),4.55–4.45(m,3H),4.29(s,1H),4.25–4.01(m,9H),3.85(dd,J＝20.1,9.8Hz, 5H),3.69–3.55(m,2H),3.29(s,3H),3.24(dd,J＝10.7,3.6Hz,1H). ¹³ CNMR(100MHz,D ₂ O)δ53.47,54.15,56.77,63.08,64.38,65.18,66.38,66.62,67.17,68.70,68.95,69.38,70.89,71.53,72.81,73.11,74.38,74.79,76.47,91.21 ,95.88,98.87,99.76, 173.43,173.79.

Example 5 Heparanase Activity Inhibition Experiment

The heparanase activity inhibition experimental method of some tetrasaccharide compounds is as follows: active heparanase is diluted to 120 ng/mL with Buffer A, biot-HS-K is diluted to 1.4 ng/mL with Buffer B (pH 5.5), SA-d2 is diluted to 1 μg/mL with Buffer C (pH 7.5), and the compound samples are diluted to different concentration gradients with Buffer A.

4 μL of sample solution or fondaparinux solution and 3 μL of heparanase solution or Buffer A were added to a micro-96 well plate. After the micro-96 well plate was incubated at 37°C for 10 min, the enzyme reaction was started by adding 3 μL of biot-HS-K and incubated at 37°C for 30 min. Then 10 μL of SA-d2 solution or Buffer C was added. After incubation at room temperature for 15 min, the HTRF signal was detected using a microplate reader, with excitation at 384 nm and emission at 620 nm and 665 nm. The fluorescence intensity ratio of 665 nm/620 nm was used to calculate the average energy transfer rate (ΔF%) for each sample. The positive control system consisting of biot-HS-K and SA-d2 should produce the maximum ΔF%, while the background fluorescence intensity of the negative control (biot-HS-K) should produce the minimum ΔF%.

The inhibition rate is based on the following formula:

R(Ratio)＝(665nm/620nm ratio of fluorescence intensity)×10,000

ΔR＝Rsample-Rnegtive

ΔF(%)=(ΔR/Rnegtive)×100

ΔFmax＝ΔF of positive control

ΔFblank＝ΔF of buffer A

Inhibition(%)=(ΔFsample-ΔFblank)/(ΔFmax-ΔFblank)×100%

The experimental results are shown in Figure 1. Compounds CV025, CV026, CV027, CV028, CV029 and CV030 all showed a certain inhibitory effect on heparanase, with _IC50 values of 22.38μM, 26.68μM, 20.30μM, 25.51μM, 30.61μM and 25.95μM, respectively.

Claims (6)

1. A compound having a structure of formula I, or a pharmaceutically acceptable salt thereof, wherein the four monosaccharides are represented by A, B, C, and D from left to right respectively: The invention is characterized in that: R ₁ is the same and is NHSO ₃ Y; R ₂ of sugar B is selected from hydrogen or SO ₃ Y, and R ₂ of sugar D is selected from hydrogen; R ₃ is the same and is SO ₃ Y; R ₄ is selected from hydrogen; R ₅ is selected from SO ₃ Y; R ₆ is hydrogen or an alkyl group of 1 to 4 carbon atoms; Y are the same or different and are independently selected from hydrogen or a monovalent cation, wherein the monovalent cation is Na ⁺ , K ⁺ , Li ⁺ or NH ₄ ⁺ ; Monosaccharide A is selected from sulfonated glucuronic acid, R represents a substituent of a hydroxyl group on the sugar ring of monosaccharide A, R includes a substituent OR ₇ at position 2, a substituent OR ₈ at position 3, and a substituent OR ₉ at position 4 of the sugar ring, wherein R ₇ and R ₈ are the same or different and are independently selected from hydrogen or SO ₃ Y, and R ₉ is selected from SO ₃ Y; Indicates that the glycosidic bond configuration is α or β. 2. The compound according to claim 1, characterized in that R ₁ is the same and is NHSO ₃ Y, B sugar R ₂ is selected from hydrogen or SO ₃ Y, D sugar R ₂ is selected from hydrogen, R ₃ , R ₅ , R ₇ and R ₉ are the same and are selected from SO ₃ Y, R ₄ is selected from hydrogen, R ₆ is selected from methyl (Me), R ₈ is selected from hydrogen or SO ₃ Y, A is selected from glucuronic acid, and Y is selected from Na ⁺ . 3. The compound according to claim 1-2, which is selected from the following structures: 4. A pharmaceutical composition comprising the compound according to claims 1-3 or a pharmaceutically acceptable salt thereof; characterized in that the pharmaceutical composition is used to treat or prevent diseases caused by overexpression of heparanase, including but not limited to inflammatory diseases and cancer. 5. Use of the compound according to claims 1-3 in the preparation of drugs, characterized in that the compound is used in the preparation of heparanase inhibitors, and the drug is used to treat or prevent diseases caused by heparanase overexpression, including but not limited to inflammatory diseases and cancer. 6. A method for preparing compound CV025, characterized in that The specific preparation method is as follows: After the fully protected tetrasaccharide compound CV025-1 is dissolved in tetrahydrofuran, 1.25N LiOH solution and 30% hydrogen peroxide aqueous solution are added dropwise at room temperature, and methanol and 6N NaOH solution are added after continuous stirring for 12 hours, and stirring is continued for at least 12 hours. After TLC detection, the pH is adjusted to 2 with 4N hydrochloric acid under ice-water bath conditions, and then the reaction solution is extracted with DCM three times and concentrated under reduced pressure and then subjected to silica gel column chromatography (DCM: MeOH = 15: 1) to obtain a white solid 1-1β. Under argon protection, the above product 1-1β and SO ₃ ·NMe ₃ are dissolved in anhydrous DMF, the reaction system is heated to 65°C and stirred for at least 12 hours, the reaction solution is taken and the reaction degree is monitored by high performance liquid chromatography, and the reaction system is allowed to naturally rise to room temperature after stopping heating. The reaction solution is concentrated and purified by Sephadex LH-20 gel column, and then Dowex-50-WX4-Na ⁺ ion exchange was performed to obtain intermediate 2-1β. Compound 2-1β was dissolved in a mixed solvent consisting of methanol, tert-butanol and water (v/v/v=2:1:1), palladium carbon was added, and the mixture was stirred for 24 hours under a hydrogen pressure of 4 atm. The palladium carbon was then filtered through filter paper to remove the palladium carbon and the reaction solution was concentrated. Dowex-50-WX4-Na+ was used to perform ion exchange to obtain intermediate 3-1β. Intermediate 3-1β was dissolved in water, and the pH was adjusted to 9-10 with a 4N NaOH solution. Then sulfur trioxide pyridine complex was added in batches. Note that sodium hydroxide solution was continuously added dropwise when sulfur trioxide pyridine complex was added to maintain the pH. When the reaction was complete by TLC, 1N hydrochloric acid was used to neutralize the reaction solution to a pH of 9. The reaction solution was then concentrated and separated and purified using a Sephadex G-25 gel column. After concentration, it was exchanged into sodium salt using a Dowex-50-WX4-Na+ column. The sugar-containing component was collected and the solvent was concentrated to obtain the target product CV025.