A macromolecular JAK inhibitor, its preparation method and application

Abstract

This invention discloses a macromolecular JAK inhibitor, its preparation method, and its applications. This macromolecular JAK inhibitor is an anti-inflammatory amphiphilic polymer of hexachlorocyclotriphosphazene or cyanuric chloride coupled with different functional groups. The anti-inflammatory amphiphilic polymer is synthesized with hexachlorocyclotriphosphazene or cyanuric chloride as the backbone structure, linked by a stepwise nucleophilic substitution reaction with different proportions of hydrophilic modules polyethylene glycol and 3-aminobenzoyl hydrazide (i.e., luminol). The preparation method of this type of anti-inflammatory macromolecular drug is simple, its structure and function are controllable, and it is easy to synthesize on a large scale. Furthermore, this type of amphiphilic anti-inflammatory macromolecular drug can self-assemble into nanomicelles in aqueous solution through intermolecular interactions. It can be applied to the treatment of various acute and chronic inflammations or diseases related to the JAK signaling pathway, and can be administered via intravenous injection, subcutaneous injection, nebulized inhalation, intramuscular injection, and any combination of the above methods. This anti-inflammatory macromolecular drug has significant therapeutic effects in acute lung injury, acute kidney injury, acute liver failure, sepsis, and asthma.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to an anti-inflammatory macromolecular JAK inhibitor that inhibits the expression of pro-inflammatory factors and the JAK pathway, its preparation method, and its application in the prevention and treatment of various acute and chronic inflammatory diseases or diseases related to the JAK signaling pathway. Background Technology

[0002] Inflammation is the body's immune response to various harmful stimuli, such as invading pathogens, damaged cells, and tissue damage. Controlled inflammatory responses benefit the host by defending against infection, promoting tissue repair, and restoring homeostasis. However, uncontrollable acute inflammation and persistent chronic inflammation are inextricably linked to the pathogenesis of many diseases, such as cytokine storms, acute pneumonia, sepsis, acute kidney injury, atherosclerosis, asthma, and neurodegenerative diseases. Therefore, anti-inflammatory treatment is crucial for inflammation-related diseases. Currently available anti-inflammatory drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and biologics. However, clinical studies have shown that most existing anti-inflammatory drugs have serious toxic side effects. For example, glucocorticoids and NSAIDs can cause gastrointestinal bleeding and perforation, acute liver / kidney injury, cardiovascular complications, osteoporosis, diabetes, and avascular necrosis of joints. Biologics may trigger cytokine storms, infections, and malignancies. Therefore, there is an urgent need to develop next-generation anti-inflammatory drugs to effectively and safely regulate pathological inflammation.

[0003] JAK is a non-receptor tyrosine protein kinase that receives signals from cytokines and transmits them through the JAK-STAT signaling pathway, making it a popular target for inflammation-related diseases. Currently, several JAK inhibitors have been approved for marketing in China, covering indications for various inflammatory diseases such as rheumatoid arthritis and atopic dermatitis. Studies have reported that JAK inhibitors can block the signal transduction of multiple inflammatory factors, effectively inhibiting the occurrence and development of inflammation. Existing JAK inhibitors are mainly in tablet form, such as tofacitinib citrate tablets. However, due to their long treatment duration, patient compliance is poor and side effects are significant. Therefore, the development of novel JAK inhibitors for the treatment of inflammatory 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 modulate 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 the anti-inflammatory effects of these natural anti-inflammatory materials are limited. By utilizing bioactive molecules for chemical modification, their anti-inflammatory effects can be effectively enhanced, exhibiting strong anti-inflammatory effects. Synthetic glycoproteins and glycopolymers have also been reported to inhibit the production of pro-inflammatory cytokines by neutrophils and macrophages. In addition, considering the pro-inflammatory effects of excessive reactive oxygen species (ROS) produced during inflammation, materials based on the regulation of the inflammatory ROS microenvironment have also been developed for the treatment of various acute / chronic inflammatory diseases, such as polymers based on Tempol, pinacol phenylborate, and bilirubin-polymer conjugates. These materials have also shown good therapeutic effects in animal models of acute kidney / liver injury, acute / chronic heart disease, non-alcoholic steatohepatitis, colitis, ischemia-reperfusion injury, asthma, atherosclerosis, and inflammatory bone disease. Although these macromolecular therapies have demonstrated promising anti-inflammatory effects in animal studies, their clinical translation remains severely limited. This is due to the complex chemical structures of these materials, the difficulty in precisely modifying their structures, and the challenges of quality control, high synthesis costs, and low reproducibility. Furthermore, the in vivo metabolic behavior and safety profile of these materials remain to be addressed. Therefore, there is an urgent need to develop novel anti-inflammatory macromolecular JAK inhibitors with well-defined chemical structures, flexible functional regulation, predictable hydrolysis / metabolic characteristics, high anti-inflammatory activity, and excellent safety profiles. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention aims to provide a method for synthesizing an anti-inflammatory macromolecular JAK pathway inhibitor, which is an anti-inflammatory amphiphilic polymer of hexachlorocyclotriphosphazene or cyanuric chloride coupled with different functional groups. This amphiphilic polymer uses a hexachlorocyclotriphosphazene or cyanuric chloride cyclic compound as its backbone structure, with hydrophilic polyethylene glycol molecular chains and the active molecule luminol linked to it, respectively.

[0006] To achieve the above objectives, the technical solution adopted in this invention is: a macromolecular JAK inhibitor, comprising an amphiphilic polymer with different functional groups coupled to a hexachlorocyclotriphosphazene or cyanuric chloride backbone molecule, wherein the coupled groups are polyethylene glycol derivative groups containing terminal amino groups and luminol groups, and its chemical structure is as follows: in:

[0007] R and R1 are polyethylene glycol derivative groups or luminol groups containing terminal amino 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 backbone molecule, the total number of R and R1 groups is 6, and in the structure with cyanuric chloride as the backbone, the total number of R and R1 groups is 3.

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

[0009] Among the aforementioned macromolecular JAK inhibitors, the skeletal structure of the functionalized hexachlorocyclotriphosphazene or cyanuric chloride amphiphilic polymers can be, in addition to hexachlorocyclotriphosphazene or cyanuric chloride, halocyclic triphosphazenes with nucleophilic substitution reactivity, such as hexafluorocyclotriphosphazene, halogenated 1,3,5-triazine, etc.

[0010] This invention also provides a method for preparing a macromolecular JAK inhibitor, comprising the following steps: under nitrogen or argon protection, a halocyclic triphosphazene or a halo-1,3,5-triazine is reacted stepwise with polyethylene glycol containing a terminal amino group and 3-amino-phthalamide hydrazide in an organic solvent containing an acid-binding agent in a nucleophilic substitution reaction. Each substitution reaction takes 4-48 hours, and the reaction temperature is -20-65°C. The liquid after the reaction contains a macromolecular JAK inhibitor. The generated salt can be further removed by vacuum filtration, the reaction solution can be concentrated by rotary evaporation, or the product can be obtained by direct dialysis and lyophilization, i.e., an amphiphilic polymer in which a hexachlorocyclotriphosphazene or cyanuric chloride backbone is simultaneously coupled with polyethylene glycol and luminol.

[0011] In the above method, the halogenated cyclotriphosphazene is selected from hexachlorocyclotriphosphazene or hexafluorocyclotriphosphazene, 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 polyethylene glycol with terminal amino groups has an average molecular weight between 200 Da and 5000 Da.

[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 hexachlorocyclotriphosphazene to amino-terminated polyethylene glycol is between 1:0.8 and 1:3; the molar ratio of halogenated 1,3,5-triazine to amino-terminated polyethylene glycol is between 1:0.8 and 1:2.

[0016] In the above method, the molar ratio of hexachlorocyclotriphosphazene to 3-amino-phthalic acid hydrazide is between 1:4 and 1:6; the molar ratio of halogenated 1,3,5-triazine to 3-amino-phthalic acid 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 application of the above-mentioned macromolecular JAK inhibitors in the preparation of drugs for treating acute and chronic inflammation or diseases related to the JAK signaling pathway.

[0019] The diseases mentioned include acute lung injury, acute kidney injury, acute liver failure, sepsis, and asthma. Administration methods include intravenous injection, subcutaneous injection, nebulized inhalation, intramuscular injection, and any combination of these methods.

[0020] A drug for treating acute or chronic inflammation or inhibiting the JAK signaling pathway, including the aforementioned macromolecular JAK inhibitors, or pharmaceutically acceptable salts thereof.

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

[0022] This invention uses acute organ injury as a model to verify the therapeutic efficacy of the anti-inflammatory macromolecular JAK inhibitor of this invention in inflammatory-related diseases. These inflammatory-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, and atherosclerosis.

[0023] The beneficial technical effects of this invention are as follows:

[0024] (1) The anti-inflammatory macromolecular JAK inhibitor designed in this invention is composed of hexachlorocyclotriphosphazene or cyanuric trichlorotriphosphazene amphiphilic polymers. These amphiphilic polymers are prepared by stepwise nucleophilic substitution reactions. 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 in this invention is formed by chemical bonding of hexachlorocyclotriphosphazene or cyanuric chloride with luminol. It can only release the active unit in a high-level reactive oxygen environment to exert the anti-inflammatory effect, thereby avoiding the premature release of the active unit in the body.

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

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

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

[0029] (6) The anti-inflammatory macromolecular JAK inhibitor designed in this invention is prepared into micelles with a particle size of about 100-200nm, which makes it easy to target and accumulate at the lesion site to exert anti-inflammatory effects, and the therapeutic effect is better than that of small molecule anti-inflammatory drugs. Attached Figure Description

[0030] Figure 1 A macromolecular anti-inflammatory drug, luminol, coupled with a hexachlorocyclotriphosphazene backbone, consisting of one amino-terminal polyethylene glycol and five luminol groups. 1 H NMR spectrum;

[0031] Figure 2 Infrared spectra of a hexachlorocyclotriphosphazene backbone coupled with one terminal amino group of polyethylene glycol and five luminol anti-inflammatory macromolecules;

[0032] Figure 3 Transmission electron microscopy image of a hexachlorocyclotriphosphazene backbone coupled with one terminal amino group of polyethylene glycol and five luminol anti-inflammatory macromolecules;

[0033] Figure 4 Mass spectra of the hydrolysis products of a hexachlorocyclotriphosphazene backbone coupled with one terminal amino group of polyethylene glycol and five luminol anti-inflammatory macromolecular drugs;

[0034] Figure 5 It exhibits in vitro anti-inflammatory activity of a hexachlorocyclotriphosphazene backbone coupled with one terminal amino group of polyethylene glycol and five luminol anti-inflammatory macromolecules;

[0035] Figure 6 It is a macromolecular drug that is a hexachlorocyclotriphosphazene skeleton coupled with one terminal amino polyethylene glycol and five luminol to inhibit lung inflammation in mice with acute lung injury;

[0036] Figure 7This is an H&E stained section of a hexachlorocyclotriphosphazene backbone coupled with one terminal amino polyethylene glycol and five luminol anti-inflammatory macromolecules to relieve symptoms of acute lung injury.

[0037] Figure 8 The effects of a hexachlorocyclotriphosphazene backbone coupled with one terminal amino group of polyethylene glycol and five luminol anti-inflammatory macromolecules on body weight and liver and kidney function in an acute toxicity experiment in mice;

[0038] Figure 9 A macromolecular anti-inflammatory drug, luminol, coupled to a cyanuric chloride backbone with one amino-terminated polyethylene glycol and two luminol groups. 1 HNMR spectrum;

[0039] Figure 10 Infrared spectra of a cyanuric chloride backbone coupled with one amino-terminated polyethylene glycol and two luminol anti-inflammatory macromolecules;

[0040] Figure 11 Particle size and distribution diagram of micelles formed by the self-assembly of a cyanuric chloride backbone coupled with one amino-terminated polyethylene glycol and two luminol anti-inflammatory macromolecules in water. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to specific embodiments, but this does not limit the invention. Simple substitutions or improvements made to the present invention by those skilled in the art are all within the scope of the technical solutions protected by the present invention.

[0042] The present invention will now be described in detail with reference to non-limiting embodiments.

[0043] Example 1

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

[0045] Example 2

[0046] Under nitrogen protection, 1 mmol of hexachlorocyclotriphosphazene was dissolved in 10 mL of ultra-dry 1,4-dioxane in a two-necked round-bottom flask and placed in ice-cold ethanol at -20°C. Separately, 1.6 mmol of terminal aminomethoxy polyethylene glycol (MW = 2000) was dissolved in 5 mL of ultra-dry dichloromethane and added dropwise to the hexachlorocyclotriphosphazene mixture. Then, 0.5 mmol of N,N-diisopropylethylamine was added, and the mixture was reacted at room temperature for 4 hours. After the reaction was complete, the 1,4-dioxane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added to the mixture containing 6 mmol of luminol and 0.5 mmol of... An ultra-dry dimethyl sulfoxide solution of N,N-diisopropylethylamine was added dropwise, and the mixture was refluxed under nitrogen protection. The reaction was carried out at 65°C for 48 hours. The reaction solution was collected, dialyzed overnight in a 2000 kDa dialysis bag, and then freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotriphosphazene skeleton coupled with one polyethylene glycol and five 3-amino-phthaloylhydrazide groups.

[0047] Example 3

[0048] Under nitrogen protection, 0.1 mmol of hexachlorocyclotriphosphazene was dissolved in 10 mL of ultra-dry dichloromethane in a two-necked round-bottom flask and placed in ice-cold ethanol at -20°C. Separately, 0.24 mmol of terminal aminomethoxy polyethylene glycol (MW=1000) was dissolved in 5 mL of ultra-dry dichloromethane and added dropwise to the above hexachlorocyclotriphosphazene. Then, 0.5 mmol of sodium carbonate was added, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the dichloromethane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added. A solution of ultra-dry dimethyl sulfoxide containing 0.4 mmol of 3-amino-phthalamide and 0.5 mmol of sodium carbonate was added dropwise. Under nitrogen protection and reflux, the reaction was carried out at 65°C for 48 h. The reaction solution was collected, dialyzed overnight in a 1000 KD dialysis bag, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotriphosphazene skeleton coupled with 3 polyethylene glycols and 3-amino-phthalamides.

[0049] Example 4

[0050] Under argon protection, 0.1 mmol of hexachlorocyclotriphosphazene was dissolved in 10 mL of ultra-dry 1,4-dioxane in a two-necked round-bottom flask and placed in ice-cold ethanol at -20°C. Separately, 0.16 mmol of terminal aminomethoxy polyethylene glycol (MW = 1000) was dissolved in 5 mL of ultra-dry dichloromethane and added dropwise to the aforementioned hexachlorocyclotriphosphazene, followed by 0.5 mmol of sodium carbonate. The reaction was carried out at room temperature for 24 h. After the reaction was complete, the 1,4-dioxane was removed by rotary evaporation. After adding the hexacyclophosphamide, 10 mL of ultra-dry dimethyl sulfoxide was added. Then, an ultra-dry dimethyl sulfoxide solution containing 0.5 mmol of 3-amino-phthalic acid hydrazide and 0.5 mmol of sodium carbonate was added dropwise. The mixture was refluxed under nitrogen protection and reacted at 65 °C for 48 h. The reaction solution was collected and dialyzed overnight in a 1000 KD dialysis bag. After freeze-drying, a macromolecular JAK inhibitor with a hexachlorocyclotriphosphazene skeleton coupled with two polyethylene glycol chains and four 3-amino-phthalic acid hydrazide groups was obtained.

[0051] Example 5

[0052] Under nitrogen protection, 0.1 mmol of hexachlorocyclotriphosphazene was dissolved in 10 mL of ultra-dry dichloromethane in a two-necked round-bottom flask and placed in ice-cold ethanol at -20°C. Separately, 0.08 mmol of terminal aminomethoxy polyethylene glycol (MW = 4000) was dissolved in 5 mL of ultra-dry dichloromethane and added dropwise to the above hexachlorocyclotriphosphazene. Then, 0.5 mmol of pyridine was added, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the dichloromethane was removed by rotary evaporation, and then 10 mL of ultra-dry dimethyl sulfoxide was added. A solution of ultra-dry dimethyl sulfoxide containing 0.6 mmol of 3-amino-phthalamide and 0.5 mmol of pyridine was added dropwise. The mixture was refluxed under nitrogen protection and reacted at 65°C for 48 h. The reaction solution was collected, dialyzed overnight in a 4000 KD dialysis bag, and freeze-dried to obtain a macromolecular JAK inhibitor with a hexachlorocyclotriphosphazene skeleton coupled with one polyethylene glycol chain and five 3-amino-phthalamide groups.

[0053] Example 6

[0054] Accurately weigh 2.5 mmol of cyanuric chloride and 1.00 mmol of amino-terminated methoxy polyethylene glycol (Mw = 2000), mix well, and dissolve in 20 mL of anhydrous dichloromethane (DCM). Then add 0.6 mL of DIPEA and stir in an ice-water bath at 0 °C for 24 h. Monitor the reaction endpoint using thin-layer chromatography. After the reaction is complete, add 100 mL of dichloromethane to the reaction solution, and extract three times with 10 mL of water. Dry the obtained organic phase with anhydrous magnesium sulfate, filter, and evaporate to dryness. Separate and purify the product by silica gel gradient elution, evaporate to dryness, and the white product (TCT-mPEG-) is obtained. 2k ).

[0055] Weigh out TCT-mPEG- 2k0.1 mmol of 3-amino-phthalamide hydrazide and 0.5 mmol of 3-amino-phthalamide hydrazide were dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO), followed by the addition of 0.3 mL of TEA. The mixture was purged with nitrogen three times. The mixture was stirred under nitrogen protection at 65 °C for 48 h. The reaction solution was then transferred to a 2000 dialysis bag and dialyzed for 72 h. After lyophilization, a macromolecular JAK inhibitor with a cyanuric chloride backbone coupled to one polyethylene glycol chain and two 3-amino-phthalamide hydrazide groups was obtained.

[0056] Example 7

[0057] Accurately weigh 2.5 mmol of cyanuric chloride and amino-terminated polyethylene glycol (mPEG- 1k Mix 1.00 mmol of (-NH2) and dissolve in 15 mL of anhydrous dichloromethane. Then add 0.5 mL of DIPEA and stir in an ice-water bath at 0 °C for 24 h. Monitor the reaction endpoint using thin-layer chromatography. After the reaction is complete, add 100 mL of dichloromethane to the reaction solution and extract three times with 10 mL of water. Dry the obtained organic phase with anhydrous magnesium sulfate, filter, and evaporate to dryness. Separate and purify the product by silica gel gradient elution, evaporate to dryness, and the white product (TCT-mPEG) is obtained. 1k ).

[0058] Weigh out TCT-mPEG 1k 0.1 mmol of 3-amino-phthalamide and 0.5 mmol of 3-amino-phthalamide hydrazide were dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF), followed by the addition of 0.2 mL of TEA. The mixture was purged with nitrogen three times. The mixture was stirred under nitrogen protection at 65 °C for 48 h. The reaction solution was then transferred to a 1000 dialysis bag and dialyzed for 72 h. After lyophilization, a macromolecular JAK inhibitor with a cyanuric chloride backbone coupled to one polyethylene glycol group and two 3-amino-phthalamide hydrazide groups was obtained.

[0059] Example 8

[0060] Accurately weigh 2.5 mmol of cyanuric chloride and amino-terminated polyethylene glycol (mPEG). 11 Mix 1.00 mmol of (-NH2) and dissolve in 10 mL of anhydrous dichloromethane. Then add 0.5 mL of DIPEA and stir in an ice-water bath at 0 °C for 24 h. Monitor the reaction endpoint using thin-layer chromatography. After the reaction is complete, add 100 mL of dichloromethane to the reaction solution and extract three times with 10 mL of water. Dry the obtained organic phase with anhydrous magnesium sulfate, filter, and evaporate to dryness. Separate and purify the product by silica gel gradient elution and evaporate to dryness to obtain a white product (TCT-mPEG). 11 ).

[0061] Weigh out TCT-mPEG 110.1 mmol of 3-amino-phthalamide hydrazide and 0.5 mmol of 3-amino-phthalamide hydrazide were dissolved in 2 mL of anhydrous dimethyl sulfoxide, followed by the addition of 0.2 mL of TEA. The mixture was purged with nitrogen three times. The mixture was stirred under nitrogen protection at 65 °C for 48 h. The reaction solution was then transferred to a 500 mL dialysis bag and dialyzed for 72 h. After lyophilization, a macromolecular JAK inhibitor with a cyanuric chloride backbone coupled to one polyethylene glycol group and two 3-amino-phthalamide hydrazide groups was obtained.

[0062] Example 9

[0063] Under nitrogen protection, 0.1 mmol of 2,4,6-tribromo-1,3,5-triazine was dissolved in 10 mL of ultra-dry tetrahydrofuran, and 1.2 mmol of N,N-diisopropylethylamine was added, along with 0.2 mmol of amino-terminated methoxy polyethylene glycol (mPEG). 500 The reaction mixture (-NH2) was reacted at 25 °C for 24 h, followed by reaction with 0.3 mmol of 3-amino-phthalic acid hydrazide at 65 °C for 48 h. After the reaction was complete, the generated salt was removed by vacuum filtration, the reaction solution was concentrated to 5 mL by rotary evaporation, and finally precipitated in 50 mL of ice-cold diethyl ether at -10 °C. After filtration, the product was obtained, namely a 2,4,6-tribromo-1,3,5-triazine amphiphilic polymer modified with one polyethylene glycol and two 3-amino-phthalic acid hydrazide molecules.

[0064] Example 10

[0065] Accurately weigh 2.5 mmol of cyanuric chloride and 1.00 mmol of amino-terminated methoxy polyethylene glycol (mPEG8-NH2), mix well, dissolve in 10 mL of ultra-dry tetrahydrofuran, then add 0.5 mL of DIPEA, and stir in an ice-water bath at 0 °C for 24 h. Monitor the reaction endpoint using thin-layer chromatography. After the reaction is complete, add 100 mL of dichloromethane to the reaction solution, and extract three times with 10 mL of water. The obtained organic phase is dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. Separate and purify the product by silica gel gradient elution, evaporate to dryness, and the product is white (TCT-mPEG8).

[0066] Weigh 0.1 mmol of TCT-mPEG8 and 0.5 mmol of 3-amino-phthalamide hydrazide, dissolve them in 2 mL of anhydrous dimethyl sulfoxide, then add 0.2 mL of TEA, and purge three times with nitrogen. Stir at 65 °C under nitrogen protection for 48 h. Transfer the reaction solution to a 500 mL dialysis bag, dialyze for 72 h, and lyophilize to obtain a macromolecular JAK inhibitor with a cyanuric chloride backbone coupled to one polyethylene glycol group and two 3-amino-phthalamide hydrazide groups.

[0067] Example 11

[0068] Under nitrogen protection, 0.1 mmol of hexabromocyclotriphosphazene was dissolved in 10 mL of ultra-dry 1,4-dioxane in a two-necked round-bottom flask and placed in ice-cold ethanol at -20°C. Separately, 0.16 mmol of terminal aminomethoxy polyethylene glycol (MW = 500) was dissolved in 5 mL of ultra-dry dichloromethane and added dropwise to the hexabromocyclotriphosphazene mixture. Then, 0.5 mmol of triethylamine was added, and the mixture was reacted at room temperature for 24 h. After the reaction was completed, the reaction system was removed, and the 1,4-dioxane was removed by rotary evaporation. Then, 10 mL of ultra-dry dimethyl sulfoxide was added to the mixture containing 0.5 mmol of 3-aminobenzoylhydrazide and 0.5 mmol of... DIPEA was added dropwise to an ultra-dry dimethyl sulfoxide solution under nitrogen protection and refluxed. The reaction was carried out at 65°C for 48 hours. The reaction solution was then removed and dialyzed overnight in a 500 kDa dialysis bag. After freeze-drying, a macromolecular JAK inhibitor with a hexabromocyclotriphosphazene skeleton coupled with two polyethylene glycol groups and four 3-amino-phthalamide hydrazide groups was obtained.

[0069] Figure 1 and Figure 2 The results showed that the anti-inflammatory macromolecular drug synthesized in Case 1 was a product of one hexachlorocyclotriphosphazene skeleton coupled with one terminal aminomethoxy polyethylene glycol and five 3-amino-benzoyl hydrazides.

[0070] Figure 3 The results showed that the obtained anti-inflammatory macromolecular JAK inhibitor self-assembled in water to form nanomicelles of uniform size and spherical shape.

[0071] Figure 4 The mass spectrum of the hydrolysis products of the obtained anti-inflammatory macromolecular drug in hydrogen peroxide is shown. The spectral results indicate that the anti-inflammatory macromolecular JAK inhibitor is hydrolyzed to produce 3-aminophthalic acid.

[0072] Figure 5 This study investigated the in vitro anti-inflammatory effects of a large-molecule JAK inhibitor. Results showed that this JAK inhibitor effectively inhibited LPS-induced secretion of pro-inflammatory factors in RAW264.7 cells.

[0073] Figure 6 This invention demonstrates the in vivo anti-inflammatory effect of the obtained large-molecule JAK inhibitor. An acute lung injury mouse model was established using LPS via nasal drops, followed by treatment with the JAK inhibitor via tail vein injection. Experimental results showed that the JAK inhibitor effectively alleviated lung inflammation in mice with acute lung injury.

[0074] Figure 7 These are H&E-stained sections of the lungs of mice with acute liver and lung injury after treatment with a large-molecule anti-inflammatory JAK inhibitor via tail vein injection. The experimental results show that, compared to the model group, JAK inhibitor treatment effectively alleviated typical pathological features of ALI, including inflammatory cell infiltration, pulmonary edema, and alveolar hemorrhage.

[0075] Figure 8 Acute toxicity was evaluated in mice by administering 500 mg / kg of HPL via tail vein injection. Results showed that the mice's body weight did not change significantly after tail vein injection of a high concentration of the anti-inflammatory macromolecular drug, and their liver and kidney function were not affected, preliminarily demonstrating that the obtained anti-inflammatory macromolecular JAK inhibitor has good biocompatibility in vivo.

[0076] Figure 9 and Figure 10 The results showed that the anti-inflammatory macromolecular JAK inhibitor synthesized in Example 6 was an anti-inflammatory macromolecular JAK inhibitor consisting of one cyanuric chloride backbone coupled with one polyethylene glycol and two 3-amino-phthalamide hydrazides. Figure 11 The results showed that the obtained anti-inflammatory macromolecular JAK inhibitor formed uniform nanomicelle solutions in water through self-assembly.

Claims

1. A macromolecular JAK inhibitor, characterized in that: Amphiphilic polymers coupled on hexachlorocyclotriphosphazene or tricyanochloride backbone molecules, the coupled groups are polyethylene glycol derivative groups containing terminal amino groups and luminol groups, the chemical structure is: or wherein: R and R1are polyethylene glycol derivative groups containing terminal amino groups or luminol groups, and at least one of the plurality of groups R and R1is a polyethylene glycol derivative group containing terminal amino groups and at least one is a luminol group; the total number of R and R1groups is 6 in the structure of the backbone molecule with hexachlorocyclotriphosphazene and 3 in the structure of the backbone with cyanuric chloride; the polyethylene glycol derivative group containing terminal amino groups is selected from where n is between 4 and 120 in size.

2. A process for the preparation of the macromolecular JAK inhibitor of claim 1, characterized in that, The method comprises the following steps: under the protection of nitrogen or argon, hexachlorocyclotriphosphazene or cyanuric chloride is subjected to step-by-step nucleophilic substitution reaction with polyethylene glycol containing terminal amino groups and 3-amino-benzene dicarboxylic hydrazide respectively in an organic solvent containing an acid-binding agent, the time for each step of the substitution reaction is 4-48 h, the reaction temperature is-20-65 ℃, and the liquid after the reaction ends contains a macromolecular JAK inhibitor.

3. The method for preparing the macromolecular JAK inhibitor according to claim 2, characterized in that: The polyethylene glycol containing terminal amino groups has an average molecular weight of 200 Da to 5000 Da.

4. The method for preparing the macromolecular JAK inhibitor according to claim 2, characterized in that: The acid-binding agent is selected from one or a combination of two of triethylamine, N,N-diisopropyl ethylamine, sodium carbonate and pyridine.

5. The method for preparing the macromolecular JAK inhibitor according to claim 2, characterized in that: The organic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide and 1,4-dioxane.

6. The method for preparing the macromolecular JAK inhibitor according to claim 2, characterized in that: The molar ratio of the amount of hexachlorocyclotriphosphazene to the amount of polyethylene glycol containing terminal amino groups is 1:0.8 to 1:3, and the molar ratio of the amount of cyanuric chloride to the amount of polyethylene glycol containing terminal amino groups is 1:0.8 to 1:

2.

7. The method for preparing the macromolecular JAK inhibitor according to claim 2, characterized in that: The molar ratio of the amount of hexachlorocyclotriphosphazene to the amount of 3-amino-benzene dicarboxylic hydrazide is 1:4 to 1:6, and the molar ratio of the amount of cyanuric chloride to the amount of 3-amino-benzene dicarboxylic hydrazide is 1:2 to 1:

4.

8. Use of the macromolecular JAK inhibitor of claim 1 in the preparation of a medicament for treating acute or chronic inflammation or a disease related to the JAK signaling pathway.

9. A medicament for treating acute or chronic inflammation or inhibiting JAK signaling pathway, characterized in that: The macromolecular JAK inhibitor of claim 1, or a pharmaceutically acceptable salt thereof.

Images