CN114957354A - Heparin pentasaccharide structural compound - Google Patents

Abstract

The present invention provides a heparin pentasaccharide compound and a preparation method thereof. The heparin pentasaccharide compound has HPA inhibitory activity, and can be used to treat or prevent pathophysiological processes or diseases that HPA participates in, or the overexpression or activity enhancement of HPA leads to pathological changes. Physiologically altered diseases, such as reducing tumor metastasis and invasion, prolonging the survival time of tumor patients, or treating renal disease, cardiovascular disease, inflammatory disease, pathological angiogenesis, etc.

Description

Heparin pentasaccharide structure compound

technical field

The invention belongs to the technical field of pharmaceutical compounds, and in particular relates to a heparin pentasaccharide structure compound, a preparation method thereof, as well as HPA inhibitory activity and pharmaceutical application.

Background technique

An important component of eukaryotic cell surface and extracellular matrix is heparansulfate proteoglycan (HSPG), which is a complex formed by a core protein and four heparansulfate (HS) side chains. It is distributed in the intima and media lining of great vessels and on the subendothelial basement membrane of capillaries. Heparanase (HPA) is the only endogenous glycosidase found in mammalian cells. It can specifically recognize HSPG and degrade HSPG into short sugar chains of 4kDa-7kDa by hydrolyzing glycosidic bonds. Function.

Heparanase is mainly distributed in platelet cells, mast cells, placental basement membrane, keratinocytes, leukocyte cell membrane or extracellular matrix in vivo, and has two natural substrates, heparin and heparan sulfate.

In cell and animal experiments, it was found that heparanase is overexpressed in various types of cancer cells such as lung cancer, breast cancer, liver cancer, and intestinal cancer. Heparanase destroys the physical barrier of cells by hydrolyzing the heparan sulfate side chain of HSPG. , release related growth factors, which are closely related to cell metastasis, angiogenesis, and lymphangiogenesis, suggesting that heparanase may be closely related to the metastasis and invasiveness of cancer, and may be a potential antitumor drug target. In theory, the competitive inhibition of heparanase may prevent the degradation of HSPG in tumor cells, thereby reducing the metastasis and invasiveness of cancer cells. Cell, animal and clinical trials have also confirmed this. By inhibiting heparanase, The ability of tumor metastasis was significantly reduced, and the survival time was effectively prolonged.

Studies have also found that heparanase is also involved in several other diseases such as diabetic nephropathy, proteinuric glomerular disease, amyloid nephropathy, osteolysis in bone metastases and other bone tissue-related pathologies, atherosclerosis. , cardiovascular disease and skin aging, as well as abnormal angiogenesis, inflammatory response processes, etc. For example, studies have found that the proliferation of smooth muscle cells (SMC) can stabilize atherosclerotic lesions, because heparan sulfate can regulate the proliferation of SMCs and affect the stability of plaques, and heparanase plays an important role in inflammation, coagulation, etc. The expression is increased in atherosclerosis associated with plaque instability, so heparanase can be involved in the regulation of atherosclerotic SMC function and is closely related to the development of atherosclerotic plaques and the transformation of unstable plaques . Membranous nephropathy (MN) is the main cause of nephrotic syndrome (NS) in adults. The common clinical manifestations of MN patients are massive proteinuria, hypoproteinemia, hyperlipidemia and venous thrombosis. , With the protracted development of the disease, it will eventually lead to renal failure and uremia. HSPGs are localized to the glomerular filtration membrane in the kidney, carry a large negative charge and play an important role in the maintenance of the charge barrier in the glomerulus. The researchers injected monoclonal antibodies against HSPG into rats to block the side chain of HSPG, and the results showed that the rats produced selective proteinuria, indicating that HSPG plays an important role in the production of proteinuria. HPA degrades HSPG on the basement membrane, destroys its integrity, reduces the negative charge on the basement membrane, weakens the charge barrier, and makes it easier for proteins to pass through the glomerulus, resulting in massive proteinuria, which can be significantly reduced by inhibitors against HPA. The occurrence of proteinuria indicates that HPA may play an important role in the pathophysiological process of MN.

Therefore, for pathophysiological processes or diseases involving HPA, or diseases with pathophysiological changes caused by HPA overexpression or enhanced activity, the use of HPA-targeting inhibitor compounds can play a role in the treatment or treatment of the corresponding pathophysiological processes or diseases. The improvement effect, for example, may be used to reduce tumor metastasis and invasion, prolong the survival time of tumor patients, or treat renal disease, cardiovascular disease, inflammatory disease, pathological angiogenesis, etc.

In the 1980s, the work of total synthesis of pentasaccharide compounds has begun. In the synthesis, the main solution is to solve the low reaction efficiency caused by the intermolecular reaction of the reducing end hemiacetal of the pentasaccharide. How to design the synthetic route includes the use of suitable protective groups. , so that the regioselectivity and stereoselectivity are conducive to the formation of correct glycosidic bonds and improve the reaction efficiency.

The groups of Petitou and van Boeckel successively synthesized the pentasaccharide sequence present in the natural heparin molecule. The synthetic route adopted a [1+4] synthesis strategy to obtain a fully protected pentasaccharide molecule in approximately 70% yield. In addition to the iduronic acid donor, the remaining several sugar group donors all use bromo sugars, and the bromo sugars are generally activated by silver salts, and the synthesis cost of the pentasaccharide is relatively high. The pentasaccharide is the first chemically synthesized heparin molecule with anticoagulant activity, which lays the foundation for the successful launch of fondaparinux sodium in the later stage, and also opens up a way for the synthesis of other heparin-like molecules.

In the later synthesis, it was found that when the pentasaccharide undergoes catalytic hydrogenation, the benzyl group at the reducing end is removed to form a hemiacetal, and all amino groups are also exposed. Under this condition, the amino group and the aldehyde group will quickly generate a stable Schiff. Base, the resulting final product always contains dimers or trimers. In order to solve this problem, Petitou's group protected the terminal position of glucosamine at the reducing end with methyl group, and used the same synthetic strategy as the above-mentioned pentasaccharide to couple it to obtain a fully protected pentasaccharide. The sulfonated heparin pentasaccharide was The sugar is fondaparinux.

In 1991, Petitou's group used the [3+2] convergent synthesis strategy to synthesize the fully protected pentasaccharide protected by the methyl group at the reducing end. The glycosylation yield was about 70%, and the glycosylation reaction was stereospecific. , the separation and purification is easier. At the same time, this strategy can save a lot of disaccharide fragments containing iduronic acid, because the synthesis steps of iduronic acid are many and the yield is not high, so it is much more economical than the above [1+4] route.

But even so, the synthetic method has more than 60 steps, and the yield is less than 0.1%, which was the longest synthetic step in the world for chemical drugs at that time. After 2008, fondaparinux sodium became a generic drug, and more and more scientists or enterprises began to develop technology for the synthesis of fondaparinux. Zhou, Ding, Qin and other research teams successively used the [3+2] convergent synthesis strategy to synthesize fondaparinux sodium, but the trisaccharide donor leaving groups used were different, and the protecting group strategies were different. There are also different characteristics when synthesizing monosaccharide modules.

In 2013, Zhou's research group reported a synthetic route for fondaparinux sodium. The whole route adopted a [3+2] convergent synthesis strategy, which required a total of 36 steps, and the total yield was 0.017%. It should be pointed out that this route directly uses cellobiose as a starting material to synthesize a disaccharide receptor composed of glucuronic acid and glucosamine. This method effectively avoids the problem of poor β-selectivity in the glucuronic acid glycosylation reaction. At the same time, the researchers synthesized the ethylthio group donor and the trichloroacetimide ester donor of iduronic acid. It was found that the disaccharide product generated when the trichloroacetimide ester donor was used for glycosylation was α/β isomers, while the disaccharide product is mono-configurational when ethylthio is used as the glycosyl donor.

In 2014, Hung's research group reported a total synthesis route of fondaparinux sodium, which adopted the [4+1] convergent synthesis strategy. The whole route has a total of 36 steps, and the total yield can reach 0.63%. Most of the monosaccharide modules used in this route are synthesized by the one-pot method invented by the research group. Moreover, the strategy of first glycosylation and then oxidation to uronic acid is adopted, which effectively avoids the disadvantages of weak uronic acid reactivity and low yield. And the 2-position of glucose is a Bz protective group, and there is an adjacent group involved, so that the newly generated glycosidic bond is completely β configuration.

In 2016, Qin's research group published a total synthesis route of fondaparinux sodium, using a [3+2] convergent synthesis strategy, starting from the coupling reaction of monosaccharides and monosaccharides to the synthesis of fully protected pentasaccharides. 14 steps are required and the overall yield of 14 steps can reach 3.5%. They did a lot of optimization work on the synthesis and purification of monosaccharides during the synthesis process, for example, using the Staudinger reaction to separate the α/β isomers of H sugars. Furthermore, they have also done a lot of work on the construction of glucuronic acid β-glycosidic bonds, prepared four glycosyl donors and two glycosyl acceptors for screening, and then proceeded to synthesize disaccharides after obtaining the optimal combination. Finally, it should be pointed out that this strategy can achieve 10g level when synthesizing fully protected pentasaccharide, which to some extent shows that this strategy is suitable for industrial scale-up production.

的两条合成路线进行整合，在合成非还原端三糖供体时，需要磺酸化的羟基均用PMB临时保护。正因如此，葡萄糖醛酸二位才可以用Bz保护，这样就可以有效构建β糖苷键。为了有效控制[3+2]糖基化的立体选择性，研究者将PMB转换成Ac，使得糖基化反应有较高的α选择性。同时证明了三糖硫苷供体和三糖三氯乙酰亚胺酯供体在合成全保护肝素五糖分子上的等效性。Ding's research group combined Alchemia and

The two synthetic routes were integrated. When synthesizing the non-reducing end trisaccharide donor, the sulfonated hydroxyl groups were temporarily protected with PMB. Because of this, the glucuronic acid 2-position can be protected with Bz, so that the β-glycosidic bond can be effectively constructed. In order to effectively control the stereoselectivity of [3+2]glycosylation, the researchers converted PMB into Ac, which made the glycosylation reaction have high α selectivity. At the same time, the equivalence of trisaccharide glucosinolate donor and trisaccharide trichloroacetimide ester donor in the synthesis of fully protected heparin pentasaccharide was demonstrated.

SUMMARY OF THE INVENTION

The invention provides a heparin pentasaccharide structure compound, which has valuable pharmacological properties, especially has inhibitory effect on HPA, and can be used to prepare HPA inhibitors, or prepare and treat pathophysiological processes or diseases involved in HPA, or Drugs for diseases whose expression or activity is enhanced to bring about pathophysiological changes, such as reducing tumor metastasis and invasion, prolonging the survival time of tumor patients, or treating kidney diseases, cardiovascular diseases, inflammatory diseases, pathological angiogenesis, etc. The present invention further provides a preparation method of the heparin pentasaccharide compound.

A heparin pentasaccharide structure compound, which has 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 monovalent cations such as H, Na ⁺ , K ⁺ , Li ⁺ , NH ₄ ⁺ and the like.

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

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

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

Those skilled in the art can understand that any one or two or more Y is selected from the compound of formula A with monovalent cations such as Na ⁺ , K ⁺ , Li ⁺ , NH ₄ ⁺ , etc., which is essentially the compound of formula A in which Y is H When Y is a monovalent cation, the corresponding group in formula A is its anion group, such as -COO ^- , -SO ₃ ^- .

The compound of formula A is a compound with a single optical activity, that is, GlcNS6S(1→4)GlcA3Sβ(1→4)GlcNS3S6S(1→4)IdoA2S(1→4)GlcNS6S, and the terminal methyl ester of the H sugar is an α structure type.

In one embodiment of the present invention, Y is H, and the structural formula of the compound is as follows, which is referred to as CV001 in the present invention.

In one embodiment of the present invention, Y is Na ⁺ , and the structural formula of the compound is as follows, which is referred to as CV122 in the present invention, which is essentially the sodium salt of CV001.

In one embodiment of the present invention, Y is K ⁺ , and the structural formula of the compound is as follows, which is referred to as CV123 in the present invention, which is essentially the potassium salt of CV001.

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

Formula A can adopt heparin pentasaccharide synthesis methods known in the art, such as 4+1 synthesis strategy, or 3+2 synthesis strategy and the like.

The present invention provides a new synthetic reaction method to synthesize a compound of formula A, which is carried out in a 3+2 synthetic route, using a trisaccharide donor leaving group different from the prior art, a different hydroxyl protecting group strategy, and a different The monosaccharide module synthesis method of the present invention uses fully protected pentasaccharide intermediate 1, successively undergoes dehydroxylation and carboxyl protecting group, sulfonation and azide reduction, and finally sulfonation to obtain the compound of formula A:

wherein R ₁ , R ₂ and R ₃ may be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl, and pivaloyl; Y is as defined above. In a specific embodiment of the present invention, R ₁ and R ₂ are both acetyl groups, R ₃ is benzoyl, and Y is H. In a specific embodiment of the present invention, R ₁ and R ₂ are both acetyl groups, R ₃ is benzoyl group, and Y is Na ⁺ . In a specific embodiment of the present invention, R ₁ and R ₂ are both acetyl groups, R ₃ is benzoyl group, and Y is K ⁺ .

The fully protected pentasaccharide intermediate 1 can be obtained by the glycosylation reaction of the trisaccharide intermediate 2 and the disaccharide acceptor 3, and the reaction formula is as follows:

The trisaccharide intermediate 2 can be coupled with the monosaccharide intermediate 7 and the disaccharide intermediate 8 through the (1→4) glycosidic bond to obtain the trisaccharide intermediate 4 with a specific stereo configuration. 1,6 ring-opening obtains trisaccharide intermediate 5, and then trisaccharide intermediate 2 is obtained. The reaction formula is as follows:

The disaccharide intermediate 8 can be obtained from the glycosylation reaction of the monosaccharides 10 and 11, and then separating the product in the β configuration. The reaction formula is as follows:

The monosaccharide 10 can be prepared by using glucose as a raw material and through a reaction comprising the following steps: 1) converting the 1-hydroxyl group of glucose into glucosinolate; 2) protecting the 4-position and 6-position hydroxyl groups of glucose and protecting them with a temporary protective group 3-hydroxyl, then use benzyl to protect the 2-hydroxyl, then remove the temporary protecting group and use R ₂ to protect the 3-hydroxyl; 3) After deprotecting the 4- and 6-hydroxyl groups of glucose, react, respectively The hydroxyl group was oxidized and methyl esterified, and the 4-hydroxyl group was protected with a chloroacetyl group; 4) The 1-position glucosinolate was converted to bromine to obtain the monosaccharide intermediate 10.

Monosaccharide 11, monosaccharide 7, disaccharide acceptor 3 used in the above preparation method can be prepared according to synthetic methods known in the art, for example: Preactivation-based, iterative one-pot synthesis of anticoagulantpentasaccharide fondaparinux Sodium.Org.Chem . Front., 2019, 6, 3116; Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. ChemMed Chem, 2014, 9, 1071-1080.

The definitions of R ₁ , R ₂ and R ₃ in each intermediate compound in the above preparation method are the same as the definitions of the corresponding groups in intermediate 1; X is a leaving group suitable for reacting with other acceptors to form between glycosides. The bond of , preferably X is hydroxyl, thioalkyl, thioaryl, halogen, trichloroimidoacetyl, phosphate, tert-butyldiphenylsilyloxy.

Therefore, the present invention also provides each intermediate in the above-mentioned synthetic method and its preparation method.

其中R₂为氯乙酰基、乙酰基、苯甲酰基或特戊酰基。A monosaccharide intermediate 10, its structure is as follows:

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

Preferably, the monosaccharide intermediate 10 is in α configuration,

In one embodiment of the present invention, R ₂ of the monosaccharide intermediate 10 is an acetyl group, which is an α configuration, named 10-1, and the structural formula is

Different protective groups are used for the 2-position hydroxyl group and the 3-position hydroxyl group of the monosaccharide intermediate 10, which is beneficial to the selective sulfonation on the 3-position hydroxyl group when the compound of formula A is finally synthesized. In addition, the 2-hydroxyl group is protected by a benzyl group, together with the 1-position bromine, which is beneficial to the selectivity of the β-glycosidic bond and the high yield of the reaction when the intermediate 10 is used to synthesize the disaccharide.

The preparation method of the monosaccharide intermediate 10 includes the following steps: 1) using glucose as a starting material, and converting the 1-hydroxyl group of glucose into glucosinolate through a reaction; 2) after protecting the 4-position and 6-position hydroxyl groups of glucose, using The temporary protecting group protects the 3-position hydroxyl group, and then uses the benzyl group to protect the 2-position hydroxyl group, and then removes the temporary protecting group and replaces the 3-position hydroxyl group with R ₂ to protect the 3-position hydroxyl group; 3) React after deprotecting the 4-position and 6-position hydroxyl groups of glucose, The 6-hydroxyl group was oxidized and methylated, respectively, and the 4-position hydroxy group was protected with chloroacetyl; 4) The 1-position glucosinolate was converted to bromine to obtain the monosaccharide intermediate 10.

According to the present invention, in the step 1), all hydroxyl groups of glucose are first protected with a hydroxyl protecting group, and then the hydroxyl group protected at the 1-position of glucose is converted into glucosinolates through a reaction. The hydroxyl protecting group can be various hydroxyl protecting groups known in the art, including but not limited to acetyl, chloroacetyl, benzoyl, pivaloyl, etc. In one embodiment of the present invention, an acetyl group is used. All hydroxyl groups of glucose are protected.

According to the present invention, in the step 1), a method known in the art can be used to convert the hydroxyl group at the 1-position of glucose into a glucosinolate. Said glucosinolate can be various types of glucosinolate-SR known in the art, wherein R is alkyl, substituted alkyl, aryl or substituted aryl, such as methyl glucosinolate-SMe, ethyl glucosinolate Set, Thiosulfan-SPh, p-toluidine Stol, etc. In one embodiment of the invention, the glucosinolate is p-tolylthio. In one embodiment of the present invention, under the action of an accelerator, the hydroxyl group protected at the 1-position of glucose is converted into a p-tolylthio group with p-toluenethiophenol. The accelerator includes but is not limited to: boron trifluoride ether, boron trifluoride, zinc chloride, tin chloride, Lewis acid, etc., the Lewis acid includes but is not limited to zirconium tetrachloride, iron trichloride or Titanium tetrachloride, etc. In one embodiment of the present invention, the accelerator is boron trifluoride diethyl ether. In another embodiment of the present invention, a tributylstannyl derivative of a thiol is used to react with a Lewis acid.

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

In one embodiment of the present invention, in the step 2), benzylidene is used to protect the 4- and 6-hydroxyl groups of glucose.

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

In an embodiment of the present invention, in the step 2), under the action of Bu ₂ SnO or Bu ₂ Sn(OMe) ₂ , dehydration or de-methanol respectively obtains a cyclodibutylstannylidene derivative, the derivative Is a convenient intermediate for the regiobenzylation of polyols. This derivative can then be reacted with a PMB donor to produce a product in which the 3-hydroxyl group is protected with PMB. In one embodiment of the invention, the hydroxyl group at the 3 position is protected with PMB in the presence of PMBCl, CsF and DMF. In another embodiment of the present 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 present invention, the 3-hydroxyl group 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), in the presence of BnBr, NaH and DMF, the 2-position hydroxyl group is protected with a benzyl group. In another embodiment of the present invention, the ₂ -hydroxyl group is protected with a benzyl group in the presence of ceric ammonium nitrate, Br2, dichloromethane and water.

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

In one embodiment of the present invention, in the step 3), TEMPO oxidation and methyl esterification are used to oxidatively methylate the 6-hydroxyl group of glucose.

According to the present invention, the step 4) can adopt a method known in the art to convert the 1-position glucosinolate into bromine, including but not limited to treatment with iodine bromide.

According to the present invention, after the step 4), the obtained intermediate 10 may be subjected to configuration separation to obtain the intermediate 10 in the α configuration for subsequent synthesis of the disaccharide intermediate.

的制备方法，包括：以葡萄糖12为起始原料，经全乙酰化得到单糖中间体13；在BF₃.Et₂O作用下，与对甲苯硫酚反应得到单糖中间体14；在催化量甲醇钠的作用下脱掉所有乙酰基，随后将4,6-二羟基用苄叉保护得到2,3-二羟基裸露的单糖中间体15；在二丁基氧化锡的作用下选择性在3位上PMB保护基作为临时保护基，得到单糖中间体16；将2位羟基苄醚化得到单糖中间体17；在DDQ的作用下将临时保护基PMB氧化脱除得到中间体18，随后将其乙酰化得到单糖中间体19；在醋酸条件下加热将苄叉保护基脱除，得到4,6-二羟基的单糖中间体20；TEMPO氧化、甲酯化得到单糖中间体21；将4位羟基用氯乙酰基保护得到单糖中间体22；最后用溴化碘处理，将β硫苷转化成α构型的单糖中间体10-1。反应式如下：In one embodiment of the present invention, the monosaccharide intermediate 10-1

The preparation method comprises the following steps: using glucose 12 as a starting material, through peracetylation to obtain monosaccharide intermediate 13; under the action of BF ₃ .Et ₂ O, reacting with p-toluene thiophenol to obtain monosaccharide intermediate 14; All acetyl groups were removed under the action of a large amount of sodium methoxide, and then the 4,6-dihydroxyl group was protected with benzylidene to obtain the 2,3-dihydroxyl naked monosaccharide intermediate 15; under the action of dibutyltin oxide, selectivity The PMB protecting group at the 3-position was used as a temporary protecting group to obtain the monosaccharide intermediate 16; the hydroxybenzyl etherification of the 2-position gave the monosaccharide intermediate 17; under the action of DDQ, the temporary protecting group PMB was oxidatively removed to obtain the intermediate 18 , and then acetylated to obtain monosaccharide intermediate 19; heating under acetic acid conditions to remove the benzylidene protecting group to obtain 4,6-dihydroxy monosaccharide intermediate 20; TEMPO oxidation and methyl esterification to obtain monosaccharide intermediate Body 21; protecting the 4-hydroxyl group with chloroacetyl group to obtain monosaccharide intermediate 22; finally treating with iodine bromide to convert β-glucosinolate into α-configuration monosaccharide intermediate 10-1. The reaction formula is as follows:

其中，R₂为氯乙酰基、乙酰基、苯甲酰基或特戊酰基。在本发明的一个实施方式中所述二糖中间体9的R₂为乙酰基，命名为9-1，结构式为

A disaccharide intermediate 9, its structural formula is as follows:

Wherein, R ₂ is chloroacetyl, acetyl, benzoyl or pivaloyl. In one embodiment of the present invention, R ₂ of the disaccharide intermediate 9 is an acetyl group, named 9-1, and the structural formula is

The preparation method of the disaccharide intermediate 9 includes the following steps: reacting the α-configuration monosaccharide intermediate 10 and the monosaccharide intermediate 11 to generate the disaccharide intermediate 9 .

The monosaccharide intermediate 11 can be synthesized according to the method described in the literature (Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharide fondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116). The use of intermediate 11 with an internal ether ring structure is beneficial to increase the β configuration ratio of intermediate 9.

According to the present invention, the Ag ₂ CO ₃ /AgOTf reaction system or the Ag ₂ O/TMSOTf reaction system can be used to carry out the above-mentioned Koenigs-Knorr glycosylation reaction to generate the disaccharide intermediate 9 . In one embodiment of the present invention, the reaction is performed under the action of insoluble silver carbonate and a catalytic amount of silver trifluoromethanesulfonate. Preferably, the amount of silver trifluoromethanesulfonate used in the reaction is 0.05-0.2 equivalents, and It is beneficial to ensure the reaction efficiency and the selectivity of β configuration.

其中，R₂为氯乙酰基、乙酰基、苯甲酰基或特戊酰基。在本发明的一个实施方式中所述二糖中间体8的R₂为乙酰基，命名为8-1，结构式为

A disaccharide intermediate 8, its structural formula is as follows:

Wherein, R ₂ is chloroacetyl, acetyl, benzoyl or pivaloyl. In one embodiment of the present invention, R ₂ of the disaccharide intermediate 8 is an acetyl group, named 8-1, and the structural formula is

The preparation method of the disaccharide intermediate 8 includes the following steps: removing the 4-position chloroacetyl group of the disaccharide intermediate 9, separating and removing the product of the (1→4) glycosidic bond configuration, and obtaining a pure β configuration The disaccharide intermediate 8.

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

其中，R₁和R₂可以相同或不同，独立地选自氯乙酰基、乙酰基、苯甲酰基或特戊酰基；X为适合与其他受体反应的离去基团，以形成糖苷之间的键。A trisaccharide intermediate 2, its structural formula is as follows:

Wherein, R ₁ and R ₂ can be the same or different, and are independently selected from chloroacetyl, acetyl, benzoyl or pivaloyl; X is a leaving group suitable for reacting with other acceptors to form a gap between glycosides key.

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

Preferably, the trisaccharide intermediate is

In one embodiment of the present invention, R ₁ of the trisaccharide intermediate is acetyl group, R ₂ is acetyl group, X is trichloroiminoacetyl group, named 2-1-1, and the structural formula is

The preparation method of described trisaccharide intermediate 2, comprises the steps:

Step 1) Monosaccharide intermediate 7 and disaccharide intermediate 8 are coupled through (1→4) glycosidic bond to obtain trisaccharide intermediate 4 with specific stereo configuration;

Step 2) Trisaccharide intermediate 4 is subjected to 1,6 ring-opening under the action of a catalyst to obtain trisaccharide intermediate 5;

Step 3) Trisaccharide intermediate 2 is obtained from trisaccharide intermediate 5.

The reaction formula is as follows, and the definitions of R ₁ , R ₂ and X are the same as the aforementioned definitions.

The trisaccharide intermediate 2 in two configurations, α-configuration and β-configuration, can be obtained by means of silica gel column chromatography.

The intermediate 2 with mixed configuration can be directly used in the next reaction to obtain the fully protected compound 1 with α configuration, or the two configurations of α configuration and β configuration obtained by the separation of intermediate 2 can be obtained separately in the next step. The fully protected compound 1 in the α configuration, that is, the intermediate 2 of which configuration or mixed configuration is used in the subsequent synthesis, does not affect the subsequent reaction and yield.

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

According to the present invention, the step 2) is carried out in a corresponding acid anhydride (such as chloroacetic anhydride, acetic anhydride, benzoic anhydride, pivalic anhydride), adding TBSOTf, TMSOTf, Et ₂ OBF 3 or Et ₃ SiOTf, etc., and the reaction temperature is -20℃～0℃.

所用反应体系可以是苄胺和THF，或者Bu₃SnOMe和二氯甲烷，或者三氟化硼-乙醚在湿乙腈中。随后，在有机溶剂中在碱和三氯乙腈存在条件下，10-30℃下，将中间体6的端基羟基转化为三氯乙酰亚胺酯供体三糖中间体2-1，所述有机溶剂可以选自二氯甲烷、四氢呋喃等；所述碱可以选自无机碱类，例如碳酸钾、碳酸氢钾、氢化钠、DBU等。According to the present invention, in the step 3), a method known in the art can be used to selectively remove the end-group protecting group of the trisaccharide intermediate 5. In one embodiment of the present invention, at 10-30 °C, the end-group protecting group of trisaccharide intermediate 5 is selectively removed to obtain trisaccharide intermediate 6 in which X is a hydroxyl group.

The reaction system used can be benzylamine and THF, or _Bu3SnOMe and dichloromethane, or boron trifluoride-diethyl ether in wet acetonitrile. Subsequently, in the presence of a base and trichloroacetonitrile in an organic solvent at 10-30 °C, the terminal hydroxyl group of intermediate 6 was converted to trichloroacetimidate ester donor trisaccharide intermediate 2-1, the described The organic solvent can be selected from dichloromethane, tetrahydrofuran, etc.; the base can be selected from inorganic bases, such as potassium carbonate, potassium bicarbonate, sodium hydride, DBU and the like.

Monosaccharide intermediate 7 can be prepared using synthetic methods known in the art, eg Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux. ChemMed Chem, 2014, 9, 1071-1080.

The use of disaccharide intermediate 8 with internal ether ring structure to synthesize trisaccharide intermediate 4 is beneficial to simultaneously protect the 1-position and 6-position hydroxyl groups of the disaccharide intermediate 8, so that monosaccharide intermediate 7 and disaccharide intermediate 8 react Only the latter has a hydroxyl group at the 4-position, which improves the selectivity of forming 1→4 glycosidic bonds; and after ring-opening to obtain trisaccharide intermediate 5, the terminal group of intermediate 5 can be converted into any available leaving group X, in particular trichloroimidoacetyl, without being limited by the type of leaving group of the monosaccharide intermediate 7.

A fully protected pentasaccharide intermediate 1, its structural formula is as follows:

wherein R ₁ , R ₂ and R ₃ may be the same or different and independently selected from chloroacetyl, acetyl, benzoyl, pivaloyl. In a specific embodiment of the present invention, R ₁ and R ₂ are both acetyl groups, and R ₃ is benzoyl group.

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

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

Without being limited by a special theory, the inventors found that the 6-position of the trisaccharide intermediate 2F sugar is an acyl group, which is beneficial to increase the proportion of α-configuration in the product when synthesizing fully protected pentasaccharide 1, and even obtain a pentasaccharide with all-α configuration. sugar 1; and the acyl group is stable to acid and will not fall off during the glycosylation reaction, ensuring a high yield of fully protected pentasaccharide 1.

The disaccharide receptor 3 can be prepared according to synthetic methods known in the art, for example: Preactivation-based, iterative one-pot synthesis of anticoagulant pentasaccharidefondaparinux Sodium. Org. Chem. Front., 2019, 6, 3116. The inventors of the present invention have found that in the Fischer glycosylation reaction, hydrogen bonding is conducive to the formation of α-configuration products, and the α-configuration of the H sugar at the reducing end of disaccharide receptor 3 is synthesized through Fischer glycosylation. The use of a Cbz protecting group on the amino group of the disaccharide acceptor 3 is beneficial to improve the yield of the methoxy α configuration in the monosaccharide H. In addition, the Cbz protecting group can be removed under the same conditions as the azide and benzyl groups, and can tolerate strong bases without adding additional reaction steps from the fully protected pentasaccharide 1 to the preparation of the compound of formula A.

In one embodiment of the present invention, the synthetic reaction formula of described fully protected pentasaccharide 1 is as follows:

When the trichloroacetimide ester is used as the sugar group donor in the reaction, the reaction conditions are mild, the thermal stability is relatively good, the reaction activity is high and the yield is high.

The present invention also provides intermediates I, II and III useful in the synthesis of the compounds of formula A.

The structure of the compound I is as follows:

The structure of the compound II is as follows:

其中Y定义与式A中相同，优选Y为H或Na⁺或K⁺。

Wherein Y is defined as in formula A, preferably Y is H or Na ⁺ or K ⁺ .

The structure of compound III is as follows:

其中Y定义与式A中相同，优选Y为H或Na⁺或K⁺。

Wherein Y is defined as in formula A, preferably Y is H or Na ⁺ or K ⁺ .

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

The dehydroxylation and carboxyl protecting group, sulfonation and azide reduction can all be carried out using reaction methods and conditions known in the art. In one embodiment of the present invention, intermediate 1 is simultaneously removed from R ₁ , R ₂ , R ₃ and methyl ester in the presence of a base to obtain intermediate I. In one embodiment of the present invention, intermediate I obtains O-sulfonated intermediate II under the action of SO ₃ ·NEt ₃ . In one embodiment of the present invention, intermediate II removes benzyl and Cbz through catalytic hydrogenation, and simultaneously reduces azide to generate amino group to obtain intermediate III. In one embodiment of the present invention, three amino groups in the compound of formula III are sulfonated under the action of SO ₃ ·Py to obtain compound CV001. CV001 was ion-exchanged with a sodium-type ion-exchange resin to obtain compound CV122. 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 the like. CV001 was ion-exchanged with potassium-type ion-exchange resin to obtain compound CV123. The potassium ion exchange resin can be resins known in the art, including but not limited to Amberlite IR120 K ⁺ , Dowex-50-WX4-K ⁺ and the like.

The present invention adopts [3+2] convergent synthesis strategy to synthesize the compound of formula A, adopts different trisaccharide donor leaving group, different hydroxyl protecting group strategy and different monosaccharide module synthesis method from the prior art . In addition to the aforementioned advantages of using trichloroacetimide ester as the trisaccharide donor leaving group and the H-glycosyl amino group using Cbz protection, the sulfonated hydroxyl group in the method of the present invention is protected by R ₁ , R ₂ , R ₃ , The protection and deprotection method is simple, the conditions are mild, and the reaction yield is high; the non-sulfonated hydroxyl group is protected by a benzyl group, and the protecting group is resistant to acid and alkali and is not affected in the glycosidation reaction, and finally can be reduced by a catalytic hydrogenation method, and the yield is obtained. high rate. In addition, the sulfonated hydroxyl group and the non-sulfonated hydroxyl group adopt different protecting groups, which ensures that the selective sulfonation can be carried out in the synthesis.

The present invention also provides a pharmaceutical composition comprising a compound of formula A of the present invention or a solvate thereof as an active ingredient, optionally further comprising one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier is various adjuvants commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrating agents, etc. . In one embodiment of the invention, the invention provides a method that inhibits HPA activity, or is suitable for the treatment or prevention of a pathophysiological process or disease or condition in which HPA is involved, or a disease in which HPA is overexpressed or affected by enhanced HPA activity or a pharmaceutical composition for a disorder comprising a compound of formula A of the present invention or a solvate thereof as an active ingredient, optionally further comprising one or more pharmaceutically acceptable carriers.

The present invention also provides the use of the compound of formula A in the preparation of HPA inhibitors.

The present invention also provides the use of the compound of formula A in the preparation of a medicament for inhibiting the activity of HPA. The medicament can be used to treat or prevent a pathophysiological process or disease or condition in which HPA is involved, or a disease or condition in which HPA is overexpressed or affected by enhanced HPA activity.

The present invention also provides the use of the compound of formula A in the preparation of a pharmaceutical composition.

The pharmaceutical composition of the present invention is used for inhibiting HPA activity, or is suitable for treating or preventing pathophysiological processes or diseases or conditions in which HPA is involved, or diseases or conditions in which HPA is overexpressed or affected by enhanced HPA activity.

The present invention also provides a method for treating or preventing a pathophysiological process or disease or condition in which HPA is involved, or a disease or condition affected by HPA overexpression or enhanced HPA activity, by administering to a patient in need the compound of formula A of the present invention or A pharmaceutical composition containing a compound of formula A.

The pathophysiological process or disease or condition in which HPA participates, or a disease or condition affected by HPA overexpression or enhanced HPA activity, mainly due to HPA overexpression or enhanced activity in vivo, for example: tumor metastasis , tumor invasion or infiltration, diabetic nephropathy, membranous nephropathy, proteinuric glomerular disease, amyloid nephropathy, osteolysis, atherosclerosis, cardiovascular disease, etc. Said HPA overexpression or activity enhancement in vivo refers to those skilled in the art using conventional detection methods in the field (including but not limited to enzymatic chemical detection, enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry, Western blotting, tissue Microarray, gene detection and other methods) to detect the expression level of HPA gene or protein or HPA enzyme activity in the individual or in corresponding tissues or cells (such as cancer or tumor tissue and/or cells, diseased kidney tissue, etc.) The expression level or enzyme activity is higher than the normal level, for example, it is higher than the normal level by more than 110%, or more than 120%, or more than 130%, or more than 150%, or more than 200%, etc.; The gene or protein expression level or enzymatic activity level of HPA in the population or in corresponding tissues and/or cells, or the expression level or enzymatic activity of HPA gene or protein in tissues and/or cells of non-diseased organs of the same patient Level.

The amount of the compound of formula A contained in the pharmaceutical composition (calculated as the compound of formula A) is 0.1-1000 mg, preferably 1-500 mg, 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 may be, for example, 0.1%-10%, 0.3-5%, or 10%- 90%, preferably 20%-80%, more preferably 30%-70% and other content ranges.

The dosage form of the pharmaceutical composition can be in the form of oral agents, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; it can also be in the form of injection administration, such as injections, powder injections, etc. , administered by intravenous, intraperitoneal, subcutaneous or intramuscular injection. All dosage forms used are well known to those of ordinary skill in the art of pharmacy. For example, the pharmaceutical composition can be an injection solution, and the concentration of the compound of formula A in the injection solution can be 1-15 mg/ml, such as 5 mg/ml, 10 mg/ml, 12.5 mg/ml and the like.

Routes of administration of the pharmaceutical compositions include, but are not limited to: oral; buccal; sublingual; transdermal; pulmonary; rectal; parenteral, eg, by injection, including subcutaneous, intradermal , intramuscular, intravenous; by implanted reservoir or reservoir.

The administered dose of the compound of formula A (based on the compound of formula A) will depend on the age, health and weight of the recipient, the type of concomitant drug, the frequency of treatment, the route of administration, and the like. 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 dosage of the compound of formula A (calculated as the compound of formula A) is 0.01-100 mg/kg/day, preferably 0.1-10 mg/kg/day, such as 0.5 mg/kg/day, 1 mg/kg/day, 2 mg/kg/day day, 5mg/kg/day, etc.

The pharmaceutical composition can be administered in combination with other therapeutic agents or made into a combination drug. The other therapeutic agent can be a tumor therapeutic drug, a kidney disease therapeutic drug, a cardiovascular therapeutic drug, etc., according to different types of diseases and conditions.

Tumor treatment drugs, such as: drugs that damage the structure and function of DNA (such as nitrogen mustard, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, etc.), nucleotide synthase inhibitors (such as 5-fluorouracil, cape Tabine, raltitrexed, 6-mercaptopurine, etc.), DNA polymerase inhibitors (such as cytarabine, gemcitabine, etc.), dihydrofolate reductase inhibitors (such as methotrexate, pemetrexed, etc.), nucleoside Acid reductase inhibitors (such as hydroxyurea), drugs that inhibit RNA synthesis (such as doxorubicin, daunorubicin, epirubicin, pirarubicin, etc.), topoisomerase inhibitors (such as hydroxycamptothecin) alkali, irinotecan, topotecan, etc.), tubulin inhibitors (such as vincristine, vindesine, vinorelbine, paclitaxel, docetaxel, etc.), drugs that affect hormone balance (such as toremifene) , exemestane, letrozole, bicalutamide, enzalutamide, medroxyprogesterone, megestrol, testosterone propionate, goserelin, leuprolide, 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, ramucirumab, etc.), immunomodulators (such as rituximab, pembrolizumab, ipilimumab, etc.).

Cardiovascular treatment drugs, such as: lipid-lowering drugs (such as: lovastatin), antihypertensive drugs (such as beta receptor antagonists, ACEI, angiotensin II receptor antagonists, etc.), anticoagulants.

Nephropathy treatment drugs, such as: ACEI (such as captopril, benazepril, enalapril, perindopril, etc.), angiotensin II receptor antagonists (such as valsartan, losartan, erin besartan, candesartan, etc.), anticoagulants (such as heparin, low molecular weight heparin, enoxaparin, fondaparinux, bivalirudin, etc.), glucocorticoids (such as methylprednisolone), cyclophosphamide , cyclosporine, chlorambucil, tripterygium glycosides, etc.

The HPA inhibitory activity of the compound of formula A of the present invention is higher than that of fondaparinux, but the anticoagulant activity is much lower than that of fondaparinux, so the bleeding risk is extremely low while being used for the therapeutic use of inhibiting HPA. The compound of formula A has a clear structure, is beneficial to preparation and quality control, and does not combine with other proteins, and has a long half-life in vivo, with a t _1/2 of 17 hours in a two-chamber model.

Description of drawings

Figure 1. Dose-response graph of the inhibitory effect of CV122 and fondaparinux on heparanase

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the intended protection scope of the present invention.

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

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

Example 1 Synthesis of Pentasaccharide Intermediate III

1. Preparation method of monosaccharide intermediate 10-1

Using glucose 12 as the starting material, the monosaccharide intermediate 13 was obtained by peracetylation; under the action of BF ₃ ^. Et ₂ O, the monosaccharide intermediate 14 was obtained by reacting with p-toluene thiophenol; under the action of catalytic sodium methoxide Removal of all acetyl groups followed by protection of the 4,6-dihydroxyl group with benzylidene to give the 2,3-dihydroxyl naked monosaccharide intermediate 15; selective PMB protection at the 3-position under the action of dibutyltin oxide As a temporary protecting group, monosaccharide intermediate 16 was obtained; 2-hydroxybenzyl etherification was used to obtain monosaccharide intermediate 17; under the action of DDQ, the temporary protecting group PMB was oxidatively removed to obtain intermediate 18, which was subsequently acetylated The monosaccharide intermediate 19 was obtained; the benzylidene protecting group was removed by heating under the condition of acetic acid to obtain the 4,6-dihydroxy monosaccharide intermediate 20; TEMPO oxidation and methyl esterification were performed to obtain the monosaccharide intermediate 21; The hydroxyl group is protected with a chloroacetyl group to give the monosaccharide intermediate 22; the final treatment with iodine bromide converts the β-glucosinolate to the α-configuration monosaccharide intermediate 10-1.

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, two-step total yield 83%; d) 1) Bu ₂ SnO, MeOH, refluxing; 2) PMBCl, CsF, DMF, 90°C, two-step total yield 75%; e) BnBr, NaH , DMF, 92%; f) DDQ, DCM, _H2O , 78%; g) _Ac2O , _Et3N , DCM, 98%; h) 80% AcOH, 90°C, 76%; i) 1) 2,2,6,6-Tetramethylpiperidinooxy(TEMPO), Iodobenzene diacetate, DCM, H ₂ O; 2) MeI, KHCO ₃ , DMF, 57% overall yield for two steps; j) ClAc ₂ O, Py, DCM, 76 %; k) IBr, DCM, 76%.

2. The preparation method of disaccharide intermediate 9-1

Monosaccharide intermediate 10-1 and monosaccharide intermediate 11-1 reacted overnight under the action of insoluble silver carbonate and catalytic amount of silver trifluoromethanesulfonate to form disaccharide intermediate 9-1. After NMR identification, the product α/ The beta ratio is approximately 1:1.

分子筛(1g,粉末)，抽真空烤瓶，通氩气保护后冷却至室温。用10mL干燥的二氯甲烷将单糖中间体10-1(300mg,0.63mmol)和单糖中间体11-1(172mg,0.75mmol)溶解，用注射器加入到反应瓶中，室温搅拌半小时，将Ag₂CO₃(260mg,0.94mmol)和催化量AgOTf(16mg,0.063mmol)加入到反应瓶中，避光，反应过夜，TLC检测供体10-1完全消失，生成一主要物质，加硅藻土过滤，滤液减压浓缩后直接柱层析纯化(石油醚/乙酸乙酯＝2:1)得到α/β构型混合的二糖单糖中间体9-1。单糖中间体11-1合成方法参见Preactivation-based,iterative one-pot synthesis of anticoagulantpentasaccharide fondaparinux Sodium.Org.Chem.Front.,2019,6,3116。Add to two bottles

Molecular sieve (1g, powder), vacuumize the baking bottle, pass through argon protection, and cool to room temperature. Monosaccharide intermediate 10-1 (300 mg, 0.63 mmol) and monosaccharide intermediate 11-1 (172 mg, 0.75 mmol) were dissolved in 10 mL of dry dichloromethane, added to the reaction flask with a syringe, and stirred at room temperature for half an hour, Ag ₂ CO ₃ (260 mg, 0.94 mmol) and catalytic amount of AgOTf (16 mg, 0.063 mmol) were added to the reaction flask, protected from light, reacted overnight, TLC detected that the donor 10-1 disappeared completely, generating a main substance, adding silicon After filtration with algae, the filtrate was concentrated under reduced pressure and purified by direct column chromatography (petroleum ether/ethyl acetate=2:1) to obtain a disaccharide monosaccharide intermediate 9-1 with a mixed α/β configuration. For the synthesis method of monosaccharide intermediate 11-1, see Preactivation-based, iterative one-pot synthesis of anticoagulantpentasaccharide fondaparinux Sodium.Org.Chem.Front., 2019,6,3116.

HRMS [M+Na] ⁺ m/z _650.1327 ( _calcd : _{C26H30ClN3NaO13} , _650.1365 ).

3. Preparation method of disaccharide intermediate 8-1

The 4-position chloroacetyl group of the disaccharide intermediate 9-1 is removed, and the α-configuration product is separated to obtain the pure β-configuration disaccharide intermediate 8-1.

The above-mentioned disaccharide intermediate 9-1 was dissolved in 15 mL of a mixed solvent of chloroform and methanol (V/V=1:1), and thiourea (192 mg, 2.52 mmol) was added and heated to 60°C. When the raw material disappears completely and three single points are formed, the heating is stopped, cooled to room temperature, and an appropriate amount of silica gel is added to spin dry, and the direct column chromatography separation (petroleum ether/ethyl acetate=1:1) obtains a colorless foamy disaccharide intermediate 8 -1 (583 mg, 42% yield in two steps).

¹ H NMR (400MHz, CDCl ₃ ) δ 7.38-7.23(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 ( _calcd : _{C24H29N3NaO12} , _574.1649 ).

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

Monosaccharide intermediate 7-1 is glycosylated and coupled with disaccharide intermediate 8-1 to obtain trisaccharide intermediate 4-1 with specific stereo configuration; acetylation gives 1,6 ring-opened trisaccharide intermediate 5- 1; Under the action of benzylamine, the terminal acetyl group is removed to obtain trisaccharide intermediate 6-1, and then trisaccharide intermediate 2-1-1 is obtained by adding trichloroacetimide ester.

分子筛(1g,粉末)，抽真空烤瓶，通氩气保护后冷却至室温。用10mL干燥的甲苯将二糖中间体8-1(430mg,0.78mmol)和单糖中间体7-1(578mg,1.0mmol)溶解，用注射器加入到反应瓶中，室温搅拌半小时，冷却至-20℃，逐滴加入TfOH(8.9μL,0.1mmol)，维持在-20℃下搅拌1.5h。TLC监测二糖中间体8-1反应完全后，加入过量三乙胺淬灭反应，用一小段硅胶将分子筛滤除，滤液减压浓缩，所得粗产品直接柱层析分离(石油醚/乙酸乙酯＝3:1)得到三糖中间体4-1(651mg,87％)。The preparation method of trisaccharide intermediate 4-1: add in two-necked bottle

Molecular sieve (1g, powder), vacuumize the baking bottle, pass through argon protection, and cool to room temperature. Dissolve disaccharide intermediate 8-1 (430 mg, 0.78 mmol) and monosaccharide intermediate 7-1 (578 mg, 1.0 mmol) with 10 mL of dry toluene, add them to the reaction flask with a syringe, stir at room temperature for half an hour, and cool to -20°C, TfOH (8.9 μL, 0.1 mmol) was added dropwise, and stirring was maintained at -20°C for 1.5 h. After monitoring the completion of the reaction of disaccharide intermediate 8-1 by TLC, excess triethylamine was added to quench the reaction, the molecular sieve was filtered off with a small section of silica gel, the filtrate was concentrated under reduced pressure, and the obtained crude product was directly separated by column chromatography (petroleum ether/ethyl acetate) Ester=3:1) to give trisaccharide intermediate 4-1 (651 mg, 87%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.30 (ddd, J=23.3, 15.2, 7.2 Hz, 16H), 5.47 (s, 1H), 5.32-5.13 (m, 2H), 4.99 (d, J=3.4 Hz, 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. , 127.98,127.91127.78,103.45,100.20,99.11,79.83,78.24,77.18,76.35,74.74.93,74.45,73.70.70.21,63.79.79,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95,95.

HRMS [M+K] ⁺ m/z _999.3036 ( _calcd : _C46H52KN6O17 , _999.3026 ).

Preparation method of trisaccharide intermediate 5-1: Dissolve trisaccharide intermediate 4-1 (370 mg, 0.39 mmol) in 10 mL of acetic anhydride, under nitrogen protection, cool to 0 °C in an ice bath, add TBSOTf (38 μL, 0.16 mmol), after maintaining the reaction at low temperature for 20 min, TLC monitoring the complete reaction of the raw materials, and adding triethylamine to quench the reaction. The reaction solution was concentrated under reduced pressure, and the obtained crude product was directly purified by column chromatography (petroleum ether/ethyl acetate=5:1) to obtain trisaccharide intermediate 5-1 (409 mg, 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) ^.13C NMR(101MHz, _CDCl3 )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 (calcd: _{C50H58NaN6O20} , _1085.3604 ).

Preparation method of trisaccharide intermediate 2-1-1: Dissolve trisaccharide intermediate 5-1 (100 mg, 0.094 mmol) in 5 mL of THF, add benzylamine (51 μL, 0.47 mmol) to react at room temperature, and monitor the completion of the reaction by TLC Then, the reaction solution was transferred to a separating funnel, the remaining benzylamine was washed away with 1N hydrochloric acid, the aqueous phase was back-extracted twice with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure. The obtained crude product was directly purified by column chromatography to obtain trisaccharide intermediate 6-1. The above trisaccharide intermediate 6-1 was dissolved in 5 mL of dichloromethane, anhydrous potassium carbonate (19 mg, 0.14 mmol) was added, and trichloroacetonitrile (14 μL, 0.14 mmol) was added dropwise at room temperature. After the addition, the reaction solution was stirred at room temperature for 2 h, and the raw materials were basically reacted completely by TLC monitoring. The potassium carbonate was filtered off with a small section of silica gel, the filtrate was concentrated under reduced pressure, and the obtained crude product was directly purified by column chromatography (petroleum ether/ethyl acetate=5 : 1) to obtain a colorless syrup trisaccharide intermediate 2-1-1 (79 mg, two-step yield 72%). Two configurations, α configuration and β configuration, can be separated by silica gel column chromatography, and the NMR data of the two configurations are as follows.

Intermediate 2-1-1 with mixed configuration can be directly used in the next reaction to obtain fully protected compound 1-1 with α configuration, or the α configuration and β configuration obtained by separating intermediate 2-1-1. Each configuration can be put into the next step to obtain the fully protected compound 1-1 with α configuration, that is, no matter which configuration or mixed configuration intermediate 2-1-1 is used in the subsequent synthesis, it will not affect the subsequent reaction and Yield.

^1H 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} ( _calcd : _{C50H56Cl3NaN7O19} , _1186.2594 ).

^1H NMR (400MHz, CDCCl ₃ ) δ8.77(s, 1H), 7.40-7.28(m, 10H), 7.27-7.18(m, 5H), 5.68(d, J=8.4 Hz, 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 trisaccharide trichloroacetimide ester donor trisaccharide compound 2-1-1 and the disaccharide intermediate 3-1 are catalyzed by trifluoromethanesulfonic acid to generate the fully protected pentasaccharide intermediate 1-1.

分子筛(1g,粉末)，抽真空烤瓶，通氩气保护后冷却至室温。用10mL干燥的二氯甲烷将三糖中间体2-1-1(110mg,0.095mmol)和二糖中间体3-1(95.7mg,0.11mmol)溶解，用注射器加入到反应瓶中，室温搅拌半小时，冷却至-20℃，逐滴加入TfOH(2μL,0.019mmol)，TLC监测三糖中间体2-1-1反应完全后，加入三乙胺淬灭反应，将分子筛用硅藻土过滤，滤液减压浓缩，所得粗产品柱层析纯化(石油醚/乙酸乙酯＝5:1)得到无色油状五糖中间体1-1(133mg,76％)。Add to two bottles

Molecular sieve (1g, powder), vacuumize the baking bottle, pass through argon protection, and cool to room temperature. The trisaccharide intermediate 2-1-1 (110 mg, 0.095 mmol) and the disaccharide intermediate 3-1 (95.7 mg, 0.11 mmol) were dissolved in 10 mL of dry dichloromethane, and added to the reaction flask with a syringe, and stirred at room temperature Half an hour, cooled to -20°C, TfOH (2 μL, 0.019 mmol) was added dropwise, TLC monitored the completion of the reaction of trisaccharide intermediate 2-1-1, triethylamine was added to quench the reaction, and the molecular sieve was filtered through celite , the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (petroleum ether/ethyl acetate=5:1) to obtain a colorless oily pentasaccharide intermediate 1-1 (133 mg, 76%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.14 (d, J=7.6 Hz, 2H), 7.58 (t, J=7.3 Hz, 1H), 7.47 (t, J=7.6 Hz, 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) _a ^_ ,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 (calcd: _{C93H103NaN7O33} , _{1868.6494 )} .

6. The preparation method of pentasaccharide intermediate III-1

The fully protected pentasaccharide 1-1 simultaneously removes Ac, Bz and methyl ester under the combined action of LiOH, H ₂ O ₂ and NaOH to obtain hexahydroxy compound I; under the action of SO ₃ ·NEt ₃ to heat to obtain O-sulfonic acid The acidified intermediate compound II-1; the benzyl group and Cbz are removed by catalytic hydrogenation, and the azide is reduced to generate an amino group at the same time to obtain the triamino compound III-1.

Preparation method of pentasaccharide intermediate III-1: After the fully protected pentasaccharide intermediate 1-1 (100 mg, 0.054 mmol) was dissolved in 4.9 mL of THF, 1.3 mL of 1.25N LiOH solution and 2.7 mL of 30% H _were added dropwise in turn _O2 solution, after 12 hours of reaction at room temperature, add 3mL of methanol and 1.6mL of 4N NaOH solution, continue to react at room temperature for 12 hours, spot plate monitoring, TLC color development will generate a long trailing strip, and there is no stray spot above. Adjust pH to 2 with 6N hydrochloric acid under ice bath, transfer the reaction solution to a separating funnel and extract three times with dichloromethane, combine the organic layers, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and perform flash column chromatography Separation (dichloromethane/methane = 15:1) afforded the pentasaccharide intermediate I (73 mg, 90%). Only one single peak was found by HPLC purity test, which proved its high purity. Pentasaccharide intermediate I (100 mg, 0.067 mmol) and SO ₃ ·NEt ₃ (1.1 g, 6.0 mmol) were dissolved in 10 mL of anhydrous DMF, and reacted at 65° C. for 24 hours. The degree of reaction was monitored by HPLC. When a single When there is one peak, stop the reaction, spin dry DMF, dissolve the oily substance in methanol, directly pass through Sephadex LH-20 gel column, and use a mixed solution composed of dichloromethane and methanol (V/V=1:1) as elution The sugar-containing components were collected, the solvent was spin-dried, and methanol was used as the eluent to pass through the Dowex-50-WX4-Na ⁺ column to exchange for sodium salt, and the sugar-containing components were collected and concentrated to obtain a pale yellow solid pentasaccharide intermediate II- 1 (136 mg, 95%). The purity of the compound was checked again by HPLC, and it was found that the peak time was consistent with the product in the reaction solution. The pentasaccharide intermediate II-1 (692 mg, 0.32 mmol) was dissolved in 2 mL of methanol, 1 mL of tert-butanol and 1 mL of water were added, Pd/C (40 mg) was added, stirred at room temperature for 2 days under a hydrogen pressure of 4 atm, and diatomaceous earth was added. After filtration, the filtrate was spin-dried to obtain gray-green solid pentasaccharide intermediate III-1 (488 mg, 100%). NMR showed that there was no hydrogen in the aromatic region.

¹ H NMR (400MHz, D ₂ O) δ 5.67 (d, J=3.8Hz, 1H), 5.51 (d, J=3.7Hz, 1H), 5.29 (s, 1H), 5.05 (d, J=3.5 Hz,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) δ 174.87, 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 ( _calcd : _{C31H47N3Na6O43S62-} , _738.4647 ).

The preparation of embodiment 2 compound CV001

The preparation method of CV001: take the pentasaccharide intermediate III-1 (200mg, 0.13mmol) prepared in Example 1 and dissolve it in 2mL of water, add dropwise 2N NaOH solution to adjust the pH between 9-10 (and in the following reaction) Continuously add 2N NaOH dropwise to maintain the pH between 9-10) add SO ₃ ·Py (110 mg, 0.7 mmol), add another batch after half an hour, repeat six times, and then stir at room temperature for 4 hours and then add in hydrochloric acid. And to pH 7-8, after concentration, use water as eluent to directly pass through Sephadex G-25 gel column, collect sugar-containing fractions, and concentrate to obtain the target compound CV001 (198 mg, 96%).

¹ H NMR (400MHz, D ₂ O) δ 5.55 (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– 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, 71.4, 6, 9.57, 4 , 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 (calcd: C ₃₁ H ₅₃ N ₃ O ₅₂ S ₉ , 527.9616)

Example 3 Synthesis of compound CV122

Synthetic method 1: The sodium salt CV122 can be obtained by exchanging the compound CV001 with sodium.

After dissolving CV001 in water, it was exchanged into sodium salt through Dowex-50-WX4-Na ⁺ column with water, the sugar-containing fraction was collected, and the solvent was concentrated to obtain CV122 as a white solid.

Synthetic method 2: take the pentasaccharide intermediate III-1 (200mg, 0.13mmol) prepared in Example 1 and dissolve it in 2mL of water, add dropwise 2N NaOH solution to adjust the pH between 9-10 (and continuously in the following reaction) Add 2N NaOH dropwise to maintain pH between 9-10) add SO ₃ ·Py (110 mg, 0.7 mmol), add another batch after half an hour, repeat six times, then stir at room temperature for 4 hours and neutralize with hydrochloric acid To pH 7-8, after concentration, use water as eluent to directly pass through Sephadex G-25 gel column, collect sugar-containing fractions, concentrate, exchange water into sodium salt through Dowex-50-WX4-Na ⁺ column, collect The sugar-containing fraction was concentrated and the solvent was concentrated to give CV122 as a white solid (235 mg, 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) ^.13C 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 ( _calcd : _{C31H45N3Na8O52S93-} , _586.5801 ).

Example 4 Synthesis of compound CV123

Synthesis method 1: Potassium salt CV123 can be obtained by exchanging compound CV001 for potassium.

After CV001 was dissolved in water, water was passed through Dowex-50-WX4-K ⁺ column to exchange into potassium salt, sugar-containing fractions were collected, and the solvent was concentrated to obtain white solid CV123.

Synthetic method 2: Take the pentasaccharide intermediate III-1 (137mg, 0.09mmol) prepared in Example 1 and dissolve it in 2mL of water, add dropwise 2N NaOH solution to adjust the pH between 9-10 (and continuously in the following reaction) 2N NaOH was added dropwise to maintain the pH between 9-10) SO ₃ ·Py (99 mg, 0.63 mmol) was added, and another batch was added after half an hour, which was repeated six times, and then neutralized with hydrochloric acid after stirring at room temperature for 4 hours. To pH 7-8, after concentration, use water as eluent to directly pass through Sephadex G-25 gel column, collect sugar-containing fractions, concentrate, exchange water into potassium salt through Dowex-50-WX4-K ⁺ column, collect The sugar-containing fraction was concentrated and the solvent was concentrated to give CV123 as a white solid (175 mg, 97%).

¹ H NMR (400MHz, D ₂ O) δ 5.48 (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 is used as an example to verify the biological activity of the compound of formula A.

Embodiment 5 Homogeneous time-resolved fluorescence (HTRF) detects the inhibitory activity test of CV122 heparanase (HPA) The experimental reagents and instruments used are as follows:

Recombinant Human Active Heparanase (Recombinant Human Active Heparanase/HPSE, R&Dsystems, Catalog Number: 7570-GH, which is an HPA isoform with enzymatic activity), Biotin-HeparanSulfate-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), special micro-96-well plate for HTRF, multi-function microplate detector.

Fondaparinux sodium (provided by Tianjin Hongri Pharmaceutical Co., Ltd.).

CV122 was prepared by synthetic method 2 of Example 3.

experimental method

The optimal concentration of heparanase was determined to be 120 ng/mL in the preliminary experiments, 1.4 ng/mL of Bio-HS-Eu, 1 μg/mL of SA-d2, and gradient dilution of CV122 samples (0-500 μM).

The experiment consisted of CV122 sample group, fondaparinux sample group, blank control group, negative control group and positive control group. In the CV122 sample group, 4 μL of CV122 sample solution (using RhHPSE dilution buffer as solvent configuration) and 3 μL of heparan were firstly prepared. Enzyme solution (prepared with RhHPSE dilution buffer as solvent) was added to micro-96 well plate and incubated at 37°C for 10min, then 3μL Bio-HS-Eu solution was added and incubated at 37°C for 30min, after incubation, 10μL SA-d2 solution was added at room temperature After being placed for 15 minutes, the HTRF signal was detected in a multi-function microplate detector; the fondaparinux sodium sample group and the CV122 group were operated in the same manner, except that the tested substance was replaced by fondaparinux sodium; in the negative control system, only Contains Bio-HS-Eu solution, and other solutions are replaced with the same volume of buffer; the positive control system only includes Bio-HS-Eu solution and SA-d2 solution, and other solutions are replaced with the same volume of buffer; the blank control group includes acetyl Heparinase solution, Bio-HS-Eu solution and SA-d2 solution, the samples were replaced with the same volume of buffer.

The detection excitation wavelength was 320 nm, and the emission wavelengths were 620 nm and 665 nm, respectively. The ratio of fluorescence intensity at 665 nm/620 nm was used to calculate the average energy transfer rate (ΔF%) of each sample. The positive control group without sample and enzyme solution should produce the maximum fluorescence energy transfer rate (ΔF _max ); the negative control group only contains Bio-HS-Eu solution, and its fluorescence energy transfer rate is the background absorption of the system, which needs to be calculated. Subtracted; the blank control group does not contain the sample solution to be tested. At this time, the enzyme has the strongest hydrolysis effect on Bio-HS-Eu and should produce the smallest fluorescence energy transfer rate (ΔF _blank ), and samples with different concentration gradients in the sample group should produce different Fluorescence energy transfer rate (ΔF _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 Figure 1. It can be seen from Figure 1 that the inhibitory effect of CV122 on heparanase has a dose-effect relationship, and the calculated IC ₅₀ of CV122 for the inhibition of this enzyme is 3.27 μM. Fondaparinux sodium also has inhibitory effect on heparanase with IC ₅₀ of 86.02 μM. The inhibitory activity of CV122 on heparanase is about 26 times that of fondaparinux sodium.

Example 6 Determination of anticoagulant activity of CV122

1. Experimental principle

Heparin (Heparin) first generates a complex (AT-Hep.) with excess ATIII, and it is theoretically believed that all heparin generates an AT-Hep. complex with thrombin-inhibiting activity. The AT-Hep. complex is then combined with excess FXa, the remaining free FXa can hydrolyze the substrate S ₂₇₆₅ , and the pNA product develops color at 405 nm, and then the titer is calculated by bs2000 software using the 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

The ATIII, FXa, and chromogenic substrate S ₂₇₆₅ required for the activity test are all commercially available from Beijing Adhoc International Biotechnology Co., Ltd. Among them, human antithrombin Antithrombin (ATIII), AG00-0132; bovine-derived activated factor X ActivatedFactor X(FXa), AG00-0121; FXa factor chromogenic substrate S ₂₇₆₅ , AG00-0102-10.

Prepare the corresponding solutions according to the following table:

Low molecular weight heparin sodium standard/sample preparation: The standard or sample is diluted to 1.0IU/ml (the starting dose can be adjusted appropriately within the range of the Pharmacopoeia), and then diluted to five concentration gradients of A-E [A] 0.1600 (or 0.2 /0.18) IU/ml×0.75=[B]0.1200IU/ml×0.75=[C]0.0900IU/ml×0.75=[D]0.0675IU/ml×0.75=[E]0.0506IU/ml, according to The operation sequence in the following table adds samples, and then the microplate reader reads the OD value at 450nm, and uses the bs2000 software to calculate the titer by applying the method of parallel line 4.4 of the amount of reaction.

Enzyme Activity Assay Operation Sequence

3. It has been determined:

The standard low molecular weight heparin sodium (H0185000, low molecular weight heparin analysis biological standard, purchased from Beijing Adhoc International Biotechnology Company) has a potency of 100IU/mg;

The measured titer of CV122 was PT=119.96IU/mg, and the confidence limit of the titer was FL=4.6284%;

The titer of fondaparinux (provided by Tianjin Hongri Pharmaceutical Co., Ltd.) was PT=923.01IU/mg, and the confidence limit of the titer was FL=6.014%.

The higher the titer, the better the anticoagulant activity. It can be seen from the above experiments that the anticoagulant activity of CV122 is lower than that of fondaparinux.

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

Claims (18)

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:

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 ⁺ 。

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:

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, 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.

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