PAD4 inhibitor and synovial membrane targeting peptide conjugate as well as preparation method and application thereof

Abstract

The invention provides a PAD4 inhibitor and synovial membrane targeting peptide conjugate as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The PAD4 inhibitor and synovial membrane targeting peptide conjugate provided by the invention has a synovial membrane targeting peptide structure, and can enter and be accumulated in joint tissues through systemic circulation after administration. The linker used in the invention has a chemical bond which can be split under an acidic or oxidation condition, and can quickly release ZT-5 in a responsive manner under low pH and high oxidation conditions in an arthritis inflammation microenvironment, so that the effect of resisting rheumatoid arthritis is achieved. Meanwhile, the ZT-5 can be enriched into the facet joint cavity by utilizing the excessively proliferated synovial tissue, so that the bioavailability of the ZT-5 is improved, the administration frequency is reduced, and the defect of poor pharmacokinetic performance of a PAD4 inhibitor based on a Cl-amidine structure is overcome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a PAD4 inhibitor-synovial-targeting peptide conjugate, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint pain, swelling, and stiffness. Although the exact cause of RA is not fully understood, research indicates that both the innate and adaptive immune systems are involved in different stages of the disease. Various cell types, including T cells, B cells, neutrophils, macrophages, and fibroblast-like synovial cells, are involved in the development and progression of RA. Among these cells, the macrophage-neutrophil axis has been shown to be a key factor in the development and progression of RA.

[0003] In the acute phase of rheumatoid arthritis, the formation of citrullinated proteins acts as a self-antigen, activating macrophages and causing them to release cytokines, thus inducing inflammation. Neutrophils are recruited to the inflamed joints by these cytokines. Neutrophils can also release a network of structures called NETs, ​​consisting of DNA, histones, and other particulate proteins. These molecules can lead to further immune responses and accelerate disease progression. Upon activation, neutrophils extensively citrullinate histones, a key step in NET release. Furthermore, in synovial tissue, NETs mediate articular cartilage damage and increase cartilage immunogenicity by promoting the citrullination of cartilage components. Studies have found that peptidyl arginine deiminase 4 (PAD4) is the only PAD isoenzyme in the PAD family to carry a nuclear localization sequence, enabling it to bind to a variety of substrates in the body. PAD4 also induces inflammation through the NFκB pathway by modifying proteins via citrullination. Mounting evidence suggests that PAD4-mediated protein citrullination and NET formation play a crucial role in rheumatoid arthritis (RA), promoting its development and progression. Therefore, developing highly effective and selective PAD4 inhibitors is of paramount importance for the treatment of RA. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a PAD4 inhibitor-synovial-targeting peptide conjugate, its preparation method, and its application. The PAD4 inhibitor-synovial-targeting peptide conjugate provided by this invention, after administration, can enter and accumulate in small joint tissues via systemic circulation, and rapidly release ZT-5 using the low pH and high oxidation conditions of the arthritic inflammatory microenvironment, thereby achieving an anti-rheumatoid arthritis effect.

[0005] To achieve the aforementioned objectives, this invention provides a PAD4 inhibitor conjugate with a synovial targeting peptide, having the structure shown in Formula I, abbreviated as STP2. The PAD4 inhibitor conjugate with a synovial targeting peptide provided by this invention has a drug molecule-linker-targeting peptide structure, wherein the drug molecule is ZT-5, a peptidyl arginine deiminase 4 (PAD4) inhibitor containing a Cl-amidine structure, and the targeting peptide is a synovial targeting peptide with the amino acid sequence Phe-Ile-Glu-Trp (SEQ ID NO.1: FIEW). In rheumatoid arthritis, synovial tissue exhibits aggressive behavior characterized by excessive proliferation, erosion, and damage to the joint cavity, playing a positive role in the pathogenesis and progression of the disease. Due to its synovial targeting peptide structure, this invention, after administration, can enter and accumulate in the small joints of the hands and feet (especially the proximal interphalangeal joints, metacarpophalangeal joints, wrist joints, and metatarsophalangeal joints of the foot, and can also gradually affect larger joints such as the elbow, shoulder, and knee). The linker of this invention has a hydrazone bond structure, which is a chemical bond that can be broken under acidic or oxidative conditions. Under the low pH and high oxidative conditions of the inflammatory microenvironment of arthritis, it can rapidly and responsively release ZT-5, achieving an anti-rheumatoid arthritis effect. Simultaneously, this invention can utilize excessively proliferating synovial tissue to enrich ZT-5 into the small joint cavity, improving the bioavailability of ZT-5, reducing the frequency of administration, and overcoming the poor pharmacokinetic performance of PAD4 inhibitors based on the Cl-amidinium structure.

[0006] The results of the examples show that the PAD4 inhibitor ZT-5 of the present invention and its conjugate with the synovial targeting peptide STP2 exhibit strong anti-inflammatory activity in the LPS-induced RAW264.7 cell line inflammation model, and show good anti-inflammatory effects in the mouse collagen-induced arthritis model and the carrageenan-induced rat paw edema model. Attached Figure Description

[0007] Figure 1 A synthetic route for the fully protective synovial targeting peptide STP; Figure 2 Synthetic route for the PAD4 inhibitor ZT-5; Figure 3 ZT-5, a PAD4 inhibitor 1 H NMR spectrum; Figure 4 Mass spectrum of AD4 inhibitor conjugate with synovial-targeting peptide STP2; Figure 5 The results of MTT assay for the cytotoxicity of different concentrations of STP2 on RAW264.7 cells were obtained. Figure 6 Results of the effect of STP2 on the transcription of inflammatory factors; Figure 7The results of ZT-5 and STP2 inhibiting oxidative stress in RAW264.7 cells; Figure 8 Changes in the transcriptional level of the iNOS gene in cells; Figure 9 NO level in cell supernatant; Figure 10 The preparation process and self-healing effect of PT hydrogel; Figure 11 The swelling and degradation results of the PT hydrogel; Figure 12 Establishing a CIA mouse model and administering medication to CIA mice; Figure 13 Clinical manifestations and scores of CIA mice; Figure 14 This describes the clinical manifestation of carrageenan-induced paw edema in rats. Figure 15 H&E staining was performed on the epithelial tissue of the mouse paw. Detailed Implementation

[0008] This invention provides a PAD4 inhibitor conjugate with a synovial-targeting peptide, having the structure shown in Formula I: Formula I.

[0009] This invention provides a method for preparing the above-mentioned PAD4 inhibitor-synovial-targeting peptide conjugate, comprising the following steps: Under acidic conditions, the fully deprotected synovial targeting peptide STP with the structure shown in Formula 1 is coupled with the PAD4 inhibitor ZT-5 with the structure shown in Formula 2 to obtain a PAD4 inhibitor-synovial targeting peptide conjugate with the structure shown in Formula 1. Formula 1; Formula 2.

[0010] In this invention, the molar ratio of the synovial deprotection targeting peptide STP to the PAD4 inhibitor ZT-5 is preferably 1:1~2, more preferably 0.58:0.7. In this invention, the acidic environment is preferably provided by trifluoroacetic acid, and the pH value of the acidic environment is preferably 0~1. In this invention, the coupling reaction temperature is 20~25°C, more preferably room temperature, and the time is 48~72 h, more preferably 48~60 h.

[0011] In this invention, the method for preparing the fully deprotected synovial targeting peptide STP having the structure shown in Formula 1 preferably includes the following steps: A compound having the structure shown in formula a undergoes a deBoc protection reaction to give a compound having the structure shown in formula b. A compound having the structure shown in formula b undergoes a ring-opening amidation reaction with succinic anhydride to obtain a compound having the structure shown in formula c. In the presence of an activator, a compound having the structure shown in formula c undergoes an acylation reaction with benzyl hydrazide formate to obtain a fully protected synovial targeting peptide STP having the structure shown in formula d. The fully protected synovial target peptide STP with the structure shown in Formula d was subjected to a complete deprotection reaction to obtain the fully deprotected synovial target peptide STP with the structure shown in Formula 1. Formula a; Formula b; Formula c; Formula d.

[0012] In this invention, a compound having the structure shown in formula a undergoes a deprotection reaction to obtain a compound having the structure shown in formula b. In this invention, the deprotection reagent for the deprotection reaction is preferably an HCl / EA solution, and the deprotection reaction is preferably carried out under ice bath conditions for a time preferably 2-6 h, more preferably 4 h.

[0013] In this invention, a compound having the structure shown in formula b undergoes a ring-opening amidation reaction with succinic anhydride to obtain a compound having the structure shown in formula c. In this invention, the molar ratio of the compound having the structure shown in formula b to succinic anhydride is preferably 1:1 to 4, more preferably 1:1.5 to 2; the solvent used in the ring-opening amidation reaction is preferably dichloromethane; the pH value of the ring-opening amidation reaction is preferably 8 to 10; the reaction temperature is preferably room temperature; and the reaction time is preferably 1 to 6 h, more preferably 3 to 4 h.

[0014] In this invention, in the presence of an activator, a compound having the structure shown in formula c undergoes an acylation reaction with benzyl hydrazide to obtain a fully protected synovial targeting peptide STP having the structure shown in formula d. In this invention, the activator is preferably HOBt and EDCI, and the molar ratio of HOBt to EDCI is preferably 1:1. Preferably, the invention first performs an activation reaction, followed by an acylation reaction. The activation reaction is preferably carried out under ice bath conditions for 30 minutes. The molar ratio of the compound having the structure shown in formula c to benzyl hydrazide is preferably 0.2~1:1, more preferably 0.5~0.8:1. The pH value of the acylation reaction is preferably 6~8, more preferably 7~8. The temperature of the acylation reaction is preferably room temperature, and the time is preferably overnight.

[0015] In this invention, a fully protected synovial targeting peptide STP having the structure shown in Formula d undergoes a complete deprotection reaction to obtain a fully deprotected synovial targeting peptide STP having the structure shown in Formula 1. In this invention, the deprotection catalyst used in the complete deprotection reaction is preferably Pd / C, and the complete deprotection is preferably carried out under hydrogen conditions for a time preferably 2-6 hours, more preferably 4-5 hours.

[0016] In this invention, the preparation method of the PAD4 inhibitor ZT-5 having the structure shown in Formula 2 preferably includes the following steps: In the presence of an activator, Cbz-Orn(Boc)-OH undergoes a first condensation reaction with benzylamine to give compound ZT-1. Compound ZT-1 underwent a deCbz protection reaction to yield compound ZT-2; In the presence of an activator, compound ZT-2 undergoes a second condensation reaction with p-formylbenzoic acid to obtain compound ZT-3; Compound ZT-3 underwent a deBoc protection reaction to yield compound ZT-4; Under alkaline conditions, compound ZT-4 undergoes a substitution reaction with ethyl 2-chloroacetylimine to obtain ZT-5, a PAD4 inhibitor with the structure shown in Formula 2. ZT-1; ZT-2; ZT-3; ZT-4.

[0017] In the presence of an activator, Cbz-Orn(Boc)-OH undergoes a first condensation reaction with benzylamine to yield compound ZT-1. In this invention, the activator is preferably HOBt and DCC, with a preferred molar ratio of HOBt to DCC of 1:1; the solvent for the first condensation reaction is preferably tetrahydrofuran; the molar ratio of Cbz-Orn(Boc)-OH to benzylamine is preferably 8-10 mmol:1-1.2 mL; the pH of the first condensation reaction is preferably 8-10, more preferably 8-9; the temperature is preferably room temperature; and the reaction time is preferably overnight.

[0018] In this invention, compound ZT-1 undergoes a deprotection reaction to obtain compound ZT-2. In this invention, the deprotection catalyst used in the deprotection reaction is preferably Pd / C, and the deprotection reaction is preferably carried out under hydrogen conditions for a time of 2-6 hours, more preferably 4-5 hours.

[0019] In this invention, compound ZT-2 undergoes a second condensation reaction with p-formylbenzoic acid in the presence of an activator to obtain compound ZT-3. In this invention, the activator is preferably HOBt and DCC, with a preferred molar ratio of HOBt to DCC of 1:1; the solvent used in the second condensation reaction is preferably tetrahydrofuran. In this invention, the molar ratio of compound ZT-2 to p-formylbenzoic acid is preferably 1:1 to 3, more preferably 1:2; the pH value of the second condensation reaction is preferably 8 to 10, more preferably 8 to 9; the temperature is preferably room temperature; and the time is preferably overnight.

[0020] In this invention, compound ZT-3 undergoes a deprotection reaction to obtain compound ZT-4. The deprotection reagent used in this invention is preferably an HCl / EA solution, and the deprotection reaction is preferably carried out under ice bath conditions for a time of 2-6 h, more preferably 3-5 h.

[0021] In this invention, compound ZT-4 undergoes a substitution reaction with ethyl 2-chloroacetylimine under an alkaline environment to obtain the PAD4 inhibitor ZT-5 having the structure shown in Formula 2. In this invention, the molar ratio of compound ZT-4 to ethyl 2-chloroacetylimine is preferably 1:1 to 3, more preferably 1:2; the solvent for the substitution reaction is preferably methanol; the pH of the alkaline environment is preferably 8 to 10, more preferably 9 to 10; and the alkaline reagent providing the alkaline environment is preferably N,N-diisopropylethylamine. In this invention, the temperature of the substitution reaction is preferably room temperature, and the reaction time is preferably overnight.

[0022] This invention provides the application of the above-mentioned PAD4 inhibitor and synovial-targeting peptide conjugate in the preparation of an anti-rheumatoid arthritis drug. In this invention, the anti-rheumatoid arthritis drug is preferably a drug for the prevention or treatment of anti-rheumatoid arthritis.

[0023] The present invention provides a pharmaceutical composition comprising the above-mentioned PAD4 inhibitor and synovial-targeting peptide conjugate, and a pharmaceutically acceptable carrier, diluent or excipient.

[0024] In this invention, the dosage form of the pharmaceutical composition is an injectable hydrogel formulation. Preferably, the injectable hydrogel is a PT hydrogel, and the preparation method of the PT hydrogel preferably includes the following steps: PVA aqueous solution and TSPBA aqueous solution were mixed to obtain PT hydrogel.

[0025] In this invention, the degree of alcoholysis of the PVA is preferably 87.0~89.0% (mol / mol), and the mass concentration of the PVA aqueous solution is preferably 9%; the mass concentration of the TSPBA (N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine) aqueous solution is preferably 3%; and the volume ratio of the PVA aqueous solution to the TSPBA aqueous solution is preferably 2:1~1:3, more preferably 1:1.

[0026] The following detailed description, in conjunction with embodiments, illustrates the PAD4 inhibitor and synovial-targeting peptide conjugate provided by the present invention, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 (1) Synthesis of PAD4 inhibitor ZT-5 according to Figure 2 The route shown is for synthesizing the PAD4 inhibitor ZT-5, and the method is as follows: ① Synthesis of Cbz-Orn(Boc)-NBzl(ZT-1) Accurately weigh 3.660 g (10 mmol) of Cbz-Orn(Boc)-OH using the weight reduction method and place it in a flask. Dissolve it completely in 100 mL of anhydrous tetrahydrofuran (THF) until the solution is clear and transparent. Add a stir bar and, under ice bath conditions, add 1.620 g (12 mmol) of 1-hydroxybenzotriazole (HOBt) and 2.472 g (12 mmol) of dicyclohexylcarbodiimide (DCC) sequentially. Activate for 30 min, and the solution will become white and turbid. Then, add 1.2 mL of benzylamine to the flask and adjust the pH to 8 with N-methylmorpholine (NMM). Remove the ice bath and stir the reaction overnight at room temperature (25 °C). Monitor the reaction by thin-layer chromatography (TLC) with a developing solvent of petroleum ether (PE): ethyl acetate (EA): glacial acetic acid (HAc) = 2:1:0.1 and Rf = 0.4. When the reaction of the starting material Cbz-Orn(Boc)-OH is observed to be basically complete, the reaction is considered complete. After the reaction was completed, the product was concentrated to dryness under reduced pressure to remove THF, and then reconstituted with 150 mL of EA. The product was then sonicated to completely dissolve it, but a white solid remained insoluble. The solution was filtered under reduced pressure using a vacuum circulating water pump, and the filter cake was rinsed with EA. The filtrate was transferred to a 250 mL separatory funnel and successively extracted with saturated NaHCO3 solution (60 mL / time), saturated NaCl solution (60 mL / time), 5 wt% KHSO4 solution (60 mL / time), saturated NaCl solution (60 mL / time), 5 wt% NaHCO3 solution (60 mL / time), and saturated NaCl solution (60 mL / time). During the extraction process, the EA layer gradually changed from yellow to colorless. The EA layer was dried with anhydrous sodium sulfate for 2 hours, and the sodium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a colorless oily substance, which was recrystallized with a PE:EA mixed solvent of 2:1 to obtain 4.111 g (90.3%) of the target product Cbz-Orn(Boc)-NBzl, a white solid powder. ESI-MS (m / z): 456.78 [M+H] + .

[0028] ② Synthesis of H-Orn(Boc)-NBzl (ZT-2) 2.000 g (4.4 mmol) of Cbz-Orn(Boc)-NBzl was dissolved in 150 mL of methanol (MeOH) until colorless, clear, and transparent. Under stirring, 0.200 g of Pd / C was added to a flask, a single-way valve was connected, and the air in the flask was evacuated as much as possible using a vacuum circulating water pump. Then, a hydrogen line was connected, and the pressure was adjusted to 0.02 MPa. The reaction was allowed to proceed for 6 h. The reaction progress was monitored by TLC (developing solvent: PE:EA:HAc = 2:1:0.1, product Rf < 0.1). The reaction was considered complete when the starting material spot disappeared. Pd / C was removed by vacuum filtration, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.285 g (90.9%) of the target product Orn(Boc)-NBzl, a white solid. ESI-MS (m / z): 322.34 [M+H] + .

[0029] ③ Synthesis of ZT3 Accurately weigh 0.720 g (4.8 mmol) of p-formylbenzoic acid (FBA) using the weight reduction method and place it in a flask. Dissolve it completely in 100 mL of THF until the solution is clear and transparent. Add a stir bar and, under ice bath conditions, add 0.648 g (4.8 mmol) of HOBt and 0.998 g (4.8 mmol) of DCC sequentially. Activate for 30 min until the solution becomes white and turbid. Then add 1.285 g of Orn(Boc)-NBzl (4 mmol) to the flask and adjust the pH to 8 with NMM. Remove the ice bath and stir the reaction overnight at room temperature (25°C). Monitor the reaction by thin-layer chromatography (TLC) with a developing solvent of PE:EA:HAc = 2:1:0.1 and Rf = 0.6. Ninhydrin staining showed that Orn(Boc)-NBzl had reacted almost completely, indicating the end of the reaction. After the reaction was completed, the product was concentrated to dryness under reduced pressure to remove THF, and then reconstituted with 150 mL of EA. The product was then sonicated to completely dissolve it, but a white solid remained insoluble. The solution was filtered under reduced pressure using a vacuum circulating water pump, and the filter cake was rinsed with EA. The filtrate was transferred to a 250 mL separatory funnel and successively washed three times with saturated NaHCO3 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% KHSO4 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% NaHCO3 solution (60 mL / time), and three times with saturated NaCl solution (60 mL / time). During the extraction process, the EA layer gradually changed from yellow to colorless. The EA layer was dried with anhydrous sodium sulfate for 2 hours, and the sodium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a colorless oily substance, which was then purified by a medium-pressure preparative column (the purification reagent used was a mixture of MeOH and dichloromethane, with a MeOH volume fraction of 6%) to obtain 1.126 g (62.1%) of the target product ZT-3, which was a white solid. ESI-MS (m / z): 454.71 [M+H] + .

[0030] ④ Synthesis of ZT-4 Weigh 0.906 g (2 mmol) of dried ZT-3 and place it in a flask. In a fume hood, add 20 mL of 4 N HCl / EA solution under ice bath conditions. The compound is initially soluble in 4 N HCl / EA solution. Attach a drying tube to the flask and react for 4 h with stirring in an ice bath. A white solid precipitates in the flask. Scrape a portion from the flask with a plastic spoon, dissolve it in MeOH, and monitor the reaction progress by TLC (by volume ratio, with CH2Cl2:CH3OH = 15:1 as the developing solvent, Rf < 0.1). Once the starting material spot disappears, the reaction is considered complete. After the reaction was completed, the reaction solution was depressurized and dried under warm water bath conditions by connecting a single universal vacuum circulating water pump with a valve to the flask. The residue was added to 30 mL of dry EA and dried again under stirring in a 50 ℃ water bath. This operation was repeated 3 times. Then, 30 mL of dry petroleum ether was added and dried under stirring in a 50 ℃ water bath. This operation was repeated 3 times until there was no obvious acid gas residue, and 0.729 g (93.5%) of the target product HCl·ZT-4 was obtained as a pale yellow solid powder.

[0031] ⑤ Synthesis of ZT-5 0.729 g (1.87 mmol) of HCl·ZT-4, after being dried, was dissolved in 50 mL of anhydrous MeOH until clear and transparent. A stir bar was added, and 0.591 g (3.74 mmol) of 2-chloroacetylimine ethyl ester (CAS No.: 36743-66-5) was added under ice bath conditions. The pH was adjusted to 10 with N,N-diisopropylethylamine (DIPEA). The ice bath was removed, and the reaction was allowed to proceed overnight at room temperature. The reaction progress was monitored by TLC (by volume, with EA:H2O:HAc = 4:1:1 as the developing solvent and Rf = 0.3). The reaction was considered complete after the ninhydrin spot disappeared. After the reaction solution was concentrated under reduced pressure, it was separated and purified by a C18 medium-pressure preparative column (the purification reagent used was a mixture of MeOH and deionized water, with a volume fraction of 20% for MeOH). After removing MeOH by concentration under reduced pressure, it was freeze-dried in a vacuum freeze dryer to remove water, yielding 0.514 g (64.2%) of the target product ZT-5, which was a pale yellow solid powder.

[0032] ESI-MS (m / z): 429.71 [M+H] +, 1H NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H),9.64 (s, 1H), 9.24 (s, 1H), 8.64 (dd, J = 6.9, 2.8 Hz, 2H), 8.04 – 7.41 (m,4H), 7.35 – 7.18 (m, 5H), 4.53 (dt, J = 8.2, 4.2 Hz, 1H), 4.46 (s, 2H), 4.30 (d, J = 5.9 Hz, 2H), 1.99 – 1.79 (m, 2H), 1.63 (dd, J = 19.1, 11.5 Hz, 2H).

[0033] The obtained PAD4 inhibitor ZT-5 1 H NMR image as follows Figure 3 As shown (300 MHz, DMSO-d6) (2) Synthesis of STP, a fully protective synovial targeting peptide according to Figure 1 The route shown is for synthesizing the fully protective synovial targeting peptide STP, and the method is as follows: ① Synthesis of Boc-FI-OBzl 2.651 g (10 mmol) of Boc-F-OH was accurately weighed and placed in a flask using the weight reduction method. It was completely dissolved in 100 mL of anhydrous THF until the solution was clear and transparent. A stir bar was added, and under ice bath conditions, 1.620 g (12 mmol) of HOBt and 2.472 g (12 mmol) of DCC were added sequentially. Activation was performed for 30 min, and the solution became white and turbid. Then, 4.552 g (12 mmol) of Tos·HI-OBzl was added to the flask, and the pH was adjusted to 8 using NMM. The ice bath was removed, and the reaction was stirred overnight at room temperature (25°C). The reaction was monitored by TLC, with the developing solvent being PE:EA:HAc = 2:1:0.1 and Rf = 0.7. The reaction was considered complete when the starting material Boc-F-OH was observed to have reacted substantially. After the reaction, the solution was concentrated to dryness under reduced pressure to remove THF, and then reconstituted with 150 mL of EA. The product was then sonicated to completely dissolve, although a white solid remained undissolved. The solution was filtered under reduced pressure using a vacuum circulating water pump, and the filter cake was rinsed with EA. The filtrate was transferred to a 250 mL separatory funnel and successively washed three times with saturated NaHCO3 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% KHSO4 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% NaHCO3 solution (60 mL / time), and three times with saturated NaCl solution (60 mL / time). During the extraction process, the EA layer gradually changed from yellow to colorless. The EA layer was dried with anhydrous sodium sulfate for 2 hours, and the sodium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a colorless oily substance, which was then purified by separation using a medium-pressure preparative column (eluent: EA:PE = 1:2) to obtain 3.740 g (79.9%) of the target product Boc-FI-OBzl, which was a white solid.

[0034] ②Synthesis of Boc-FI-OH 2.060 g (4.4 mmol) of Boc-FI-OBzl was dissolved in 150 mL of MeOH until it became colorless, clear, and transparent. Under stirring, 0.200 g of Pd / C was added to a flask, a single-way valve was connected, and the air in the flask was evacuated as much as possible using a vacuum circulating water pump. Then, a hydrogen pipeline was connected, and the pressure reducing valve was adjusted to 0.02 MPa. The reaction was allowed to proceed for 6 h. The reaction progress was monitored by TLC (developing solvent: PE:EA:HAc = 2:1:0.1, product Rf < 0.1). The reaction was considered complete when the starting material spot disappeared. Pd / C was removed by vacuum filtration, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.590 g (95.6%) of the target product Boc-FI-OH, which was a white solid.

[0035] ③ Synthesis of Boc-E(OBzl)W-OBzl 3.371 g (10 mmol) of Boc-E(OBzl)-OH was accurately weighed and placed in a flask using the weight reduction method. It was completely dissolved in 100 mL of THF until the solution was clear and transparent. A stir bar was added, and under ice bath conditions, 1.620 g (12 mmol) of HOBt and 2.472 g (12 mmol) of DCC were added sequentially. After activation for 30 min, the solution became white and turbid. Then, 3.960 g (12 mmol) of HCl·HW-OBzl was added to the flask, and the pH was adjusted to 8 with NMM. The ice bath was removed, and the reaction was stirred overnight at room temperature (25°C). The reaction was monitored by TLC, with the developing solvent being PE:EA:HAc = 2:1:0.1 and Rf = 0.5. The reaction was considered complete when the starting material Boc-E(OBzl)-OH was observed to have reacted almost completely. After the reaction, the solution was concentrated to dryness under reduced pressure to remove THF, and then reconstituted with 150 mL of EA. The product was then sonicated to completely dissolve, although a white solid remained undissolved. The solution was filtered under reduced pressure using a vacuum circulating water pump, and the filter cake was rinsed with EA. The filtrate was transferred to a 250 mL separatory funnel and successively washed three times with saturated NaHCO3 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% KHSO4 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% NaHCO3 solution (60 mL / time), and three times with saturated NaCl solution (60 mL / time). During the extraction process, the EA layer gradually changed from yellow to colorless. The EA layer was dried with anhydrous sodium sulfate for 2 hours, and the sodium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a colorless oily substance, which was then purified by separation using a medium-pressure preparative column (eluent: EA:PE = 1:2) to obtain 5.361 g (87.4%) of the target product Boc-E(OBzl)W-OBzl, which was a light pink solid.

[0036] ④ Synthesis of H-E(OBzl)W-OBzl Weigh 3.066 g (5 mmol) of dried Boc-E(OBzl)W-OBzl into a flask. In a fume hood, under ice bath conditions, add 50 mL of 4 N HCl / EA solution. The compound is initially soluble in the 4 N HCl / EA solution. Attach a drying tube to the flask and react for 4 h with stirring in an ice bath. A pale red solid precipitates in the flask, and the solution turns purplish-red. Scrape a portion from the flask with a plastic spoon, dissolve it in MeOH, and monitor the reaction progress by TLC (by volume, with PE:EA:HAc = 2:1:0.1, Rf < 0.1). Once the starting material spot disappears, the reaction is considered complete. After the reaction was completed, the reaction solution was depressurized and dried under warm water bath conditions by connecting a single universal vacuum circulating water pump with a valve to the flask. The residue was added to 30 mL of dry EA and dried again under stirring in a 50 ℃ water bath. This operation was repeated 3 times. Then, 30 mL of dry petroleum ether was added and dried under stirring in a 50 ℃ water bath. This operation was repeated 3 times until there was no obvious acid gas residue. 2.483 g (90.3%) of the target product HCl·HE(OBzl)W-OBzl was obtained as a purple-red solid powder.

[0037] ⑤ Synthesis of Boc-FIE(OBzl)W-OBzl 1.512 g (4 mmol) of Boc-FI-OH was accurately weighed and placed in a flask using the weight reduction method. It was completely dissolved in 100 mL of THF until the solution was clear and transparent. A stir bar was added, and under ice bath conditions, 0.648 g (4.8 mmol) of HOBt and 0.988 g (4.8 mmol) of DCC were added sequentially. After activation for 30 min, the solution became white and turbid. Then, 2.309 g (4.2 mmol) of HCl·HE(OBzl)W-OBzl was added to the flask, and the pH was adjusted to 8 with NMM. The ice bath was removed, and the reaction was stirred overnight at room temperature (25°C). The reaction was monitored by TLC, with the developing solvent being PE:EA:HAc = 2:1:0.1 and Rf = 0.65. The reaction was considered complete when the starting material Boc-FI-OH was observed to have reacted almost completely. After the reaction, the solution was concentrated to dryness under reduced pressure to remove THF, and then reconstituted with 150 mL of EA. The product was then sonicated to completely dissolve, but a white solid remained undissolved. The solution was filtered under reduced pressure using a vacuum circulating water pump, and the filter cake was rinsed with EA. The filtrate was transferred to a 250 mL separatory funnel and successively washed three times with saturated NaHCO3 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% KHSO4 solution (60 mL / time), three times with saturated NaCl solution (60 mL / time), three times with 5 wt% NaHCO3 solution (60 mL / time), and three times with saturated NaCl solution (60 mL / time). During the extraction process, the EA layer gradually changed from yellow to colorless. The EA layer was dried with anhydrous sodium sulfate for 2 hours, and the sodium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a colorless oily substance, which was then purified by separation using a medium-pressure preparative column (eluent: EA:PE = 1:2) to obtain 3.040 g (87.0%) of the target product Boc-FIE(OBzl)W-OBzl, which was a white solid.

[0038] ⑥ Synthesis of D-FIE(OBzl)W-OBzl Weigh 0.903 g (1.03 mmol) of dried Boc-FIE(OBzl)W-OBzl into a flask. In a fume hood, under ice bath conditions, add 10 mL of 4 N HCl / EA solution. The compound is initially soluble in the 4 N HCl / EA solution. Attach a drying tube to the flask and react for 4 h with stirring in an ice bath. A pale red solid precipitates in the flask, and the solution turns purplish-red. Scrape a portion from the flask with a plastic spoon, dissolve it in MeOH, and monitor the reaction progress by TLC (by volume ratio, with PE:EA:HAc = 2:1:0.1, Rf < 0.1). Once the starting material spot disappears, the reaction is considered complete. After the reaction was completed, the reaction solution was depressurized and dried under warm water bath conditions by connecting a single universal vacuum circulating water pump with a valve to the flask. The residue was then added to 30 mL of dry EA and dried again under stirring in a 50 ℃ water bath. This operation was repeated 3 times. Then, 30 mL of dry petroleum ether was added and dried under stirring in a 50 ℃ water bath. This operation was repeated 3 times until there was no obvious acid gas residue.

[0039] ⑦ Add 30 mL of anhydrous dichloromethane (DCM) and dissolve it completely until the solution is clear and transparent. Add 0.150 g (1.5 mmol) of succinic anhydride under ice bath conditions and adjust the pH to 10 with DIPEA. Remove the ice bath and react at room temperature for 3 h. Monitor the reaction progress by TLC (by volume ratio, developing solvent is DCM:MeOH:HAc = 15:1:1, Rf = 0.33). The reaction is considered complete when the starting material spot disappears after ninhydrin is observed to develop color. The reaction solution was transferred to a 100 mL separatory funnel and washed three times (20 mL each time) with 5 wt% KHSO4 solution and three times (20 mL each time) with saturated NaCl solution. A white flocculent precipitate will appear during the extraction process. The precipitate can be collected by centrifugation. The reaction solution was dried with anhydrous sodium sulfate for 0.5 h and filtered to remove sodium sulfate. The filtrate was concentrated under reduced pressure to obtain a colorless solid. The solid collected by centrifugation and the solid obtained by concentration were separated and purified by a medium-pressure preparative column (the eluent used was a mixed solution of DCM:MeOH:HAc = 90:9.9:0.1). After concentration under reduced pressure, 0.669 g (74.1%) of the target product D-FIE(OBzl)W-OBzl was obtained as a pale yellow solid powder.

[0040] ⑧ Synthesis of STP, a fully protective synovial targeting peptide Accurately weigh 0.669 g (0.77 mmol) of D-FIE(OBzl)W-OBzl into a flask, dissolve it completely in 25 mL of THF until the solution is clear and transparent, add a stir bar, and under ice bath conditions, add 0.135 g (1 mmol) of HOBt and 0.191 g (1 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) sequentially, activate for 30 min, then add 0.166 g (1 mmol) of benzyl hydrazide formate to the flask, and adjust the pH to 8 with NMM. Remove the ice bath, and stir the reaction overnight at room temperature (25℃); monitor the reaction by TLC, with the developing solvent being DCM:MeOH:HAc = 15:1:1, Rf = 0.57; observe that the starting material D-FIE(OBzl)W-OBzl has basically reacted completely, and judge the reaction to be finished. After the reaction was completed, the reaction solution was transferred to a 100 mL separatory funnel and successively washed three times (20 mL each time) with saturated NaHCO3 solution, three times (20 mL each time) with saturated NaCl solution, three times (20 mL each time) with 5 wt% KHSO4 solution, three times (20 mL each time) with saturated NaCl solution, three times (20 mL each time) with 5 wt% NaHCO3 solution, and three times (20 mL each time) with saturated NaCl solution. During the extraction process, a white flocculent precipitate will appear. The precipitate can be collected by centrifugation. The organic layer is dried with anhydrous sodium sulfate for 0.5 h by stirring. The sodium sulfate is removed by filtration. The filtrate is concentrated under reduced pressure to obtain a colorless solid. The solid collected by centrifugation and the solid obtained by concentration are separated and purified by medium-pressure preparative column (the eluent used is a mixed solution of DCM:MeOH:HAc = 90:9.9:0.1). After concentration under reduced pressure, 0.671 g (85.8%) of the target product STP is obtained, which is a light pink solid powder.

[0041] 1H NMR (300 MHz, DMSO-d6) δ 10.86 (s, 1H), 9.67 (s, 1H), 9.11 (s,1H), 8.42 (d, J = 6.9 Hz, 1H), 8.11 (d, J = 8.2 Hz, 1H), 8.03 (d, J = 7.8 Hz,1H), 7.93 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 3.0 Hz,6H), 7.34 – 7.14 (m, 14H), 7.14 – 7.10 (m, 3H), 7.10 – 7.02 (m, 2H), 6.96(ddd, J 4.38 (q, J =7.4 Hz, 1H), 4.16 (t, J = 8.1 Hz, 1H), 3.20 – 3.03 (m, 2H), 2.98 (dd, J =13.9, 4.2 Hz, 1H), 2.71 (dd, J = 14.0, 9.7 Hz, 1H), 2.35 (dt, J = 16.5, 7.9Hz, 3H), 2.24 (d, J = 13.1 Hz, 3H), 1.87 (ddd, J = 30.5, 14.0, 7.6 Hz, 3H), 1.71 (d, J = 8.3 Hz, 1H), 1.05 (dt, J = 14.3, 7.8 Hz, 1H), 0.78 (t, J = 6.6Hz, 6H).

[0042] (3) Synthesis of PAD4 inhibitor and synovial-targeting peptide conjugate STP2 ① STP deprotection 0.671 g (0.65 mmol) of STP was dissolved in 20 mL of MeOH until it became colorless, clear, and transparent. Under stirring, 0.060 g of Pd / C was added to a flask, a single-way valve was connected, and the air in the flask was evacuated as much as possible using a vacuum circulating water pump. Then, a hydrogen line was connected, and the pressure reducing valve was adjusted to 0.02 MPa. The reaction was allowed to proceed for 6 hours. The reaction progress was monitored by TLC (developing solvent: DCM:MeOH:HAc = 15:1:1, product Rf < 0.1). The reaction was considered complete when the starting material spot disappeared. Pd / C was removed by vacuum filtration, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.411 g (89.2%) of the target product, completely deprotected STP, as a pink solid.

[0043] ②Synthesis of STP2 0.410 g (0.58 mmol) of completely deprotected STP was dissolved in chromatographic MeOH until clear and transparent. A stir bar was added, and 0.300 g (0.7 mmol) of ZT-5 and 0.500 g of anhydrous sodium sulfate were added under ice bath conditions. The pH was adjusted to 1 with trifluoroacetic acid. The ice bath was removed, and the reaction mixture was reacted at room temperature for 48 h. The reaction solution was filtered to remove sodium sulfate, concentrated under reduced pressure, and reconstituted with a small amount of dimethyl sulfoxide (DMSO). A large amount of pure water was added, resulting in the precipitation of a pink solid. The solution and solid were transferred to a dialysis bag (M... W =500 Da), dialyzed with pure water for 96 h, the dialysate was freeze-dried in a vacuum freeze dryer to remove water, yielding 0.331 g (51.0%) of the target product STP2, as a light pink solid powder. ESI-MS (m / z): 1118.40 [M+H] + .

[0044] The mass spectrum of the obtained PAD4 inhibitor conjugate with synovial-targeting peptide STP2 is shown below. Figure 4 As shown.

[0045] Test Example 1: Evaluation of in vitro anti-inflammatory activity (a) MTT assay to detect the toxicity of the conjugate to RAW264.7 cells ①Resuscitation and culture of RAW264.7 cells (1) Add 5 mL of PBS or DMEM-HG basal medium to a 15 mL centrifuge tube; (2) Take the frozen cells out of the liquid nitrogen tank or -80℃ freezer and quickly put them into 37℃ water for water bath thawing. Transfer the thawed cells quickly to a 15 mL centrifuge tube containing PBS or DMEM-HG basic culture medium and centrifuge at 1000 rpm for 5 min at room temperature. (3) After centrifuging, discard the supernatant of the cells, add 2 mL of complete culture medium along the wall of the centrifuge tube, gently blow away the cell pellet to make a single-cell suspension, transfer it to a cell culture flask, add 2-3 mL of complete culture medium containing 10% (v / v) fetal bovine serum (FBS) to make the culture system 4-5 mL, gently shake the cell flask horizontally in the manner of "up, down, left and right" to make the cells evenly distributed in the culture medium, and put it into a 37℃ incubator containing 5% CO2 for culture.

[0046] (4) After culturing for 2-3 days, observe the cell status and the color change of the cell culture medium. If the cell supernatant contains cell debris or the culture medium is yellow, discard the original culture medium, rinse with PBS, add 4-5 mL of fresh complete culture medium, and put it back into a 37℃ incubator containing 5% CO2 for culturing.

[0047] ②Passaging of RAW264.7 cells (1) When the cell density is 80%~90%, passage the cells, discard the old culture medium in the cell culture flask, add 2 mL of PBS to each flask to rinse twice and then discard it; (2) Add 3 mL of complete culture medium, use a 1 mL pipette to gently blow the cells from the bottom of the bottle and transfer them to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature; (3) After centrifugation, resuspend and culture the cells according to the cell resuscitation method in 2.1.1.

[0048] ③Lamination of RAW264.7 cells When the cell confluence reaches 80% to 90%, cells are collected according to the cell passage method in 2.1.2. 10 μL of cell resuspension is transferred to each well of a cell counting plate, and cell counting is performed using a microscope. The cell suspension is then diluted with complete culture medium, and 8000 cells are evenly seeded into each well of a 96-well plate. Finally, the plates are incubated at 37°C in a 5% CO2 incubator for 12 h.

[0049] ④ Cryopreservation of RAW264.7 cells Once the cell confluence reaches approximately 90%, cryopreservation can be performed. Collect the cell pellet according to the cell passage method described above. Add 2 mL of serum-free cryopreservation solution to each T25 cell culture flask, gently disperse the pellet, and then transfer it to cryovials. Add 1 mL of cell suspension to each cryovial. Label the cryovials with the cell name, passage number, and cryopreservation date. Transfer the cryovials to a -80°C freezer for storage, or after the cryopreservation solution has completely frozen, transfer the cryovials to a liquid nitrogen tank for long-term storage.

[0050] ⑤ MTT assay for cell viability (1) Prepare 100 ng / mL LPS basal medium using DMEM-HG basal medium and lipopolysaccharide (LPS). Prepare working solutions of different concentrations of the compound using the conjugate STP2 with DMEM-HG basal medium and LPS basal medium. After removing the old medium, add the newly prepared working solution and incubate for 24 h.

[0051] (2) Prepare MTT solution (50 mg / mL) using DMSO and thiazolyl blue (MTT). Prepare MTT working solution (1 mg / mL) using DMEM-HG basal medium and MTT solution. Remove the old working solution containing the compound, add 100 μL of fresh MTT working solution to each well, and continue incubation for 4 h.

[0052] (3) The absorbance at 490 and 570 nm was measured using a multi-functional microplate reader, and the results were statistically analyzed using Graphpad Prism 9.5 software. Data for each group are expressed as mean ± standard error (x ± SD). The Student's-test (unpaired, two-tailed) was used to compare significant differences between two groups, and the One-way ANOVA test was used to compare significant differences between more than two groups. <0.05, <0.01, <0.001, <0.0001. P<0.05 indicates that the difference is statistically significant.

[0053] Different concentrations of STP2 working solution without LPS and different concentrations of STP2 working solution containing LPS were added to 96-well cell culture plates, and cell proliferation activity was detected using MTT assay. The results of MTT assay for the cytotoxicity of different concentrations of STP2 on RAW264.7 cells are shown in the figure. Figure 5 As shown, Figure 5 In the table, A represents the cytotoxicity results of normal RAW264.7 cells, and B represents the cytotoxicity results of RAW264.7 cells stimulated with LPS. Figure 5 In the n=6 group, compared with the 0 μM group, <0.05, <0.01, P < 0.001, <0.0001. Figure 5 A in the figure shows that STP2 concentrations below 200 μM do not exhibit toxicity to normal RAW264.7 cells; Figure 5 The results showed that at concentrations below 200 μM, it did not exhibit toxicity to RAW264.7 inflammatory cells stimulated with 100 ng / mL LPS.

[0054] (ii) The conjugate STP2 inhibits the transcription of inflammatory factors in RAW264.7 cells. To investigate the effect of the conjugate STP2 on the transcriptional levels of inflammatory cytokines in an LPS-stimulated RAW264.7 cell inflammation model, RAW264.7 cells were stimulated with 10 μM and 20 μM STP2 working solutions containing LPS, respectively. Cellular RNA was collected after 24 h. Changes in the mRNA transcriptional levels of typical inflammatory cytokines IL-1β, IL-6, and TNF-α were detected using qRT-PCR.

[0055] The specific method is as follows: ① Passaging, seeding, and drug administration of RAW264.7 cells RAW264.7 cells were passaged, cultured, and seeded into plates according to the method described above. RAW264.7 cells were seeded into 12-well plates at a density of 1 × 10⁶ cells / well. 6 / well seed plate. Prepare 10 μM and 20 μM STP2 working solutions using 100 ng / mL LPS basal medium and incubate as described above.

[0056] ②Trizol method for extracting cellular RNA Chloroform, isopropanol, and 75% ethanol were all pre-cooled at 4°C, and the following operations were all performed on ice.

[0057] (1) Discard the culture medium in the cell culture plate; (2) Add 1000 μL Trizol Reagent to each well of a 12-well plate to completely wet the bottom of the well with the lysis buffer. Let stand for 5 min, then use a pipette to blow the cells off the bottom of the plate to completely remove them and transfer the cell lysis buffer to a 1.5 mL enzyme-free centrifuge tube. (3) Add 200 μL of pre-cooled chloroform to each tube, vortex the mixture for 30 s to ensure that the lysis solution and chloroform are thoroughly mixed, and let stand at 4℃ for 3 min until the mixture shows stratification. (4) Centrifuge at 12000 rpm for 15 min at 4℃. At this time, the liquid in the enzyme-free tube is divided into the upper colorless aqueous phase, the middle white gel and the lower pink organic phase. (5) Use a 200 μL pipette to aspirate 200 μL of the colorless upper aqueous phase twice along the liquid surface (total 400 μL) and transfer it to a new enzyme-free centrifuge tube. Be careful not to aspirate any liquid other than the aqueous phase. (6) Add an equal volume of isopropanol to each tube as to the water drawn up, invert and mix 10-15 times, and let stand at room temperature for 10 min. (7) Centrifuge at 12,000 rpm for 10 min at 4℃, discard the supernatant, add 500 μL of 75% ethanol to each tube, invert and shake to make the precipitate float; repeat twice. (8) Centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, centrifuge briefly for 30 s, use a 10 μL pipette to remove as much of the remaining 75% ethanol as possible and discard it, let it stand at room temperature to allow the ethanol to evaporate fully; (9) Add 50 μL of enzyme-free water to dissolve and mix the RNA, place it in an ice box to continue subsequent experiments or store it in a -20℃ / -80℃ refrigerator for long-term storage. (10) When using Nanodrop to detect RNA concentration and purity, RNA OD is generally required. 260 / OD 280 >1.8, and OD 260 / OD 230 ≥2.0.

[0058] ③ mRNA reverse transcription (1) Removal of genomic DNA: Prepare the working solution for removing genomic DNA in a 200 μL enzyme-free tube according to Table 1.

[0059] Table 1 Genomic DNA Removal Working Solution System

[0060] Mix all components in the enzyme-free tube and incubate at 42°C for 2 min.

[0061] (2) Reverse transcription: Prepare the reverse transcription reaction system in the enzyme-free tube after step (1) according to Table 2.

[0062] Table 2 Genomic DNA Removal Working Solution System

[0063] After mixing, set the PCR instrument to 37℃ for 15 min and 85℃ for 5 s. Place the enzyme-free tube on the PCR instrument. After the program is completed, the product can be used for qPCR reaction or stored at -20℃ for a short period of time.

[0064] ④ qRT-PCR detection of the effect of the conjugate on the transcriptional expression of inflammatory factors in LPS-stimulated RAW264.7 cells (1) mRNA primer design: The NM numbers of mouse IL-1β, IL-6, TNF-α, iNOS and mouse β-actin mRNA were found on the NCBI Gene website. The primer sequences were then searched on the NCBI primer-BLAST website and their specificity was verified. The primer sequences are shown in Table 3.

[0065] (2) The qRT-PCR reaction working solution was prepared according to Table 4. The cDNA was from the reverse transcription product of 2.2.3. The working solution was added to the qRT-PCR 96-well plate, and three auxiliary wells were set for each sample.

[0066] (3) Centrifuge the 96-well plate at 2500×g for 5 min, set the program on the qRT-PCR instrument, and the reaction conditions are shown in Table 5.

[0067] (4) Data processing: Using β-actin as an internal reference, the ΔCt value is obtained by subtracting the internal reference cycle threshold from the cycle threshold of each gene (Ct). The ΔCt value is obtained by subtracting the control group ΔCt value from the experimental group ΔCt value. Finally, 2 (-△△Ct) The value represents the relative expression level of the gene. The results were compared using a one-way ANOVA test to determine statistical significance.

[0068] Table 3 qRT-PCR primer sequences

[0069] Table 4 qRT-PCR reaction system

[0070] Table 5 qRT-PCR reaction conditions

[0071] The results of STP2's effect on the transcription of inflammatory factors are as follows: Figure 6 As shown, Figure 6 In the table, A represents the mRNA expression level of interleukin-1, B represents the mRNA expression level of interleukin-6, and C represents the mRNA expression level of tumor necrosis factor-α. Figure 6 In the group with n=3, compared to the LPS group, <0.05, <0.01, <0.001, <0.0001.

[0072] Depend on Figure 6 It can be seen that STP2 significantly reduced the mRNA transcription levels of IL-1β (P<0.0001), IL-6 (P<0.0001), and TNF-α (P<0.01) in RAW264.7 cells stimulated with 100 ng / mL LPS; and the degree of reduction in mRNA transcription levels was correlated with the concentration of STP2.

[0073] (III) ZT-5 and STP2 can inhibit oxidative stress in RAW264.7 cells. To investigate the cellular oxidative stress levels of LPS-stimulated RAW264.7 cell inflammation model induced by ZT-5 and STP2, the cells were stained with DCFH-DA fluorescent probe and then quantitatively detected using the FITC fluorescence channel of a flow cytometer. The specific method was as follows: (1) DCFH-DA working solution (10 μM) was prepared using DMEM-HG basal medium and DCFH-DA fluorescent probe solution (10 mM). The old working solution containing the compound was removed, and 1 mL of fresh DCFH-DA working solution was added to each well. The cells were then incubated for 30 min.

[0074] (2) Remove the DCFH-DA working solution, rinse the cells twice with preheated PBS, gently blow the cells down with 1 mL of PBS per well, and transfer the cells to a 5 mL flow cytometer.

[0075] (3) Cell fluorescence intensity was detected by the FITC fluorescence channel of a flow cytometer, and the results were statistically analyzed using Graphpad Prism 9.5 software. Data for each group are expressed as mean ± standard error (x ± SD).

[0076] Test results are as follows Figure 7 As shown, Figure 7 In the table, A represents cell fluorescence intensity, and B represents the statistical value of cell fluorescence intensity (n=3, compared with the LPS group). <0.05, <0.01, <0.001, <0.0001). The results showed that after 6 h of stimulation with 1 μg / mL LPS, the cell fluorescence intensity increased significantly (P < 0.0001), while the intervention of ZT-5 and STP2 (20 μM) significantly inhibited the increase in cell fluorescence intensity (P < 0.0001), indicating that both ZT-5 and STP2 can inhibit cellular oxidative stress induced by LPS stimulation. Statistical analysis of the mean fluorescence intensity of the ZT-5 group and the STP2 group revealed that STP2 had a significantly stronger inhibitory effect on cellular oxidative stress induced by LPS stimulation than ZT-5 (P < 0.05).

[0077] (iv) The PAD4 inhibitor ZT-5 and its conjugate STP2 can reduce NO levels in the supernatant of RAW264.7 cells. To investigate the mechanism by which ZT-5 and STP2 inhibit LPS-induced cellular oxidative stress, RAW264.7 cells were stimulated with 20 μM ZT-5 and STP2 working solutions containing 100 ng / mL LPS, respectively. Cellular RNA was collected after 24 h. Changes in cellular iNOS mRNA transcription levels were detected using qRT-PCR. The specific methods were as follows: RAW264.7 cells were passaged, cultured, and seeded into plates. RAW264.7 cells were seeded into 12-well plates at a density of 1 × 10⁶ cells / well. 6 / well seeding plate. Prepare 20 μM ZT-5 and STP2 working solutions using 1 μg / mL LPS basal medium and incubate for 24 h as described in 2.1. Transfer cell supernatant to 1.5 mL centrifuge tubes, centrifuge at 1200 rpm for 5 min, and then aspirate the cell supernatant. Detect the NO content in the cell supernatant directly using a Griess kit, or aliquot and store at -20°C.

[0078] Changes in cellular iNOS gene transcription levels, such as Figure 8 As shown, Figure 8 In the group with n=3, compared to the LPS group, <0.05, <0.01, <0.001, <0.0001. It can be seen that ZT-5 and STP2 significantly reduced the mRNA transcription level of iNOS in cells stimulated by LPS (P<0.001), and STP2 showed a better inhibitory trend than ZT-5, but no statistical difference was observed.

[0079] RAW264.7 cells were stimulated with 20 μM ZT-5 and STP2 working solutions containing 1 μg / mL LPS, respectively. After 24 h, cell supernatants were collected, and the NO levels in the cell supernatants were measured using a cellular nitric oxide assay kit (Griess method). The NO levels in the cell supernatants were as follows: Figure 9 As shown, Figure 9 In the group with n=3, compared to the LPS group, <0.05, <0.01, <0.001, <0.0001. It can be seen that ZT-5 and STP2 significantly reduced the NO level in the supernatant of RAW264.7 cells stimulated by LPS (P<0.0001), and STP2 had a better ability to inhibit NO production than ZT-5 (P<0.001).

[0080] Test Example 2: Preparation and Characterization of Injectable Hydrogels (1) Preparation of PT hydrogel ① Preparation of blank PT hydrogel A. Weigh 10 g of PVA1788 into a beaker, add 100 mL of distilled water, heat in a 90℃ water bath and stir until the PVA is completely dissolved to obtain a 9 wt% PVA solution.

[0081] B. Weigh 3 g of TSPBA into a beaker, add 100 mL of distilled water, dissolve by sonication, and then filter through a 0.22 μm filter membrane to obtain a 3 wt% TSPBA solution.

[0082] C. Mix TSPBA (3 wt%) and PVA (9 wt%) in a 1:1 (v / v) ratio to form a PT hydrogel.

[0083] ② Preparation of drug-loaded PT hydrogel Weigh 16.1 mg (37.5 μmol) ZT-5, 41.9 mg (37.5 μmol) STP2 and 13.4 mg (37.5 μmol) indomethacin (INM) and disperse them evenly in 1.5 mL of 9 wt% PVA solution. Mix them with TSPBA solution at a ratio of 1:1 (v / v) to form a drug-loaded PT hydrogel.

[0084] The preparation process and self-healing effect of PT hydrogel are as follows: Figure 10 As shown, Figure 10 In the diagram, A is the preparation process flow chart for PT hydrogel, and B is the self-healing effect of PT hydrogel.

[0085] The 9% (w / v) PVA-3% (w / v) TSPBA complex exhibited a rapid sol-gel transition, quickly forming a hydrogel. Figure 10 As can be seen from B in the figure, the hydrogel also has self-healing properties and can maintain good self-sustaining properties in joints, thereby giving it a highly biocompatible and highly hydrated state. PT hydrogel can serve as an ideal platform for drug delivery in vivo.

[0086] (2) 3.2 PVA-TSPBA hydrogel can be rapidly released in the inflammatory microenvironment. To verify pH-triggered PT hydrogel lysis, the hydrogel was first dried and then soaked in a buffer solution at pH 7.2. Its swelling rate and degradation results were then measured, as follows: ① Swelling rate of P-T hydrogel A. Mix 200 μL of TSPBA (3 wt%) and PVA (9 wt%) in a 1.5 mL centrifuge tube at a ratio of 1:1 (v / v) to form a PT hydrogel.

[0087] B. Centrifuge at 12000 rpm for 10 min to remove air bubbles from the hydrogel.

[0088] C. Dry in an oven at 40 ℃ until constant weight.

[0089] D. Carefully peel off the dried hydrogel and weigh it to obtain W0. E. Add 10 mL of PBS to a 50 mL centrifuge tube and place the dried hydrogel inside. Stir at 37 ℃ and 300 rpm. Record the weight of the hydrogel at time t as Wt. The swelling percentage (%) = (Wt / Wt) / Wt. t -W0) / W0×100%.

[0090] ② Degradation of P-T hydrogels A. Swell the PT hydrogel to constant weight according to the method in 3.2.1, and weigh it to obtain W0.

[0091] B. Add a buffer solution with pH=6.0 to a beaker and place the hydrogel, which has swollen to constant weight, into the beaker.

[0092] Stir at 37 ℃ and 300 rpm, and record the weight of the hydrogel at time t as Wt. The hydrogel residue percentage (%) is calculated as Wt. t / W0×100%.

[0093] The swelling and degradation results of PT hydrogel are as follows Figure 11 As shown, Figure 11 In the diagram, A represents the swelling result, and B represents the degradation result. For example... Figure 11 As shown in Figure A, after soaking the hydrogel in PBS for 480 minutes, swelling kinetics analysis showed a significant swelling rate approaching 950%, indicating that the hydrogel has good cross-linking properties. The hydrogel was then immersed in a solution at pH 6.0 simulating an inflammatory microenvironment, and changes in its morphology and weight were monitored. The results are shown in Figure A. Figure 11 As shown in Figure B, at pH 6, the hydrogel rapidly disintegrates and loses weight, becoming undetectable after 1 h. In contrast, the morphological changes caused by immersion in PBS are negligible, suggesting that the hydrogel can release drugs on demand in the low pH environment of the inflammatory microenvironment.

[0094] Test Example 3: Evaluation of in vivo anti-inflammatory activity (I) Establishment and evaluation of collagen-induced arthritis mice ①Preparation of Type II Collagen Emulsion (1) Type II collagen and Complete Freund's adjuvant (CFA) emulsifier (IIC / CFA): In a clean bench, use a 10 mL sterile syringe to draw 5 mL of 2 mg / mL chicken type II collagen solution and 5 mL of Complete Freund's adjuvant containing 4 mg / mL inactivated Mycobacterium tuberculosis. Use a three-way tube to emulsify the chicken type II collagen at a ratio of 1:1 (v:v) until a viscous emulsion is formed. The emulsification process is carried out on ice.

[0095] (2) Type II collagen and incomplete Freund's adjuvant (IFA) emulsifier (IIC / IFA): In a clean bench, use a 10 mL sterile syringe to draw 5 mL of 2 mg / mL chicken type II collagen solution and 5 mL of incomplete Freund's adjuvant without inactivated Mycobacterium tuberculosis, and emulsify according to the preparation method of complete Freund's adjuvant emulsifier.

[0096] ② Establishment and treatment of CIA mouse model (1) Select DBA / 1J male mice susceptible to RA. When the mice are 8-10 weeks old, inject 100 μL of IIC / CFA intradermally at a distance of 2-3 cm from the base of the tail for the first immunization. The time of the first immunization is recorded as Day 0. (2) On day 21, 100 μL of IIC / IFA was injected intradermally at a distance of 2 cm from the base of the mouse tail for a second immunization.

[0097] (3) The mice were observed from day 0 to day 27, twice a week. After day 27, they were observed every two days. During the observation, the mice in each group were given relevant drug treatment and scored.

[0098] ③ Evaluation of the total clinical score of the limbs in the CIA mouse model Changes in mouse symptoms were recorded starting on day 21. From day 21 to day 27, mice were observed twice a week. From day 27 until the end of the experiment, mice were observed every two days. The severity of the disease was assessed by scoring the mice's limbs (0 points: no symptoms; 1 point: slight redness and swelling of the toes or soles; 2 points: slight redness and swelling of the toes, soles, and ankles; 3 points: significant redness and swelling of the toes, soles, and ankles; 4 points: severe redness and swelling of the toes, soles, and ankles). The total score for all four limbs was the mouse's clinical score.

[0099] ④ Evaluation of average foot thickness and ankle joint diameter in CIA mouse models (1) Average foot thickness of the four limbs: Starting from day 21, the thickness of the middle part of the foot of each mouse was measured once using an electronic vernier caliper. The average thickness of the foot of the four limbs is the average foot thickness. The measurement frequency is consistent with the scoring frequency.

[0100] (2) Average ankle joint diameter of the four limbs: Starting from day 21, the ankle joint diameter of each mouse was measured once using an electronic vernier caliper. The average ankle joint diameter of the four limbs is the average ankle joint diameter. The measurement frequency is consistent with the scoring frequency.

[0101] CIA mouse modeling and CIA mouse administration methods, such as Figure 12As shown. On day 21, CIA-induced model mice were randomly divided into four groups of three: Model group, Indomethacin (INM) group, ZT-5 group, and STP2 group. Three mice that were not induced were retained as the Control group. Starting from day 27, the INM, ZT-5, and STP2 groups were administered the drug via in situ injection at a dose of 50 μmol / kg, twice a day, for a total of eight injections. The experiment ended on day 42.

[0102] Clinical manifestations and scores of CIA mice are as follows: Figure 13 As shown, Figure 13 In the diagram, A is a photograph of the CIA mice, B is the clinical score of the mice's arthritis, and C is a statistical graph of the average ankle joint diameter of the mice (n=3, compared with the CIA group). <0.05, <0.01, <0.001, <0.0001).

[0103] As can be seen, photographs were taken of the mouse's right hind paw on day 42, and the results were as follows: Figure 13 As shown in Figure A, the model group still exhibited significant swelling, while the swelling in the three drug-treated groups was significantly reduced compared to the model group. Clinical arthritis scores were assessed in mice every two days, and the results are as follows: Figure 13 As shown in Figure B, from day 21 to day 27, there was no significant difference in clinical scores between the three treatment groups and the model group, and the scores increased, indicating the occurrence of inflammation and the rationality of the randomization of the mice. From day 29 to day 35, the clinical scores of the three treatment groups gradually increased, reaching a peak on day 35. From day 37 to the end of treatment, the clinical scores showed a downward trend, and the clinical scores of the STP2 treatment group were significantly different from those of the model group (P < 0.0001), indicating that the STP2 treatment group could significantly improve the arthritis symptoms of CIA mice. Figure 13 The statistical analysis of the average ankle joint diameter in mice by C in the data also confirms this point.

[0104] (II) Evaluation of the inhibitory effects of ZT-5 and STP2 on carrageenan-induced paw edema in rats Subcutaneous injection of carrageenan-induced edema is a commonly used animal model of acute inflammation. The model construction method is as follows: (1) Select male rats with a weight of 150-180 g, prepare ZT-5 and STP2 working solutions (14 mM) with physiological saline, administer them via tail vein at a dose of 14 μmol / kg, and record the time as min 0.

[0105] (2) Record the thickness of the rat's feet at 10 min.

[0106] (3) At 50 min, 100 μL of 1% (w / v) carrageenan solution was injected subcutaneously into the base of the palm, which was recorded as h0. The degree of paw swelling of the rat was observed and recorded every 30 min.

[0107] (4) Take rat paw epithelium at 4 h, fix it with 4% paraformaldehyde fixative, and then perform H&E pathological section staining to evaluate the infiltration of immune cells.

[0108] One hour before subcutaneous injection of 1% (w / v) carrageenan solution, rats were given prophylactic treatment with 14 μmol / kg ZT-5 and STP2. Photos were taken 4 hours after carrageenan injection. The clinical manifestations of carrageenan-induced paw edema in rats are as follows: Figure 14 As shown, Figure 14 In the table, A shows photographs of rat paw swelling, and B shows statistical analysis of the swelling rate (n=4, compared with the Model group). <0.05, <0.01, <0.001, <0.0001). By Figure 14 As shown in Figure A, the carrageenan-injected Model group exhibited significant redness and swelling, while the two groups receiving drug intervention showed no significant redness or swelling. Statistical analysis of the swelling rate showed that STP2 intervention significantly reduced paw swelling in rats (P < 0.01).

[0109] Four hours after carrageenan injection, rat paw epithelium was collected for H&E pathological section staining, and the results are as follows. Figure 15 As shown, after injection of carrageenan, immune cell infiltration appeared in the dermis and subcutaneous tissue of rats, indicating that carrageenan injection successfully induced acute inflammation in the rat paws. However, intervention with ZT-5 and STP2 could inhibit the immune cell infiltration in this area, indicating that ZT-5 and STP2 can suppress acute inflammation in rats.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A conjugate of a PAD4 inhibitor and a synovial-targeting peptide, characterized in that, It has the structure shown in Equation I: Formula I.

2. The method for preparing the PAD4 inhibitor and synovial-targeting peptide conjugate according to claim 1, characterized in that, Includes the following steps: Under acidic conditions, the fully deprotected synovial targeting peptide STP with the structure shown in Formula 1 is coupled with the PAD4 inhibitor ZT-5 with the structure shown in Formula 2 to obtain a PAD4 inhibitor-synovial targeting peptide conjugate with the structure shown in Formula 1. Formula 1; Formula 2.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the synovial protection-targeting peptide STP to the PAD4 inhibitor ZT-5 is 1:1~2.

4. The preparation method according to claim 2, characterized in that, The acidic environment is provided by trifluoroacetic acid, and the pH value of the acidic environment is 0 to 1.

5. The preparation method according to claim 2 or 4, characterized in that, The coupling reaction is carried out at a temperature of 20-25°C for 48-72 hours.

6. The preparation method according to claim 2, characterized in that, The preparation method of the STP, a fully deprotected synovial targeting peptide having the structure shown in Formula 1, includes the following steps: A compound having the structure shown in formula a undergoes a deBoc protection reaction to give a compound having the structure shown in formula b. A compound having the structure shown in formula b undergoes a ring-opening amidation reaction with succinic anhydride to obtain a compound having the structure shown in formula c. In the presence of an activator, a compound having the structure shown in formula c undergoes an acylation reaction with benzyl hydrazide formate to obtain a fully protected synovial targeting peptide STP having the structure shown in formula d. The fully protected synovial target peptide STP with the structure shown in Formula d was subjected to a complete deprotection reaction to obtain the fully deprotected synovial target peptide STP with the structure shown in Formula 1. Formula a; Formula b; Formula c; Formula d.

7. The preparation method according to claim 2, characterized in that, A method for preparing the PAD4 inhibitor ZT-5 having the structure shown in Formula 2 includes the following steps: In the presence of an activator, Cbz-Orn(Boc)-OH undergoes a first condensation reaction with benzylamine to give compound ZT-1. Compound ZT-1 underwent a deCbz protection reaction to yield compound ZT-2; In the presence of an activator, compound ZT-2 undergoes a second condensation reaction with p-formylbenzoic acid to obtain compound ZT-3; Compound ZT-3 underwent a deBoc protection reaction to yield compound ZT-4; Under alkaline conditions, compound ZT-4 undergoes a substitution reaction with ethyl 2-chloroacetylimine to obtain ZT-5, a PAD4 inhibitor with the structure shown in Formula 2. ZT-1; ZT-2; ZT-3; ZT-4。 8. The use of the PAD4 inhibitor and synovial-targeting peptide conjugate according to claim 1 or the PAD4 inhibitor and synovial-targeting peptide conjugate prepared by any one of claims 2 to 7 in the preparation of anti-rheumatoid arthritis drugs.

9. A pharmaceutical composition, characterized in that, It includes the PAD4 inhibitor and synovial-targeting peptide conjugate as described in claim 1 or the PAD4 inhibitor and synovial-targeting peptide conjugate prepared by any one of the preparation methods described in claims 2 to 7, as well as pharmaceutically acceptable carriers, diluents or excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is an injectable hydrogel formulation.

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