WO2021128253A1 - 一种糖胺聚糖衍生物及其应用 - Google Patents

一种糖胺聚糖衍生物及其应用 Download PDF

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WO2021128253A1
WO2021128253A1 PCT/CN2019/129110 CN2019129110W WO2021128253A1 WO 2021128253 A1 WO2021128253 A1 WO 2021128253A1 CN 2019129110 W CN2019129110 W CN 2019129110W WO 2021128253 A1 WO2021128253 A1 WO 2021128253A1
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glycosaminoglycan
reaction
group
glycosaminoglycan derivative
alkyl
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PCT/CN2019/129110
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English (en)
French (fr)
Inventor
张运好
王景文
任丽鸽
林森茂
李锂
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深圳市海普瑞药业集团股份有限公司
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Priority to CN201980103149.3A priority Critical patent/CN114901702B/zh
Priority to PCT/CN2019/129110 priority patent/WO2021128253A1/zh
Publication of WO2021128253A1 publication Critical patent/WO2021128253A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof

Definitions

  • This application belongs to the technical field of biological materials, and relates to a glycosaminoglycan derivative and its application.
  • Heparan sulfate proteoglycans are a class of sugar complexes, which have two parts: a core protein and one or more heparan sulfate sugar chains covalently connected to the core protein.
  • Extracellular matrix refers to a substance secreted by cells and located in the lower layer of epithelial or endothelial cells and around connective tissue cells to provide mechanical support and physical strength for tissues and organs.
  • the cell membrane is a membrane structure located on the periphery of the protoplast and close to the cell wall, which can prevent extracellular materials from freely entering the cell and ensure the relative stability of the cell environment.
  • HSPGs are one of the main components of ECM and cell membranes, and the heparan sulfate on HSPGs is combined with a large number of growth factors, such as fibroblast growth factor, vascular endothelial growth factor, transforming growth factor and hepatocyte growth factor. HSPGs play an important role in different physiological and pathological processes such as growth, development, inflammation, invasion and infection of microorganisms and viruses, and the occurrence and development of tumors. In the body, heparan sulfate sugar chains are generally cleaved specifically by endogenous heparanase.
  • Heparanase is an endogenous ⁇ (1 ⁇ 4) endoglycosidase, and it is the only endogenous glycosidase that can degrade HSPGs.
  • heparanase In normal tissue cells, heparanase is mainly distributed in placenta, spleen, platelets, neutrophils, monocytes, activated T/B lymphocytes, and in the heart, brain, lung, skeletal muscle, kidney, and pancreas. It is not expressed in metastatic malignant tumor cells. Heparanase can promote tumor invasion and metastasis, can also inhibit tumor cell apoptosis, and participate in a series of physiological and pathological activities such as nerve axon growth, autoimmunity, and tumor angiogenesis.
  • heparanase The expression of heparanase is abnormally increased in pancreatic cancer, breast cancer, melanoma and other tumors, and its overexpression is usually positively correlated with the poor prognosis of the tumor.
  • the biological behavior of tumor cells is to promote tumor cell invasion And transfer. Heparanase can specifically cleave heparan sulfate sugar chains located on the cell surface and in the ECM, destroy the stable structure of the extracellular matrix and basement membrane, thereby making the invasion and metastasis of tumor cells easier.
  • heparanase cuts long heparan sulfate sugar chains into small fragments, which are generally composed of 20-30 sugar residues. There is evidence that these oligosaccharide chains have more biological functions than complete heparan sulfate sugar. With strong chains, different degradation products have different roles in the development of tumors.
  • heparanase is A suitable target for cancer treatment.
  • Heparin also known as unfractionated heparin, is named after it was first discovered in the liver. It is a mucopolysaccharide composed of glucuronic acid or iduronic acid and glucosamine alternately connected by ⁇ (1 ⁇ 4) glycosidic bonds. Heparin belongs to Heparin is a type of polyanionic glycosaminoglycan that is heterogeneous in structure and highly dispersed in the degree of polymerization. Heparin has a large amount of negative charge and the relative molecular mass is 1200-40000 Da. In addition to its anticoagulant effect, heparin also has a variety of biological activities and clinical uses, including anti-inflammatory, anti-angiogenesis and anti-tumor effects.
  • Heparin is similar in structure to HS (heparan sulfate), the natural substrate of heparanase, and can competitively bind to heparanase and inhibit the expression of heparanase activity in tumor cells.
  • HS heparan sulfate
  • tumor cells are inhibited from degrading the extracellular matrix, thereby reducing the ability of tumor cells to invade surrounding tissues.
  • the application of heparin in the treatment of tumors, especially tumor metastasis is severely limited by the anticoagulant activity of heparin.
  • many studies have chemically modified heparin to reduce its anticoagulant ability while preserving its anti-tumor activity.
  • CN102924627A discloses a method for preparing low-anticoagulant heparin with anti-tumor activity. It uses heparin as a starting material for complete desulfation, and then resulfation at the N position.
  • the PAPS regeneration system is adopted through AST-IV, 6 -OST-1 and 2-OST combined enzymatic synthesis of sulfated heparin at a specific site, and finally the heparin derivative is desulfated at the N position to finally obtain a heparin derivative with a specific anti-tumor effect with low anticoagulant activity.
  • CN1547477A discloses a partially desulphated glycosaminoglycan derivative and its preparation method. It uses heparin as a raw material to desulfonate a part of the 2-position of its glucosamine residue, and then partially N-desulfonate The free amino group generated after the acid group undergoes N-reacetylation, and then sodium periodate is used to oxidize the vicinal diol structure at the 2 and 3 positions in the uronic acid residue to generate an aldehyde group, and then the aldehyde group is reduced to a primary alcohol. A heparanase inhibitor with reduced anticoagulant activity is obtained. The heparanase inhibitor can be further obtained by acid hydrolysis or enzymatic hydrolysis to obtain a low molecular weight heparanase inhibitor.
  • CN105744940A discloses a carboxylated glycosaminoglycan derivative and a preparation method and application thereof. At least part of the residues of the derivative are cleaved residues with three carboxyl groups.
  • the method for preparing the carboxylated glycosaminoglycan derivative includes: efficiently converting adjacent diols and optionally adjacent OH/NH 2 on the sensitive non-sulfated residues of glycosaminoglycans into aldehydes, Then, under suitable conditions, the aldehyde group is oxidized to obtain two new carboxyl groups.
  • the starting glycosaminoglycan is a natural or synthetic glycosaminoglycan, preferably from heparin, low molecular weight heparin or heparan sulfate.
  • the heparin and low molecular weight heparin derivatives prepared by the method show effective inhibition of heparanase activity in vitro and in vivo in multiple myeloma experimental models.
  • CN105814086A discloses a glycosaminoglycan derivative and a preparation method and application thereof.
  • the starting material of the derivative is a natural or synthetic glycosaminoglycan, which is firstly N-desulfated and optionally 2-O desulfurized Acidification, then at least part of the adjacent diols and OH/NH 2 are converted to the corresponding aldehydes, and finally the aldehydes are reduced to the corresponding alcohols.
  • the resulting glycosaminoglycan derivatives can be used as active pharmaceutical ingredients for the treatment of pathological conditions such as multiple myeloma and other cancers.
  • the purpose of this application is to provide a glycosaminoglycan derivative.
  • the glycosaminoglycan derivatives of the present application have high heparanase inhibitor activity, low anticoagulant activity, and good anti-tumor metastasis activity.
  • the present application provides a glycosaminoglycan derivative, the glycosaminoglycan derivative has the structure shown in the following formula I:
  • the W ring contains the following structures represented by formula A, formula B, formula C, formula D and formula E:
  • R 1 is SO 3 - or H
  • R 2 is SO 3 - or H
  • R 3 is SO 3 -, acetyl group or H
  • the R is methyl, trifluoromethyl, hydroxymethyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, cyclopropyl, Cyclobutyl, cyclopentyl, cyclohexyl, phenyl, cyclopropylmethyl, cyclobutylmethyl, cyclohexylmethyl, benzyl, phenethyl, phenylpropyl, p-methylbenzyl, p-hydroxybenzyl , P-methylphenethyl, -CH 2 -COOH or -CH(CH 3 )-COOH.
  • the glycosaminoglycan is unfractionated heparin, low molecular weight heparin or heparan sulfate.
  • the molecular weight of the glycosaminoglycan derivative is 4000-20000, for example 4000, 4300, 4500, 5000, 6000, 8000, 9000, 10000, 12000, 14000, 16000, 18000 or 20000.
  • the molecular weight of the glycosaminoglycan derivative is 8000-18000, and in other embodiments, the molecular weight of the glycosaminoglycan derivative is 10000-16000.
  • the ratio of the structure represented by the formula E to the structure represented by the W ring is 20% to 90%, such as 20%, 23%, 25%, 28%, 30%, 35%, 38%, 40%, 43%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.
  • the structure represented by the formula E accounts for 30% to 70% of the structure represented by the W ring. In other embodiments, the structure represented by the formula E accounts for 40% to 60% of the structure represented by the W ring.
  • this application provides a glycosaminoglycan derivative, which is prepared by the following method:
  • step (3) The reaction product of step (2) and the amine compound NH 2 -R undergo an aldehyde amine condensation reaction, and then reduced to obtain the glycosaminoglycan derivative;
  • the R is methyl, trifluoromethyl, hydroxymethyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, cyclopropyl Cyclobutyl, cyclopentyl, cyclohexyl, phenyl, cyclopropylmethyl, cyclobutylmethyl, cyclohexylmethyl, benzyl, phenethyl, phenylpropyl, p-methylbenzyl, p-hydroxy Benzyl, p-methylphenethyl, -CH 2 -COOH or -CH(CH 3 )-COOH.
  • the alkaline condition in step (1) is 0.5M ⁇ 2.0M (for example, 0.5M, 0.8M, 1.0M, 1.2M, 1.5M, 1.8M or 2.0M) sodium hydroxide aqueous solution or hydrogen Potassium oxide aqueous solution.
  • the reaction temperature of the epoxidation reaction in step (1) is 20°C to 100°C (for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C) , 60°C, 70°C, 80°C, 90°C or 100°C), the reaction time is 20 minutes to 120 minutes (for example, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes) , 100 minutes, 110 minutes or 120 minutes).
  • the reaction temperature of the epoxidation reaction is 40°C to 80°C, and the reaction time is 20 minutes to 60 minutes; in other preferred embodiments, the reaction temperature of the epoxidation reaction is The temperature is 40°C, 50°C or 60°C, and the reaction time is 20 minutes, 30 minutes, 45 minutes or 60 minutes.
  • the pH value of the epoxy group ring-opening reaction in step (1) is 5.0 to 9.0 (for example, 5.0, 5.3, 5.5, 5.8, 6.0, 6.4, 6.8, 7.0, 7.4, 7.8, 8.0, 8.3, 8.5, 8.8 or 9.0), the reaction temperature is 30°C to 100°C (for example, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C or 100°C).
  • the pH of the epoxy group ring-opening reaction in step (1) is 6.0 to 8.0, and the reaction temperature is 40°C to 80°C; in a further preferred embodiment, step (1) The pH of the epoxy group ring-opening reaction in) is 6.5 to 7.5, and the reaction temperature is 40°C, 50°C, 60°C or 70°C.
  • the oxidant used in the oxidation reaction in step (2) is sodium periodate.
  • the amount of sodium periodate calculated by mass is 10-30 times (for example, 10 times, 12 times, 15 times, 18 times, 20 times the amount of glycosaminoglycan compound in step (1)). , 22 times, 25 times, 28 times or 30 times).
  • the amount of sodium periodate calculated by mass is 15 times, 18 times, 20 times, 22 times or 25 times the amount of glycosaminoglycan compound in step (1).
  • the amount of the amine compound NH 2 -R in step (3) and the amount of glycosaminoglycan compound in step (1) are calculated as 5-20 mmol/g in mole/mass ratio, for example 5 mmol/g , 6mmol/g, 7mmol/g, 8mmol/g, 9mmol/g, 10mmol/g, 12mmol/g, 14mmol/g, 16mmol/g, 18mmol/g or 20mmol/g.
  • the amount of amine compound NH 2 -R in step (3) and the amount of glycosaminoglycan compound in step (1) are calculated as 8-20 mmol/g by mole/mass ratio, wherein , R has the meaning described in this application.
  • the reducing agent used in the reduction reaction in step (3) is sodium borohydride.
  • the amount of sodium borohydride calculated by mass is 0.1-0.5 times the amount of glycosaminoglycan compound in step (1), such as 0.1 times, 0.15 times, 0.18 times, 0.2 times, 0.23 times, 0.25 times, 0.28 times, 0.3 times, 0.35 times, 0.38 times, 0.4 times, 0.45 times, 0.48 times or 0.5 times.
  • the amount of sodium borohydride is calculated by mass as the sugar in step (1)
  • the dosage of aminoglycan compound is 0.15-0.3 times.
  • the glycosaminoglycan compound is unfractionated heparin, low molecular weight heparin or heparan sulfate.
  • the low molecular weight heparin is enoxaparin sodium, dalteparin sodium or nadroparin calcium.
  • the present application provides the use of the glycosaminoglycan derivatives as described in the first or second aspect in the preparation of drugs with heparanase inhibitory activity.
  • glycosaminoglycan derivatives described in this application have good heparanase inhibitory activity and can be used for the development of drugs with heparanase inhibitory activity.
  • anticoagulant activity of the glycosaminoglycan derivatives described in this application is far less than that of glycosaminoglycan compounds such as heparin sodium.
  • the present application provides a pharmaceutical composition, which includes the glycosaminoglycan derivative as described in the first or second aspect as an active ingredient.
  • the pharmaceutical composition further includes a pharmaceutical carrier and/or excipient.
  • the present application provides the application of the glycosaminoglycan derivative according to the first aspect or the second aspect or the pharmaceutical composition according to the fourth aspect in the preparation of antineoplastic drugs.
  • glycosaminoglycan derivatives described in this application have good anti-tumor metastasis activity, and can be used as anti-tumor drugs or in the development of anti-tumor drugs.
  • the glycosaminoglycan derivative of the present application has good heparanase inhibitory activity and can be used for the development of drugs with heparanase inhibitory activity.
  • the anticoagulant activity of the glycosaminoglycan derivatives described in this application is far less than that of glycosaminoglycan compounds such as heparin sodium.
  • the glycosaminoglycan derivatives described in the present application have good anti-tumor metastasis activity and can be used as anti-tumor drugs or in the development of anti-tumor drugs.
  • alkyl means a saturated linear or branched monovalent hydrocarbon radical. Unless otherwise specified, the alkyl group contains 1-10 carbon atoms; in some embodiments, the alkyl group contains 1-8 carbon atoms; in other embodiments, the alkyl group contains 1-6 carbon atoms. In some embodiments, the alkyl group contains 1-4 carbon atoms; in other embodiments, the alkyl group contains 1-3 carbon atoms.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), isopropyl (i-Pr, -CH(CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl (i-Bu, -CH 2 CH (CH 3 ) 2 ), sec-butyl (s-Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-Bu, -C(CH 3 ) 3 ), n-pentyl (-CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl -2-butyl (-C(CH 3 ) 2
  • cycloalkyl means a monovalent or multivalent saturated monocyclic, bicyclic or tricyclic ring system containing 3-12 carbon atoms.
  • Examples of cycloalkyl groups further include, but are by no means limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl , Cyclododecyl and so on.
  • cycloalkylalkyl means that an alkyl group can be substituted with one or more cycloalkyl groups, where alkyl and cycloalkyl groups have the meanings described in this application. Examples of such include, But it is not limited to cyclopropylethyl, cyclopentylmethyl, cyclohexylmethyl and so on.
  • aryl means a monocyclic, bicyclic or tricyclic carbocyclic ring system containing 6-14 membered rings, wherein at least one ring system is aromatic, and each ring system contains 3-7 membered rings, and only An attachment point is connected to the rest of the molecule.
  • Aryl groups may include, but are not limited to, phenyl, naphthyl, and the like.
  • arylalkyl means that the alkyl group can be substituted with one or more aryl groups, where the alkyl and aryl groups have the meanings described in this application. Examples of such include, but not Limited to benzyl, 2-phenylethyl and so on.
  • the C2 and C3 positions of the uronic acid in the fragment a of the glycosaminoglycan compound are reacted in a suitable solvent to form the fragment b containing an epoxy structure under the action of a base;
  • the base includes an organic base and an inorganic base, such as hydroxide Sodium, potassium hydroxide, etc.
  • the suitable solvent includes but is not limited to water; the reaction can be carried out at room temperature or heated to 40°C-120°C, for example 60°C.
  • Fragment b is heated and stirred in a suitable solvent to obtain fragment c.
  • the suitable solvent includes but is not limited to water.
  • Fragment a'and fragments c and d in the glycosaminoglycan compound are reacted in a suitable solvent under the action of an oxidizing agent to obtain fragment e with C2-C3 bond cleavage and ring opening;
  • the oxidizing agent includes but is not limited to NaIO 4 ;
  • Suitable solvents include but are not limited to water.
  • Fragment e is reacted with an amine compound in a suitable solvent to obtain fragment f;
  • the suitable solvent includes but is not limited to water.
  • Fragment f is reduced under the action of a reducing agent to obtain fragment g;
  • the reducing agent includes but is not limited to sodium borohydride;
  • the suitable solvent includes but is not limited to water.
  • the NMR spectra were collected using a Bruker Avance 600MHz NMR spectrometer equipped with a 5mm TCI cryoprobe at 25°C. Use Bruker TopSpin 3.0 software to process the spectrum and integrate the peaks.
  • the MTC series of compounds in this application have the characteristic structure of secondary amines obtained by condensation reduction of aldehyde groups and primary amines.
  • the aliphatic and aromatic hydrocarbons linked to the secondary amines have specific chemical shifts.
  • the glycosaminoglycan derivative MTC1 is prepared, and the preparation method includes the following steps:
  • the product solution obtained by the reaction in step (2) was cooled to 4°C, NaIO 4 aqueous solution (0.2mol/L, 200.87g) was added, and then the reaction was stirred at 4°C in the dark for 16 hours; ethylene glycol (20.0mL) was added, Continue to stir for 1 hour to terminate the reaction, and finally the reaction solution is passed through a hollow fiber column to remove salt.
  • n-pentylamine (12 mL, 9.0 g, 100 mmol) to the solution after the reaction in step (3) to remove salt, adjust the pH of the reaction solution to 7.0 with HCl solution (6M), and stir and react at room temperature for 7 hours. After the reaction is over, the reaction solution is subjected to salt removal treatment.
  • Sodium borohydride (2.0 g) was added to the solution after the reaction in step (4) was demineralized, and the reaction was stirred at room temperature for 16 hours. After the reaction, the pH was adjusted to 4 with HCl solution (6M), stirring was continued for 1 hour, and finally the pH was adjusted to neutral with NaOH solution (0.1M).
  • the glycosaminoglycan derivative MTC2 is prepared, and the preparation method includes the following steps:
  • the product solution obtained by the reaction in step (2) was cooled to 4°C, NaIO 4 aqueous solution (0.2mol/L, 256.32g) was added, and then the reaction was stirred at 4°C in the dark for 20 hours; ethylene glycol (20.0mL) was added, Continue to stir for 1 hour to terminate the reaction, and finally the reaction solution is passed through a hollow fiber column to remove salt.
  • Sodium borohydride (2.0 g) was added to the solution after salt removal in step (4), and the reaction was stirred at room temperature for 16 hours. After the reaction, the pH was adjusted to 4 with HCl solution (6M), stirring was continued for 1 hour, and finally the pH was adjusted to neutral with NaOH solution (0.1M).
  • the glycosaminoglycan derivative MTC3 is prepared, and the preparation method includes the following steps:
  • the product solution obtained by the reaction in step (2) was cooled to 4°C, NaIO 4 aqueous solution (0.2mol/L, 198.36g) was added, and then the reaction was stirred at 4°C in the dark for 16 hours; ethylene glycol (20.0mL) was added, Continue to stir for 1 hour to terminate the reaction, and finally the reaction solution is passed through a hollow fiber column to remove salt.
  • Sodium borohydride (1.9 g) was added to the solution after salt removal in step (4), and the reaction was stirred at room temperature for 16 hours. After the reaction, the pH was adjusted to 4 with HCl solution (6M), stirring was continued for 30 minutes, and finally the pH was adjusted to neutral with NaOH solution (0.1M).
  • the glycosaminoglycan derivative MTC4 is prepared, and the preparation method includes the following steps:
  • the product solution obtained by the reaction in step (2) was cooled to 4°C, NaIO 4 aqueous solution (0.2mol/L, 200.87g) was added, and then the reaction was stirred at 4°C in the dark for 16 hours; ethylene glycol (20.0mL) was added, Continue to stir for 1 hour to terminate the reaction, and finally the reaction solution is passed through a hollow fiber column to remove salt.
  • Phenethylamine (5 mL, 4.82 g, 40 mmol) was added to half of the salt-removed solution in step (3), the pH of the reaction solution was adjusted to 7.0 with HCl solution (6M), and the reaction was stirred at room temperature for 19 hours. After the reaction is over, the reaction solution is subjected to salt removal treatment.
  • the glycosaminoglycan derivative MTC5 is prepared, and the preparation method includes the following steps:
  • the product solution obtained by the reaction in step (2) was cooled to 4°C, NaIO 4 aqueous solution (0.2mol/L, 200.06g) was added, and then the reaction was stirred at 4°C in the dark for 18 hours; ethylene glycol (20.0mL) was added, Continue to stir for 1 hour to terminate the reaction, and finally the reaction solution is passed through a hollow fiber column to remove salt.
  • Phenethylamine (6 mL, 5.78 g, 48 mmol) was added to half of the salt-removed solution in step (3), the pH of the reaction solution was adjusted to 7.0 with HCl solution (6M), and the reaction was stirred at room temperature for 20 hours. After the reaction is over, the reaction solution is subjected to salt removal treatment.
  • Sodium borohydride (1.1 g) was added to the solution after salt removal in step (4), and the reaction was stirred at room temperature for 6 hours. After the reaction, the pH was adjusted to 4 with HCl solution (6M), stirring was continued for 1 hour, and finally the pH was adjusted to neutral with NaOH solution (0.1M).
  • the glycosaminoglycan derivative MTC6 is prepared, and the preparation method includes the following steps:
  • the product solution obtained by the reaction in step (2) was cooled to 4°C, NaIO 4 aqueous solution (0.2mol/L, 199.85g) was added, and then the reaction was stirred at 4°C in the dark for 20 hours; ethylene glycol (20.0mL) was added, Continue to stir for 1 hour to terminate the reaction, and finally the reaction solution is passed through a hollow fiber column to remove salt.
  • Phenethylamine (6 mL, 5.78 g, 48 mmol) was added to half of the salt-removed solution in step (3), the pH of the reaction solution was adjusted to 7.0 with HCl solution (6M), and the reaction was stirred at room temperature for 24 hours. After the reaction is over, the reaction solution is subjected to salt removal treatment.
  • the glycosaminoglycan derivative MTC7 is prepared, and the preparation method includes the following steps:
  • step (2) The solution obtained in step (2) was cooled to 5°C, NaIO 4 solution (0.2mol/L, 100g) was added, and the reaction was stirred at 4°C in the dark for 16h; ethylene glycol (10.0mL) was added, and stirring was continued for 1h , Terminate the reaction, dialysis to remove salt.
  • the heparanase inhibitory activity test was performed on the prepared glycosaminoglycan derivatives.
  • the assay solution (100 ⁇ L) contains 40 mM sodium acetate buffer pH 5.0 and 100 mM Fondaparinux (GlaxoSmithKline), with or without the test sample. Heparanase was added to a final concentration of 140 pM to start the assay. Seal the plate with adhesive tape and incubate at 37°C for 2-24 hours. By adding 100 ⁇ L of 1.69mM 4-[3-(4-iodophenyl)-1H-5tetrazole]-1,3-benzenedisulfonate (WST-1, Aspep, Melbourne, Australia) in 0.1M NaOH Stop the measurement. The plate was resealed with adhesive tape and developed at 60°C for 60 minutes.
  • the absorbance was measured at 584 nm (Fluostar, BMG, Labtech). In each plate, in the same buffer and volume, prepare a standard curve constructed with D-galactose as a reducing sugar standard in the range of 2-100 ⁇ M, and determine the IC 50 value.
  • the test result data is shown in Table 2. According to the results of Table 2, it can be seen that the IC 50 value of the MTC series compound of the present application is 7-53ng/mL, and even can reach 7-22ng/mL, with good heparan Enzyme inhibitory activity.
  • the prepared glycosaminoglycan derivatives were tested for anticoagulation potency, using the APTT assay method.
  • APTT Activated partial thromboplastin time, activated partial thromboplastin time
  • the plasma sample is incubated with an appropriate amount of phospholipids and a negatively charged catalase agent that can activate the endogenous coagulation pathway, and incubated at 37°C for a certain period of time , Adding calcium ions, start the clotting process, and record the time required for plasma clotting, which is the activated partial thromboplastin time.
  • Stago's STA-R automatic blood coagulation analyzer is used to detect the anticoagulant activity of heparin derivatives by the APTT method.
  • the specific method is: take 50 ⁇ L of heparin sodium or the saline solution of the test substance and 50 ⁇ L of plasma and react in a water bath at 37 ⁇ 1°C for 30 seconds. Add 50 ⁇ L APTT reagent (preheated in 37 ⁇ 1°C water bath), mix well and immediately start the stopwatch on the coagulometer. After the stopwatch reads for 3 minutes, add 50 ⁇ L of calcium chloride (0.025M) (preheated in 37 ⁇ 1°C water bath) through the automatic sampler. STA-R automatic blood coagulation analyzer automatically records the clotting time. The measured coagulation time is converted into a logarithm, and the titer is calculated according to the quantitative response parallel line method in the bioassay statistical method.
  • the prepared glycosaminoglycan derivatives were subjected to the pharmacodynamic study on the B16 mouse melanoma lung metastasis model, and the method was as follows:
  • B16 cells (purchased from the cell bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured and passaged in RPMI-1640 complete medium containing 10% fetal bovine serum in a 37°C incubator containing 5% CO 2. When the number of growth period, you can prepare for vaccination.
  • mice Females, were randomly grouped according to body weight: model control group and experimental group, the corresponding dose (2.5mg/kg) of the sample was injected into the tail vein, and the model control group was replaced by normal saline.
  • the melanoma cells were inoculated into the tail vein. Observe the mice's activity status, weight changes, and whether there are any abnormalities every day.
  • mice were sacrificed, fresh lungs were taken, and the lung tissues of the mice were fixed with Bouin fixative to observe the distribution of tumor metastases in the lungs of the mice, and calculate the inhibition rate of tumor metastasis in the mice. .
  • Tumor metastasis inhibition rate (number of tumor metastases in the model control group-number of metastases in the administration group)/number of tumor metastases in the model control group ⁇ 100%
  • the inhibitory rates of MTC1, MTC2, MTC5, and MTC6 on melanoma lung metastasis were 54%, 56%, 72%, and 63% at a dose of 2.5 mg/kg, respectively, showing good tumor metastasis inhibitory activity.

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Abstract

本申请提供一种糖胺聚糖衍生物及其应用，本申请所述的糖胺聚糖衍生物具有如式I所示结构。本申请的糖胺聚糖衍生物具有良好的乙酰肝素酶抑制活性，可用于具有乙酰肝素酶抑制活性的药物的开发。此外，本申请所述糖胺聚糖衍生物具有良好的抗肿瘤转移活性，可用作抗肿瘤药物。

Description

一种糖胺聚糖衍生物及其应用 技术领域
本申请属于生物材料技术领域，涉及一种糖胺聚糖衍生物及其应用。
背景技术
硫酸乙酰肝素蛋白聚糖(HSPGs)是一类糖复合物，该类物质具有两部分：核心蛋白和以共价键方式连接在核心蛋白上的一条或者多条硫酸乙酰肝素糖链。细胞外基质(Extracellular matrix,ECM)是指由细胞分泌的，位于上皮或内皮细胞下层和***细胞周围，为组织、器官提供力学支持和物理强度的物质。细胞膜是位于原生质体***、紧贴细胞壁的膜结构，能够防止细胞外物质自由进入细胞，保证细胞内环境的相对稳定。HSPGs是ECM和细胞膜的主要成分之一，且HSPGs上的硫酸乙酰肝素结合有大量的生长因子，比如成纤维生长因子、血管内皮生长因子、转化生长因子和肝细胞生长因子。HSPGs在生长、发育、炎症反应、微生物和病毒的侵袭和感染以及肿瘤的发生发展等不同的生理病理过程中均有重要作用。在体内，硫酸乙酰肝素糖链一般被内源性乙酰肝素酶特异性切割。
乙酰肝素酶是一种内源性的β(1→4)糖苷内切酶，是唯一能降解HSPGs的内源糖苷酶。在正常组织细胞中，乙酰肝素酶主要分布于胎盘、脾脏、血小板及中性粒细胞、单核细胞、活化T/B淋巴细胞内，而在心脏、脑、肺、骨骼肌、肾脏、胰腺中不表达，普遍存在于转移性恶性肿瘤细胞中。乙酰肝素酶可以促进肿瘤侵袭和转移，还可抑制肿瘤细胞凋亡，参与神经轴突生长、自身免疫以及肿瘤血管形成等一系列生理、病理活动。
乙酰肝素酶在胰腺癌、乳腺癌、黑色素瘤等多种肿瘤中表达异常升高，其过表达通常与肿瘤的预后不良正相关，表现在肿瘤细胞的生物学行为上就是促进肿瘤细胞的侵袭和转移。乙酰肝素酶能够特异性地切割位于细胞表面和ECM中的硫酸乙酰肝素糖链，破坏细胞外基质和基底膜的稳定结构，从而使得肿瘤细胞的侵袭和转移变得容易。
同时，乙酰肝素酶将长的硫酸乙酰肝素糖链切割成小的片段，这些片段一般由20-30个糖残基组成，有证据表明这些寡糖链的生物功能比完整的硫酸乙酰肝素糖链强，不同的降解产物在肿瘤的发展过程中的作用不同。
此外，细胞外基质和细胞膜上的硫酸乙酰肝素被乙酰肝素酶切割成小片段之后，结合在糖链上的各种生成因子被释放出来，促进肿瘤细胞生长和肿瘤血管生成。
早在20世纪80年代，人们就开始研究乙酰肝素酶引起的糖链降解在肿瘤发生发展过程中的作用，目前已经有了比较明确的认识，越来越多的证据证实乙酰肝素酶是一个癌症治疗的合适靶标。
肝素又称普通肝素，因首先从肝脏发现而得名，是一种由葡萄糖醛酸或艾杜糖醛酸与葡萄糖胺交替组成的粘多糖，通过α(1→4)糖苷键连接，肝素属于在结构上不均一，在聚合 程度上高度分散的一类聚阴离子糖胺聚糖，肝素带有大量负电荷，相对分子质量在1200-40000Da。肝素除具有抗凝作用外，还具有多种生物活性和临床用途，包括抗炎、抗血管生成及抗肿瘤作用等。肝素与乙酰肝素酶的天然底物HS(硫酸乙酰肝素)结构相似，能够竞争性结合乙酰肝素酶，抑制肿瘤细胞乙酰肝素酶活性表达。通过抑制乙酰酶活性，一方面抑制肿瘤细胞降解细胞外基质，从而降低肿瘤细胞侵入周围组织的能力。另一方面阻断由乙酰肝素酶从细胞外基质中释放生长因子的能力，进而抑制了肿瘤细胞的生长和肿瘤血管的生长。但是，肝素在***，特别是肿瘤转移方面的应用受到了肝素抗凝活性的严重限制。目前已有较多研究对肝素进行了化学修饰，以降低其抗凝能力，同时保存其抗肿瘤活性。
CN102924627A公开了一种具有抗肿瘤活性的低抗凝肝素的制备方法，其以肝素为起始物，进行完全脱硫酸化，然后进行N位重新硫酸化，采用PAPS再生***，通过AST-IV、6-OST-1和2-OST组合酶法合成特定位点硫酸化肝素，最后再将上述肝素衍生物进行N位脱硫酸化，最终得到具有低抗凝活性的专一抗肿瘤作用的肝素衍生物。
CN1547477A公开了一种部分脱硫酸糖胺聚糖衍生物及其制备方法，其以肝素为原料，将其葡糖胺残基中2位部分N-脱磺酸基，再将部分N-脱磺酸基后生成的游离氨基进行N-重乙酰化，随后使用高碘酸钠氧化糖醛酸残基中的2、3位邻二醇结构生成醛基，然后将醛基还原成伯醇，从而得到抗凝活性降低的乙酰肝素酶抑制剂。此乙酰肝素酶抑制剂可进一步通过酸水解或者酶解获得低分子量的乙酰肝素酶抑制剂。
CN105744940A公开一种羧基化糖胺聚糖衍生物及其制备方法和应用，该衍生物至少部分残基为具有三个羧基的裂解的残基。用于制备所述羧基化糖胺聚糖衍生物的方法包括：将糖胺聚糖的敏感非硫酸化残基上相邻的二醇和任选的相邻的OH/NH 2有效转化为醛，随后在适宜条件下，将醛基继续氧化得到两个新羧基。起始糖胺聚糖是天然或合成的糖胺聚糖，优选自肝素、低分子肝素或硫酸类肝素。由该方法制备的肝素和低分子量肝素衍生物在体外和在体内多发性骨髓瘤实验模型中表现出对类肝素酶活性的有效抑制。
CN105814086A公开了一种糖胺聚糖衍生物及其制备方法和应用，所述衍生物的起始原料是天然或合成的糖胺聚糖，首先进行N-脱硫酸化和任选地2-O脱硫酸化，然后其中至少部分相邻的二醇和OH/NH 2被转化为相应的醛，最后将所述醛还原为相应的醇。所得糖胺聚糖衍生物可作为用于治疗诸如多发性骨髓瘤和其他癌症的病理病况的药物活性成分。
然而，如上这些现有技术中乙酰肝素酶抑制剂的活性还有待进一步提高。
发明内容
针对现有技术的不足，本申请的目的在于提供一种糖胺聚糖衍生物。本申请的糖胺聚糖衍生物具有较高的乙酰肝素酶抑制剂活性，抗凝活性低，具有良好的抗肿瘤转移活性。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种糖胺聚糖衍生物，所述糖胺聚糖衍生物具有如下式I所示结构：
Figure PCTCN2019129110-appb-000001
其中，W环包含下列式A、式B、式C、式D和式E所示的结构：
Figure PCTCN2019129110-appb-000002
其中R 1为SO 3 -或H；R 2为SO 3 -或H；R 3为SO 3 -、乙酰基或H；
R为烷基、环烷基、芳基、环烷基烷基、芳基烷基或-CH(R 4)-COOH；其中，R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代。
在一些实施方案中，所述R为C 1-6烷基、C 3-8环烷基、C 6-10芳基、C 3-8环烷基-C 1-6烷基、C 6-10芳基-C 1-6烷基或-CH(R 4)-COOH；其中，所述R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代。
在另一些实施方案中，所述R为甲基、三氟甲基、羟甲基、乙基、正丙基、异丙基、正丁基、叔丁基、正戊基、环丙基、环丁基、环戊基、环己基、苯基、环丙基甲基、环丁基甲基、环己基甲基、苄基、苯乙基、苯丙基、对甲基苄基、对羟基苄基、对甲基苯乙基、-CH 2-COOH 或-CH(CH 3)-COOH。
在本申请中，所述糖胺聚糖为未分级肝素、低分子肝素或硫酸乙酰肝素。
在本申请中，所述糖胺聚糖衍生物的分子量为4000-20000，例如4000、4300、4500、5000、6000、8000、9000、10000、12000、14000、16000、18000或20000。
在一些实施方案中，所述糖胺聚糖衍生物的分子量为8000-18000，在另一些实施方案中，所述糖胺聚糖衍生物的分子量为10000-16000。
在本申请中，所述式E所示的结构占W环所示结构的比例为20％～90％，例如20％、23％、25％、28％、30％、35％、38％、40％、43％、45％、50％、55％、60％、65％、70％、75％、80％、85％或90％。在一些实施方案中，所述式E所示的结构占W环所示结构的比例为30％～70％。在另一些实施方案中，所述式E所示的结构占W环所示结构的比例为40％～60％。
第二方面，本申请提供了一种糖胺聚糖衍生物，所述糖胺聚糖衍生物通过以下方法制备得到：
(1)糖胺聚糖的2-O-脱硫酸化：在碱性条件下，糖胺聚糖化合物2-位磺酸化艾杜糖醛酸C2-C3位经过环氧化反应形成环氧基团，然后环氧基团开环生成C2-C3位邻二醇结构；
(2)将糖醛酸C2-C3位邻二醇结构氧化开环并生成二醛；和
(3)将步骤(2)的反应产物与胺类化合物NH 2-R发生醛胺缩合反应，然后还原得到所述糖胺聚糖衍生物；
其中，所述R为烷基、环烷基、芳基、环烷基-烷基-、芳基-烷基-或-CH(R 4)-COOH；其中，R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代。
在一些实施方案中，所述R为C 1-6烷基、C 3-8环烷基、C 6-10芳基、C 3-8环烷基-C 1-6烷基-、C 6-10芳基-C 1-6烷基-或-CH(R 4)-COOH；其中，所述R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代。
在另一些优选的实施方案中，所述R为甲基、三氟甲基、羟甲基、乙基、正丙基、异丙基、正丁基、叔丁基、正戊基、环丙基、环丁基、环戊基、环己基、苯基、环丙基甲基、环 丁基甲基、环己基甲基、苄基、苯乙基、苯丙基、对甲基苄基、对羟基苄基、对甲基苯乙基、-CH 2-COOH或-CH(CH 3)-COOH。
在本申请中，步骤(1)所述碱性条件为0.5M～2.0M(例如0.5M、0.8M、1.0M、1.2M、1.5M、1.8M或2.0M)的氢氧化钠水溶液或氢氧化钾水溶液。
在本申请中，步骤(1)中所述环氧化反应的反应温度为20℃至100℃(例如20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、70℃、80℃、90℃或100℃)，反应时间为20分钟至120分钟(例如20分钟、25分钟、30分钟、40分钟、50分钟、60分钟、70分钟、80分钟、100分钟、110分钟或120分钟)。在一些优选的实施方案中，所述环氧化反应的反应温度为40℃至80℃，反应时间为20分钟至60分钟；在另一些优选的实施方案中，所述环氧化反应的反应温度为40℃、50℃或60℃，反应时间为20分钟、30分钟、45分钟或60分钟。
在本申请中，步骤(1)中所述环氧基团开环反应的pH值为5.0至9.0(例如5.0、5.3、5.5、5.8、6.0、6.4、6.8、7.0、7.4、7.8、8.0、8.3、8.5、8.8或9.0)，反应温度为30℃至100℃(例如30℃、40℃、50℃、60℃、70℃、80℃、90℃或100℃)。在一些优选的实施方案中，步骤(1)中所述环氧基团开环反应的pH值为6.0至8.0，反应温度为40℃至80℃；在进一步优选的实施方案中，步骤(1)中所述环氧基团开环反应的pH值为6.5至7.5，反应温度为40℃、50℃、60℃或70℃。
在本申请中，步骤(2)中氧化反应使用的氧化剂为高碘酸钠。在一些优选的实施方案中，高碘酸钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的10～30倍(例如10倍、12倍、15倍、18倍、20倍、22倍、25倍、28倍或30倍)。在进一步优选的实施方案中，高碘酸钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的15倍、18倍、20倍、22倍或25倍。
在本申请中，步骤(3)中胺类化合物NH 2-R的用量与步骤(1)中糖胺聚糖化合物的用量按摩尔量/质量比计算为5～20mmol/g，例如5mmol/g、6mmol/g、7mmol/g、8mmol/g、9mmol/g、10mmol/g、12mmol/g、14mmol/g、16mmol/g、18mmol/g或20mmol/g。在一些优选的实施方案中，步骤(3)中胺类化合物NH 2-R的用量与步骤(1)中糖胺聚糖化合物的用量按摩尔量/质量比计算为8～20mmol/g，其中，R具有本申请所述的含义。
在本申请中，步骤(3)中还原反应使用的还原剂为硼氢化钠。
在一些实施方案中，所述硼氢化钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的0.1～0.5倍，例如0.1倍、0.15倍、0.18倍、0.2倍、0.23倍、0.25倍、0.28倍、0.3倍、0.35倍、0.38倍、0.4倍、0.45倍、0.48倍或0.5倍，在一些优选的实施方案中，硼氢化钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的0.15～0.3倍。
在本申请中，所述糖胺聚糖化合物为未分级肝素、低分子肝素或硫酸乙酰肝素。在一些实施方案中，所述低分子肝素为依诺肝素钠、达肝素钠或那曲肝素钙。
第三方面，本申请提供了如第一方面或者第二方面所述的糖胺聚糖衍生物在制备具有乙 酰肝素酶抑制活性的药物中的应用。
本申请所述的糖胺聚糖衍生物具有良好的乙酰肝素酶抑制活性，可用于具有乙酰肝素酶抑制活性的药物的开发。并且本申请所述的糖胺聚糖衍生物抗凝活性远远小于肝素钠等糖胺聚糖化合物。
第四方面，本申请提供一种药物组合物，所述药物组合物包括如第一方面或者第二方面所述的糖胺聚糖衍生物作为活性成分。
在一些实施方案中，所述药物组合物还包括药用载体和/或赋形剂。
第五方面，本申请提供了如第一方面或者第二方面所述的糖胺聚糖衍生物或者第四方面所述的药物组合物在制备抗肿瘤药物中的应用。
本申请所述糖胺聚糖衍生物具有良好的抗肿瘤转移活性，可用作抗肿瘤药物或者用于抗肿瘤药物的开发。
相对于现有技术，本申请取得了以下有益效果：
本申请的糖胺聚糖衍生物具有良好的乙酰肝素酶抑制活性，可用于具有乙酰肝素酶抑制活性的药物的开发。并且本申请所述的糖胺聚糖衍生物抗凝活性远远小于肝素钠等糖胺聚糖化合物。此外，本申请所述糖胺聚糖衍生物具有良好的抗肿瘤转移活性，可用作抗肿瘤药物或者用于抗肿瘤药物的开发。
术语定义
除非有相反陈述，下列用在说明书和权利要求书中的术语具有下述含义。
术语“烷基”表示饱和直链或支链一价碳氢化合物原子团。除非另外详细说明，烷基基团含有1-10个碳原子；在一些实施例中，烷基基团含有1-8个碳原子；在另一些实施例中，烷基基团含有1-6个碳原子；再在一些实施例中，烷基基团含有1-4个碳原子；在另一些实施例中，烷基基团含有1-3个碳原子。烷基基团的实例包含，但并不限于，甲基(Me，-CH 3)、乙基(Et，-CH 2CH 3)、正丙基(n-Pr，-CH 2CH 2CH 3)、异丙基(i-Pr，-CH(CH 3) 2)、正丁基(n-Bu，-CH 2CH 2CH 2CH 3)、异丁基(i-Bu，-CH 2CH(CH 3) 2)、仲丁基(s-Bu，-CH(CH 3)CH 2CH 3)、叔丁基(t-Bu，-C(CH 3) 3)，正戊基(-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH 3)CH 2CH 2CH 3)、3-戊基(-CH(CH 2CH 3) 2)、2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)，3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)，2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、正己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3)、正庚基、正辛基，等等。
术语“环烷基”表示含有3-12个碳原子的，单价或多价的饱和单环、双环或三环体系。环烷基基团的实例进一步包括，但绝不限于环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基、环癸基、环十一烷基、环十二烷基等等。
术语“环烷基烷基”表示烷基基团可以被一个或多个环烷基基团所取代，其中烷基和环烷基基团具有如本申请所述的含义，这样的实例包括，但并不限于，环丙基乙基，环戊基甲基，环己基甲基等等。
术语“芳基”表示含有6-14元环的单环、双环或三环的碳环体系，其中，至少一个环体系是芳香族的，其中每一个环体系包含3-7元环，且只有一个附着点与分子的其余部分相连。芳基可以包括但不限于苯基和萘基等等。
术语“芳基烷基”表示烷基基团可以被一个或多个芳基基团所取代，其中烷基和芳基基团具有如本申请所述的含义，这样的实例包括，但并不限于，苯甲基、2-苯基乙基等等。
术语“任选地被……所取代”，可以与术语“未取代或被……所取代”交换使用，即所述结构是未取代的或者被一个或多个本申请所述的取代基取代。
一般合成方法
一般地，本申请的化合物可以通过本申请所描述的方法制备得到，除非有进一步的说明，其中取代基的定义如式I所示。
Figure PCTCN2019129110-appb-000003
(1)C2、C3环氧化
糖胺聚糖化合物的片段a中糖醛酸C2、C3位在碱的作用下，在合适的溶剂中反应生成含环氧结构的片段b；所述碱包含有机碱和无机碱，例如氢氧化钠、氢氧化钾等；所述合适的溶剂包含但不限于水；所述反应可在室温下或加热至40℃-120℃进行，例如60℃。
(2)环氧结构开环
片段b在合适的溶剂中加热搅拌反应得到片段c，所述合适的溶剂包含但不限于水。
(3)C2-C3键氧化开环
糖胺聚糖化合物中片段a’和片段c和d在氧化剂的作用下，在合适的溶剂中反应得到C2-C3键断裂开环的片段e；所述氧化剂包含但不限于NaIO 4；所述合适的溶剂包含但不限于水。
(4)醛胺缩合
片段e在合适的溶剂中与胺类化合物反应得到片段f；所述合适的溶剂包含但不限于水。
(5)还原
片段f在还原剂的作用下被还原得到片段g；所述还原剂包含但不限于硼氢化钠；所述合适的溶剂包含但不限于水。
检测方法
核磁测定在25℃下，利用配备5mm TCI冷冻探头的Bruker Avance 600MHz核磁共振波谱仪采集核磁谱图。使用Bruker TopSpin 3.0软件处理谱图并对谱峰进行积分。本申请MTC系列化合物具有醛基与伯胺缩合还原得到仲胺的特征结构，在 1H- 13C HSQC谱图中仲胺连接的脂肪烃及芳香烃具有特定的化学位移，参考“Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins”(Journal of Pharmaceutical and Biomedical Analysis 136(2017)92–105)对核磁谱图进行分析，通过核磁检测可确定MTC系列化合物的结构特征，结构特征的说明如表1所示。
表1
Figure PCTCN2019129110-appb-000004
Figure PCTCN2019129110-appb-000005
分子量(Mw)测试分子量通过HPLC-GPC(高效液相色谱-凝胶渗透层析)测定。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例1
在本实施例中，制备糖胺聚糖衍生物MTC1，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(10.0g，分子量Mw＝17086Da)溶于水(60mL)中，搅拌使其完全溶解，然后加入NaOH溶液(2mol/L，60mL)，加热至60℃，反应30分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)得到的产物溶液中加水(360mL)稀释，然后加热至70℃，反应24小时；中间体分子量Mw＝14671Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的产物溶液冷却至4℃，加入NaIO 4水溶液(0.2mol/L，200.87g)，然后于4℃下避光搅拌反应16小时；加入乙二醇(20.0mL)，继续搅拌1小时终止反应，反应溶液最后经中空纤维柱除盐。中间体分子量Mw＝13315Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液中加入正戊胺(12mL，9.0g，100mmol)，用HCl溶液(6M)将反应液调pH至7.0，室温下搅拌反应7小时。反应结束后，反应溶液做除盐处理。中间体分子量Mw＝12791Da。
(5)硼氢化钠还原
向步骤(4)反应除盐后的溶液中加入硼氢化钠(2.0g)，室温下搅拌反应16h。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌1小时，最后用NaOH溶液(0.1M)调pH至中性。采用多次醇沉法除盐，在480g溶液中加入25％NaCl溶液(20g)，然后加入乙醇(1000g)进行沉淀；所得沉淀再用水(96g)重新溶解，加入25％NaCl溶液(4g)，然后加入乙醇(200g)进行沉淀，沉淀冻干得到MTC1；分子量Mw＝12091Da。通过核磁测定gsU-NHR(R＝-(CH 2) 4CH 3,46％)、G/I+GalA(9％)、C2/C3epox(7％)和I2S(38％)占总糖醛酸单糖残基的百分比。
实施例2
在本实施例中，制备糖胺聚糖衍生物MTC2，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(10.0g，分子量Mw＝17498Da)溶于水(60mL)中，搅拌使其完全溶解，然后加入NaOH溶液(2mol/L，60mL)，加热至80℃，反应30分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)得到的产物溶液中加水(360mL)稀释，然后加热至70℃，反应24小时；中间体分子量Mw＝13892Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的产物溶液冷却至4℃，加入NaIO 4水溶液(0.2mol/L，256.32g)，然后于4℃下避光搅拌反应20小时；加入乙二醇(20.0mL)，继续搅拌1小时终止反应，反应溶液最后经中空纤维柱除盐。中间体分子量Mw＝12536Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液中加入正戊胺(12mL，9.0g，100mmol)，用HCl溶液(6M)将反应液调pH至7.0，室温下搅拌反应12小时。反应结束后，反应溶液做除盐处理。中间体分子量Mw＝13005Da。
(5)硼氢化钠还原
向步骤(4)反应除盐后的溶液中加入硼氢化钠(2.0g)，室温下搅拌反应16小时。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌1小时，最后用NaOH溶液(0.1M)调pH至中性。采用多次醇沉法除盐，在480g溶液中加入25％NaCl溶液(20g)，然后加入乙醇(1004g)进行沉淀；所得沉淀再用水(96g)重新溶解，加入25％NaCl溶液(4g)，然后加入乙醇(210g)进行沉淀，沉淀冻干得到MTC2；分子量Mw＝13843Da。通过核磁测定gsU-NHR(R＝-(CH 2) 4CH 3,55％)、G/I+GalA(7％)、C2/C3epox(6％)和I2S(32％)占总糖醛酸单糖残基的百分比。
实施例3
在本实施例中，制备糖胺聚糖衍生物MTC3，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(10g，分子量Mw＝16533Da)溶于水(60mL)中，搅拌使其完全溶解，然后加入NaOH溶液(2mol/L，60mL)，加热至40℃，反应30分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)得到的产物溶液中加水(360mL)稀释，然后加热至70℃，反应24小时；中间体分子量Mw＝15560Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的产物溶液冷却至4℃，加入NaIO 4水溶液(0.2mol/L，198.36g)，然后于4℃下避光搅拌反应16小时；加入乙二醇(20.0mL)，继续搅拌1小时终止反应，反应溶液最后经中空纤维柱除盐。中间体分子量Mw＝14098Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液中加入正戊胺(12mL，9.0g，100mmol)，用HCl溶液(6M)将反应液调pH至7.0，室温下搅拌反应7小时。反应结束后，反应溶液做除盐处理。中间体分子量Mw＝14923Da。
(5)硼氢化钠还原
向步骤(4)反应除盐后的溶液中加入硼氢化钠(1.9g)，室温下搅拌反应16小时。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌30min，最后用NaOH溶液(0.1M)调pH至中性。采用多次醇沉法除盐，在480g溶液中加入25％NaCl溶液(19.8g)，然后加入乙醇(1024g)进行沉淀；所得沉淀再用水(92g)重新溶解，加入25％NaCl溶液(3.8g)，然后加入乙醇(218g)进行沉淀，沉淀冻干得到MTC3；分子量Mw＝15026Da。通过核磁测定gsU-NHR(R＝-(CH 2) 4CH 3,39％)、G/I+GalA(8％)、C2/C3epox(5％)和I2S(48％)占总糖醛酸单糖残基的百分比。
实施例4
在本实施例中，制备糖胺聚糖衍生物MTC4，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(10.0g，分子量Mw＝16582Da)溶于水(60mL)中，搅拌使其完全溶解，然后加入NaOH溶液(2mol/L，60mL)，加热至60℃，反应20分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)得到的产物溶液中加水(360mL)稀释，然后加热至70℃，反应24小时；中间体分子量Mw＝14671Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的产物溶液冷却至4℃，加入NaIO 4水溶液(0.2mol/L，200.87g)，然后于4℃下避光搅拌反应16小时；加入乙二醇(20.0mL)，继续搅拌1h终止反应，反应 溶液最后经中空纤维柱除盐。中间体分子量Mw＝13315Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液一半加入苯乙胺(5mL，4.82g，40mmol)，用HCl溶液(6M)将反应液调pH至7.0，室温下搅拌反应19小时。反应结束后，反应溶液做除盐处理。中间体分子量Mw＝13678Da。
(5)硼氢化钠还原
向步骤(4)反应除盐后的溶液中加入硼氢化钠(1.1g)，室温下搅拌反应6小时。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌1小时，最后用NaOH溶液(0.1M)调pH至中性。采用多次醇沉法除盐，在240g溶液中加入25％NaCl溶液(10g)，然后加入乙醇(500g)进行沉淀；所得沉淀再用水(48g)重新溶解，加入25％NaCl溶液(2g)，然后加入乙醇(100g)进行沉淀，沉淀冻干得到MTC4；分子量Mw＝13256Da。通过核磁测定gsU-NHR(R＝-CH 2Ph,37％)、G/I+GalA(7％)、C2/C3epox(5％)和I2S(51％)占总糖醛酸单糖残基的百分比。
实施例5
在本实施例中，制备糖胺聚糖衍生物MTC5，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(10.0g，分子量Mw＝17452Da)溶于水(60mL)中，搅拌使其完全溶解，然后加入NaOH溶液(2mol/L，60mL)，加热至60℃，反应45分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)得到的产物溶液中加水(360mL)稀释，然后加热至70℃，反应24小时；中间体分子量Mw＝14552Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的产物溶液冷却至4℃，加入NaIO 4水溶液(0.2mol/L，200.06g)，然后于4℃下避光搅拌反应18小时；加入乙二醇(20.0mL)，继续搅拌1小时终止反应，反应溶液最后经中空纤维柱除盐。中间体分子量Mw＝12305Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液一半加入苯乙胺(6mL，5.78g，48mmol)，用HCl溶液(6M)将反应液调pH至7.0，室温下搅拌反应20小时。反应结束后，反应溶液做除盐处理。中间体分子量Mw＝12598Da。
(5)硼氢化钠还原
向步骤(4)反应除盐后的溶液中加入硼氢化钠(1.1g)，室温下搅拌反应6小时。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌1小时，最后用NaOH溶液(0.1M)调pH至中性。采用多次醇沉法除盐，在240g溶液中加入25％NaCl溶液(10g)，然后加入乙醇(500g)进行沉淀；所得沉淀再用水(48g)重新溶解，加入25％NaCl溶液(2g)， 然后加入乙醇(100g)进行沉淀，沉淀冻干得到MTC5；分子量Mw＝12078Da。通过核磁测定gsU-NHR(R＝-CH 2Ph,53％)、G/I+GalA(8％)、C2/C3epox(6％)和I2S(33％)占总糖醛酸单糖残基的百分比。
实施例6
在本实施例中，制备糖胺聚糖衍生物MTC6，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(10.0g，分子量Mw＝17358Da)溶于水(60mL)中，搅拌使其完全溶解，然后加入NaOH溶液(2mol/L，60mL)，加热至60℃，反应60分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)得到的产物溶液中加水(360mL)稀释，然后加热至70℃，反应24小时；中间体分子量Mw＝13799Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的产物溶液冷却至4℃，加入NaIO 4水溶液(0.2mol/L，199.85g)，然后于4℃下避光搅拌反应20小时；加入乙二醇(20.0mL)，继续搅拌1小时终止反应，反应溶液最后经中空纤维柱除盐。中间体分子量Mw＝10989Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液一半加入苯乙胺(6mL，5.78g，48mmol)，用HCl溶液(6M)将反应液调pH至7.0，室温下搅拌反应24小时。反应结束后，反应溶液做除盐处理。中间体分子量Mw＝11537Da。
(5)硼氢化钠还原
向步骤(4)反应除盐后的溶液中加入硼氢化钠(1.1g)，室温下搅拌反应6小时。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌1小时，最后用NaOH溶液(0.1M)调pH至中性。采用多次醇沉法除盐，在240g溶液中加入25％NaCl溶液(10g)，然后加入乙醇(500g)进行沉淀；所得沉淀再用水(48g)重新溶解，加入25％NaCl溶液(2g)，然后加入乙醇(100g)进行沉淀，沉淀冻干得到MTC6；分子量Mw＝11096Da。通过核磁测定gsU-NHR(R＝-CH 2Ph,57％)、G/I+GalA(7％)、C2/C3epox(8％)和I2S(28％)占总糖醛酸单糖残基的百分比。
实施例7
在本实施例中，制备糖胺聚糖衍生物MTC7，制备方法包括以下步骤：
(1)C2、C3环氧化
将肝素钠成品(5.0g，分子量Mw＝16680Da)溶于水(30mL)中，搅拌使其完全溶解，溶液升温至60℃左右，加入NaOH溶液(2mol/L，30mL)，继续升温至60℃后，反应30分钟。反应结束后，冷却至室温，用HCl溶液(6M)调pH至7.0。
(2)环氧结构开环
向步骤(1)反应得到的产物溶液中加水(180mL)，所得溶液加热至70℃，搅拌反应24小时。中间体分子量Mw＝15592Da。
(3)高碘酸钠氧化开环
将步骤(2)反应得到的溶液降温至5℃，加入NaIO 4溶液(0.2mol/L，100g)，然后于4℃下避光搅拌反应16h；加入乙二醇(10.0mL)，继续搅拌1h，终止反应，透析除盐。中间体分子量Mw＝12405Da。
(4)醛胺缩合
向步骤(3)反应除盐后的溶液中分批加入甘氨酸(3.5g,47mmol)，室温下搅拌5h。反应结束后做除盐处理。中间体分子量Mw＝12162Da。
(5)硼氢化钠还原
向步骤(4)反应初验后的溶液中分批加入硼氢化钠(1.2g)，室温下搅拌3h。反应结束后，用HCl溶液(6M)调pH至4，继续搅拌10分钟，最后用NaOH溶液(0.1M)调pH至中性。然后中空纤维柱除盐，浓缩冻干得到MTC7；分子量Mw＝12289Da。通过核磁测定gsU-NHR(R＝-CH 2COOH,35％)、G/I+GalA(9％)、C2/C3epox(8％)和I2S(48％)占总糖醛酸单糖残基的百分比。
实施例8
在本实施例中，对于制备得到的糖胺聚糖衍生物进行乙酰肝素酶抑制活性测试。
参照“Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening”(Anal Biochem.396(1),2010.,112-116)中公开的体外测定乙酰肝素酶抑制活性的方法，对本申请MTC系列化合物的乙酰肝素酶抑制活性进行测试，方法如下：
测定溶液(100μL)包含40mM乙酸钠缓冲液pH 5.0和100mM磺达肝素(GlaxoSmithKline)，其含有或不含有测试样品。加入乙酰肝素酶至140pM的终浓度以开始所述测定。将板用黏带密封，并在37℃下孵育2-24小时。通过加入100μL的1.69mM4-[3-(4-碘苯基)-1H-5四唑]-1,3-苯二磺酸盐(WST-1，Aspep，Melbourne，澳大利亚)在0.1M NaOH溶液中停止测定。将板用黏带再密封，并在60℃下显影60分钟。在584nm下测量吸光度(Fluostar,BMG,Labtech)。在各板中，在相同的缓冲液和体积下，在2-100μM的范围下制备用D-半乳糖作为还原糖标准物构建的标准曲线，测定IC 50值。
测试结果数据如表2所示，根据表2的结果可以看出，本申请MTC系列化合物其IC 50值在7-53ng/mL，甚至是能够达到7-22ng/mL，具有良好的乙酰肝素酶抑制活性。
表2
化合物 IC 50(ng/mL)
MTC1 13
MTC2 9
MTC3 16
MTC4 22
MTC5 7
MTC6 12
MTC7 53
实施例9
在本实施例中，对制备得到的糖胺聚糖衍生物进行抗凝效价测试，使用APTT测定法。
APTT(Activated partial thromboplastin time，活化部分凝血活酶时间)测定原理为血浆样本与适量的磷脂以及能激活内源性凝血途径的带负电荷的触酶剂进行共同孵育，在37℃孵育一定时间后，加入钙离子，启动凝血过程，并记录血浆凝固所需时间，即为活化部分凝血活酶时间。本申请使用Stago公司的STA-R全自动血凝分析仪，通过APTT法检测肝素衍生物的抗凝活性。
具体方法为：取50μL肝素钠或供试品的盐水溶液和50μL血浆于37±1℃温度下水浴反应30秒。加入50μL APTT试剂(37±1℃水浴锅预热)，混匀并立即启动血凝仪上的秒表。秒表读数为3分钟后，通过自动加样器立即加入50μL的氯化钙(0.025M)(37±1℃水浴锅预热)。STA-R全自动血凝分析仪自动记录凝血时间。测得的凝结时间换算成对数，照生物检定统计法中的量反应平行线测定法计算效价。
实验结果如表3所示，由表3数据可以看出，本申请MTC系列化合物的抗凝效价在22-47IU/mg，表明本申请MTC系列化合物抗凝作用显著低于肝素钠。
表3
化合物 抗凝效价(IU/mg)
MTC1 30
MTC2 26
MTC3 26
MTC4 30
MTC5 24
MTC6 22
MTC7 47
肝素钠 200
实施例10
在本实施例中，对制备得到的糖胺聚糖衍生物进行B16小鼠黑色素瘤肺转移模型上的药效研究，方法如下：
B16细胞(购买于中科院上海生命科学研究院细胞库)用含10％胎牛血清的RPMI-1640完全培养基、在含5％CO 2的37℃培养箱中培养、传代，当细胞增殖处于对数增长期时，即 可进行接种准备。
取C57小黑鼠，雌性，根据体重随机分组：模型对照组和实验组，尾静脉注射相应剂量(2.5mg/kg)的样品，模型对照组药物以生理盐水代替。
给药30分钟后进行尾静脉接种黑色素瘤细胞。每日观察小鼠活动状态、体重变化情况、是否有异常表现。
接种后第14天结束实验，处死所有实验小鼠，取鲜肺称重，用Bouin固定液固定小鼠肺组织，观察小鼠肺部肿瘤转移灶分布情况，计算小鼠肺部肿瘤转移抑制率。
肿瘤转移抑制率＝(模型对照组肿瘤转移灶数量-给药组转移灶数量)/模型对照组肿瘤转移灶数量×100％
实验结果：
MTC1、MTC2、MTC5、MTC6在2.5mg/kg的给药剂量下对黑色素瘤肺转移的抑制率分别为54％、56％、72％和63％，显示出良好的肿瘤转移抑制活性。
申请人声明，本申请通过上述实施例来说明本申请的糖胺聚糖衍生物及其应用，但本申请并不局限于上述实施例，即不意味着本申请必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (20)

  1. 一种糖胺聚糖衍生物，其具有如式I所示结构：
    Figure PCTCN2019129110-appb-100001
    其中，W环包含下列式A、式B、式C、式D和式E所示的结构：
    Figure PCTCN2019129110-appb-100002
    其中，R 1为SO 3 -或H；R 2为SO 3 -或H；R 3为SO 3 -、乙酰基或H；
    R为烷基、环烷基、芳基、环烷基烷基、芳基烷基或-CH(R 4)-COOH；其中，所述R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基或三氟甲氧基的基团所取代。
  2. 根据权利要求1所述的糖胺聚糖衍生物，其中，所述R为C 1-6烷基、C 3-8环烷基、C 6-10芳基、C 3-8环烷基-C 1-6烷基、C 6-10芳基-C 1-6烷基或-CH(R 4)-COOH；其中，所述R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代；
    优选地，所述R为甲基、三氟甲基、羟甲基、乙基、正丙基、异丙基、正丁基、叔丁基、 正戊基、环丙基、环丁基、环戊基、环己基、苯基、环丙基甲基、环丁基甲基、环己基甲基、苄基、苯乙基、苯丙基、对甲基苄基、对羟基苄基、对甲基苯乙基、-CH 2-COOH或-CH(CH 3)-COOH。
  3. 根据权利要求1或2所述的糖胺聚糖衍生物，其中，所述糖胺聚糖为未分级肝素、低分子肝素或硫酸乙酰肝素。
  4. 根据权利要求1-3中任意一项所述的糖胺聚糖衍生物，其中，所述糖胺聚糖衍生物的分子量为4000-20000；
    优选地，所述糖胺聚糖衍生物的分子量为8000-18000；
    更优选地，所述糖胺聚糖衍生物的分子量为10000-16000。
  5. 根据权利要求1-4中任意一项所述的糖胺聚糖衍生物，其中，所述式E所示的结构占W环所示结构的摩尔百分比为20％～90％；
    优选地，所述式E所示的结构占W环所示结构的比例为30％～70％；
    更优选地，所述式E所示的结构占W环所示结构的比例为40％～60％。
  6. 一种糖胺聚糖衍生物，其通过以下方法制备得到：
    (1)糖胺聚糖的2-O-脱硫酸化：在碱性条件下，糖胺聚糖化合物2-位磺酸化艾杜糖醛酸C2-C3位经过环氧化反应形成环氧基团，然后环氧基团开环生成C2-C3位邻二醇结构；
    (2)将糖醛酸C2-C3位邻二醇结构氧化开环并生成二醛；和
    (3)将步骤(2)的反应产物与胺类化合物NH 2-R发生醛胺缩合反应，然后经还原反应得到所述糖胺聚糖衍生物；
    其中，所述R为烷基、环烷基、芳基、环烷基-烷基-、芳基-烷基-或-CH(R 4)-COOH；其中，R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代。
  7. 根据权利要求6所述的糖胺聚糖衍生物，其中，所述R为C 1-6烷基、C 3-8环烷基、C 6-10芳基、C 3-8环烷基-C 1-6烷基-、C 6-10芳基-C 1-6烷基-或-CH(R 4)-COOH；其中，所述R 4为H、CH 3-、CH 3-CH 2-CH(CH 3)-、(CH 3) 2CH-、(CH 3) 2CH-CH 2-、HOOC-CH 2-、H 2N-CO-CH 2-、HN＝C(NH 2)-NH-(CH 2) 3-、HS-CH 2-、H 2N-CO-(CH 2) 2-、H 2N-(CH 2) 4-、CH 3-S-(CH 2) 2-、Ph-CH 2-、HO-CH 2-、CH 3-CH(OH)-、HO-p-Ph-CH 2-或HOOC-(CH 2) 2-；所述烷基、环烷基、芳基、环烷基烷基和芳基烷基任选地被1、2或3个选自氢原子、氟原子、氯原子、溴原子、碘原子、羟基、氨基、氰基、硝基、甲基、乙基、叔丁基、三氟甲基、甲氧基和三氟甲氧基的基团所取代；
    优选地，所述R为甲基、三氟甲基、羟甲基、乙基、正丙基、异丙基、正丁基、叔丁基、 正戊基、环丙基、环丁基、环戊基、环己基、苯基、环丙基甲基、环丁基甲基、环己基甲基、苄基、苯乙基、苯丙基、对甲基苄基、对羟基苄基、对甲基苯乙基、-CH 2-COOH或-CH(CH 3)-COOH。
  8. 根据权利要求6或7所述的糖胺聚糖衍生物，其中，步骤(1)所述碱性条件为0.5M～2.0M的氢氧化钠水溶液或氢氧化钾水溶液。
  9. 根据权利要求6-8中任意一项所述的糖胺聚糖衍生物，其中，步骤(1)中所述环氧化反应的反应温度为20℃至100℃，反应时间为20分钟至120分钟；
    优选地，所述环氧化反应的反应温度为40℃至80℃，反应时间为20分钟至60分钟；
    更优选地，所述环氧化反应的反应温度为40℃、50℃或60℃，反应时间为20分钟、30分钟、45分钟或60分钟。
  10. 根据权利要求6-9中任意一项所述的糖胺聚糖衍生物，其中，步骤(1)中所述环氧基团开环反应的pH值为5.0至9.0，反应温度为30℃至100℃；
    优选地，步骤(1)中所述环氧基团开环反应的pH值为6.0至8.0，反应温度为40℃至80℃；
    更优选地，步骤(1)中所述环氧基团开环反应的pH值为6.5至7.5，反应温度为40℃、50℃、60℃或70℃。
  11. 根据权利要求6-10中任意一项所述的糖胺聚糖衍生物，其中，步骤(2)中氧化反应使用的氧化剂为高碘酸钠。
  12. 根据权利要求11所述的糖胺聚糖衍生物，其中，高碘酸钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的10～30倍；
    优选地，高碘酸钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的15倍、18倍、20倍、22倍或25倍。
  13. 根据权利要求6-12中任意一项所述的糖胺聚糖衍生物，其中，步骤(3)中胺类化合物NH 2-R的用量与步骤(1)中糖胺聚糖化合物的用量按摩尔量/质量比计算为5～20mmol/g；
    优选地，步骤(3)中胺类化合物NH 2-R的用量与步骤(1)中糖胺聚糖化合物的用量按摩尔量/质量比计算为8～20mmol/g。
  14. 根据权利要求6-13中任意一项所述的糖胺聚糖衍生物，其中，步骤(3)中还原反应使用的还原剂为硼氢化钠。
  15. 根据权利要求14所述的糖胺聚糖衍生物，其中，所述硼氢化钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的0.1～0.5倍；
    优选地，所述硼氢化钠的用量按质量计算为步骤(1)中糖胺聚糖化合物用量的0.15～0.3倍。
  16. 根据权利要求6-15中任意一项所述的糖胺聚糖衍生物，其中，所述糖胺聚糖化合物为未分级肝素、低分子肝素或硫酸乙酰肝素。
  17. 根据权利要求16所述的糖胺聚糖衍生物，其中，所述低分子肝素为依诺肝素、达肝 素钠或那曲肝素钙。
  18. 根据权利要求1-17中任意一项所述的糖胺聚糖衍生物在制备具有乙酰肝素酶抑制活性的药物中的应用。
  19. 一种药物组合物，其包含权利要求1-17中任意一项所述的糖胺聚糖衍生物；
    优选地，所述药物组合物还包括药用载体和/或赋形剂。
  20. 根据权利要求1-17中任意一项所述的糖胺聚糖衍生物或权利要求19所述的药物组合物在制备抗肿瘤药物中的应用。
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