Bipyridine derivative and preparation method and application thereof, and bipyridine ruthenium complex and preparation method and application thereof

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a bipyridine derivative and a preparation method and application thereof, and a bipyridine ruthenium complex and a preparation method and application thereof. The invention provides a bipyridine derivative which has a structure as shown in a formula 1. The invention provides a bipyridine ruthenium complex which has a structure as shown in a formula 2. The BM-Cl provided by the invention has excellent anti-tumor activity through mouse tumor model caudal vein administration, and can be used as a PAD4 inhibitor. The BM-Ru1-Cl shows the excellent anti-tumor activity of dose dependence through intragastric administration of a mouse tumor model. Meanwhile, BM-Ru1-Cl can still play an anti-tumor role after being orally taken and absorbed into blood, and cannot be damaged by the stomach environment.

Description

Technical Field

[0001] This invention belongs to the field of anticancer drug technology, specifically relating to bipyridine derivatives and their preparation methods and applications, and bipyridine ruthenium complexes and their preparation methods and applications. Background Technology

[0002] Peptidyl-arginine deiminase (PAD) is an important post-translational modification enzyme that mediates citrullination of proteins in various cells (tumor cells, neutrophils, and macrophages, etc.) and different components (nuclear and cytoplasmic proteins, etc.), participating in gene regulation. Citrullination alters the structure and function of target proteins, participating in various physiological and pathological processes. To date, five PAD isoenzymes have been identified in humans and mammals. These enzymes are highly conserved, with sequence similarity exceeding 50%, but they differ in tissue localization and substrate specificity, thus performing different functions. Notably, PAD4 is the only PAD isoenzyme carrying a nuclear localization sequence, enabling it to bind to a variety of substrates in vivo. Among these, histones are the most studied substrates. By citrullinizing histones at different sites, altering their interactions with DNA and other nuclear proteins, PAD4 activates or inhibits gene transcription. PAD4 also participates in the inhibition of p53 activity and regulates the expression of p53 target genes through citrullination modification of histones. In addition, histone citrullination can promote the formation of extracellular traps (NETs) in neutrophils. Pathological studies show that PAD4 is overexpressed in various malignant tumors, such as breast cancer, lung cancer, colorectal cancer, ovarian cancer, bladder cancer, and other metastatic cancers. Increasing evidence suggests that PAD4-mediated protein citrullination and NET formation play a crucial role in cancer progression, not only promoting the development of the primary tumor but also promoting distant metastasis of cancer cells and the formation of cancer-related thrombi.

[0003] Currently, the anti-cancer drugs approved by the FDA have relatively poor anti-tumor activity. Summary of the Invention

[0004] The purpose of this invention is to provide bipyridine derivatives and their preparation methods and applications, as well as bipyridine-ruthenium complexes and their preparation methods and applications. The bipyridine derivatives provided by this invention exhibit excellent antitumor activity, and the bipyridine-ruthenium complexes exhibit excellent dose-dependent antitumor activity. The bipyridine-ruthenium complexes can still exert antitumor effects after oral absorption into the bloodstream and are not destroyed by the gastric environment, making them an excellent orally administered PAD4 inhibitor.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] This invention provides a bipyridine derivative having the structure shown in Formula 1:

[0007]

[0008] This invention provides a method for preparing the bipyridine derivative described in the above technical solution, comprising the following steps:

[0009] Z-Orn(Boc)-OH was activated in an organic solvent and then reacted with benzylamine under alkaline conditions to obtain Z-Orn(Boc)-NBzl;

[0010] In a reducing gas atmosphere, Z-Orn(Boc)-NBzl is subjected to a catalytic reduction reaction in an organic solvent to obtain Orn(Boc)-NBzl;

[0011] 4'-Methyl-2,2'-bipyridine-4-carboxylic acid was activated in an organic solvent and then reacted with Orn(Boc)-NBzl under alkaline conditions to obtain BM-Orn(Boc)-NBzl;

[0012] The BM-Orn(Boc)-NBzl was reacted in an organic solution of hydrogen chloride to obtain the hydrochloride salt of BM-Orn-NBzl.

[0013] The hydrochloride of BM-Orn-NBzl and ethyl 2-chloroacetylimine were reacted in an organic solvent under alkaline conditions to obtain a bipyridine derivative with the structure shown in Formula 1.

[0014] The structures of Z-Orn(Boc)-OH, Z-Orn(Boc)-NBzl, Orn(Boc)-NBzl, BM-Orn(Boc)-NBzl, BM-Orn-NBzl, and 2-chloroacetylimine ethyl ester are as follows:

[0015]

[0016] This invention provides the application of the bipyridine derivatives described in the above technical solutions or the bipyridine derivatives prepared by the preparation methods described in the above technical solutions in the preparation of PAD4 inhibitors or anticancer drugs.

[0017] This invention provides a PAD4 inhibitor or anticancer drug, comprising the bipyridine derivative described in the above technical solution or the bipyridine derivative prepared by the preparation method described in the above technical solution.

[0018] This invention provides a bipyridine ruthenium complex having the structure shown in Formula 2:

[0019]

[0020] This invention provides a method for preparing the bipyridine-ruthenium complex described in the above technical solution, comprising the following steps:

[0021] The bipyridine derivatives described in the above technical solutions or the bipyridine derivatives prepared by the above technical solutions are reacted with cis-dichlorobis(2,2'-bipyridine)ruthenium to obtain the bipyridine-ruthenium complex with the structure shown in Formula 2;

[0022] The structure of the cis-dichlorobis(2,2'-bipyridine)ruthenium is as follows:

[0023]

[0024] Preferably, the method for preparing the bipyridine ruthenium includes the following steps:

[0025] The bipyridine ruthenium was obtained by reacting 4'-methyl-2,2'-bipyridine-4-carboxylic acid and cis-dichlorobis(2,2'-bipyridine)ruthenium dihydrate in a polar solvent.

[0026] This invention provides the application of the bipyridine-ruthenium complex prepared by the preparation method described in the above technical solution in the preparation of PAD4 inhibitors or anticancer drugs.

[0027] This invention provides a PAD4 inhibitor or anticancer drug, comprising the bipyridine-ruthenium complex described in the above technical solution or the bipyridine-ruthenium complex prepared by the preparation method described in the above technical solution.

[0028] Preferably, the dosage form of the PAD4 inhibitor or anticancer drug is an oral dosage form.

[0029] This invention provides a bipyridine derivative (BM-Cl) having the structure shown in Formula 1. This invention also provides a bipyridine-ruthenium complex (BM-Ru1-Cl) having the structure shown in Formula 2. The BM-Cl provided by this invention exhibits excellent antitumor activity when administered via tail vein in a mouse tumor model and can be used as a PAD4 inhibitor. This invention obtains BM-Ru1-Cl by complexing the PAD4 inhibitor BM-Cl with cis-dichlorobis(2,2'-bipyridine)ruthenium. Compared to cisplatin and its derivatives, the BM-Ru1-Cl provided by this invention forms a six-coordinate octahedral configuration with a wider oxidation state range than platinum; secondly, since ruthenium and iron belong to the same group, it can be transported in vivo through transferrin in the blood, similar to iron, resulting in faster absorption and metabolism, higher bioavailability, and lower systemic toxicity. Furthermore, BM-Ru1-Cl exhibits excellent dose-dependent antitumor activity when administered via gavage in a mouse tumor model. Meanwhile, BM-Ru1-Cl can still exert anti-tumor effects after oral absorption into the bloodstream. It can remain stable in the gastric environment and is not destroyed by the gastric environment, making it an excellent oral PAD4 inhibitor. Attached Figure Description

[0030] Figure 1 The synthetic routes for preparing BM-Cl and BM-Ru1-Cl in Examples 1 and 2 of this invention are shown below;

[0031] Figure 2 This is a synthetic route diagram for preparing BM-Ru1 in Comparative Example 1 of the present invention;

[0032] Figure 3 The mass spectrum of the BM-Ru1 ruthenium complex prepared in Comparative Example 1 of this invention;

[0033] Figure 4 The BM-Cl mass spectrometer of the PAD4 inhibitor prepared in Example 1 of this invention;

[0034] Figure 5 The PAD4 inhibitor BM-Cl prepared in Example 1 of this invention 1 HNMR (300MHz, DMSO-d6);

[0035] Figure 6 The mass spectrometry of the BM-Ru1-Cl complex, a PAD4 inhibitor prepared in Example 2 of this invention;

[0036] Figure 7 The ruthenium complex BM-Ru1-Cl, a PAD4 inhibitor prepared in Example 2 of this invention, is... 1 HNMR (300MHz, DMSO-d6);

[0037] Figure 8 This describes the effect of the compounds in this invention on the formation of NETs. Detailed Implementation

[0038] This invention provides a bipyridine derivative having the structure shown in Formula 1:

[0039]

[0040] The bipyridine derivative with the structure shown in Formula 1 provided by this invention has shown excellent antitumor activity when administered via the tail vein in a mouse tumor model.

[0041] This invention provides a method for preparing the bipyridine derivative described in the above technical solution, comprising the following steps:

[0042] Z-Orn(Boc)-OH was activated in an organic solvent and then reacted with benzylamine under alkaline conditions to obtain Z-Orn(Boc)-NBzl;

[0043] In a reducing gas atmosphere, Z-Orn(Boc)-NBzl is subjected to a catalytic reduction reaction in an organic solvent to obtain Orn(Boc)-NBzl;

[0044] 4'-Methyl-2,2'-bipyridine-4-carboxylic acid was activated in an organic solvent and then reacted with Orn(Boc)-NBzl under alkaline conditions to obtain BM-Orn(Boc)-NBzl;

[0045] The BM-Orn(Boc)-NBzl was reacted in an organic solution of hydrogen chloride to obtain the hydrochloride salt of BM-Orn-NBzl.

[0046] The hydrochloride of BM-Orn-NBzl and ethyl 2-chloroacetylimine were reacted in an organic solvent under alkaline conditions to obtain a bipyridine derivative with the structure shown in Formula 1.

[0047] The structures of Z-Orn(Boc)-OH, Z-Orn(Boc)-NBzl, Orn(Boc)-NBzl, BM-Orn(Boc)-NBzl, BM-Orn-NBzl, and 2-chloroacetylimine ethyl ester are as follows:

[0048]

[0049] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0050] This invention involves activating Z-Orn(Boc)-OH in an organic solvent and then reacting it with benzylamine under alkaline conditions to obtain Z-Orn(Boc)-NBzl. In this invention, the organic solvent is preferably tetrahydrofuran (THF), and in the examples, anhydrous tetrahydrofuran can be used. The activation preferably includes the following steps: mixing the Z-Orn(Boc)-OH, the organic solvent, and an activating reagent for activation to obtain an activated reaction solution containing the activated Z-Orn(Boc)-OH. In this invention, the activating reagent preferably includes 1-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). The molar ratio of Z-Orn(Boc)-OH to HOBt is preferably 1:1.2. The molar ratio of Z-Orn(Boc)-OH to DCC is preferably 1:1.2. The preferred mixing order of the Z-Orn(Boc)-OH, organic solvent, and activating reagent is as follows: dissolve the Z-Orn(Boc)-OH in the organic solvent, and then add the activating reagent. Dissolution is preferably carried out under stirring conditions. Dissolution is preferably carried out under ice-water bath conditions. Activation is preferably carried out under ice-water bath conditions, and the activation time is preferably 20–30 minutes. After activation, a white turbidity appears in the resulting activated reaction solution.

[0051] After activation, an activation reaction solution is obtained. The present invention reacts the activation reaction solution with benzylamine under alkaline conditions to obtain Z-Orn(Boc)-NBzl. In the present invention, the preferred ratio of Z-Orn(Boc)-OH to benzylamine is 10 mmol: 1.2 mL. The alkaline conditions are preferably adjusted using an alkaline reagent, preferably N-methylmorpholine (NMM). The preferred pH value of the alkaline conditions is 8. The present invention preferably mixes the activation reaction solution, benzylamine, and alkaline reagent before reacting. The mixing is preferably carried out under ice-water bath conditions, and the preferred mixing order is: adding benzylamine dropwise to the activation reaction solution, followed by adding the alkaline reagent. The reaction is preferably carried out at room temperature (25–30 °C). The reaction is preferably carried out under stirring conditions. The present invention preferably detects the reaction progress by TLC, and the developing solvent used in the TLC is preferably CH2Cl2 and CH3OH, with a preferred volume ratio of CH2Cl2 to CH3OH of 20:1. The R-value of the TLC is... f The preferred value is 0.25. During the TLC detection process, the present invention preferably determines the end of the reaction after observing that the Z-Orn(Boc)-OH reaction is basically complete.

[0052] In this invention, after the reaction is completed, the reaction solution is directly obtained. Preferably, the reaction solution is desolventized to obtain a crude product. The crude product is redissolved in ethyl acetate (EA) and then subjected to solid-liquid separation to obtain a liquid product. The liquid product is then sequentially extracted, concentrated, and purified to obtain Z-Orn(Boc)-NBzl. The extraction is preferably performed sequentially using a saturated NaHCO3 solution, a first saturated NaCl solution, a 5wt% KHSO4 solution, a second saturated NaCl solution, a 5wt% NaHCO4 solution, and a third saturated NaCl solution. Each solution is preferably extracted three times. The extraction yields an organic extract phase. The organic extract phase is dried and concentrated to obtain a concentrated liquid product. The concentrated liquid product is then purified by column chromatography to obtain Z-Orn(Boc)-NBzl. The drying is preferably performed using solid Na2SO4. The concentration is preferably performed under reduced pressure. The column chromatography purification is preferably performed using a medium-pressure preparative column. The roughening reagent used for column chromatography purification is preferably a mixture of methanol and dichloromethane, wherein the volume fraction of methanol in the mixture of methanol and dichloromethane is preferably 6%.

[0053] After obtaining Z-Orn(Boc)-NBzl, the present invention performs a catalytic reduction reaction of Z-Orn(Boc)-NBzl in an organic solvent under a reducing gas atmosphere to obtain Orn(Boc)-NBzl. In the present invention, the reducing gas preferably includes hydrogen, and in the embodiments, the reducing gas is preferably hydrogen. The organic solvent is preferably methanol. The catalytic reduction reaction is preferably carried out under the condition of a catalyst, and the catalyst is preferably a palladium on carbon (Pd / C) catalyst. The present invention preferably mixes Z-Orn(Boc)-NBzl, organic solvent and catalyst, and carries out the catalytic reduction reaction in a hydrogen atmosphere. The mass ratio of Z-Orn(Boc)-NBzl to catalyst is preferably 1:0.1. Before the catalytic reduction reaction begins, the present invention preferably replaces air with hydrogen. The catalytic reduction reaction is preferably carried out in a fume hood, and the catalytic reduction reaction time is preferably 5-6 hours. The catalytic reduction reaction is preferably monitored by TLC, and the developing solvent used in the TLC is preferably CH2Cl2 and CH3OH, with a volume ratio of CH2Cl2 to CH3OH preferably of 15:1. The R-value of the TLC is... f Preferably, the value is <0.1. During the TLC detection process, the present invention preferably determines that the reaction is complete after the Z-Orn(Boc)-NBzl raw material spot disappears.

[0054] In this invention, after the catalytic reduction reaction is completed, a reaction solution is obtained. Preferably, the reaction solution undergoes solid-liquid separation to obtain a liquid product; the liquid product is then concentrated to dryness to obtain Orn(Boc)-NBzl. In this invention, the solid-liquid separation is preferably performed by vacuum filtration. The solid-liquid separation is for catalyst removal. The concentration is preferably performed under reduced pressure.

[0055] After obtaining Orn(Boc)-NBzl, this invention activates 4'-methyl-2,2'-bipyridine-4-carboxylic acid in an organic solvent and then reacts it with Orn(Boc)-NBzl under alkaline conditions to obtain BM-Orn(Boc)-NBzl. In this invention, the organic solvent is preferably tetrahydrofuran (THF), and in the examples, anhydrous tetrahydrofuran can be used. The activation preferably includes the following steps: mixing the 4'-methyl-2,2'-bipyridine-4-carboxylic acid, the organic solvent, and the activating reagent for activation to obtain an activated reaction solution containing activated 4'-methyl-2,2'-bipyridine-4-carboxylic acid. In this invention, the activating reagent preferably includes 1-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). The molar ratio of 4'-methyl-2,2'-bipyridine-4-carboxylic acid to HOBt is preferably 1:1.1. The molar ratio of 4'-methyl-2,2'-bipyridine-4-carboxylic acid to DCC is preferably 1:1.1. The preferred mixing order of the 4'-methyl-2,2'-bipyridine-4-carboxylic acid, organic solvent, and activating reagent is: dissolving the 4'-methyl-2,2'-bipyridine-4-carboxylic acid in the organic solvent, followed by adding the activating reagent. Dissolution is preferably carried out under stirring conditions. Dissolution is preferably carried out under ice-water bath conditions. Activation is preferably carried out under ice-water bath conditions, and the activation time is preferably 20–30 min. A white turbidity appears in the activated reaction solution after activation.

[0056] After activation, an activated reaction solution is obtained. In this invention, the activated reaction solution is reacted with Orn(Boc)-NBzl under alkaline conditions to obtain BM-Orn(Boc)-NBzl. In this invention, the molar ratio of 4'-methyl-2,2'-bipyridine-4-carboxylic acid and Orn(Boc)-NBzl is preferably 1:1.1. The alkaline conditions are preferably adjusted using an alkaline reagent, preferably N-methylmorpholine (NMM). The pH value of the alkaline conditions is preferably 8. In this invention, the activated reaction solution, Orn(Boc)-NBzl, and the alkaline reagent are preferably mixed before the reaction. The mixing is preferably carried out under ice-water bath conditions, and the preferred mixing order is: adding Orn(Boc)-NBzl to the activated reaction solution, followed by adding the alkaline reagent. The reaction is preferably carried out at room temperature (25–30°C). The reaction is preferably carried out under stirring conditions. The present invention preferably uses TLC to detect the reaction progress. The developing solvent used in the TLC is preferably CH2Cl2 and CH3OH, and the volume ratio of CH2Cl2 to CH3OH is preferably 15:1. The R-value of the TLC... fThe preferred value is 0.35. During the TLC detection process, the present invention preferably determines the end of the reaction after observing that the 4'-methyl-2,2'-bipyridine-4-carboxylic acid has basically reacted completely.

[0057] In this invention, after the reaction is completed, the reaction solution is directly obtained. Preferably, the reaction solution is desolventized to obtain a crude product. The crude product is redissolved in ethyl acetate (EA) and then subjected to solid-liquid separation to obtain a liquid product. The liquid product is then sequentially extracted, concentrated, and purified to obtain Z-Orn(Boc)-NBzl. The extraction is preferably performed sequentially using saturated NaHCO3 solution, a first saturated NaCl solution, a 5wt% KHSO4 solution, a second saturated NaCl solution, a 5wt% NaHCO4 solution, and a third saturated NaCl solution. Each solution is preferably extracted three times. The extraction yields an organic extract phase. The organic extract phase is dried and concentrated to obtain a concentrated liquid product. The concentrated liquid product is then purified by column chromatography to obtain Z-Orn(Boc)-NBzl. The drying is preferably performed using solid Na2SO4. The concentration is preferably performed under reduced pressure. The column chromatography purification is preferably performed using a medium-pressure preparative column. The roughening reagent used for column chromatography purification is preferably a mixture of methanol and dichloromethane, wherein the volume fraction of methanol in the mixture of methanol and dichloromethane is preferably 7%.

[0058] After obtaining BM-Orn(Boc)-NBzl, the present invention reacts BM-Orn(Boc)-NBzl in an organic solution of hydrogen chloride to obtain the hydrochloride salt of BM-Orn-NBzl. In the present invention, the organic solution of hydrogen chloride is preferably an ethyl acetate (EA) solution of hydrogen chloride (HCl / EA solution). The molar concentration of hydrogen chloride in the organic solution of hydrogen chloride is preferably 3-4 mol / L. The reaction is carried out under ice-water bath conditions and under stirring. The reaction time is preferably 5-6 h. The present invention preferably detects the reaction progress by TLC, and preferably dissolves the product obtained from the reaction in methanol (MeOH) before TLC detection. The developing solvent used in the TLC is preferably CH2Cl2 and CH3OH, and the volume ratio of CH2Cl2 to CH3OH is preferably 15:1. The R-value of the TLC is... f Preferably, the value is <0.1. During the TLC detection process, the present invention preferably determines the end of the reaction after the BM-Orn(Boc)-NBzl raw material spot disappears.

[0059] In this invention, after the reaction is completed, a reaction solution is obtained. Preferably, the reaction solution is vacuum dried under a warm water bath to obtain a residual product. The residual product is then washed sequentially with EA and petroleum ether to obtain HCl·BM-Orn-NBz. The EA is preferably dried EA. The petroleum ether is preferably dried petroleum ether. Preferably, the two solvents are used for washing three times each.

[0060] After obtaining the hydrochloride salt of BM-Orn-NBzl (HCl·BM-Orn-NBzl), the present invention reacts the hydrochloride salt of BM-Orn-NBzl with ethyl 2-chloroacetylimine under alkaline conditions in an organic solvent to obtain a bipyridine derivative with the structure shown in Formula 1. In the present invention, the alkaline conditions are preferably adjusted by an alkaline reagent, preferably N,N-diisopropylethylamine (DIPEA). The pH value of the alkaline conditions is preferably 9-10. The present invention preferably involves mixing the HCl·BM-Orn-NBzl, the organic solvent, ethyl 2-chloroacetylimine, and the alkaline reagent for the reaction. The preferred mixing order is: dissolving the HCl·BM-Orn-NBzl in the organic solvent, then sequentially adding ethyl 2-chloroacetylimine and the alkaline reagent. The mixing is preferably carried out in an ice-water bath. The reaction is preferably carried out at room temperature (25-30°C). The reaction is carried out under stirring. The present invention preferably uses TLC to detect the reaction process. The developing solvent used in the TLC is preferably EA, H2O, and acetic acid (HAc), and the volume ratio of EA, H2O, and HAc is preferably 4:1:1. The R-value of the TLC... f The preferred value is 0.1. The TLC detection preferably uses ninhydrin for color development. During the TLC detection process, the present invention preferably determines the end of the reaction after the starting material spot disappears.

[0061] In this invention, after the reaction is completed, a reaction solution is obtained. Preferably, the reaction solution is concentrated and then purified by column chromatography to obtain a purified solution. The purified solution is then concentrated and dried to obtain a bipyridine derivative with the structure shown in Formula 1. The concentration is preferably carried out under reduced pressure. The column chromatography purification is preferably carried out using a C18 medium-pressure preparative column. The eluent used in the column chromatography purification is preferably a mixture of methanol and deionized water. The volume fraction of methanol in the mixture of methanol and deionized water is preferably 25%. The purified solution is concentrated under reduced pressure, and the methanol in the eluent is removed by concentration. The drying is preferably vacuum cooling drying.

[0062] This invention provides the application of the bipyridine derivatives described in the above technical solutions or the bipyridine derivatives prepared by the preparation methods described in the above technical solutions in the preparation of PAD4 inhibitors or anticancer drugs.

[0063] This invention provides a PAD4 inhibitor or anticancer drug, comprising the bipyridine derivative described in the above technical solution or the bipyridine derivative prepared by the preparation method described in the above technical solution.

[0064] In this invention, the anticancer drugs preferably include anti-breast cancer drugs, anti-lung cancer drugs, anti-rectal cancer drugs, anti-ovarian cancer drugs, anti-bladder cancer drugs, or anti-other metastatic cancer drugs.

[0065] This invention provides a bipyridine ruthenium complex having the structure shown in Formula 2:

[0066]

[0067] The bipyridine ruthenium complex (BM-Ru1-Cl) with the structure shown in Formula 2 provided by this invention exhibits excellent dose-dependent antitumor activity when administered orally to a mouse tumor model. Furthermore, BM-Ru1-Cl retains its antitumor effect after oral absorption into the bloodstream, without being destroyed by the gastric environment.

[0068] This invention provides a method for preparing the bipyridine-ruthenium complex described in the above technical solution, comprising the following steps:

[0069] The bipyridine derivatives described in the above technical solutions or the bipyridine derivatives prepared by the above technical solutions are reacted with cis-dichlorobis(2,2'-bipyridine)ruthenium to obtain the bipyridine-ruthenium complex with the structure shown in Formula 2;

[0070] The structure of the cis-dichlorobis(2,2'-bipyridine)ruthenium is as follows:

[0071]

[0072] In this invention, the cis-dichlorobis(2,2'-bipyridine)ruthenium is preferably cis-dichlorobis(2,2'-bipyridine)ruthenium dihydrate.

[0073] In this invention, the solvent used in the reaction is preferably an aqueous ethanol solution. The volume ratio of ethanol to water in the aqueous ethanol solution is preferably 1:1. Preferably, the bipyridine derivative with the structure shown in Formula 1, the solvent, and cis-dichlorobis(2,2'-bipyridine)ruthenium dihydrate are mixed for the reaction. The molar ratio of the bipyridine derivative to the cis-dichlorobis(2,2'-bipyridine)ruthenium dihydrate is preferably 1:1. The reaction is carried out under reflux conditions. Preferably, the reaction progress is monitored by TLC, and the developing solvent used in the TLC is preferably EA, H2O, and acetic acid (HAc), with the volume ratio of EA, H2O, and HAc preferably being 4:1:2. The R-value of the TLC is... fThe preferred value is 0.15. During the TLC detection process, the product spots show red fluorescence. In this invention, it is preferable to observe the disappearance of the starting material spots to determine the end of the reaction.

[0074] In this invention, after the reaction is completed, a reaction solution is obtained. Preferably, the reaction solution is concentrated and then purified by column chromatography to obtain a purified solution. The purified solution is then concentrated and dried to obtain a bipyridine-ruthenium complex with the structure shown in Formula 2. The concentration is preferably carried out under reduced pressure. The column chromatography purification is preferably carried out using a C18 medium-pressure preparative column. The eluent used in the column chromatography purification is preferably a mixture of methanol and deionized water. The volume fraction of methanol in the mixture of methanol and deionized water is preferably 15%. The purified solution is concentrated under reduced pressure, and the methanol in the eluent is removed by concentration. The drying is preferably vacuum cooling drying.

[0075] This invention provides the application of the bipyridine-ruthenium complex prepared by the preparation method described in the above technical solution in the preparation of PAD4 inhibitors or anticancer drugs.

[0076] This invention provides a PAD4 inhibitor or anticancer drug, comprising the bipyridine-ruthenium complex described in the above technical solution or the bipyridine-ruthenium complex prepared by the preparation method described in the above technical solution.

[0077] In this invention, the anticancer drug preferably includes anti-breast cancer drugs, anti-lung cancer drugs, anti-rectal cancer drugs, anti-ovarian cancer drugs, anti-bladder cancer drugs, or anti-other metastatic cancer drugs.

[0078] In this invention, the dosage form of the PAD4 inhibitor or anticancer drug is preferably an oral dosage form.

[0079] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0080] Example 1:

[0081] according to Figure 1The synthetic route shown in this embodiment provides a method for preparing the bipyridine derivative with the structure shown in Formula 1. The bipyridine derivative with the structure shown in Formula 1 is named N-(1-(benzylamino)-5-(2-chloroacetimidamido)-1-oxopentan-2-yl)-4'-methyl-[2,2'-bipyridine]-4-carboxamide (N-(1-(benzylamino)-5-(2-chloroacetimidamido)-1-oxopentan-2-yl)-4'-methyl-[2,2'-bipyridine]-4-carboxamide, BM-Orn(Cl), BM-Cl)

[0082] (1) Synthesis of Z-Orn(Boc)-NBzl

[0083] Accurately weigh 3.660 g (10 mmol) of benzyloxycarbonyl amino acid (Z-Orn(Boc)-OH) into a flask using the weight reduction method. Dissolve it completely in 100 mL of anhydrous 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 until the solution becomes white and turbid. Then, add 1.2 mL of benzylamine dropwise 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). Analyze by TLC (volume ratio, developing solvent: CH2Cl2:CH3OH = 20:1, R...) f=0.25) Monitor the reaction progress and observe that the reactant Z-Orn(Boc)-OH is basically completely reacted, indicating that the reaction is complete. After the reaction is complete, concentrate to dryness under reduced pressure to remove THF, then reconstitute with 150mL EA, and sonicate to completely dissolve the product, although a white solid remains 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 extracted three times sequentially 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% NaHCO4 solution (60 mL / time), and saturated NaCl solution (60 mL / time). During the extraction process, the EA layer gradually became colorless. The EA layer was dried with Na2SO4 for 2 hours, and Na2SO4 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 methanol and dichloromethane, with a methanol volume fraction of 6%) to obtain 3.894 g (85.6%) of the target product Z-Orn(Boc)-NBzl, which was a white solid powder. ESI-MS (m / z): 911.78 [2M+H] + .

[0084] (2) Synthesis of Orn(Boc)-NBzl

[0085] 3.894 g (8.6 mmol) of Z-Orn(Boc)-NBzl, after being evaporated to dryness, was dissolved in 150 mL of methanol until it became colorless, clear, and transparent. Under stirring, 0.390 g of palladium on carbon (Pd / C) catalyst was added to a flask, and a three-way valve and hydrogen gas bag were connected. The air in the flask was first evacuated as much as possible using a vacuum circulating water pump, then hydrogen gas was introduced. This process was repeated three times, keeping the hydrogen gas bag connected to the flask. The flask was placed in a fume hood and reacted for 6 hours. TLC (by volume, with CH2Cl2:CH3OH = 15:1 as the developing solvent) was performed. f <0.1) Monitor the reaction progress; once the starting material spot disappears, the reaction is considered complete. Remove Pd / C by vacuum filtration, and concentrate the filtrate to dryness under reduced pressure to obtain 2.509 g (90.5%) of the target product Orn(Boc)-NBzl, a white solid. ESI-MS (m / z): 322.34 [M+H] + .

[0086] (3) Synthesis of BM-Orn(Boc)-NBzl

[0087] Accurately weigh 0.428 g (2 mmol) of 4'-methyl-[2,2'-bipyridine]-4-carboxylic acid using the weight reduction method and place it in a 100 mL flask. Add a stir bar and dissolve the solution in 50 mL of anhydrous THF until it becomes colorless, clear, and transparent. While stirring in an ice bath, add 0.297 g (2.2 mmol) of HOBt and 0.453 g (2.2 mmol) of DCC sequentially. Activate for 30 min; the solution becomes white and turbid. Add 0.996 g (2.2 mmol) of Orn(Boc)-NBzl to the flask and adjust the pH to 8 with NMM. Remove the ice bath and react overnight at room temperature (25°C) with stirring. Analyze by TLC (by volume, with CH2Cl2:CH3OH = 15:1 as the developing solvent). f =0.35) Monitor the reaction progress and determine the end of the reaction when the reactants have basically reacted completely. After the reaction is complete, concentrate to dryness under reduced pressure to remove THF, then reconstitute with 50 mL of EA, and sonicate to completely dissolve the product, leaving a white solid that remains 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 100 mL separatory funnel and extracted three times sequentially with saturated NaHCO3 solution (30 mL / time), saturated NaCl solution (30 mL / time), 5 wt% KHSO4 solution (30 mL / time), saturated NaCl solution (30 mL / time), 5 wt% NaHCO4 solution (30 mL / time), and saturated NaCl solution (30 mL / time). During the extraction process, the EA layer gradually became colorless. The EA layer was dried with Na2SO4 for 2 hours, and Na2SO4 was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a white solid. The solid was purified by separation using a medium-pressure preparative column (the purification reagent used was a mixture of methanol and dichloromethane, with a methanol volume fraction of 7%) to obtain 0.761 g (73.5%) of the target product BM-Orn(Boc)-NBzl, which was a white solid. ESI-MS (m / z): 518.42 [M+H] + .

[0088] (4) Synthesis of BM-Orn-NBzl hydrochloride (HCl·BM-Orn-NBzl)

[0089] After BM-Orn(Boc)-NBzl was evaporated to dryness, 20 mL of a 4 mol / L ethyl acetate solution of hydrogen chloride (4N HCl / EA solution) was added in a fume hood under ice bath conditions. The BM-Orn(Boc)-NBzl compound is insoluble in the 4N HCl / EA solution. A drying tube was attached to the bottle neck, and the reaction was carried out for 6 hours with stirring in an ice bath. A portion was scraped from the bottle with a plastic spoon, dissolved in MeOH, and then analyzed by TLC (by volume, with CH2Cl2:CH3OH = 15:1 as the developing solvent).f <0.1) Monitor the reaction progress. Once the starting material spot disappears, the reaction is considered complete. After the reaction is complete, the reaction solution is depressurized and dried under a vacuum circulating water pump connected to a valve in a warm water bath. Add 30 mL of dry petroleum ether (EA) to the residue, stir in a warm water bath (40°C), and dry again. Repeat this operation three times. Then add 30 mL of dry petroleum ether, stir in a warm water bath, and dry again. Repeat this operation three times until no obvious acid gas remains, yielding 0.606 g (90.8%) of the target product HCl·BM-Orn-NBzl, a pale pink solid powder. ESI-MS (m / z): 418.31 [M+H] + .

[0090] (5) Synthesis of BM-Cl

[0091] 0.606 g (1.33 mmol) of HCl·BM-Orn-NBzl, after being dried, was dissolved in 50 mL of anhydrous methanol until clear and transparent. A stir bar was added, and 1.044 g (6.65 mmol) of 2-chloroacetylimine ethyl ester 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 carried out overnight at room temperature. TLC (by volume, with EA:H2O:HAc = 4:1:1, R) was performed. f =0.1) Monitor the reaction progress; ninhydrin shows color change, and the reaction is considered complete when the starting material spot disappears. The reaction solution was concentrated under reduced pressure and then purified by a C18 medium-pressure preparative column (the purification reagent used was a mixture of methanol and deionized water, with methanol at a volume fraction of 25%). After removing methanol by reduced pressure concentration, the solution was freeze-dried to remove water, yielding 0.168 g (25.6%) of the target product BM-Orn(Cl)-NBzl(BM-Cl), a pale pink solid powder. ESI-MS (m / z): 493.29 [M+H] +,1 HNMR (300MHz, DMSO) δ10.36 (s, 1H), 9.67 (s, 1H), 9.14 (d, J = 7.8Hz, 1H), 9.10-8.77 (m, 2H), 8. 75(t,J=6.0Hz,1H),8.60(dd,J=4.9,0.8Hz,1H),8.28(dt,J=1.7,0.9Hz,1H),7.98(dd,J=4.8 ,1.9Hz,1H),7.54-7.15(m,6H),4.58(ddd,J=9.7,7.7,4.8Hz,1H),4.48(s,2H),4.33(d,J=6. 0Hz, 2H), 3.32 (d, J = 7.1Hz, 2H), 2.44 (s, 3H), 2.13-1.80 (m, 2H), 1.67 (tt, J = 15.4, 7.6Hz, 2H).

[0092] Example 2:

[0093] according to Figure 1 The synthetic route shown in this embodiment provides a method for preparing the bipyridine ruthenium complex (BM-Ru1-Cl) with the structure shown in Formula 2.

[0094] The BM-Cl prepared in Example 1 and cis-dichlorobis(2,2'-bipyridine)ruthenium(II) dihydrate were dissolved in ethanol and water at a molar ratio of 1:1 and heated to reflux. The solution was then analyzed by TLC (by volume ratio, with EA:H2O:HAc = 4:1:2, R...). f =0.15) Monitor the reaction progress; the product spot shows red fluorescence. The reaction is considered complete when the starting material spot disappears. The reaction solution is concentrated under reduced pressure and then purified by a C18 medium-pressure preparative column (the purification reagent used is a mixture of methanol and deionized water, with methanol at a volume fraction of 15%). After removing methanol by reduced pressure concentration, the solution is freeze-dried under vacuum to remove water, yielding a red solid powder BM-Ru1-Cl. ESI-MS (m / z): 453.16 [1 / 2M+H] +,1 H NMR (300MHz, DMSO) δ9.75(d,J=3.7Hz,1H),9.59(t,J=6.4Hz,1H),9.43(d,J=8.6Hz,1H),9.32(d,J=13.7H z,0H),8.90(d,J=8.3Hz,5H),8.27-8.11(m,5H),7.97(tt,J=6.8,3.4Hz,1H),7.87-7.75(m,1H),7.75(s, 1H),7.56(ddd,J=7.4,5.7,2.8Hz,6H),7.42(dd,J=6.1,1.6Hz,1H),7.34-7.18(m,4H),4.54(d,J=2.6Hz, 2H), 4.51 (d, J = 3.4Hz, 0H), 4.30 (d, J = 8.9Hz, 2H), 2.56 (s, 3H), 2.03 (d, J = 34.1Hz, 1H), 1.90-1.59 (m, 1H).

[0095] Comparative Example 1:

[0096] This comparative example provides a method for synthesizing BM-Ru1, the synthetic route is as follows: Figure 2 As shown:

[0097] Accurately weigh 428.44 mg (2 mmol) of 4'-methyl-[2,2'-bipyridine]-4-carboxylic acid using the weight reduction method and place it in a 100 mL flask. Dissolve it in 40 mL of methanol; the solution is white and turbid. Add a stir bar. Then add 968.68 mg (2 mmol) of cis-dichlorobis(2,2'-bipyridine)ruthenium(II) dihydrate, add 15 mL of deionized water, reflux for 4 h, and perform TLC (by volume ratio, with EA:H2O:HAc = 4:1:2, R...). f =0.1) Monitor the reaction progress. When the starting material spot disappears and the solution turns dark red and clear, the reaction is considered complete. Concentrate the reaction solution to dryness and remove the solvent. Purify by separation using a C18 medium-pressure preparative column (using a mixture of methanol and deionized water, with methanol at 5% by volume), yielding 1.213 g (96.6%) of the target product BM-Ru1 ruthenium complex, a dark red solid powder. ESI-MS (m / z): 314.07 [1 / 2M+H] + .

[0098] Structural characterization

[0099] Figure 3 The BM-Ru1 ruthenium complex mass spectrum prepared for Comparative Example 1.

[0100] Figure 4 BM-Cl mass spectrometry of the PAD4 inhibitor prepared in Example 1.

[0101] Figure 5 BM-Cl, the PAD4 inhibitor prepared in Example 1 1 HNMR (300MHz, DMSO-d6) image.

[0102] Figure 6 The mass spectrometry of the ruthenium complex BM-Ru1-Cl, a PAD4 inhibitor prepared in Example 2.

[0103] Figure 7 The ruthenium complex BM-Ru1-Cl, a PAD4 inhibitor prepared in Example 2, 1 ¹H NMR (300MHz, DMSO-d6) image.

[0104] Evaluation of in vitro antitumor activity

[0105] 1. Inhibits tumor cell proliferation

[0106] Mouse breast cancer cells (4T1 cells) were digested and seeded at 3000 cells per well in 96-well plates. After overnight culture in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), different concentrations of the compound were added and incubated for 24 h. MTT (5 mg / mL) was added, and incubation continued for 4 h. The absorbance at 490 and 570 nm was measured using a multi-mode microplate reader, and the data were analyzed using a one-way ANOVA with Graphpad Prism 10. The results are shown in Table 1. Table 1 shows that BM-Ru1 and BM-Ru1-Cl had very poor killing effects on 4T1 cells in vitro.

[0107] Table 1 IC50 of the test compounds against the in vitro antiproliferative activity of 4T1 50 Value (Mean±SD)

[0108] Group <![CDATA[IC 50 (μM)]]> BM-Ru1 >200 BM-Cl 6.04±0.17 BM-Ru1-Cl >200

[0109] 2. Anti-tumor cell metastasis

[0110] One day prior to incubation, 4T1 cells were starved using serum-free medium. After digestion, the cells were resuspended in serum-free DMEM and counted. 50,000 cells were seeded in the upper chamber of a transwell. BM-Cl (5 μM), BM-Ru1 (5 μM), and BM-Ru1-Cl (5 μM) were added sequentially, while 600 μL of medium containing 10% serum was added to the lower chamber. Cells were divided into control, BM-Cl (5 μM), BM-Ru1 (5 μM), and BM-Ru1-Cl (5 μM). After 24 h of incubation, the upper chamber medium was removed, cells were washed twice with PBS, fixed with 4% paraformaldehyde at 4°C for 30 min, and stained with crystal violet at room temperature for 15 min. After washing off the crystal violet stain, the cells were air-dried and photographed using a Zeiss optical microscope. Cell migration was counted using ImageJ, expressed as Mean ± SD. As shown in Table 2, the number of 4T1 cells crossing the transwell membrane was significantly reduced after treatment with different doses of BM-Cl, BM-Ru1, and BM-Ru1-Cl. The inhibitory effect of BM-Cl-5 was significantly different from that of the positive groups RGDS (20 μM) and NAMI-A (20 μM) (p values ​​were <0.0001 and 0.0023, respectively). The inhibitory effect of BM-Ru1-Cl-5 was not significantly different from that of the positive groups RGDS (20 μM) and NAMI-A (20 μM) (p values ​​were 0.2455 and 0.9969, respectively). In Table 2, RGDS is an abbreviation for Arg-Gly-Asp-Ser polypeptide, a common integrin binding sequence.

[0111] Table 2. In vitro anti-transfer activity of the test compounds against 4T1

[0112]

[0113]

[0114] 3. Inhibition of extracellular neutrophil trap (NET) formation in vitro

[0115] The effects of BM-Cl, BM-Ru1-Cl, and BM-Ru1 on the release of extracellular traps (NETs) from neutrophils were evaluated. ICR mice were euthanized by cervical dislocation, and the hind legs were separated, the bone ends were removed, and the bone marrow cavity was exposed. The bone marrow cavity was rinsed with PBS and then filtered through a 70 μm nylon membrane to obtain a single-cell suspension. Neutrophils were isolated using a mouse neutrophil isolation kit (TBDSceicge, LZS1100), counted, and seeded into confocal culture dishes. The following groups were selected: 1) blank control group; 2) phorbol ester model group; 3) BM-Ru1-Cl-5 (5 μM); 4) BM-Cl-5 (5 μM); 5) BM-Ru1-Cl-1 (1 μM); 6) BM-Cl-1 (1 μM). After two hours of incubation, 0.15 μM of the inducer phorbol ester (PMA) was added, and incubation continued for another two hours. After centrifugation, the culture medium was aspirated, and the sample was washed with PBST and PBS, then fixed with 4% paraformaldehyde for 15 minutes, followed by washing with PBST and PBS, and then blocked with 5% BSA for 30 minutes. H3cit antibody (Abcam:ab5103) was added and incubated overnight at 4°C. The primary antibody was recovered by centrifugation, and the sample was washed with PBST and PBS, then incubated with secondary antibody at room temperature for 2 hours. The secondary antibody was recovered, and the sample was washed with PBST and PBS. DAPI staining was then performed for 3 minutes, followed by washing with PBST and PBS, and finally, an anti-fluorescence quenching blocking agent was added. The sample was observed using a laser confocal microscope (TCS SP8 STED, Leica GmbH, Germany). Figure 8 The effect of the compound on NET formation. Figure 8 In the model group, BM-Ru1-Cl-1 and BM-Cl-1 showed obvious filaments (red fluorescence) and intracellular DNA leakage (blue fluorescence) under the induction of PMA inducer. However, the red fluorescence of BM-Ru1-Cl-5 and BM-Cl-5 groups was significantly weakened, which proved that BM-Ru1-Cl and BM-Cl can effectively inhibit the expression of H3cit and the formation of NETs at a dose of 5 μM.

[0116] Evaluation of in vivo antitumor activity

[0117] 1. Preliminary evaluation of in situ antitumor activity

[0118] Female BALB / c mice were purchased from Beijing VitalLiver Laboratory Animal Technology Co., Ltd. All animal experiments were conducted according to procedures approved by the Animal Care and Use Committee of Capital Medical University. Mice were randomly assigned to: 1) Control; 2) N-(1-(benzylamino)-5-(2-chloroacetimidinyl)-1-oxopentan-2-yl)-6-(dimethylamino)-2-naphthylcarboxamide (YW3-56); 3) Cisplatin; 4) BM-Ru1-iv-5μmol / kg; 5) BM-Ru1-Cl-iv-5μmol / kg; 6) BM-Cl-iv-5μmol / kg; 7) BM-Ru1-ig-20μmol / kg;

[0119] 8) BM-Ru1-Cl-ig-20μmol / kg; 9) BM-Cl-ig-20μmol / kg, 12 cells per group. Luciferase-labeled 4T1 cells were cultured in high-glucose DMEM medium containing 10% FBS. Cells were collected after reaching the appropriate passage number. Cells were washed three times with PBS and resuspended at 1×10⁻⁶. 7 Cells were stored at a density of [number] cells / mL on ice. 0.1 mL of cell suspension was subcutaneously injected into the third pair of mammary pads on the left side of mice. Tumor volume was measured using calipers after 5 days, and mice were administered the appropriate drugs according to their assigned groups. Drug administration was repeated every two days, with mouse weight and tumor volume measured again. Mice were sacrificed on day 15 for organ harvesting. Table 3 shows that, compared with the control group, 4T1 mice treated with BM-Ru1-Cl-iv-5μmol / kg, BM-Cl-iv-5μmol / kg, and BM-Ru1-Cl-ig-20μmol / kg had significantly smaller tumors (p values ​​were 0.0083; <0.0001; <0.0001, respectively). This indicates that BM-Ru1-Cl and BM-Cl have a good inhibitory effect on in situ tumor growth. Compared with the BM-Ru1-Cl-iv-5μmol / kg group, the tumor weight of the BM-Cl-iv-5μmol / kg and BM-Ru1-Cl-ig-20μmol / kg groups was significantly different (p values ​​were 0.0383 and 0.0336, respectively). However, there was no significant difference in tumor weight between the BM-Ru1-Cl-ig-20μmol / kg and BM-Cl-iv-5μmol / kg groups, suggesting that BM-Ru1-Cl can exert its effect through gavage and that the dosage can be increased.

[0120] Table 3. Inhibitory effects of the tested compounds on tumor growth in BABL / c 4T1 tumor-bearing mice.

[0121] Group Dosage (μmol / kg) Average tumor weight (g) Tumor inhibition rate (%) Control / 0.687±0.151 / YW3-56 5 0.435±0.144 36.8 Cisplatin 2.5 0.264±0.077 61.6 BM-Ru1-iv 5 0.765±0.119 0 BM-Ru1-Cl-iv 5 0.475±0.080 30.9 BM-Cl-iv 5 0.372±0.114 45.9 BM-Ru1-ig 20 0.672±0.155 2.3 BM-Ru1-Cl-ig 20 0.369±0.066 46.3 BM-Cl-ig 20 0.642±0.221 6.5

[0122] 2. Evaluation of dose-response relationship

[0123] Female BALB / c mice were purchased from Beijing VitalLiver Laboratory Animal Technology Co., Ltd. All animal experiments were conducted according to procedures approved by the Animal Care and Use Committee of Capital Medical University. Mice were randomly assigned to: 1) Control; 2) Cisplatin-ig-20μmol / kg; 3) BM-Ru1-Cl-ig-40μmol / kg; 4) BM-Ru1-Cl-ig-20μmol / kg; 5) BM-Ru1-Cl-ig-10μmol / kg; 6) BM-Ru1-Cl-ig-5μmol / kg, with 12 mice in each group. Luciferase-labeled 4T1 cells were cultured in high-glucose DMEM medium containing 10% FBS. Cells were collected after reaching the appropriate passage number. After washing three times with PBS, the cells were resuspended at 1×10⁻⁶. 7 Cells were stored at a density of [number] cells / mL on ice. 0.1 mL of cell suspension was subcutaneously injected into the third pair of mammary pads on the left side of mice. Tumor volume was measured using calipers 7 days later, and appropriate drugs were administered according to the groups. The cisplatin group received the drug every three days, while the other groups received the drug every two days. Mice were weighed and tumor volume was measured. Mice were sacrificed on day 15 for organ harvesting. Table 4 shows that, compared with the control group, the 4T1 tumor-bearing mice in the BM-Ru1-Cl-ig-40 μmol / kg, BM-Ru1-Cl-ig-20 μmol / kg, and BM-Ru1-Cl-ig-10 μmol / kg groups had significantly lighter tumors (p<0.0001), indicating that gavage administration can achieve better tumor growth inhibition with increased dosage without causing mouse mortality.

[0124] Table 4. Inhibitory effect of different doses of BM-Ru1-Cl on tumor growth in BABL / c tumor-bearing mice.

[0125] Group Dosage (μmol / kg) Average tumor weight (g) Tumor inhibition rate (%) Control / 0.726±0.209 / Cisplatin-ig 20 0.305±0.096 57.9 BM-Ru1-Cl-ig-40 40 0.156±0.074 78.6 BM-Ru1-Cl-ig-20 20 0.242±0.078 66.7 BM-Ru1-Cl-ig-10 10 0.285±0.074 60.8 BM-Ru1-Cl-ig-5 5 0.540±0.268 25.6

[0126] As demonstrated by the above embodiments, the PAD4 inhibitor BM-Cl and its bipyridine ruthenium complex BM-Ru1-Cl provided by this invention exhibit excellent antitumor activity when administered via tail vein in a mouse tumor model, and dose-dependent excellent antitumor activity when administered via gavage in the same mouse tumor model. Furthermore, BM-Ru1-Cl retains its antitumor effect after oral absorption into the bloodstream, remaining unaffected by the gastric environment, making it an excellent orally administered PAD4 inhibitor. In contrast, BM-Cl exhibits no antitumor activity when orally administered.

[0127] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bipyridine derivative, characterized in that, It has the structure shown in Equation 1:

2. The method for preparing the bipyridine derivative according to claim 1, characterized in that, Includes the following steps: Z-Orn(Boc)-OH was activated in an organic solvent and then reacted with benzylamine under alkaline conditions to obtain Z-Orn(Boc)-NBzl; In a reducing gas atmosphere, Z-Orn(Boc)-NBzl is subjected to a catalytic reduction reaction in an organic solvent to obtain Orn(Boc)-NBzl; 4'-Methyl-2,2'-bipyridine-4-carboxylic acid was activated in an organic solvent and then reacted with Orn(Boc)-NBzl under alkaline conditions to obtain BM-Orn(Boc)-NBzl; The BM-Orn(Boc)-NBzl was reacted in an organic solution of hydrogen chloride to obtain the hydrochloride salt of BM-Orn-NBzl. The hydrochloride of BM-Orn-NBzl and ethyl 2-chloroacetylimine were reacted in an organic solvent under alkaline conditions to obtain a bipyridine derivative with the structure shown in Formula 1. The structures of Z-Orn(Boc)-OH, Z-Orn(Boc)-NBzl, Orn(Boc)-NBzl, BM-Orn(Boc)-NBzl, BM-Orn-NBzl, and 2-chloroacetylimine ethyl ester hydrochloride are as follows:

3. The use of the bipyridine derivative of claim 1 or the bipyridine derivative prepared by the preparation method of claim 2 in the preparation of PAD4 inhibitors or anticancer drugs.

4. A PAD4 inhibitor or anticancer drug, characterized in that, This includes the bipyridine derivative as described in claim 1 or the bipyridine derivative prepared by the preparation method described in claim 2.

5. A bipyridine-ruthenium complex, characterized in that, It has the structure shown in Equation 2:

6. The method for preparing the bipyridine-ruthenium complex according to claim 5, characterized in that, Includes the following steps: The bipyridine derivative of claim 1 or the bipyridine derivative prepared by the preparation method of claim 2 is reacted with cis-dichlorobis(2,2'-bipyridine)ruthenium to obtain the bipyridine-ruthenium complex with the structure shown in Formula 2; The structure of the cis-dichlorobis(2,2'-bipyridine)ruthenium is as follows:

7. The preparation method according to claim 6, characterized in that, The preparation method of the bipyridine ruthenium includes the following steps: The bipyridine ruthenium was obtained by reacting 4'-methyl-2,2'-bipyridine-4-carboxylic acid and cis-dichlorobis(2,2'-bipyridine)ruthenium dihydrate in a polar solvent.

8. The use of the bipyridine-ruthenium complex of claim 5 or the bipyridine-ruthenium complex prepared by the preparation method of claim 6 or 7 in the preparation of PAD4 inhibitors or anticancer drugs.

9. A PAD4 inhibitor or anticancer drug, characterized in that, This includes the bipyridine-ruthenium complex as described in claim 5 or the bipyridine-ruthenium complex prepared by the preparation method described in claim 6 or 7.

10. The PAD4 inhibitor or anticancer drug according to claim 9, characterized in that, The PAD4 inhibitor or anticancer drug is in the form of an oral administration.

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