Macromolecular JAK inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a macromolecular JAK inhibitor as well as a preparation method and application thereof. The macromolecular JAK inhibitor is a phosphonitrilic chloride trimer or cyanuric chloride anti-inflammatory amphiphilic polymer coupled with different functional groups. The anti-inflammatory amphiphilic polymer is synthesized by taking phosphonitrilic chloride trimer or cyanuric chloride as a skeleton structure and connecting hydrophilic modules polyethylene glycol and 3-amino-phthalylhydrazine (namely luminol) in different proportions through gradual nucleophilic substitution reaction. The preparation method of the anti-inflammatory macromolecular drug is simple, the structure and the function are controllable, and large-scale synthesis is easy. In addition, the amphiphilic anti-inflammatory macromolecular drug can be self-assembled in a water solution through intermolecular interaction to form nano-micelles. The compound can be applied to treatment of various acute and chronic inflammations or JAK signal channel related diseases, and the administration modes comprise intravenous injection, subcutaneous injection, aerosol inhalation, intramuscular injection and any combination of the modes. The anti-inflammatory macromolecule has a remarkable treatment effect on acute lung injury, acute kidney injury, acute hepatic failure, sepsis and asthma.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anti-inflammatory macromolecular JAK inhibitor capable of inhibiting the expression of pro-inflammatory factors and the JAK pathway, a preparation method and application thereof in preventing and treating various acute and chronic inflammatory diseases or diseases related to the JAK signaling pathway. Background Art

[0002] Inflammation is an immune response caused by the body to different harmful stimuli (such as invading pathogens, damaged cells and tissue damage). Controllable inflammatory responses are beneficial to the host's defense against infection, promoting tissue repair and restoring body homeostasis. However, uncontrollable acute inflammation and persistent chronic inflammation are inseparable from the pathogenesis of many diseases, such as cytokine storm, acute pneumonia, sepsis, acute kidney injury, atherosclerosis, asthma and neurodegenerative diseases. Therefore, anti-inflammation is crucial for the treatment of inflammation-related diseases. Existing drugs used in clinical anti-inflammatory treatment include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids and biologics. However, clinical studies have shown that most of the existing anti-inflammatory drugs have serious toxic side effects. For example, glucocorticoids and NSAIDs can cause gastrointestinal bleeding and perforation, acute liver / kidney damage, cardiovascular complications, osteoporosis, diabetes and aseptic joint necrosis. Biologics may trigger inflammatory storms, infections and malignant tumors. Therefore, it is urgent to develop a new generation of anti-inflammatory drugs to effectively and safely regulate pathological inflammation.

[0003] JAK is a non-receptor tyrosine protein kinase that can receive signals produced by cytokines and transmit them through the JAK-STAT signaling pathway. It has become a hot target for inflammation-related diseases. At present, a variety of JAK inhibitors have been approved for marketing in China, and their indications cover a variety of inflammation-related diseases such as rheumatoid arthritis and atopic dermatitis. Studies have reported that JAK inhibitors can block the signal transduction of a variety of inflammatory-related factors and effectively inhibit the occurrence and development of inflammation. Existing JAK inhibitors are mainly in the form of tablets, such as tofacitinib citrate tablets. However, due to its long course of treatment, patient compliance is poor and toxic side effects are large. Therefore, the development of new JAK inhibitors for the treatment of inflammation-related diseases is of great significance.

[0004] In recent years, with the rapid development of nanotechnology and materials science, researchers have begun to explore the development of drug-free anti-inflammatory macromolecular materials to treat inflammatory diseases or regulate the inflammatory microenvironment to prevent and treat inflammation-related diseases. For example, natural polysaccharides and their derivatives have shown anti-inflammatory effects in different animal models, but these natural anti-inflammatory materials have limited anti-inflammatory effects. By chemically modifying with bioactive molecules, their anti-inflammatory effects can be effectively enhanced, showing a strong anti-inflammatory effect. Synthetic glycoproteins and glycopolymers have also been reported to inhibit the production of proinflammatory cytokines by neutrophils and macrophages. In addition, considering that the excessive production of reactive oxygen species during inflammation has a pro-inflammatory effect, materials based on the regulation of the inflammatory reactive oxygen microenvironment have also been developed for the treatment of various acute / chronic inflammatory diseases, such as polymers based on Tempol, phenylboronic acid pinacol ester, and bilirubin-polymer conjugates. These materials also show good therapeutic effects in animal models of acute renal / liver injury, acute / chronic heart disease, non-alcoholic steatohepatitis, colitis, ischemia-reperfusion injury, asthma, atherosclerosis, and inflammatory bone disease. Although these macromolecular therapies have shown desirable anti-inflammatory effects in animal studies, their clinical translation is still greatly limited. This is because these materials have complex chemical structures and are difficult to precisely modify. They also face problems such as difficult quality control, high synthesis costs, and low reproducibility. In addition, the in vivo metabolic behavior and safety performance of these materials remain to be resolved. Therefore, there is an urgent need to develop new anti-inflammatory macromolecular JAK inhibitors with clear chemical structure composition, flexible functional regulation, predictable hydrolysis / metabolism characteristics, efficient anti-inflammatory activity, and excellent safety performance. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a method for synthesizing an anti-inflammatory macromolecular JAK pathway inhibitor, which is an anti-inflammatory amphiphilic polymer of hexachlorocyclotrimer phosphazene or cyanuric chloride coupled with different functional groups. The amphiphilic polymer uses hexachlorocyclotrimer phosphazene or cyanuric chloride cyclic compound as a skeleton structure, and is respectively connected with a hydrophilic polyethylene glycol molecular chain and an active molecule luminol.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a macromolecular JAK inhibitor, an amphiphilic polymer with different functional groups coupled to a hexachlorocyclotrimer phosphazene or cyanuric chloride skeleton molecule, the coupled groups are a polyethylene glycol derivative group containing an amino terminal and a luminol group, and the chemical structure thereof is: in:

[0007] R and R1 are polyethylene glycol derivative groups containing terminal amino groups or luminol groups, and at least one of the multiple groups composed of R and R1 is a polyethylene glycol derivative group containing terminal amino groups, and at least one is a luminol group; in the structure with hexachlorocyclotriphosphazene as the skeleton molecule, the total number of R and R1 groups is 6, and in the structure with cyanuric chloride as the skeleton molecule, the total number of R and R1 groups is 3.

[0008] In the above-mentioned macromolecular JAK inhibitor, the polyethylene glycol derivative group containing a terminal amino group is selected from Where n is between 4 and 120. The luminol group is

[0009] In the above-mentioned macromolecular JAK inhibitors, the skeleton structure of the functionally modified hexachlorocyclotrimer phosphazene or cyanuric chloride amphiphilic polymer may be, in addition to hexachlorocyclotrimer phosphazene or cyanuric chloride, a halogenated cyclotrimer phosphazene having nucleophilic substitution reaction activity, such as hexafluorocyclotrimer phosphazene, halogenated 1,3,5-triazine, etc.

[0010] The present invention also provides a method for preparing a macromolecular JAK inhibitor, comprising the following steps: under the protection of nitrogen or argon, halogenated cyclotrimer phosphazene or halogenated 1,3,5-triazine, respectively, with polyethylene glycol containing terminal amino groups and 3-amino-benzenedicarboxylic acid hydrazide, stepwise nucleophilic substitution reaction in an organic solvent containing an acid binding agent, the time of each substitution reaction is 4-48 hours, the reaction temperature is -20-65°C, and the liquid after the reaction contains the macromolecular JAK inhibitor. The generated salt can be further removed by vacuum filtration, the reaction liquid can be concentrated by rotary evaporation, or directly dialyzed and freeze-dried to obtain the product, that is, an amphiphilic polymer having polyethylene glycol and luminol coupled to the hexachlorocyclotrimer phosphazene or cyanuric chloride skeleton.

[0011] In the above method, the halogenated cyclotrimer phosphazene is selected from hexachlorocyclotrimer phosphazene or hexafluorocyclotrimer phosphazene, and the halogenated 1,3,5-triazine is selected from cyanuric chloride or 2,4,6-tribromo-1,3,5-triazine.

[0012] In the above method, the average molecular weight of the amino-terminated polyethylene glycol is between 200Da and 5000Da.

[0013] In the above method, the acid binding agent is selected from one or a combination of two of triethylamine, N,N-diisopropylethylamine, sodium carbonate and pyridine.

[0014] In the above method, the organic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, and 1,4-dioxane.

[0015] In the above method, the molar ratio of the hexachlorocyclotrimer phosphazene to the amino-terminated polyethylene glycol is between 1:0.8 and 1:3; the molar ratio of the halogenated 1,3,5-triazine to the amino-terminated polyethylene glycol is between 1:0.8 and 1:2.

[0016] In the above method, the molar ratio of the hexachlorocyclotrimer phosphazene to 3-amino-phthaloyl hydrazide is between 1:4 and 1:6; the molar ratio of the halogenated 1,3,5-triazine to 3-amino-phthaloyl hydrazide is between 1:2 and 1:4.

[0017] In the above synthesis method, the concentration of the hexachlorocyclotriphosphazene or cyanuric chloride in the organic solvent is between 0.01 mmol / mL and 1 mmol / mL.

[0018] The use of the above-mentioned macromolecular JAK inhibitor in the preparation of drugs for treating acute and chronic inflammation or JAK signaling pathway related diseases.

[0019] The diseases include acute lung injury, acute kidney injury, acute liver failure, sepsis and asthma. The administration methods include intravenous injection, subcutaneous injection, aerosol inhalation, intramuscular injection, and any combination of the above methods.

[0020] A drug for treating acute and chronic inflammation or inhibiting the JAK signaling pathway, comprising the above-mentioned macromolecular JAK inhibitor, or a pharmaceutically acceptable salt thereof.

[0021] This macromolecular JAK pathway inhibitor has significant therapeutic effects in acute lung injury, acute kidney injury, acute liver failure, sepsis and asthma.

[0022] The present invention uses acute organ injury as a model to verify the therapeutic effect of the anti-inflammatory macromolecular JAK inhibitor of the present invention in inflammation-related diseases. The inflammation-related diseases include but are not limited to acute lung injury, acute kidney injury, acute liver failure, sepsis, asthma, heart failure, ischemic stroke, inflammatory bowel disease, atherosclerosis, etc.

[0023] The beneficial technical effects of the present invention are:

[0024] (1) The anti-inflammatory macromolecular JAK inhibitor designed by the present invention is composed of hexachlorocyclotrimer phosphazene or cyanuric chloride amphiphilic polymers, which are prepared by stepwise nucleophilic substitution reaction. The synthesis method is simple, the structure and function are controllable, and it is easy to synthesize on a large scale.

[0025] (2) The anti-inflammatory macromolecular JAK inhibitor designed by the present invention is formed by chemically bonding hexachlorocyclotrimer phosphazene or cyanuric chloride with luminol. It can only hydrolyze into active units in a high-level active oxygen environment to exert anti-inflammatory effects, thereby avoiding the premature release of active units in the body.

[0026] (3) The anti-inflammatory macromolecular JAK inhibitor designed by the present invention is an amphiphilic polymer drug, which has both hydrophilic and hydrophobic structures. It can directly self-assemble in water to form micelles and load hydrophobic drugs to achieve the purpose of synergistic treatment.

[0027] (4) The anti-inflammatory macromolecular JAK inhibitor designed by the present invention has good anti-inflammatory effects in vivo and in vitro, and can effectively alleviate the pathological changes of acute lung injury, acute kidney injury, acute liver failure and sepsis, and achieve the purpose of treating inflammation-related diseases.

[0028] (5) The anti-inflammatory macromolecular JAK inhibitor designed by the present invention has good in vitro and in vivo biocompatibility and can be degraded into organic / inorganic small molecule hydrolysis products such as polyethylene glycol, 3-aminophthalic acid, phosphate ions and ammonium ions in the in vivo inflammatory microenvironment, thereby ensuring the in vivo safety of the nanomicelles.

[0029] (6) After the anti-inflammatory macromolecular JAK inhibitor designed by the present invention is prepared into micelles, its particle size is about 100-200 nm, which is easy to accumulate in the lesion site to exert anti-inflammatory effects, and the therapeutic effect is better than that of small molecule anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A method for coupling a hexachlorocyclotrimer phosphazene backbone with an amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecule drugs 1 H NMR spectrum;

[0031] Figure 2 The infrared spectrum of the hexachlorocyclotrimer phosphazene backbone coupled with one amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecule drugs;

[0032] Figure 3 This is a transmission electron microscopy image of a hexachlorocyclotrimer phosphazene backbone coupled with an amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecule drugs;

[0033] Figure 4 The mass spectrum of the hydrolysis product of the hexachlorocyclotrimer phosphazene backbone coupled with one amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecular drugs;

[0034] Figure 5 It is the in vitro anti-inflammatory activity of a hexachlorocyclotrimer phosphazene backbone coupled with one amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecular drugs;

[0035] Figure 6 It is a hexachlorocyclotrimer phosphazene backbone coupled with one amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecule drugs to inhibit lung inflammation in mice with acute lung injury;

[0036] Figure 7This is a H&E stained section of a hexachlorocyclotrimer phosphazene backbone coupled with one amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecule drugs to relieve symptoms of acute lung injury;

[0037] Figure 8 The effect of a hexachlorocyclotrimer phosphazene backbone coupled with an amino-terminated polyethylene glycol and five luminol anti-inflammatory macromolecules on body weight and liver and kidney function in an acute toxicity experiment in mice;

[0038] Fig. 9 A method for coupling a cyanuric chloride backbone with an amino-terminated polyethylene glycol and two luminol anti-inflammatory macromolecular drugs 1 HNMR spectrum;

[0039] Fig.10 The infrared spectrum of the anti-inflammatory macromolecular drug of coupling one amino-terminated polyethylene glycol and two luminols to the cyanuric chloride backbone;

[0040] Fig.11 This is the particle size and distribution diagram of micelles formed by self-assembly of one amino-terminated polyethylene glycol and two luminol anti-inflammatory macromolecular drugs in water coupled with a cyanuric chloride backbone. DETAILED DESCRIPTION

[0041] The following is a further detailed description of the invention content of the present invention in conjunction with specific embodiments, but does not limit the present invention. In the art, simple replacement or improvement of the present invention made by technicians all fall within the technical solution protected by the present invention.

[0042] The present invention is described in detail below with reference to non-limiting examples.

[0043] Example 1

[0044] Under nitrogen protection, 0.1 mmol of hexachlorocyclotrimer phosphazene was added to two round-bottom flasks and dissolved in 10 mL of dichloromethane, and placed in -20°C ice ethanol; 0.08 mmol of aminomethoxy-terminated polyethylene glycol (average molecular weight 2000) was weighed and dissolved in 5 mL of dichloromethane, and added dropwise to the above hexachlorocyclotrimer phosphazene solution, and then 2 mmol of triethylamine was added, and the reaction was carried out at room temperature for 6 hours; after the reaction was completed, the dichloromethane was removed by rotary evaporation, and then 10 mL of dimethyl sulfoxide was added, and a dimethyl sulfoxide solution containing 0.6 mmol of 3-amino-phenylenedicarboxylic acid hydrazide and 2 mmol of triethylamine was added dropwise, and the reaction was carried out at 65°C for 48 hours under nitrogen protection, and the reaction solution was collected, placed in a 2000Da dialysis bag, dialyzed in ultrapure water overnight, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotrimer phosphazene skeleton coupled with one polyethylene glycol chain and five 3-amino-phenylenedicarboxylic acid hydrazide groups.

[0045] Example 2

[0046] Under nitrogen protection, 1 mmol of hexachlorocyclotrimer phosphazene was added to two round-bottom flasks and dissolved in 10 mL of ultra-dry 1,4-dioxane, and placed in -20°C ice ethanol; 1.6 mmol of aminomethoxy-terminated polyethylene glycol (MW=2000) was weighed and dissolved in 5 mL of ultra-dry dichloromethane, and added dropwise to the above hexachlorocyclotrimer phosphazene, and then 0.5 mmol of N,N-diisopropylethylamine was added, and the reaction was carried out at room temperature for 4 hours; after the reaction was completed, 1,4-dioxane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added, and 6 mmol of luminol and 0.5 mmol of An ultra-dry dimethyl sulfoxide solution of N,N-diisopropylethylamine was added dropwise, refluxed under nitrogen protection, and reacted at 65°C for 48 hours. The reaction solution was collected, placed in a 2000KD dialysis bag for dialyzation overnight, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotrimer phosphazene skeleton coupled with one polyethylene glycol and five 3-amino-phthalic acid hydrazide groups.

[0047] Example 3

[0048] Under nitrogen protection, 0.1 mmol of hexachlorocyclotrimer phosphazene was added to two round-bottom flasks and dissolved in 10 mL of ultra-dry dichloromethane, and placed in -20°C ice ethanol; 0.24 mmol of aminomethoxy-terminated polyethylene glycol (MW=1000) was weighed and dissolved in 5 mL of ultra-dry dichloromethane, and added dropwise to the above hexachlorocyclotrimer phosphazene, and then 0.5 mmol of sodium carbonate was added, and the reaction was carried out at room temperature for 24 hours; after the reaction was completed, the dichloromethane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added, and an ultra-dry dimethyl sulfoxide solution containing 0.4 mmol of 3-amino-phthalic acid hydrazide and 0.5 mmol of sodium carbonate was added dropwise, and the reaction was carried out under nitrogen protection and reflux at 65°C for 48 hours, and the reaction solution was collected, placed in a 1000KD dialysis bag for dialyzing overnight, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotrimer phosphazene skeleton coupled with three polyethylene glycols and three 3-amino-phthalic acid hydrazides.

[0049] Example 4

[0050] Under argon protection, 0.1 mmol of hexachlorocyclotrimer phosphazene was added to two round-bottom flasks and dissolved in 10 mL of ultra-dry 1,4-dioxane, and placed in -20°C ice ethanol; 0.16 mmol of aminomethoxy-terminated polyethylene glycol (MW=1000) was weighed and dissolved in 5 mL of ultra-dry dichloromethane, and added dropwise to the above hexachlorocyclotrimer phosphazene, and then 0.5 mmol of sodium carbonate was added, and the reaction was carried out at room temperature for 24 hours; after the reaction was completed, 1,4-dioxane was removed by rotary evaporation. After the six rings, 10 mL of ultra-dry dimethyl sulfoxide was added, and an ultra-dry dimethyl sulfoxide solution containing 0.5 mmol 3-amino-phthalic acid hydrazide and 0.5 mmol sodium carbonate was added dropwise, and refluxed under nitrogen protection. The reaction was carried out at 65 ° C for 48 hours. The reaction solution was collected, placed in a 1000KD dialysis bag for dialyzation overnight, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotrimer phosphazene skeleton coupled with two polyethylene glycol chains and four 3-amino-phthalic acid hydrazide groups.

[0051] Example 5

[0052] Under nitrogen protection, 0.1 mmol of hexachlorocyclotrimer phosphazene was added to two round-bottom flasks and dissolved in 10 mL of ultra-dry dichloromethane, and placed in -20°C ice ethanol; 0.08 mmol of aminomethoxy-terminated polyethylene glycol (MW=4000) was weighed and dissolved in 5 mL of ultra-dry dichloromethane, and added dropwise to the above hexachlorocyclotrimer phosphazene, and then 0.5 mmol of pyridine was added, and the reaction was carried out at room temperature for 24 hours; after the reaction was completed, the dichloromethane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added, and an ultra-dry dimethyl sulfoxide solution containing 0.6 mmol of 3-amino-phthalic acid hydrazide and 0.5 mmol of pyridine was added dropwise, and refluxed under nitrogen protection, reacted at 65°C for 48 hours, and the reaction solution was collected, placed in a 4000KD dialysis bag for dialyzing overnight, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotrimer phosphazene skeleton coupled with one polyethylene glycol chain and five 3-amino-phthalic acid hydrazide groups.

[0053] Example 6

[0054] Accurately weigh 2.5mmol of cyanuric chloride and 1.00mmol of aminomethoxy-terminated polyethylene glycol (Mw=2000), mix well, dissolve in 20mL of anhydrous dichloromethane (DCM), then add 0.6mL of DIPEA, stir in an ice-water bath at 0℃ for 24h. Monitor the end point of the reaction by thin layer chromatography. When the reaction is complete, add 100mL of dichloromethane to the reaction solution, add 10mL of water to extract three times, and the obtained organic phase is dried with anhydrous magnesium sulfate, filtered, and spin-dried. The reactant is separated and purified by silica gel chromatography gradient elution, spin-dried, and the white product (TCT-mPEG- 2k ).

[0055] Weigh TCT-mPEG- 2k0.1mmol, 3-amino-benzenedicarboxylic acid hydrazide 0.5mmol, dissolved in 3mL anhydrous dimethyl sulfoxide (DMSO), then added 0.3mL TEA, nitrogen replacement three times. Stirred for 48h under nitrogen protection at 65℃. The reaction solution was placed in a 2000 dialysis bag, dialyzed for 72h, and lyophilized to obtain a macromolecular JAK inhibitor with a cyanuric chloride skeleton coupled with a polyethylene glycol chain and two 3-amino-benzenedicarboxylic acid hydrazide groups.

[0056] Example 7

[0057] Accurately weigh 2.5mmol of cyanuric chloride and aminomethoxy-terminated polyethylene glycol (mPEG- 1k -NH2) 1.00mmol was mixed and dissolved in 15mL anhydrous dichloromethane, followed by adding 0.5mL DIPEA, and stirred in an ice-water bath at 0℃ for 24h. The reaction end point was monitored by thin layer chromatography. When the reaction was complete, 100mL dichloromethane was added to the reaction solution, and 10mL water was added to extract three times. The obtained organic phase was dried over anhydrous magnesium sulfate, filtered, and dried by spin drying. The reactants were separated and purified by silica gel chromatography gradient elution, and the white product (TCT-mPEG 1k ).

[0058] Weigh TCT-mPEG 1k 0.1mmol, 3-amino-benzenedicarboxylic acid hydrazide 0.5mmol, dissolved in 2ml anhydrous N,N-dimethylformamide (DMF), then added 0.2mL TEA, nitrogen replacement three times. Stirred for 48h under nitrogen protection at 65℃. The reaction solution was placed in a 1000 dialysis bag, dialyzed for 72h, and lyophilized to obtain a macromolecular JAK inhibitor with a cyanuric chloride skeleton coupled with 1 polyethylene glycol and 2 3-amino-benzenedicarboxylic acid hydrazide groups.

[0059] Example 8

[0060] Accurately weigh 2.5mmol of cyanuric chloride and aminomethoxy-terminated polyethylene glycol (mPEG 11 -NH2) 1.00mmol was mixed and dissolved in 10mL anhydrous dichloromethane, followed by adding 0.5mL DIPEA, and stirred in an ice-water bath at 0°C for 24h. The reaction end point was monitored by thin layer chromatography. When the reaction was complete, 100mL dichloromethane was added to the reaction solution, and 10mL water was added to extract three times. The obtained organic phase was dried over anhydrous magnesium sulfate, filtered, and spin-dried. The reactant was separated and purified by silica gel chromatography gradient elution, and the product was spin-dried to obtain a white product (TCT-mPEG 11 ).

[0061] Weigh TCT-mPEG 110.1mmol, 3-amino-benzenedicarboxylic acid hydrazide 0.5mmol, dissolved in 2ml anhydrous dimethyl sulfoxide, then added 0.2mL TEA, nitrogen replacement three times. Stirred for 48h under nitrogen protection at 65℃. The reaction solution was placed in a 500 dialysis bag, dialyzed for 72h, and lyophilized to obtain a macromolecular JAK inhibitor with a cyanuric chloride skeleton coupled with 1 polyethylene glycol and 2 3-amino-benzenedicarboxylic acid hydrazide groups.

[0062] Example 9

[0063] Under nitrogen protection, 0.1mmol 2,4,6-tribromo-1,3,5-triazine was dissolved in 10mL ultra-dry tetrahydrofuran, 1.2mmol N,N-diisopropylethylamine was added, and 0.2mmol terminal aminomethoxy polyethylene glycol (mPEG 500 -NH2) was reacted at 25°C for 24h, and then reacted with 0.3mmol 3-amino-benzenedicarboxylic acid hydrazide at 65°C for 48h. After the reaction was completed, the generated salt was removed by vacuum filtration, and the reaction solution was concentrated to 5mL by rotary evaporation, and finally precipitated in 50mL of ice ether at -10°C, and the product was obtained after filtration, that is, 1 polyethylene glycol and 2 3-amino-benzenedicarboxylic acid hydrazide modified 2,4,6-tribromo-1,3,5-triazine amphiphilic polymer.

[0064] Example 10

[0065] Accurately weigh 2.5mmol of cyanuric chloride and 1.00mmol of aminomethoxy-terminated polyethylene glycol (mPEG8-NH2), mix well, dissolve in 10mL of ultra-dry tetrahydrofuran, then add 0.5mL of DIPEA, stir and react in an ice-water bath at 0℃ for 24h. Monitor the end point of the reaction by thin layer chromatography. When the reaction is complete, add 100mL of dichloromethane to the reaction solution, add 10mL of water to extract three times, and the obtained organic phase is dried with anhydrous magnesium sulfate, filtered, and spin-dried. The reactant is separated and purified by silica gel chromatography gradient elution, spin-dried, and a white product (TCT-mPEG8) is obtained.

[0066] Weigh 0.1mmol of TCT-mPEG8 and 0.5mmol of 3-amino-benzenedicarboxylic acid hydrazide, dissolve in 2ml of anhydrous dimethyl sulfoxide, then add 0.2mL of TEA, replace with nitrogen three times. Stir for 48h under nitrogen protection at 65℃. Put the reaction solution into a 500 dialysis bag, dialyze for 72h, and freeze-dry to obtain a macromolecular JAK inhibitor with a cyanuric chloride skeleton coupled with one polyethylene glycol and two 3-amino-benzenedicarboxylic acid hydrazide groups.

[0067] Embodiment 11

[0068] Under nitrogen protection, 0.1 mmol of hexabromocyclotrimer phosphazene was added to two round-bottom flasks and dissolved in 10 mL of ultra-dry 1,4-dioxane, and placed in -20°C ice ethanol; 0.16 mmol of amino-terminated methoxy polyethylene glycol (MW=500) was weighed and dissolved in 5 mL of ultra-dry dichloromethane, and added dropwise to the above hexabromocyclotrimer phosphazene, and then 0.5 mmol of triethylamine was added, and the reaction was carried out at room temperature for 24 hours; after the reaction was completed, the above reaction system was taken out, 1,4-dioxane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added, and 0.5 mmol of 3-amino-phenyl dicarboxylic acid hydrazide and 0.5 mmol of The ultra-dry dimethyl sulfoxide solution of DIPEA was added dropwise, refluxed under nitrogen protection, reacted at 65°C for 48 hours, the reaction solution was taken out, dialyzed in a 500KD dialysis bag overnight, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexabromocyclotrimer phosphazene skeleton coupled with two polyethylene glycols and four 3-amino-phthalhydrazide groups.

[0069] Figure 1 and Figure 2 The results showed that the anti-inflammatory macromolecular drug synthesized in Example 1 was the product of a hexachlorocyclotrimer phosphazene skeleton coupled with a terminal aminomethoxy polyethylene glycol and five 3-amino-phthalic acid hydrazides.

[0070] Figure 3 The results showed that the nanomicelle solution formed by self-assembly of the obtained anti-inflammatory macromolecular JAK inhibitor in water was uniform in size and spherical.

[0071] Figure 4 The mass spectrum of the hydrolysis product of the obtained anti-inflammatory macromolecular drug in hydrogen peroxide shows that the anti-inflammatory macromolecular JAK inhibitor generates 3-aminophthalic acid after hydrolysis.

[0072] Figure 5 The results showed that the JAK inhibitor could effectively inhibit the secretion of pro-inflammatory factors by RAW264.7 cells induced by LPS.

[0073] Figure 6 The in vivo anti-inflammatory effect of the obtained anti-inflammatory macromolecular JAK inhibitor. The present invention uses LPS to construct a mouse acute lung injury model by nasal drops, and the JAK inhibitor is injected through the tail vein for treatment. The experimental results show that the JAK inhibitor can effectively alleviate inflammation in the lungs of mice with acute lung injury.

[0074] Figure 7 This is an H&E-stained section of the mouse lung after the anti-inflammatory macromolecule JAK inhibitor was injected into the tail vein to treat acute liver and lung injury in mice. The experimental results showed that compared with the model group, treatment with JAK inhibitors can effectively alleviate the typical pathological characteristics of ALI, including inflammatory cell infiltration, pulmonary edema and alveolar hemorrhage.

[0075] Figure 8 The mice were given 500 mg / kg of HPL by tail vein injection for acute toxicity evaluation. The results showed that after tail vein injection of high-concentration anti-inflammatory macromolecule drugs, the weight of the mice did not change significantly, and the liver and kidney functions of the mice were not affected, which preliminarily proved that the obtained anti-inflammatory macromolecule JAK inhibitor has good biosafety in vivo.

[0076] Fig. 9 and Fig.10 The results showed that the anti-inflammatory macromolecular JAK inhibitor synthesized in Example 6 was an anti-inflammatory macromolecular JAK inhibitor composed of a cyanuric chloride backbone coupled with a polyethylene glycol and two 3-amino-phthalic acid hydrazides. Fig.11 The results showed that the nanomicelle solution formed by self-assembly of the obtained anti-inflammatory macromolecular JAK inhibitor in water had uniform particle size.

Claims

1. A macromolecular JAK inhibitor, characterized in that: An amphiphilic polymer with different functional groups coupled to the hexachlorocyclotriphosphazene or cyanuric chloride skeleton molecule, wherein the coupled groups are a polyethylene glycol derivative group containing an amino terminal and a luminol group, and the chemical structure thereof is: in: R and R1 are polyethylene glycol derivative groups containing terminal amino groups or luminol groups, and at least one of the multiple groups composed of R and R1 is a polyethylene glycol derivative group containing terminal amino groups, and at least one is a luminol group; in the structure with hexachlorocyclotriphosphazene as the skeleton molecule, the total number of R and R1 groups is 6, and in the structure with cyanuric chloride as the skeleton molecule, the total number of R and R1 groups is 3.

2. A macromolecular JAK inhibitor according to claim 1, characterized in that: The polyethylene glycol derivative group containing a terminal amino group is selected from The value of n is between 4 and 120.

3. The method for preparing the macromolecular JAK inhibitor according to claim 1 or 2, characterized in that: The method comprises the following steps: under the protection of nitrogen or argon, halogenated cyclotrimer phosphazene or halogenated 1,3,5-triazine is respectively subjected to stepwise nucleophilic substitution reaction with polyethylene glycol containing terminal amino groups and 3-amino-phthalic acid hydrazide in an organic solvent containing an acid binding agent, the time of each substitution reaction is 4-48 hours, the reaction temperature is -20-65°C, and the liquid after the reaction contains a macromolecular JAK inhibitor.

4. The method for preparing the macromolecular JAK inhibitor according to claim 3, characterized in that: The average molecular weight of the amino-terminated polyethylene glycol is between 200Da and 5000Da, the halogenated cyclotrimer phosphazene is hexachlorocyclotrimer phosphazene or hexafluorocyclotrimer phosphazene, and the halogenated 1,3,5-triazine is cyanuric chloride or 2,4,6-tribromo-1,3,5-triazine.

5. The method for preparing the macromolecular JAK inhibitor according to claim 3, characterized in that: The acid binding agent is selected from one or a combination of triethylamine, N,N-diisopropylethylamine, sodium carbonate and pyridine.

6. The method for preparing the macromolecular JAK inhibitor according to claim 3, characterized in that: The organic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, and 1,4-dioxane.

7. The method for preparing the macromolecular JAK inhibitor according to claim 3, characterized in that: The molar ratio of the hexachlorocyclotrimer phosphazene to the amino-terminated polyethylene glycol is between 1:0.8 and 1:3; the molar ratio of the halogenated 1,3,5-triazine to the amino-terminated polyethylene glycol is between 1:0.8 and 1:

2.

8. The method for preparing the macromolecular JAK inhibitor according to claim 3, characterized in that: The molar ratio of the hexachlorocyclotrimer phosphazene to 3-amino-phthaloyl hydrazide is between 1:4 and 1:6; the molar ratio of the halogenated 1,3,5-triazine to 3-amino-phthaloyl hydrazide is between 1:2 and 1:

4.

9. Use of the macromolecular JAK inhibitor according to claim 1 in the preparation of drugs for treating acute and chronic inflammation or diseases related to the JAK signaling pathway.

10. A drug for treating acute and chronic inflammation or inhibiting the JAK signaling pathway, characterized in that: It comprises the macromolecular JAK inhibitor according to claim 1, or a pharmaceutically acceptable salt thereof.

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