CN114957354A

CN114957354A - Heparin pentasaccharide structural compound

Info

Publication number: CN114957354A
Application number: CN202111544675.9A
Authority: CN
Inventors: 赵炜; 张国强; 金洪真; 王凯旋
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-02-25
Filing date: 2021-12-16
Publication date: 2022-08-30
Anticipated expiration: 2041-12-16
Also published as: CN114957354B

Abstract

The invention provides a heparin pentasaccharide compound and a preparation method thereof, the heparin pentasaccharide compound has HPA inhibitory activity, and can be used for treating or preventing pathophysiological processes or diseases in which HPA participates, or diseases in which the pathophysiological changes are brought by HPA overexpression or activity enhancement, such as reducing tumor metastasis and invasion, prolonging the survival time of tumor patients, or treating kidney diseases, cardiovascular diseases, inflammatory diseases, pathological angiogenesis and the like.

Description

Heparin pentasaccharide structural compound

Technical Field

The invention belongs to the technical field of pharmaceutical compounds, and particularly relates to a heparin pentasaccharide structure compound, a preparation method thereof, and HPA (lipocalins inhibitor) inhibitory activity and pharmaceutical application thereof.

Background

An important component of the surface and extracellular matrix of eukaryotic cells is Heparan Sulfate Proteoglycan (HSPG), which is a complex of a core protein and four Heparan Sulfate (HS) side chains, mainly distributed on the intima and media lining of large blood vessels and the subcutaneous basement membrane of capillary blood vessels. Heparanase (HPA) is the only endoglycosidase found in mammalian cells to date and is capable of specifically recognizing HSPG and performing biological functions by hydrolyzing glycosidic bonds to degrade HSPG into short sugar chains of 4kDa-7 kDa.

Heparanase is distributed in vivo mainly on the cell membrane or extracellular matrix of platelet cells, mast cells, placental basement membrane, keratinocytes, leukocytes, with two natural substrates, heparin and heparan sulfate.

The heparanase is found to be over-expressed in various cancer cells such as lung cancer, breast cancer, liver cancer, intestinal cancer and the like in cell and animal experiments, and is closely related to cell metastasis, angiogenesis and lymphangiogenesis by hydrolyzing heparanase side chains of HSPG to destroy physical barriers of cells and release related growth factors, so that the heparanase is suggested to be closely related to the metastasis and invasiveness of cancer and possibly be a potential anti-tumor drug target. Theoretically, after competitive inhibition of heparanase, degradation of tumor cells HSPG can be prevented, so that the metastasis and invasiveness of cancer cells are reduced.

Heparanase has also been found to be involved in other diseases such as diabetic nephropathy, proteinuric glomerular disease, amyloidosis nephropathy, osteolysis in bone metastasis and other bone tissue-related pathologies, atherosclerosis, cardiovascular disease and skin aging, as well as abnormal angiogenesis, inflammatory response processes, etc. For example, it has been found that Smooth Muscle Cell (SMC) proliferation can stabilize atherosclerotic lesions, and heparanase can participate in the regulation of atherosclerotic SMC function, closely related to the development of atherosclerotic plaques and the turnover of unstable plaques, since heparan sulfate can regulate SMC proliferation and affect plaque stability, and heparanase expression is increased in atherosclerosis associated with inflammation, coagulation and plaque instability. Membranous Nephropathy (MN) is a major cause of Nephrotic Syndrome (NS), and common clinical manifestations of MN patients include massive proteinuria, hypoproteinemia, hyperlipidemia and venous thrombosis, which eventually leads to renal failure and uremia with the delayed development of the disease. HSPGs are localized to the glomerular filtration membrane in the kidney and carry a large negative charge, playing an important role in the maintenance of the glomerular charge barrier. Researchers injected monoclonal antibodies aiming at HSPG into rats to block HSPG side chains, and the results show that selective proteinuria is generated in the rats, which indicates that HSPG plays an important role in generation of proteinuria. HPA degrades HSPG on a basement membrane, destroys the integrity of the HSPG, reduces negative charges on the basement membrane, weakens a charge barrier, enables proteins to easily permeate glomeruli, and accordingly generates a large amount of proteinuria.

Thus, for pathophysiological processes or diseases in which HPA is involved, or diseases in which the over-expression or activity of HPA is enhanced to bring about pathophysiological changes, the use of inhibitor compounds targeting HPA can serve to treat or improve the corresponding pathophysiological processes or diseases, for example, can be used to reduce tumor metastasis and invasion, prolong the survival of tumor patients, or treat renal diseases, cardiovascular diseases, inflammatory diseases, pathological angiogenesis, etc.

In the eighties of the twentieth century, the work of fully synthesizing pentasaccharide compounds has started, the problem of low reaction efficiency caused by intermolecular reaction of reduction end hemiacetal of pentasaccharide in the synthesis is mainly solved, and how to design a synthetic route comprises using a proper protecting group, so that regioselectivity and stereoselectivity are favorable for forming correct glycosidic bonds, and the reaction efficiency is improved.

The Petitou and van Boeckel groups have synthesized the pentasaccharide sequences present in the native heparin molecule sequentially, using a [1+4] synthesis strategy, to give a fully protected pentasaccharide molecule in about 70% yield. The rest glycosyl donors except the iduronic acid donor adopt bromo-sugar which is generally activated by silver salt, and the synthesis cost of the pentasaccharide is relatively high. The pentasaccharide is the first heparin molecule with anticoagulant activity in chemical synthesis, lays a foundation for successful marketing of fondaparinux sodium in the later period, and opens up a way for the synthesis of other heparin molecules.

In the subsequent synthesis, it is found that when pentasaccharide is subjected to catalytic hydrogenation, the benzyl at the reducing end is removed to form hemiacetal, and all amino groups are exposed, under the condition, the amino and aldehyde groups can rapidly generate stable Schiff base, and the obtained final product always contains dimer or trimer. In order to solve the problem, the Petitou group protects the terminal position of glucosamine at the reducing end by methyl, and couples the glucosamine by adopting the same synthetic strategy as the pentasaccharide to obtain the fully protected pentasaccharide, and the heparin pentasaccharide modified by sulfonation is fondaparinux sodium.

In 1991, the Petitou group adopts a [3+2] convergent synthesis strategy to synthesize the fully-protected pentasaccharide with the reduction end protected by methyl, the glycosylation yield is about 70%, and the glycosylation reaction is stereospecific and is simpler and more convenient to separate and purify. At the same time, this strategy allows a considerable saving of disaccharide fragments containing iduronic acid, which is much more economical than the above-mentioned [1+4] route, due to the more synthetic steps and the low yields of iduronic acid.

However, even so, the steps of the synthetic method exceed sixty steps and the yield is less than 0.1%, which is the longest chemical in the synthetic step in the world at that time. Fondaparinux sodium becomes a non-patent drug after 2008, and more scientists or enterprises begin to develop the synthesis technology of fondaparinux sodium. Research teams such as Zhou, Ding, Qin and the like successively use a [3+2] convergent synthesis strategy to synthesize fondaparinux sodium, and the method is characterized in that the used trisaccharide donor leaving groups are different, the protecting group strategies are different, and the method also has the characteristics in the process of synthesizing monosaccharide modules.

A Zhou research group in 2013 reports a synthetic route of fondaparinux sodium, the whole route adopts a [3+2] convergent synthetic strategy, 36 steps of reaction are required in total, and the total yield is 0.017%. It should be noted that this route directly uses cellobiose as a starting material to synthesize a disaccharide acceptor consisting of glucuronic acid and glucosamine. The method effectively avoids the problem of poor beta selectivity of glucuronic acid glycosylation reaction. Meanwhile, researchers have synthesized an ethylthio donor and a trichloroacetimidate donor of iduronic acid, and found that the disaccharide product generated by glycosylation with the trichloroacetimidate donor is α/β isomer, while the disaccharide product is of a single configuration when ethylthio is used as the glycosyl donor.

In 2014, Hung topic group reports a total synthesis route of fondaparinux sodium, a [4+1] convergent synthesis strategy is adopted, the total route is 36 steps, and the total yield can reach 0.63%. Most of monosaccharide modules used in the route are synthesized by the one-pot method invented by the subject group. And the strategy of firstly glycosylating and then oxidizing to uronic acid is adopted, so that the defects of weak reactivity and low yield of uronic acid are effectively overcome. And the 2-position of glucose is a Bz protecting group, and an ortho-group is involved, so that the newly generated glycosidic bond is completely in a beta configuration.

In 2016, the Qin research group published a complete synthetic route of fondaparinux sodium, and a [3+2] convergent synthesis strategy was adopted, so that 14 reactions were required in total from the start of the coupling reaction of monosaccharide and monosaccharide to the synthesis of fully protected pentasaccharide, and the total yield of 14 steps can reach 3.5%. They do a lot of optimization work on the synthesis and purification of monosaccharides during the synthesis process, e.g. the separation of alpha/beta isomers of H sugars using the Staudinger reaction. Furthermore, they have done much work on the construction of glucuronic acid beta glycosidic linkages, and prepared four glycosyl donors and two glycosyl acceptors for screening, and the synthesis of disaccharides after obtaining the optimal combination. Finally, it is pointed out that this strategy can be made on the order of 10g when synthesizing the fully protected pentasaccharide, which to some extent indicates that this strategy is suitable for industrial scale-up.

The Ding group will Alchemia and

the two synthetic routes of (a) are integrated, and when a non-reducing terminal trisaccharide donor is synthesized, the hydroxyl group needing sulfonation is temporarily protected by PMB. As such, only the two positions of glucuronic acid can be protected with Bz, so that the beta-glycosidic bond can be efficiently constructed. To effectively control [3+2]]Stereoselectivity of glycosylation, researchers converted PMB to Ac, resulting in glycosylation reactions with higher alpha selectivity. Meanwhile, the equivalence of the trisaccharide thioglycoside donor and the trisaccharide trichloroacetimidate donor in synthesizing the fully-protected heparin pentasaccharide molecules is proved.

Disclosure of Invention

The invention provides a heparin pentasaccharide structure compound, which has valuable pharmacological properties, especially has an effect of inhibiting HPA, can be used for preparing HPA inhibitors, or preparing medicaments for treating pathophysiological processes or diseases in which HPA participates, or diseases with pathophysiological changes caused by HPA overexpression or activity enhancement, such as reducing tumor metastasis and invasion, prolonging the survival time of tumor patients, or treating kidney diseases, cardiovascular diseases, inflammatory diseases, pathological angiogenesis and the like. The invention further provides a preparation method of the heparin pentasaccharide compound.

A heparin pentasaccharide structural compound having the structure of formula a (5 monosaccharides from left to right are represented by D, E, F, G, H respectively):

wherein each Y is the same or different and is independently selected from H, Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ And monovalent cations.

Preferably, each Y is the same and is selected from H, Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ And monovalent cations.

Preferably, each Y is the same or different and is independently selected from Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ 。

In one embodiment of the invention, each Y is the same and is selected from Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ 。

It will be understood by those skilled in the art that any one or two or more of Y are selected from Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ Isovalent cationic compound of formula A, which is essentially a salt of a compound of formula A wherein Y is H, and when Y is a monovalent cation, the corresponding group in formula A is its anionic group, e.g. -COO ^- 、-SO ₃ ^- 。

The compound of formula a is a compound having a single optical activity, i.e., glns 6S (1 → 4) GlcA3S β (1 → 4) GlcNS3S6S (1 → 4) IdoA2S (1 → 4) GlcNS6S, and the H-sugar-terminated methyl ester is in the α configuration.

In one embodiment of the invention, Y is H and the structural formula of the compound is as follows, herein designated CV 001.

In one embodiment of the present invention, Y is Na ⁺ The structural formula of the compound is shown as follows, and is denoted by CV122 in the invention, and the compound is essentially a sodium salt of CV 001.

In one embodiment of the invention, Y is K ⁺ The structural formula of the compound is shown as follows, and is denoted by CV123 in the invention, and the compound is essentially a potassium salt of CV 001.

The invention also provides a preparation method of the compound with the structure of the formula A.

Formula a may employ methods known in the art for the synthesis of heparin pentasaccharides, such as a 4+1 synthetic strategy, or a 3+2 synthetic strategy, etc.

The invention provides a new synthesis reaction method for synthesizing a compound with a structure shown in a formula A, which is carried out by a 3+2 synthesis route, selects a trisaccharide donor leaving group different from the prior art, different hydroxyl protecting group strategies and different monosaccharide module synthesis methods, protects a pentasaccharide intermediate 1 completely, and obtains the compound shown in the formula A through dehydroxylation and carboxyl protecting group, sulfonation and azide reduction in sequence and sulfonation:

wherein R is ₁ 、R ₂ And R ₃ May be the same or different and is independently selected from the group consisting of chloroacetyl, acetyl, benzoyl, pivaloyl; y is as defined above. In one embodiment of the invention, R ₁ And R ₂ Are all acetyl, R ₃ Is benzoyl and Y is H. In one embodiment of the invention, R ₁ And R ₂ Are all acetyl, R ₃ Is benzoyl and Y is Na ⁺ . In one embodiment of the invention, R ₁ And R ₂ Are all acetyl, R ₃ Is benzoyl, Y is K ⁺ 。

The fully-protected pentasaccharide intermediate 1 can be obtained by carrying out glycosylation reaction on a trisaccharide intermediate 2 and a disaccharide acceptor 3, and has the following reaction formula:

the trisaccharide intermediate 2 can be coupled through a (1 → 4) glycosidic bond between a monosaccharide intermediate 7 and a disaccharide intermediate 8 to obtain a trisaccharide intermediate 4 with a specific stereoconfiguration, and the trisaccharide intermediate 5 is obtained by ring opening of 1 and 6 of the trisaccharide intermediate 4 to obtain the trisaccharide intermediate 2, wherein the reaction formula is as follows:

disaccharide intermediate 8 can be obtained by isolating the product of the β configuration after the glycosylation of monosaccharides 10 and 11, according to the following reaction scheme:

the monosaccharide 10 can be prepared by taking glucose as a raw material through a reaction comprising the following steps: 1) converting the hydroxyl at the 1-position of the glucose into thioglycoside; 2) protecting hydroxyl at 4-position and 6-position of glucose, protecting hydroxyl at 3-position with a temporary protecting group, protecting hydroxyl at 2-position with benzyl, removing the temporary protecting group, and replacing with R ₂ Protecting the hydroxyl at the 3-position; 3) deprotection is carried out on hydroxyl at the 4-position and the 6-position of glucose, then reaction is carried out, the hydroxyl at the 6-position is oxidized and methyl esterified, and the hydroxyl at the 4-position is protected by chloroacetyl; 4) converting thioglycoside at position 1 to bromine to give monosaccharide intermediate 10.

The monosaccharide 11, monosaccharide 7, disaccharide acceptor 3 used in the above preparation method may be prepared according to synthetic methods known in the art, for example: preactive-based, iterative one-point synthesis of antimicrobial pentasaccharide fondaparinux sodium org. chem. front.,2019,6, 3116; total Synthesis of antimicrobial pentasaccharade Fondaparinux. ChemMedChem,2014,9, 1071-1080.

R in each intermediate compound in the above preparation method ₁ 、R ₂ And R ₃ Is defined byThe definition of the corresponding groups in the intermediate 1 is the same; x is a leaving group suitable for reacting with other acceptors to form a bond between glycosides, preferably X is hydroxy, thioalkyl, thioaryl, halogen, trichloroacetimidoyl, phosphate, tert-butyldiphenylsilyloxy.

Therefore, the invention also provides various intermediates in the synthesis method and a preparation method thereof.

A monosaccharide intermediate 10 having the structure:

wherein R is ₂ Is chloroacetyl, acetyl, benzoyl or pivaloyl.

Preferably the monosaccharide intermediate 10 is in the alpha configuration,

in one embodiment of the invention R of the monosaccharide intermediate 10 ₂ Is acetyl, is in alpha configuration, is named as 10-1, and has a structural formula

Different protecting groups are used for the 2-position hydroxyl group and the 3-position hydroxyl group of the monosaccharide intermediate 10, so that selective sulfonation is facilitated on the 3-position hydroxyl group during final synthesis of the compound of the formula A. In addition, the 2-hydroxyl is protected by benzyl and is combined with 1-bromine, which is beneficial to selectivity of beta glycosidic bond and high yield of reaction when the intermediate 10 is used for synthesizing disaccharide.

The preparation method of the monosaccharide intermediate 10 comprises the following steps: 1) taking glucose as a starting material, and converting 1-hydroxyl of the glucose into thioglycoside through reaction; 2) protecting hydroxyl at 4-position and 6-position of glucose, protecting hydroxyl at 3-position with a temporary protecting group, protecting hydroxyl at 2-position with benzyl, removing the temporary protecting group, and replacing with R ₂ Protecting the hydroxyl at the 3-position; 3) deprotection is carried out on hydroxyl at the 4-position and the 6-position of glucose, then reaction is carried out, the hydroxyl at the 6-position is oxidized and methyl esterified, and the hydroxyl at the 4-position is protected by chloroacetyl; 4) conversion of thioglycoside at position 1 to bromineA monosaccharide intermediate 10 is obtained.

According to the invention, in the step 1), after all hydroxyl groups of glucose are protected by adopting a hydroxyl protecting group, the hydroxyl group protected at the 1-position of glucose is converted into thioglycoside through reaction. The hydroxyl protecting group may be any of various hydroxyl protecting groups known in the art, including, but not limited to, acetyl, chloroacetyl, benzoyl, pivaloyl, and the like, and in one embodiment of the present invention, all hydroxyl groups of glucose are protected with acetyl groups.

According to the present invention, in the step 1), the hydroxyl group at the 1-position of glucose can be converted into thioglycoside in a manner known in the art. The glucosinolate can be of various types known in the art as glucosinolate-SR, where R is alkyl, substituted alkyl, aryl, or substituted aryl, such as, for example, methylthioglycoside-SMe, ethylglucosinolate Set, phenylglucosinolate-SPh, p-tolylglucosinolate Stol, and the like. In one embodiment of the invention, the thioglycoside is p-tolylthio. In one embodiment of the present invention, the protected hydroxyl group at the 1-position of glucose is converted to a p-toluenesulfonyl group with p-toluenesulfonyl phenol under the action of a promoter. Such promoters include, but are not limited to: boron trifluoride etherate, boron trifluoride, zinc chloride, stannic chloride, Lewis acids including, but not limited to, zirconium tetrachloride, ferric trichloride, titanium tetrachloride, or the like. In one embodiment of the invention, the promoter is boron trifluoride diethyl etherate. In another embodiment of the invention, tributylstannyl derivatives of thiols are used to react with Lewis acids.

According to the present invention, after the hydroxyl group protected at the 1-position of glucose is converted into glucosinolate in the step 1), a process of deprotecting other hydroxyl groups of glucose is further included. In one embodiment of the invention, the protecting group is removed under the action of a catalytic amount of sodium methoxide.

In one embodiment of the present invention, the hydroxyl groups at the 4-position and the 6-position of glucose are protected with benzylidene in the step 2).

According to the present invention, the temporary protecting group in step 2) needs to be capable of selectively reacting with the 3-position hydroxyl group to protect it when both the 3-position hydroxyl group and the 2-position hydroxyl group of glucose are present, and the deprotection conditions thereof are different from those of other hydroxyl protecting groups already present on glucose. The temporary protecting group may be, for example, PMB.

In one embodiment of the present invention, in the step 2), Bu is added ₂ SnO or Bu ₂ Sn(OMe) ₂ Under the action of (a) to dehydrate or demethanol, respectively, to give a cyclodibutylmethanotinylalkylene derivative which is a convenient intermediate for regiobenzylation of polyols. The derivative can then be reacted with a PMB donor to produce a product in which the hydroxyl group at the 3-position is protected with PMB. In one embodiment of the invention, the hydroxyl group at position 3 is protected with PMB in the presence of PMBCl, CsF and DMF. In another embodiment of the invention, the hydroxyl group at position 3 is protected with PMB in the presence of PMBO-4-methylquinoline, CsA and DMF. In yet another embodiment of the invention, the hydroxyl group at the 3-position is protected with PMB by reaction in benzene or toluene in the presence of tetrabutylammonium bromide.

In one embodiment of the present invention, in the step 2), the hydroxyl group at the 2-position is protected with a benzyl group in the presence of BnBr, NaH and DMF. In another embodiment of the invention, cerium ammonium nitrate, Br ₂ The 2-hydroxyl group is protected with a benzyl group in the presence of dichloromethane and water.

In one embodiment of the present invention, in the step 2), the protecting group PMB can be removed by oxidative cleavage with DDQ in dichloromethane/water, or ammonium ceric nitrate in acetonitrile/water.

In one embodiment of the present invention, the hydroxyl group at the 6-position of glucose is oxidatively methyl-esterified in step 3) using TEMPO oxidation and methyl esterification.

According to the present invention, said step 4) may convert the thioglycoside in position 1 to bromine in a manner known in the art, including but not limited to treatment with iodine bromide.

According to the invention, said step 4) may be followed by a conformational separation of the obtained intermediate 10, obtaining the intermediate 10 in alpha configuration for the subsequent synthesis of the disaccharide intermediate.

In one embodiment of the invention, the monosaccharide intermediate 10-1

The preparation method comprises the following steps: glucose 12 is used as an initial raw material, and a monosaccharide intermediate 13 is obtained through total acetylation; in BF ₃ .Et ₂ Reacting with p-toluene thiophenol under the action of O to obtain a monosaccharide intermediate 14; removing all acetyl under the action of catalytic amount of sodium methoxide, and then protecting 4, 6-dihydroxy with benzylidene to obtain 2, 3-dihydroxy naked monosaccharide intermediate 15; under the action of dibutyltin oxide, selectively taking a PMB protecting group on the 3-position as a temporary protecting group to obtain a monosaccharide intermediate 16; etherifying the 2-hydroxy benzyl to obtain a monosaccharide intermediate 17; oxidizing and removing the temporary protecting group PMB under the action of DDQ to obtain an intermediate 18, and then acetylating the intermediate 18 to obtain a monosaccharide intermediate 19; heating under the condition of acetic acid to remove a benzylidene protecting group to obtain a monosaccharide intermediate 20 of 4, 6-dihydroxy; TEMPO oxidation and methyl esterification are carried out to obtain a monosaccharide intermediate 21; protecting the hydroxyl at the 4-position with chloroacetyl to obtain a monosaccharide intermediate 22; finally, the beta-thioglycoside is converted into a monosaccharide intermediate 10-1 with alpha configuration by treatment with iodine bromide. The reaction formula is as follows:

a disaccharide intermediate 9 of the formula:

wherein R is ₂ Is chloroacetyl, acetyl, benzoyl or pivaloyl. In one embodiment of the present invention R of the disaccharide intermediate 9 ₂ Is acetyl, named as 9-1, and has the structural formula

The preparation method of the disaccharide intermediate 9 comprises the following steps: monosaccharide intermediate 10 in the alpha configuration is reacted with monosaccharide intermediate 11 to produce disaccharide intermediate 9.

The monosaccharide intermediate 11 may be synthesized in a literature-based manner (preactive one-point synthesis of antisense oligosaccharide fondaparinux sodium, org. chem. front.,2019,6, 3116). The intermediate 11 with an internal ether ring structure is adopted, so that the beta configuration ratio of the intermediate 9 is improved.

According to the invention, Ag may be used ₂ CO ₃ /AgOTf reaction System or Ag ₂ The O/TMSOTf reaction system was subjected to the Koenigs-Knorr glycosylation reaction to give disaccharide intermediate 9 described above. In one embodiment of the present invention, the reaction is performed under the action of insoluble silver carbonate and catalytic amount of silver trifluoromethanesulfonate, and preferably, the amount of silver trifluoromethanesulfonate used in the reaction is 0.05 to 0.2 equivalent, which is favorable for ensuring the reaction efficiency and selectivity of beta configuration.

A disaccharide intermediate 8 of the formula:

wherein R is ₂ Is chloroacetyl, acetyl, benzoyl or pivaloyl. In one embodiment of the invention R of disaccharide intermediate 8 ₂ Is acetyl, named as 8-1, and has the structural formula

The preparation method of the disaccharide intermediate 8 comprises the following steps: the 4-position chloroacetyl group of the disaccharide intermediate 9 is removed, and the product with the (1 → 4) glycosidic bond configuration is separated and removed to obtain the pure disaccharide intermediate 8 with the beta configuration.

The isolation of the α and β configurations of disaccharide intermediate 8 can be performed in the present invention using techniques known in the art for isolating different configurations, including but not limited to: silica gel column chromatography, high performance liquid chromatography, etc.

A trisaccharide intermediate 2 having the formula:

wherein R is ₁ And R ₂ May be the same or different and is independently selected from chloroacetyl, acetyl, benzoyl or pivaloyl; x is a leaving group suitable for reacting with other receptors to form a bond between glycosides.

Preferably, X is hydroxy, thioalkyl, thioaryl, halogen, trichloroacetimidate, phosphate, tert-butyldiphenylsilyloxy.

Preferably, the trisaccharide intermediate is

In one embodiment of the present invention, R of the trisaccharide intermediate ₁ Is acetyl, R ₂ Is acetyl, X is trichloroacetimidoyl, named as 2-1-1, and has the structural formula

The preparation method of the trisaccharide intermediate 2 comprises the following steps:

step 1), coupling a monosaccharide intermediate 7 and a disaccharide intermediate 8 through (1 → 4) glycosidic bonds to obtain a trisaccharide intermediate 4 with a specific stereoconfiguration;

step 2), opening the ring of the trisaccharide intermediate 4 under the action of a catalyst by 1,6 to obtain a trisaccharide intermediate 5;

and 3) obtaining a trisaccharide intermediate 2 from the trisaccharide intermediate 5.

The reaction formula is as follows, R ₁ 、R ₂ And X is as defined above.

The trisaccharide intermediate 2 with alpha configuration and beta configuration can be separated and obtained by means of silica gel column chromatography and the like.

The intermediate 2 with the mixed configuration can be directly used for the next reaction to obtain the fully protected compound 1 with the alpha configuration, or the alpha configuration and the beta configuration obtained by separating the intermediate 2 are respectively put into the next reaction to obtain the fully protected compound 1 with the alpha configuration, namely, the subsequent reaction and the yield are not influenced no matter which configuration or mixed configuration of the intermediate 2 is used in the subsequent synthesis.

According to the invention, the step 1) is carried out in an organic solvent under an acidic condition, and the reaction temperature is-10 ℃ to-25 ℃. The organic solvent may be selected from benzene, toluene, dichloromethane, and the like. The acidic condition can be formed by adding acidic substances such as TfOH, TMSOTf or TBSOTf.

According to the invention, said step 2) is carried out in the corresponding anhydride (for example chloroacetic anhydride, acetic anhydride, benzoic anhydride, pivalic anhydride) to which TBSOTf, TMSOTf, Et are added ₂ OBF3 or Et ₃ SiOTf, etc., the reaction temperature is-20 ℃ to 0 ℃.

According to the present invention, said step 3) can selectively remove the terminal protecting group of trisaccharide intermediate 5 by a method known in the art. In one embodiment of the present invention, selective removal of the terminal protecting group of trisaccharide intermediate 5 at 10 to 30 ℃ gives trisaccharide intermediate 6 in which X is hydroxyl

The reaction system used may be benzylamine and THF, or Bu ₃ SnOMe and dichloromethane, or boron trifluoride-diethyl ether in wet acetonitrile. Subsequently, the terminal hydroxyl group of intermediate 6 is converted to trichloroacetimidate donor trisaccharide intermediate 2-1 in an organic solvent, which may be selected from dichloromethane, tetrahydrofuran, etc., in the presence of a base and trichloroacetonitrile at 10-30 ℃; the base may be selected from inorganic bases such as potassium carbonate, potassium bicarbonate, sodium hydride, DBU, and the like.

Monosaccharide intermediate 7 may be prepared using synthetic methods known in the art, such as Total Synthesis of antibacterial pentasaccharade fondaparinux, chem med chem,2014,9, 1071-.

The disaccharide intermediate 8 with an internal ether ring structure is adopted to synthesize the trisaccharide intermediate 4, which is beneficial to simultaneously protecting the hydroxyl at the 1-position and the 6-position of the disaccharide intermediate 8, so that only the hydroxyl at the 4-position of the latter is obtained when the monosaccharide intermediate 7 and the disaccharide intermediate 8 react, and the selectivity of forming a 1 → 4 glycosidic bond is improved; and after ring opening to give trisaccharide intermediate 5, the intermediate 5 end group can be converted to any available leaving group X, in particular trichloroacetimidate, without being limited by the kind of leaving group of monosaccharide intermediate 7.

A fully protected pentasaccharide intermediate 1 has the following structural formula:

wherein R is ₁ 、R ₂ And R ₃ May be the same or different and is independently selected from the group consisting of chloroacetyl, acetyl, benzoyl, pivaloyl. In one embodiment of the invention, R ₁ And R ₂ Are all acetyl, R ₃ Is benzoyl.

The preparation method of the pentasaccharide intermediate 1 comprises the following steps: the trisaccharide intermediate 2 and the disaccharide acceptor 3 are subjected to glycosylation reaction to obtain a compound 1, and the reaction formula is as follows:

the reaction temperature is-10 ℃ to-25 ℃. The reaction may be carried out under strong acid conditions, such as trifluoromethanesulfonic acid, TBSOTf, TMSOTf, and the like.

Without being limited by a particular theory, the inventor finds that acyl is formed at the 6-position of the trisaccharide intermediate 2F sugar, which is beneficial to improving the proportion of alpha configuration in a product when the full-protection pentasaccharide 1 is synthesized, and even obtaining the pentasaccharide 1 with the full-alpha configuration; and the acyl group is stable to acid and cannot fall off in the glycosylation reaction, so that the high yield of the fully-protected pentasaccharide 1 is ensured.

The disaccharide receptor 3 can be prepared according to synthetic methods known in the art, for example: preactive-based, iterative one-point synthesis of antimicrobial pentasaccharide fondaparinux sodium org. chem. front.,2019,6, 3116. The inventor of the invention researches and discovers that hydrogen bonding in a Fischer glycosylation reaction is favorable for generating a product with alpha configuration, alpha configuration methyl glycoside of H sugar at the reducing end of a disaccharide acceptor 3 is synthesized by Fischer glycosylation, and a Cbz protective group is adopted on an amino group of the disaccharide acceptor 3, so that the yield of the methoxy alpha configuration in monosaccharide H is favorably improved. In addition, the Cbz protecting group can be removed under the same conditions as the azide and benzyl groups and is tolerant to strong basicities without adding additional reaction steps from fully protecting pentasaccharide 1 to preparing the compound of formula a.

In one embodiment of the invention, the fully protected pentasaccharide 1 is synthesized according to the following reaction scheme:

when trichloroacetimidate is used as glycosyl donor, the reaction has the advantages of mild reaction conditions, good thermal stability, high reaction activity and high yield.

The present invention also provides intermediates I, II and III for the synthesis of the compound of formula a.

The structure of the compound I is as follows:

the structure of the compound II is as follows:

wherein Y is as defined for formula A, preferably Y is H or Na ⁺ Or K ⁺ 。

The structure of the compound III is as follows:

wherein Y is as defined for formula A, preferably Y is H or Na ⁺ Or K ⁺ 。

Intermediates I, II and III were generated from pentasaccharide intermediate 1 via dehydroxylation and carboxyl protecting groups, sulfonation and azide reduction in that order. The deprotection efficiency of the reaction is high, all hydroxyl groups to be sulfonated can be deprotected at one time, and all hydroxyl groups which are not sulfonated can be deprotected at one time after sulfonation, and the reaction formula is as follows:

the dehydroxylation and carboxyl protecting groups, the sulfonation and the azide reduction can all be carried out using reaction methods and conditions known in the art. In one embodiment of the invention, intermediate 1 is subjected to simultaneous removal of R in the presence of a base ₁ 、R ₂ 、R ₃ And the methyl ester to give intermediate I. In one embodiment of the invention, intermediate I is in SO ₃ ·NEt ₃ To obtain the intermediate II after O-sulfonation. In one embodiment of the invention, intermediate II is subjected to catalytic hydrogenation to remove benzyl and Cbz while reducing azide to generate amino groups, resulting in intermediate III. In one embodiment of the invention, at SO ₃ Py sulfonation of three amino groups in the compound of the formula III gives the compound CV 001. CV001 was ion-exchanged with a sodium ion exchange resin to obtain compound CV 122. The sodium ion exchange resin may be a resin known in the art, including but not limited to Amberlite IR120 Na ⁺ 、Dowex-50-WX4-Na ⁺ And so on. CV001 was ion-exchanged with a potassium ion-exchange resin to obtain compound CV 123. The potassium ion exchange resin may be a resin known in the art, including but not limited to Amberlite IR 120K ⁺ 、Dowex-50-WX4-K ⁺ And the like.

The invention adopts [3+2]]The convergent synthesis strategy is used for synthesizing the compound of the formula A, and different trisaccharide donor leaving group, different hydroxyl protecting group strategies and different monosaccharide module synthesis methods are adopted compared with the prior art. In addition to the aforementioned advantages of using trichloroacetimidate as the trisaccharide donor leaving group and Cbz protection of the H sugar amino group, the sulfonated hydroxy group in the process of the invention is R ₁ 、R ₂ 、R ₃ Protection of whichThe protecting and deprotection method is simple, the condition is mild, and the reaction yield is high; the non-sulfonated hydroxyl is protected by benzyl, the protecting group is acid and alkali resistant and is not influenced in the glycosidation reaction, and finally the non-sulfonated hydroxyl can be reduced by a catalytic hydrogenation method, so that the yield is high. And the sulfonated hydroxyl and the non-sulfonated hydroxyl adopt different protecting groups, so that the selective sulfonation in the synthesis process is ensured.

The present invention also provides a pharmaceutical composition comprising a compound of formula a or a solvate thereof according to the present invention as an active ingredient, optionally together with one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier is a variety of excipients commonly used or known in the pharmaceutical art, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants and the like. In one embodiment of the present invention, the present invention provides a pharmaceutical composition for inhibiting HPA activity, or for treating or preventing a pathophysiological process or disease or disorder in which HPA is involved, or a disease or disorder in which HPA is overexpressed or affected by increased HPA activity, comprising a compound of formula a of the present invention or a solvate thereof, as an active ingredient, optionally together with one or more pharmaceutically acceptable carriers.

The invention also provides the use of a compound of formula a for the preparation of an HPA inhibitor.

The invention also provides the application of the compound of the formula A in preparing medicines for inhibiting the activity of HPA. The medicament can be used for treating or preventing pathophysiological processes or diseases or disorders in which HPA is involved, or diseases or disorders in which HPA is overexpressed or affected by enhanced HPA activity.

The invention also provides the use of a compound of formula a for the preparation of a pharmaceutical composition.

The pharmaceutical compositions of the invention are useful for inhibiting HPA activity, or for treating or preventing a pathophysiological process or a disease or disorder in which HPA is involved, or a disease or disorder in which HPA is overexpressed or affected by increased HPA activity.

The invention also provides a method for treating or preventing a pathophysiological process or a disease or disorder in which HPA is involved, or a disease or disorder in which HPA is overexpressed or affected by increased activity of HPA, by administering to a patient in need thereof a compound of formula a of the present invention or a pharmaceutical composition comprising a compound of formula a.

The pathophysiological processes or diseases or disorders in which HPA is involved, or diseases or disorders in which HPA is overexpressed or affected by increased HPA activity, are mainly caused by overexpression or increased activity of HPA in vivo, for example: metastasis of tumor, invasion or infiltration of tumor, diabetic nephropathy, membranous nephropathy, proteinuric glomerular disease, amyloidosis nephropathy, osteolysis, atherosclerosis, cardiovascular disease, etc. The overexpression or activity enhancement of HPA in vivo refers to the detection of the expression level of gene or protein of HPA or the activity of HPA enzyme in vivo or in corresponding tissue or cell (such as tissue and/or cell of cancer or tumor, diseased kidney tissue, etc.) of HPA or in the individual or corresponding tissue or cell by using the conventional detection methods in the field (including but not limited to enzymatic chemical detection, enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry, Western blotting, tissue chip, gene detection, etc.), wherein the expression level or the activity of HPA enzyme is higher than the normal level, such as higher than 110% or higher than the normal level, or higher than 120% or higher than 130% or higher than 150% or higher than 200% or higher; the normal level may be the expression level or the enzymatic activity level of the gene or protein of HPA in the body of the general human body or in the corresponding tissue and/or cell, or the expression level or the enzymatic activity level of the gene or protein of HPA in the tissue and/or cell of a non-diseased organ of the same patient.

The amount of the compound of formula A contained in the pharmaceutical composition (based on the compound of formula A) is 0.1-1000mg, preferably 1-500mg, more preferably 5-100 mg.

The mass percentage of the compound of formula a (calculated as the compound of formula a) in the pharmaceutical composition is 0.01-95%, and the content ranges may be, for example, 0.1-10%, 0.3-5%, or 10-90%, preferably 20-80%, and more preferably 30-70% according to different dosage forms.

The dosage form of the pharmaceutical composition may be in the form of oral preparations such as tablets, capsules, pills, powders, granules, suspensions, syrups, and the like; it can also be made into injection, such as injection solution, powder for injection, etc., and can be injected by intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts. For example, the pharmaceutical composition may be an injection and the concentration of the compound of formula A in the injection may be 1-15mg/ml, e.g. 5mg/ml, 10mg/ml, 12.5mg/ml etc.

Routes of administration of the pharmaceutical composition include, but are not limited to: orally administered; the medicine is taken orally; sublingual; transdermal; of the lung; of the rectum; parenteral, e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous; by implantation into a reservoir or reservoir.

The dosage of the compound of formula a administered (based on the compound of formula a) will depend on the age, health and weight of the recipient, the type of drug combination, the frequency of treatment, the route of administration, etc. The drug may be administered in a single daily dose, once daily, once every two days, once every three days, once every four days, or the total daily dose may be administered in divided doses of two, three or four times daily. The compound of formula A is administered in an amount (based on the compound of formula A) of 0.01 to 100 mg/kg/day, preferably 0.1 to 10 mg/kg/day, e.g., 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 made into a combined drug. The other therapeutic agents may be drugs for tumor therapy, drugs for renal disease therapy, drugs for cardiovascular therapy, etc., depending on the type of disease and disorder.

Tumor treating drugs, for example: drugs that disrupt the structure and function of DNA (e.g., mechlorethamine, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, etc.), nucleotide synthase inhibitors (e.g., 5-fluorouracil, capecitabine, raltitrexed, 6-mercaptopurine, etc.), DNA polymerase inhibitors (e.g., cytarabine, gemcitabine, etc.), dihydrofolate reductase inhibitors (e.g., methotrexate, pemetrexed, etc.), nucleotide reductase inhibitors (e.g., hydroxyurea), drugs that inhibit RNA synthesis (e.g., doxorubicin, daunorubicin, epirubicin, pirarubicin, etc.), topoisomerase inhibitors (e.g., hydroxycamptothecin, irinotecan, topotecan, etc.), tubulin inhibitors (e.g., vincristine, vindesine, vinorelbine, paclitaxel, docetaxel, etc.), drugs that influence hormonal balance (e.g., toremifene, exemestane, letrozole, bicalutamide, paclitaxel, etc.), drugs that influence hormone balance (e, etc.) Enzalutamide, medroxyprogesterone, megestrol, testosterone propionate, goserelin, leuprorelin, etc.), tyrosine kinase inhibitors (such as imatinib, gefitinib, erlotinib, sorafenib, sunitinib, lapatinib, apatinib, etc.), epidermal growth factor receptor inhibitors (such as trastuzumab, panitumumab, cetuximab, pertuzumab), vascular endothelial growth factor receptor inhibitors (such as bevacizumab, ramucizumab, etc.), immunomodulators (such as rituximab, pembrolizumab, ipilimumab, etc.).

Cardiovascular therapeutic agents, for example: lipid-lowering drugs (such as lovastatin), blood-pressure lowering drugs (such as beta receptor antagonist, ACEI, angiotensin II receptor antagonist, etc.), and anticoagulant drugs.

Renal disease treatment drugs, for example: ACEI (e.g., captopril, benazepril, enalapril, perindopril, etc.), angiotensin II receptor antagonists (e.g., valsartan, losartan, irbesartan, candesartan, etc.), anticoagulants (e.g., heparin, low molecular heparin, enoxaparin, fondaparinux, bivalirudin, etc.), glucocorticoids (e.g., nylon), cyclophosphamide, cyclosporine, chlorambucil, triptolide, and the like.

The HPA inhibiting activity of the compound of the formula A is higher than that of fondaparinux sodium, but the anticoagulant activity is far lower than that of fondaparinux sodium, so that the compound has extremely low bleeding risk when being used for treating the HPA. The compound of formula A has definite structure, is beneficial to preparation and quality control, is not combined with other proteins, has long half-life in vivo, and is t in a double-chamber model _1/2 It was 17 hours.

Drawings

FIG. 1 is a graph showing the relationship between CV122 and fondaparinux sodium for the dose-effect inhibition of heparanase

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

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

Ac: acetyl; AgOTf: silver triflate; bn: a benzyl group; bz: a benzoyl group; ClAc: a monochloroacetyl group; CSA: camphorsulfonic acid; cbz: a benzyloxycarbonyl group; CsF: cesium fluoride; DBU: 1, 8-diazabicycloundec-7-ene; DCM: dichloromethane; DDQ: 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone; DMF: n, N-dimethylformamide; PMB: p-methoxybenzyl; TfOH: trifluoromethanesulfonic acid; TBSOTf: tert-butyldimethylsilyl trifluoromethanesulfonate; TEMPO: 2,2,6, 6-tetramethylpiperidine oxide; TMSOTf: trimethylsilyl trifluoromethanesulfonate; tol: toluene.

Example 1 Synthesis of pentasaccharide intermediate III

1. Preparation method of monosaccharide intermediate 10-1

Glucose 12 is used as an initial raw material, and a monosaccharide intermediate 13 is obtained through total acetylation; in BF ₃ ^. Et ₂ Reacting with p-toluene thiophenol under the action of O to obtain a monosaccharide intermediate 14; removing all acetyl under the action of catalytic amount of sodium methoxide, and then protecting 4, 6-dihydroxy with benzylidene to obtain 2, 3-dihydroxy naked monosaccharide intermediate 15; under the action of dibutyltin oxide, selectively taking a PMB protecting group on the 3-position as a temporary protecting group to obtain a monosaccharide intermediate 16; etherifying 2-hydroxy benzyl to obtain monosaccharide intermediate 17; oxidizing and removing the temporary protecting group PMB under the action of DDQ to obtain an intermediate 18, and then acetylating the intermediate 18 to obtain a monosaccharide intermediate 19; heating under the condition of acetic acid to remove a benzylidene protecting group to obtain a monosaccharide intermediate 20 of 4, 6-dihydroxy; TEMPO oxidation and methyl esterification are carried out to obtain a monosaccharide intermediate 21; protecting the hydroxyl at the 4-position with chloroacetyl to obtain a monosaccharide intermediate 22; finally, the beta-thioglycoside is converted into a monosaccharide intermediate 10-1 with alpha configuration by treatment with iodine bromide.

The reaction conditions and yields in the respective steps are as follows: a) ac of ₂ O,HClO ₄ ,77％；b)TolSH,BF ₃ ·Et ₂ O, DCM, 86%; c)1) MeONa, MeOH, DCM; 2) benzaldehyde dimethyl, CSA, DMF, two-step total yield 83%; d)1) Bu ₂ SnO, MeOH, refluxing; 2) PMBCl, CsF, DMF,90 ℃, and the total yield of the two steps is 75%; e) BnBr, NaH, DMF, 92%; f) DDQ, DCM, H ₂ O,78％；g)Ac ₂ O,Et ₃ N,DCM,98％；h)80％AcOH,90℃,76％；i)1)2,2,6,6-Tetramethylpiperidinooxy(TEMPO),Iodobenzene diacetate,DCM,H ₂ O；2)MeI,KHCO ₃ DMF, overall yield of two steps 57%; j) ClAc ₂ O,Py,DCM,76％；k)IBr,DCM,76％。

2. Process for preparing disaccharide intermediate 9-1

The monosaccharide intermediate 10-1 and the monosaccharide intermediate 11-1 react overnight under the action of insoluble silver carbonate and catalytic amount of silver trifluoromethanesulfonate to generate a disaccharide intermediate 9-1, and the alpha/beta ratio of the product is about 1:1 through NMR identification.

Adding into a two-mouth bottle

And (3) performing vacuum-pumping on the molecular sieve (1g, powder), baking the bottle, introducing argon for protection, and cooling to room temperature. Dissolving monosaccharide intermediate 10-1(300mg,0.63mmol) and monosaccharide intermediate 11-1(172mg,0.75mmol) with 10mL of dry dichloromethane, adding into reaction flask with syringe, stirring at room temperature for half an hour, adding Ag ₂ CO ₃ (260mg,0.94mmol) and catalytic amount of AgOTf (16mg,0.063mmol) are added into a reaction bottle, the reaction is carried out overnight in a dark place, TLC detection donor 10-1 is completely disappeared to generate a main substance, diatomite is added for filtration, and the filtrate is directly purified by column chromatography after decompression and concentration (petroleum ether/ethyl acetate is 2:1) to obtain the disaccharide monosaccharide intermediate with mixed alpha/beta configuration9-1. The monosaccharide intermediate 11-1 may be synthesized by methods such as preactive-based, iterative one-pot synthesis of antisense oligosaccharide fondaparinux sodium, org, chem, front, 2019,6, 3116.

HRMS[M+Na] ⁺ m/z 650.1327 (calculation: C) ₂₆ H ₃₀ ClN ₃ NaO ₁₃ ,650.1365)。

3. Method for preparing disaccharide intermediate 8-1

Removing the 4-chloroacetyl of the disaccharide intermediate 9-1, and separating the product with alpha configuration to obtain the pure disaccharide intermediate 8-1 with beta configuration.

The disaccharide intermediate 9-1 was dissolved in 15mL of a mixed solvent of chloroform and methanol (V/V ═ 1:1), thiourea (192mg,2.52mmol) was added and heated to 60 ℃, heating was stopped when the starting material disappeared completely and three single points were formed, the mixture was cooled to room temperature, and a suitable amount of silica gel was added and spin-dried, and direct column chromatography (petroleum ether/ethyl acetate ═ 1:1) was performed to obtain a colorless foamy disaccharide intermediate 8-1(583mg, 42% yield in two steps).

¹ H NMR(400MHz,CDCl ₃ )δ7.38–7.23(m,6H),5.49(s,1H),5.28(s,1H),5.11(t,J＝9.3Hz,1H),4.92(d,J＝11.8Hz,1H),4.69(dd,J＝12.9,9.8Hz,2H),4.58(d,J＝5.3Hz,1H),4.01(d,J＝7.6Hz,1H),3.94(d,J＝9.8Hz,1H),3.87(t,J＝9.6Hz,1H),3.81(s,3H),3.80–3.73(m,1H),3.63(s,1H),3.51(dd,J＝9.4,7.7Hz,1H),3.34(s,1H),3.21(s,1H),2.11(s,3H),2.01(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ171.07,169.27,169.21,138.06,128.41,127.98,127.78,103.50,100.17,78.28,76.22,75.18,74.63,74.22,74.03,70.61,70.07,64.88,58.73,52.88,21.03,20.94。

HRMS[M+Na] ⁺ m/z 574.1642 (calculation: C) ₂₄ H ₂₉ N ₃ NaO ₁₂ ,574.1649)。

4. Preparation method of trisaccharide intermediate 2-1-1

The monosaccharide intermediate 7-1 and the disaccharide intermediate 8-1 are subjected to glycosylation coupling to obtain a trisaccharide intermediate 4-1 with a specific spatial configuration; performing acetylhydrolysis to obtain a 1,6 ring-opened trisaccharide intermediate 5-1; removing terminal acetyl under the action of benzylamine to obtain trisaccharide intermediate 6-1, and adding trichloroacetimidate to obtain trisaccharide intermediate 2-1-1.

The preparation method of the trisaccharide intermediate 4-1 comprises the following steps: adding into a two-mouth bottle

And (3) performing vacuum-pumping on the molecular sieve (1g, powder), baking the bottle, introducing argon for protection, and cooling to room temperature. Disaccharide intermediate 8-1(430mg,0.78mmol) and monosaccharide intermediate 7-1(578mg,1.0mmol) were dissolved in 10mL dry toluene, added to the reaction flask with a syringe, stirred at room temperature for half an hour, cooled to-20 deg.C, TfOH (8.9. mu.L, 0.1mmol) was added dropwise, and stirred at-20 deg.C for 1.5 h. After TLC monitoring disaccharide intermediate 8-1 reacting completely, adding excess triethylamine to quench reaction, filtering molecular sieve with a small section of silica gel, concentrating the filtrate under reduced pressure, and subjecting the obtained crude product to column chromatography to obtain trisaccharide intermediate 4-1(651mg, 87%).

¹ H NMR(400MHz,CDCl ₃ )δ7.30(ddd,J＝23.3,15.2,7.2Hz,16H),5.47(s,1H),5.32–5.13(m,2H),4.99(d,J＝3.4Hz,1H),4.90(d,J＝11.9Hz,1H),4.81(d,J＝13.4Hz,3H),4.69(t,J＝9.2Hz,2H),4.59(d,J＝5.6Hz,1H),4.54(d,J＝10.9Hz,1H),4.28(d,J＝11.3Hz,1H),4.19(dd,J＝12.2,2.4Hz,1H),4.11–3.98(m,2H),3.94(d,J＝9.5Hz,1H),3.86(t,J＝9.6Hz,1H),3.82–3.72(m,4H),3.69(d,J＝10.0Hz,1H),3.63(s,1H),3.54(d,J＝9.6Hz,1H),3.49(d,J＝9.4Hz,1H),3.34(dd,J＝10.3,3.4Hz,1H),3.20(s,1H),2.09(s,3H),2.01(s,3H),1.98(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ170.55,169.75,169.12,167.49,137.94,137.62,137.53,128.52,128.40,128.13,128.11,128.02,127.98,127.91,127.78,103.45,100.20,99.11,79.83,78.24,77.18,76.35,75.56,74.99,74.93,74.45,73.95,73.66,70.41,70.21,64.93,63.79,62.00,20.95,20.94,20.80。

HRMS[M+K] ⁺ m/z 999.3036 (calculation: C) ₄₆ H ₅₂ KN ₆ O ₁₇ ,999.3026)。

The preparation method of the trisaccharide intermediate 5-1 comprises the following steps: dissolving trisaccharide intermediate 4-1(370mg,0.39mmol) in 10mL acetic anhydride, cooling to 0 ℃ under nitrogen protection, adding TBSOTf (38 uL, 0.16mmol) dropwise, reacting for 20min under low temperature condition, monitoring by TLC that the raw materials are completely reacted, and adding triethylamine to quench the reaction. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography directly (petroleum ether/ethyl acetate 5:1) to give trisaccharide intermediate 5-1(409mg, 100%) as a colorless oily product.

¹ H NMR(400MHz,CDCl ₃ )δ7.87–7.03(m,15H),6.41–3.17(m,28H),2.23–1.89(m,15H). ¹³ C NMR(101MHz,CDCl ₃ )δ170.54,170.03,169.98,169.59,168.58,167.58,137.54,137.50,137.42,128.52,128.45,128.15,128.11,128.07,128.04,128.01,127.92,127.88,103.34,99.07,89.91,79.83,78.96,77.30,77.20,75.94,75.61,74.99,74.94,73.67,70.97,70.33,69.97,63.79,61.91,61.01,60.37,52.83,29.69,20.99,20.86,20.79,20.64。

HRMS[M+Na] ⁺ m/z 1085.3638 (calculation: C) ₅₀ H ₅₈ NaN ₆ O ₂₀ ,1085.3604)。

The preparation method of the trisaccharide intermediate 2-1-1 comprises the following steps: dissolving trisaccharide intermediate 5-1(100mg,0.094mmol) in 5mL THF, adding benzylamine (51 mu L, 0.47mmol) to react at room temperature, monitoring by TLC after the reaction is completed, transferring the reaction solution to a separating funnel, washing the rest benzylamine with 1N hydrochloric acid, back-extracting the water phase with ethyl acetate twice, combining the organic phases, drying with anhydrous sodium sulfate, carrying out suction filtration, and concentrating under reduced pressure to obtain a crude product, which is directly subjected to column chromatography purification to obtain trisaccharide intermediate 6-1. The trisaccharide intermediate 6-1 was dissolved in 5mL of dichloromethane, anhydrous potassium carbonate (19mg,0.14mmol) was added, and trichloroacetonitrile (14. mu.L, 0.14mmol) was added dropwise at room temperature. After the addition was complete, the reaction was stirred at room temperature for 2h, TLC monitored the starting material essentially reacted completely, filtered off potassium carbonate over a short section of silica gel, the filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography (petroleum ether/ethyl acetate 5:1) to give colorless syrup trisaccharide intermediate 2-1-1(79mg, 72% yield over two steps). The two configurations of alpha configuration and beta configuration can be separated by silica gel column chromatography, and the following are nuclear magnetic data of the two configurations.

The intermediate 2-1-1 with the mixed configuration can be directly used for the next reaction to obtain the fully-protected compound 1-1 with the alpha configuration, or the alpha configuration and the beta configuration obtained by separating the intermediate 2-1-1 are respectively put into the next reaction to obtain the fully-protected compound 1-1 with the alpha configuration, namely no matter which configuration or mixed configuration of the intermediate 2-1-1 is used in the subsequent synthesis, the subsequent reaction and the yield are not influenced.

Nuclear magnetism of the product of beta configuration: ¹ H NMR(400MHz,CDCl ₃ )δ8.81(s,1H),7.49–7.09(m,17H),6.42(s,1H),5.53(t,J＝9.7Hz,1H),5.21(t,J＝9.1Hz,1H),4.94(s,1H),4.82(d,J＝9.9Hz,3H),4.69(d,J＝11.5Hz,1H),4.62–4.51(m,2H),4.46(dd,J＝15.5,10.2Hz,2H),4.30(d,J＝12.0Hz,1H),4.23–4.02(m,4H),3.98(t,J＝9.0Hz,1H),3.87(d,J＝9.8Hz,3H),3.77(s,3H),3.64(d,J＝9.1Hz,2H),3.53(t,J＝9.1Hz,1H),3.44–3.24(m,2H),2.05(s,7H),2.02(s,3H),1.93(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ170.55,169.92,169.76,169.60,167.56,160.50,137.53,137.50,137.33,128.54,128.46,128.17,128.05,127.94,127.89,103.33,99.11,94.09,79.83,78.82,77.19,76.01,75.62,74.99,73.68,71.31,70.33,69.84,63.79,61.91,60.95,60.66,52.84,20.89,20.85,20.81,20.64。

HRMS[M+Na] ⁺ m/z 1186.2578 (calculation: C) ₅₀ H ₅₆ Cl ₃ NaN ₇ O ₁₉ ,1186.2594)。

Nuclear magnetism of product of alpha configuration: ¹ H NMR(400MHz,CDCCl ₃ )δ8.77(s,1H),7.40–7.28(m,10H),7.27–7.18(m,5H),5.68(d,J＝8.4Hz,1H),5.19(t,J＝9.4Hz,1H),5.10(t,J＝9.7Hz,1H),4.94(d,J＝3.5Hz,1H),4.82(m,2H),4.80(s,1H),4.69(d,J＝11.9Hz,1H),4.53(m,3H),4.42(d,J＝7.7Hz,1H),4.29(dd,J＝12.3,2.1Hz,1H),4.16(d,J＝11.7,1H),4.14(s,1H),3.94(t,J＝9.3Hz,1H),3.88–3.80(m,3H),3.77(s,3H),3.73(t,J＝8.8,1H),3.68–3.60(m,2H),3.54(t,J＝9.3Hz,1H),3.3(d,J＝9.8,1H),3.29(t,J＝9.8,1H),2.06(s,3H),2.06(s,3H),2.02(s,3H),1.92(s,3H). ¹³ C NMR(101MHz,CDCl3)δ170.58,169.94,169.78,169.62,167.57,160.50,137.49,137.32,128.56,128.47,128.19,127.95,103.35,99.13,94.09,79.83,78.82,77.26,77.17,76.03,75.64,75.01,74.97,73.68,71.31,70.32,69.83,63.77,61.90,60.95,60.65,31.94,29.72,29.68,29.38,22.71,20.91,20.87,20.83,20.66.

5. preparation method of fully-protected pentasaccharide intermediate 1-1

The trichloroacetimidate donor trisaccharide compound 2-1-1 and the disaccharide intermediate 3-1 of the trisaccharide generate a fully protected pentasaccharide intermediate 1-1 under the catalysis of trifluoromethanesulfonic acid.

Adding into a two-mouth bottle

And (3) performing vacuum-pumping on the molecular sieve (1g, powder), baking the bottle, introducing argon for protection, and cooling to room temperature. The trisaccharide intermediate 2-1-1(110mg,0.095mmol) and the disaccharide intermediate 3-1(95.7mg,0.11mmol) were dissolved in 10mL of dried dichloromethane, added to a reaction flask using a syringe, stirred at room temperature for half an hour, cooled to-20 ℃, TfOH (2 μ L,0.019mmol) was added dropwise, TLC monitored by TLC for completion of the reaction of the trisaccharide intermediate 2-1-1, triethylamine was added to quench the reaction, the molecular sieve was filtered through celite, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (petroleum ether/ethyl acetate ═ 5:1) to give the pentasaccharide intermediate 1-1(133mg, 76%) as a colorless oil.

¹ H NMR(400MHz,CDCl ₃ )δ8.14(d,J＝7.6Hz,2H),7.58(t,J＝7.3Hz,1H),7.47(t,J＝7.6Hz,2H),7.42–7.07(m,30H),5.60(d,J＝5.3Hz,1H),5.37(t,J＝9.9Hz,1H),5.30–5.13(m,2H),5.15–4.46(m,18H),4.42(d,J＝7.7Hz,1H),4.31(t,J＝12.1Hz,2H),4.25–4.01(m,6H),4.01–3.60(m,1H),3.54(q,J＝9.5Hz,2H),3.40–3.14(m,6H),2.10(s,3H),2.07–1.96(m,9H),1.92(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ170.95,170.55,170.01,169.88,169.64,167.62,165.42,155.88,138.47,137.55,137.51,137.33,137.28,136.31,133.51,130.04,129.11,128.71,128.56,128.52,128.37,128.25,128.18,128.04,127.95,127.92,127.67,127.37,103.47,99.11,98.84,98.21,97.48,79.82,78.55,77.35,76.64,76.48,76.42,75.62,75.01,74.92,74.74,74.69,74.33,73.55,72.88,71.78,71.10,70.32,69.65,69.47,68.88,66.91,63.76,62.13,61.91,61.08,60.71,60.40,55.27,54.40,52.83,52.43,20.93,20.85,20.63。

HRMS[M+Na] ⁺ m/z 1868.6488 (calculation: C) ₉₃ H ₁₀₃ NaN ₇ O ₃₃ ,1868.6494)。

6. Preparation method of pentasaccharide intermediate III-1

Protecting pentasaccharide 1-1 in LiOH and H ₂ O ₂ Ac, Bz and methyl ester are simultaneously removed under the combined action of NaOH to obtain a hexahydroxy compound I; in SO ₃ ·NEt ₃ Heating under the action of (1) to obtain an O-sulfonated intermediate compound II-1; removing benzyl and Cbz through catalytic hydrogenation reaction, and reducing azide to generate amino to obtain the triamino compound III-1.

The preparation method of the pentasaccharide intermediate III-1 comprises the following steps: after the fully protected pentasaccharide intermediate 1-1(100mg,0.054mmol) was dissolved in 4.9mL of THF, 1.3mL of 1.25N LiOH solution and 2.7mL of 30% H were added dropwise ₂ O ₂ After the solution was allowed to react at room temperature for 12 hours, 3mL of methanol and 1.6mL of 4N NaOH solution were added and the reaction was continued at room temperature for 12 hours, and the reaction was monitored by a dot plate, and TLC color development resulted in a streaky stripe with no top spot. The pH was adjusted to 2 with 6N hydrochloric acid while cooling on ice, the reaction solution was transferred to a separatory funnel and extracted three times with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and flash column chromatography was performed (dichloromethane/methane ═ 15:1) to give pentasaccharide intermediate I (73mg, 90%). Only one single peak is found by HPLC purity detection, and the purity is proved to be very high. Pentasaccharide intermediate I (100mg,0.067mmol) and SO ₃ ·NEt ₃ (1.1g,6.0mmol) was dissolved in 10mL of anhydrous DMF and reacted at 65 ℃ for 24 h, the extent of reaction was monitored by HPLC, and when a single peak was formed, the reaction was stopped, the DMF was spun off, and the oil was taken upDissolving with methanol, passing through Sephadex LH-20 gel column, eluting with mixed solution of dichloromethane and methanol (V/V ═ 1:1), collecting saccharide-containing component, spin drying, eluting with methanol, and passing through Dowex-50-WX4-Na column ⁺ Column exchange to sodium salt, collect sugar containing fractions and concentrate to give pentasaccharide intermediate II-1 as a pale yellow solid (136mg, 95%). The purity of the compound was checked again by HPLC, and the peak time was found to be consistent with the product in the reaction solution. Pentasaccharide intermediate II-1(692mg,0.32mmol) was dissolved in 2mL of methanol, 1mL of t-butanol and 1mL of water were added, Pd/C (40mg) was added, the mixture was stirred at room temperature under a hydrogen pressure of 4atm for 2 days, celite was added, and the filtrate was filtered by spin-drying to give a pale green solid pentasaccharide intermediate III-1(488mg, 100%) without any hydrogen in the aromatic region.

¹ H NMR(400MHz,D ₂ O)δ5.67(d,J＝3.8Hz,1H),5.51(d,J＝3.7Hz,1H),5.29(s,1H),5.05(d,J＝3.5Hz,1H),4.92(s,1H),4.63(dd,J＝20.4,9.8Hz,3H),4.53(d,J＝10.2Hz,1H),4.36(dd,J＝30.1,11.2Hz,7H),4.26–3.69(m,16H),3.62(dd,J＝13.4,6.0Hz,2H),3.55–3.36(m,6H). ¹³ C NMR(101MHz,D ₂ O)δ174.87,174.29,101.21,99.10,96.00,94.88,91.35,83.58,76.95,76.52,74.96,74.06,73.19,72.42,72.15,71.12,70.63,69.44,69.32,69.02,68.80,68.48,67.43,66.68,66.02,65.68,63.31,55.51,54.27,53.92,53.38。

HRMS[M-2Na] ⁺ m/z 738.4654 (calculation: C) ₃₁ H ₄₇ N ₃ Na ₆ O ₄₃ S ₆ ^2- ,738.4647)。

EXAMPLE 2 preparation of Compound CV001

Preparation method of CV 001: the pentasaccharide intermediate III-1 prepared in example 1(200mg,0.13mmol) was dissolved in 2mL of water, the pH adjusted between 9-10 by the addition of 2N NaOH solution (and the pH maintained between 9-10 by the continuous addition of 2N NaOH in the following reaction) and SO was added ₃ Py (110mg, 0.7mmol), added in one more batch half an hour and repeated six more times, then stirred at room temperature for 4 hours, neutralized with hydrochloric acid to pH 7-8, concentrated and passed directly through a Sephadex G-25 gel column with water as eluent, collecting the sugar-containing fraction, concentrated to give the target compound CV001(198mg, 96%).

¹ H NMR(400MHz,D ₂ O)δ5.55(d,J＝3.5Hz,1H),5.37(d,J＝3.5Hz,1H),5.13(d,J＝3.1Hz,1H),5.02(d,J＝3.1Hz,1H),4.89(d,J＝3.6Hz,1H),4.64(s,3H),4.48(t,J＝7.3Hz,1H),4.36(s,1H),4.35–3.98(m,11H),3.82-3.57(m,8H),3.47(d,J＝9.4Hz,1H),3.43-3.22(m,5H),3.15(d,J＝5.4Hz,1H). ¹³ C NMR(101MHz,D ₂ O)δ176.25,175.38,103.82,101.28,99.44,99.30,98.29,82.34,78.43,77.65,76.78,76.09,75.98,75.46,74.99,73.34,71.46,70.87,69.95,69.57,69.45,69.09,68.87,67.36,66.87,66.13,58.65,57.89,56.23,56.35。

HRMS[M–3H] ^3- m/z 527.9675 (calculation: C) ₃₁ H ₅₃ N ₃ O ₅₂ S ₉ ,527.9616)

EXAMPLE 3 Synthesis of Compound CV122

The synthesis method 1 comprises the following steps: after sodium exchange of compound CV001, sodium salt CV122 can be obtained.

Dissolving CV001 in water, and passing through Dowex-50-WX4-Na ⁺ Column exchange to sodium salt, collection of sugar containing components, concentration of solvent, white solid CV 122.

The synthesis method 2 comprises the following steps: the pentasaccharide intermediate III-1 prepared in example 1(200mg,0.13mmol) was dissolved in 2mL of water, the pH adjusted between 9-10 by the addition of 2N NaOH solution (and the pH maintained between 9-10 by the continuous addition of 2N NaOH in the following reaction) and SO was added ₃ Py (110mg, 0.7mmol), adding one more batch after half an hour, repeating six times, stirring at room temperature for 4 hours, neutralizing with hydrochloric acid to pH 7-8, concentrating, passing through Sephadex G-25 gel column with water as eluent, collecting sugar-containing fraction, concentrating, passing through Dowex-50-WX4-Na with water ⁺ Column exchange to sodium salt, collection of sugar containing fractions, concentration of solvent, yield white solid CV122(235mg, 98%).

¹ H NMR(400MHz,D ₂ O)δ5.45(d,J＝3.3Hz,1H),5.33(d,J＝3.3Hz,1H),5.21(d,J＝2.9Hz,1H),5.08(d,J＝2.9Hz,1H),4.99(d,J＝3.4Hz,1H),4.72(s,3H),4.53(t,J＝7.8Hz,1H),4.44(s,1H),4.41–4.08(m,11H),3.92(dd,J＝19.1,9.2Hz,4H),3.79–3.60(m,4H),3.57(d,J＝9.6Hz,1H),3.47–3.31(m,4H),3.30–3.22(m,1H),3.22–3.11(m,1H). ¹³ C NMR(101MHz,D ₂ O)δ174.87,174.29,101.74,99.38,98.57,98.30,97.31,80.65,76.97,76.45,76.17,76.13,75.78,75.35,74.69,72.12,70.73,70.59,69.82,69.24,69.11,68.53,68.50,66.64,66.27,65.88,57.92,57.79,56.74,55.50。

HRMS[M–3Na] ^3- m/z 586.5803 (calculation: C) ₃₁ H ₄₅ N ₃ Na ₈ O ₅₂ S ₉ ^3- ,586.5801)。

EXAMPLE 4 Synthesis of Compound CV123

The synthesis method 1: the compound CV001 was subjected to potassium exchange to obtain potassium salt CV 123.

Dissolving CV001 in water, and passing through Dowex-50-WX4-K ⁺ Column exchange to potassium salt, collection of sugar containing fractions, concentration of solvent to obtain white solid CV 123.

The synthesis method 2 comprises the following steps: the pentasaccharide intermediate III-1 prepared in example 1(137mg,0.09mmol) was dissolved in 2mL of water, the pH adjusted between 9 and 10 by the addition of 2N NaOH solution (and the pH maintained between 9 and 10 by the continuous addition of 2N NaOH in the following reaction) and SO was added ₃ Py (99mg, 0.63mmol), adding one more batch after half an hour, repeating six times, stirring at room temperature for 4 hours, neutralizing with hydrochloric acid to pH 7-8, concentrating, passing through Sephadex G-25 gel column with water as eluent, collecting sugar-containing fraction, concentrating, passing through Dowex-50-WX4-K with water ⁺ Column exchange to potassium salt, collection of sugar containing fractions, concentration of solvent, yielded CV123 as a white solid (175mg, 97%).

¹ H NMR(400MHz,D ₂ O)δ5.48(d,J＝3.4Hz,1H),5.35(d,J＝3.4Hz,1H),5.24(d,J＝2.8Hz,1H),5.09(d,J＝2.7Hz,1H),4.93(d,J＝3.6Hz,1H),4.75(m,3H),4.55(t,J＝7.6Hz,1H),4.49(s,1H),4.40–4.09(m,11H),4.23-3.84(m,4H),3.74–3.62(m,4H),3.55(d,J＝9.4Hz,1H),3.49–3.34(m,4H),3.32–3.24(m,2H),3.22–3.11(m,2H). ¹³ C NMR(101MHz,D ₂ O)δ175.35,174.32,101.85,99.68,98.54,98.26,97.43,81.77,77.23,76.69,76.13,76.10,75.98,75.35,74.57,73.42,70.64,70.56,69.96,69.37,69.26,68.36,68.22,66.57,66.46,65.79,57.65,57.34,56.74,55.26。

In the following examples, CV122 was used as an example to verify the biological activity of the compound of formula a.

Example 5 homogeneous phase time-resolved fluorescence (HTRF) detection of CV122 Heparanase (HPA) inhibitory activity assay the experimental reagents and instrumentation used were as follows:

recombinant Human Heparanase (Recombinant Human Active Heparanase/HPSE, R & D systems, Catalog Number: 7570A of the composition, a-GH, it is an HPA subtype with enzymatic activity), Biotin-heparin Sulfate-Eu cryptate (Bio-HS-Eu), Streptavidin-d2(SA-d2), 3- [ (3-cholesterol (propyl) -dimethylammonium ] -1-propane-sulfonate hydrate (CHAPS), potassium fluoride (KF), Bovine Serum Albumin (BSA), RhHPSE dilution buffer (including 20mM TrisHCl, 0.15M NaCl, and 0.1% CHAPS, pH 7.5), Bio-HS-Eu dilution buffer (0.2M NaCH3CO2, pH 5.5), SA-d2 dilution buffer (including 0.1M PBS, 0.8M KF, and 0.1% BSA, pH 7.5), HTRF dedicated micro-96 well plates, multifunctional detector.

Fondaparinux sodium (available from Hongri pharmaceutical Co., Ltd., Tianjin).

CV122 was prepared by synthesis method 2 of example 3.

Experimental methods

The optimal concentration of heparanase is 120ng/mL, the optimal concentration of Bio-HS-Eu is 1.4ng/mL, the optimal concentration of SA-d2 is 1 mu g/mL, and CV122 samples are diluted in a gradient way (0-500 mu M) through pre-experiments.

The experimental design comprises a CV122 sample group, a fondaparinux sodium sample group, a blank control group, a negative control group and a positive control group, wherein in the CV122 sample group, 4 mu LCV122 sample solution (prepared by adopting RhHPSE dilution buffer solution as a solvent) and 3 mu L heparanase solution (prepared by adopting RhHPSE dilution buffer solution as a solvent) are firstly added into a micro-96 pore plate to be incubated for 10min at 37 ℃, then 3 mu LBio-HS-Eu solution is added and incubated for 30min at 37 ℃,10 mu L SA-d2 solution is added after the incubation is finished, the mixture is placed for 15min at room temperature and then placed in a multifunctional micropore plate detector to detect HTRF signals; the operation modes of the fondaparinux sodium sample group and the CV122 group are the same, except that the tested substance is replaced by fondaparinux sodium; the negative control group system only contains Bio-HS-Eu solution, and other solutions are replaced by buffer solution with the same volume; the positive control group system only comprises a Bio-HS-Eu solution and an SA-d2 solution, and other solutions are replaced by buffer solution with the same volume; blank controls included heparanase solution, Bio-HS-Eu solution, and SA-d2 solution, with samples replaced with the same volume of buffer.

The fluorescence intensity ratio of the excitation wavelength of 320nm, the emission wavelengths of 620nm and 665nm, respectively, 665nm/620nm was used to calculate the average energy transfer rate (. DELTA.F%) for each sample. Wherein the positive control group contains no sample and the enzyme solution should produce the maximum fluorescence energy transfer rate (Δ F) _max ) (ii) a The negative control group only contains Bio-HS-Eu solution, the fluorescence energy transfer rate of the negative control group is the background absorption of the system, and deduction is needed during calculation; the blank control group does not contain the sample solution to be detected, and the hydrolysis of enzyme to Bio-HS-Eu is the strongest at the moment, so that the smallest fluorescence energy transfer rate (delta F) is generated _blank ) The samples with different concentration gradients in the sample set should produce different fluorescence energy transfer rates (Δ F) _sample )。

The inhibition rate was calculated according to the following formula:

Inhibition＝(ΔF _sample -ΔF _blank )/(ΔF _max -ΔF _blank )×100％

the results are shown in FIG. 1. As can be seen from FIG. 1, there is a dose-effect relationship between the inhibition effect of CV122 on heparanase, and the inhibition IC of CV122 on the heparanase is calculated ₅₀ At 3.27. mu.M. Fondaparinux sodium also has inhibitory effect on heparanase, IC ₅₀ 86.02 μ M. CV122 has about 26 times the inhibitory activity of fondaparinux sodium on heparanase.

Example 6 measurement of anticoagulant Activity of CV122

1. Principle of experiment

Heparin (Heparin) first forms a complex (AT-Hep.) with an excess of ATIII, and it is theorized that Heparin produces all AT-Hep. The AT-hep. complex is then bound to excess FXa, and the remaining free FXa can hydrolyze the substrate S ₂₇₆₅ The pNA product was developed at 405nm and then the titre calculated by the bs2000 software application volume reaction parallel line 4.4 method.

Heparin+AT III→[AT-Hep.]

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

[residual FXa]+Substrate→Peptide+pNA

2. Test method

ATIII, FXa, and chromogenic substrate S required for the Activity test ₂₇₆₅ Is commercially available from Beijing Adhoc International Biotechnology corporation, wherein, the human Antithrombin (ATIII), AG 00-0132; bovine-derived Activated Factor X (FXa), AG 00-0121; FXa factor chromogenic substrate S ₂₇₆₅ ，AG00-0102-10。

The corresponding solutions were prepared according to the following table:

low molecular weight heparin sodium standards/sample preparation: the standards or samples were diluted to 1.0IU/ml (starting dose can be adjusted as appropriate in pharmacopoeia), and then diluted to a-E five concentration gradients [ a ]0.1600 (or 0.2/0.18 both) IU/ml × 0.75 ═ B ]0.1200IU/ml × 0.75 ═ C ]0.0900IU/ml × 0.75 ═ D ]0.0675IU/ml × 0.75 ═ E ] 0.0506/ml, respectively, and the samples were loaded in the order of the following table operation, then the OD at 450nm was read by a microplate reader, and titers were calculated by bs2000 software dose reaction parallel line 4.4 method.

Enzyme activity determination sequence

3. Through determination:

the titer of standard low molecular weight heparin sodium (H0185000, biological standard substance for analyzing low molecular weight heparin, purchased from Beijing Adhoc International Biotechnology corporation) is 100 IU/mg;

the CV122 measures the potency PT of 119.96IU/mg, and the confidence limit rate of the potency is FL of 4.6284%;

sundaparinux sodium (provided by Hongyi pharmaceutical industry Co., Ltd., Tianjin) was found to have a potency PT of 923.01IU/mg and a potency confidence rate of FL of 6.014%.

The greater the titer, the better the anticoagulation activity. As can be seen from the above experiments, CV122 has a lower anticoagulant activity than fondaparinux sodium.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A heparin pentasaccharide structural compound having the structure of formula a:

wherein each Y is the same or different and is independently selected from H, Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ An isovalent cation;

preferably, each Y is the same and is selected from H, Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ Monovalent cations are equivalent;

preferably, each Y is the same or different and is independently selected from Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ ；

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

Preferably, the structural formula of the heparin pentasaccharide structural compound is as follows:

2. use of a compound of claim 1 for the preparation of an HPA inhibitor.

3. Use of a compound of claim 1 for the preparation of a medicament for inhibiting HPA activity; preferably, the medicament is for the treatment or prevention of a pathophysiological process or disease or disorder in which HPA is involved, or a disease or disorder in which HPA is overexpressed or affected by increased HPA activity; preferably, the disease or disorder is metastasis of a tumor, invasion or infiltration of a tumor, diabetic nephropathy, membranous nephropathy, proteinuric glomerular disease, amyloidosis nephropathy, osteolysis, atherosclerosis, cardiovascular disease.

4. A pharmaceutical composition comprising a compound according to claim 1 as an active ingredient; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; preferably, in the pharmaceutical composition, the compound of formula a accounts for 0.01% to 95% of the pharmaceutical composition by mass, preferably 10% to 90%, preferably 20% to 80%, and more preferably 30% to 70%.

5. A fully protected pentasaccharide has the following structural formula:

wherein R is ₁ 、R ₂ And R ₃ May be the same or different and is independently selected from the group consisting of chloroacetyl, acetyl, benzoyl, pivaloyl; preferably, R ₁ And R ₂ Are all acetyl, R ₃ Is benzoyl.

6. The process for preparing fully protected pentasaccharide according to claim 5, wherein the trisaccharide intermediate 2 and the disaccharide acceptor 3 are subjected to glycosylation reaction to obtain the fully protected pentasaccharide:

7. a pentasaccharide has the following structure:

8. a pentasaccharide has the following structure:

，

wherein each Y is the same or different and is independently selected from H, Na ⁺ ，K ⁺ ，NH ₄ ⁺ Or/and Li ⁺ (ii) a Preferably, each Y is the same and is selected from H, Na ⁺ ，K ⁺ ，Li ⁺ ，NH ₄ ⁺ (ii) a Preferably, each Y is the same and is selected from H or Na ⁺ Or K ⁺ 。

9. A pentasaccharide has the following structure:

10. A trisaccharide having the formula:

wherein R is ₁ And R ₂ May be the same or different and is independently selected from chloroacetyl, acetyl, benzoyl or pivaloyl; x is a leaving group suitable for reacting with other receptors to form a bond between glycosides;

preferably, X is hydroxy, thioalkyl, thioaryl, halogen, trichloroacetimidate, phosphate, t-butyldiphenylsilyloxy;

preferably, R ₁ Is acetyl, R ₂ Is acetyl;

preferably, the trisaccharide is

Preferably, the trisaccharide is

11. The process for preparing the trisaccharide as set forth in claim 10, comprising the steps of:

and 3) obtaining a trisaccharide intermediate 2 from the trisaccharide intermediate 5, wherein the reaction formula is as follows:

12. a disaccharide having the structure:

wherein R is ₂ Is chloroacetyl, acetyl, benzoyl or pivaloyl;

preferably, the disaccharide is

13. The method for producing a disaccharide according to claim 12, wherein the monosaccharide intermediate 10 having an α -configuration and the monosaccharide intermediate 11 are reacted to produce the disaccharide:

14. a disaccharide of the formula:

wherein R is ₂ Is chloroacetyl, acetyl, benzoyl or pivaloyl;

preferably, the disaccharide is:

15. a process for producing a disaccharide according to claim 14, which comprises removing chloroacetyl group at the 4-position of the disaccharide according to claim 12, and separating and removing the product having a (1 → 4) glycosidic bond configuration to obtain a disaccharide according to claim 14 having a β configuration.

16. A monosaccharide having the formula:

wherein R is ₂ Is chloroacetyl, acetyl, benzoyl or pivaloyl;

preferably, the monosaccharide is

Preferably, the monosaccharide is

17. A process for the preparation of the monosaccharide of claim 16, comprising the steps of: 1) taking glucose as a starting material, and converting 1-hydroxyl of the glucose into glucosinolate through reaction; 2) protecting hydroxyl at 4-position and 6-position of glucose, protecting hydroxyl at 3-position with a temporary protecting group, protecting hydroxyl at 2-position with benzyl, removing the temporary protecting group, and replacing with R ₂ Protecting the hydroxyl at the 3-position; 3) deprotection is carried out on hydroxyl at the 4-position and the 6-position of glucose, then reaction is carried out, the hydroxyl at the 6-position is oxidized and methyl esterified, and the hydroxyl at the 4-position is protected by monochloroacetyl; 4) converting the 1-position thioglycoside into bromine to obtain the monosaccharide;

preferably, in the step 1), after all hydroxyl groups of glucose are protected by a hydroxyl protecting group, the hydroxyl group protected at the 1-position of glucose is converted into thioglycoside through reaction; preferably, the hydroxyl protecting group is acetyl, chloroacetyl, benzoyl or pivaloyl;

preferably, in the step 1), the glucosinolate is p-toluenesulfonyl, and the protected hydroxyl at the 1-position of the glucose is converted into the p-toluenesulfonyl by using p-toluenesulfonol under the action of an accelerator; preferably, the accelerator is selected from boron trifluoride diethyl etherate, boron trifluoride, zinc chloride, tin chloride or a Lewis acid;

preferably, benzylidene is adopted in the step 2) to protect hydroxyl at the 4-position and the 6-position of glucose;

preferably, the temporary protecting group in step 2) is PMB.

18. A process for the preparation of the heparin pentasaccharide compound of claim 1,

a, prepared by sulphation of the pentasaccharide III of claim 9;

preferably, the pentasaccharide III in the claim 9 is obtained by azide reduction of the pentasaccharide II in the claim 8;

preferably, the pentasaccharide II of claim 8 is obtained by sulfonating the pentasaccharide I of claim 7;

preferably, the pentasaccharide I according to claim 7 is obtained from the dehydroxylation of the fully protected pentasaccharide according to claim 5 and the carboxyl protecting group.