A mitochondrial-targeting compound, its preparation method and application, and a pharmaceutical composition thereof.

Abstract

This invention provides a mitochondrial-targeting compound—triphenylphosphine GW7647 conjugate (TPP-GW)—that can effectively enhance anti-tumor function, a pharmaceutical composition containing TPP-GW, and a method for preparing and using the TPP-GW. The triphenylphosphine GW7647 conjugate is obtained by conjugating (3-aminopropyl)triphenylphosphine with GW7647. It is a novel mitochondrial-targeting PPARα agonist that can enhance the toxic effects of the original GW7647 on tumor cells and has a synergistic effect with immune checkpoint blockade therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a mitochondrial-targeting compound—a triphenylphosphine GW7647 conjugate, its preparation method and application, and a pharmaceutical composition comprising the triphenylphosphine GW7647 conjugate. Background Technology

[0002] Immunotherapy, based on immunology, has become an effective means of clinical cancer treatment. However, the clinical response rate of immunotherapy is low, less than 30%, and drug resistance is highly likely to occur. This is due to the development of drug resistance in tumor cells and the existence of an immunosuppressive microenvironment. Tumor cells can evolve various mechanisms to reduce their own immunogenicity to evade recognition and killing by the immune system, preventing immunotherapy from achieving its intended therapeutic effect. Inducing tumor cell death has become an effective strategy to improve the immunosuppressive tumor microenvironment. Immunogenic cell death (ICD) refers to the process by which tumor cells, upon receiving external stimuli, die and simultaneously transform from non-immunogenic to immunogenic, mediating the body's anti-tumor immune response. During ICD, tumor cells release neoantigen epitopes and damage-associated molecular patterns (DAMPs), such as the exposure of calreticulin (CRT), the release of ATP (adenosine triphosphate), and the extracellular leakage of high mobility group protein 1 (HMGB1). The release of ICD markers can promote the maturation of dendritic cells (DCs) and enhance cytotoxic CD8. + T cell activation, CD8 + Once activated, T cells kill tumor cells through direct cytotoxicity by secreting granzyme B (GzmB) and interferon-γ (IFN-γ). The search for and discovery of compounds capable of inducing ICD is a new direction for tumor immunotherapy.

[0003] The chemical formula of GW7647 (chemical name: 2-(4-(2-(1-cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid) is C 29 H 46N2O3S was initially developed as a PPARα agonist. Non-patent literature 1, 2, and 3 report on the applications of GW7647 in liver and cardiovascular diseases, respectively, but reports on its anti-tumor effects are extremely limited. The inventors of this invention used GW7647 as the experimental subject, modified its structure, and conducted animal experiments via intratumoral injection to improve the drug's safety in vivo and alter its existing target, thereby enhancing its anti-tumor efficacy and enriching the research progress on this compound in anti-tumor applications.

[0004] Existing technical documents

[0005] Non-patent literature 1: van der Meer, D. Letal. Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis.

[0006] Non-patent literature 2: Lam, VHet al. Activating PPARα prevents post-ischemiccontractiledysfunction in hypertrophied neonatal hearts.

[0007] Non-patent literature 3: Lee, HY et al. PPAR-α and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal. Summary of the Invention

[0008] After conducting in-depth research on the above-mentioned problems, the inventors discovered that:

[0009] Mitochondria are the site of cellular metabolism and energy production. The Warburg effect indicates that tumor cells produce energy through glycolysis, a low-efficiency method. To meet their high energy demands, tumor cells produce more mitochondria. Compared to normal cells, tumor cells have more mitochondria, larger mitochondria, and lower membrane potential, which facilitates the accumulation of positively charged compounds. Covalently linking a lipophilic cation to a pharmacophore of interest can enable targeted delivery of the target compound to the mitochondria. Triphenylphosphine cation (TPP) + It is simple to synthesize, has high targeting efficiency, and is relatively stable in biological systems, making it a widely used mitochondrial targeting vector.

[0010] In view of this, the inventors have for the first time successfully constructed a triphenylphosphine cation (TPP) + The coupling reaction between GW7647 and GW7647 yielded the first mitochondrial-targeted triphenylphosphine GW7647 conjugate.

[0011] The problem that the invention aims to solve

[0012] The purpose of this invention is to solve the target problem of the existing drug GW7647, and to provide a method and application for constructing mitochondrial-targeted triphenylphosphine GW7647 conjugates that is simple to prepare and can effectively improve anti-tumor function.

[0013] Technical means to solve the problem

[0014] The present invention provides the following (1) to (32):

[0015] (1) A mitochondrial-targeting compound, which is a conjugate of triphenylphosphine GW7647, with the following chemical formula:

[0016]

[0017] (2) A pharmaceutical composition comprising the compound of (1).

[0018] (3) The drug composition as in (2) is an antitumor agent.

[0019] (4) The pharmaceutical composition of (3), wherein the tumor involved by the antitumor agent is selected from colon cancer, melanoma and prostate cancer.

[0020] The method for preparing the compound (5)(1) includes coupling (3-aminopropyl)triphenylphosphine with GW7647.

[0021] (6) The preparation method as in (5), wherein the molar ratio of GW7647 to (3-aminopropyl)triphenylphosphine is 1:1 to 1:2.

[0022] (7) The preparation method as in (5) or (6), wherein the molar ratio of GW7647 to (3-aminopropyl)triphenylphosphine is 1:1 to 1:1.5.

[0023] (8) The preparation method of any one of (5) to (7), wherein the molar ratio of GW7647 to (3-aminopropyl)triphenylphosphine is 1:1 to 1:1.2.

[0024] (9) The preparation method of any one of (5) to (8), wherein GW7647, organic base, condensing agent and coupling agent are dissolved in solvent, stirred and then (3-aminopropyl)triphenylphosphine is added to the reaction system.

[0025] (10) The preparation method of (9) wherein the molar ratio of GW7647 to organic base, condensing agent, coupling agent and (3-aminopropyl)triphenylphosphine is 1:1:1:1:1 to 1:2:2:2:2.

[0026] (11) The preparation method as in (9) or (10), wherein the molar ratio of GW7647 to organic base, condensing agent, coupling agent and (3-aminopropyl)triphenylphosphine is 1:1:1:1:1 to 1:1.5:1.5:1.5:1.5.

[0027] (12) The preparation method of any one of (9) to (11), wherein the molar ratio of GW7647 to organic base, condensing agent, coupling agent and (3-aminopropyl)triphenylphosphine is 1:1:1:1:1 to 1:1.1:1.1:1.1:1.2.

[0028] (13) The preparation method of any one of (9) to (12), wherein the organic base is selected from N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, 1,8-diazabicycloundec-7-ene, pyridine, 2,6-dimethylpyridine and tetramethylethylenediamine.

[0029] (14) The preparation method of any one of (9) to (13), wherein the organic base is N,N-diisopropylethylamine.

[0030] (15) The preparation method of any one of (9) to (14), wherein the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, benzotriazole-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, propylphosphonic anhydride, and N,N'-carbonyldiimidazole.

[0031] (16) The preparation method of any one of (9) to (15), wherein the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0032] (17) The preparation method of any one of (9) to (16), wherein the coupling agent is selected from 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, N-hydroxysuccinimide, ethyl 2-oxime cyanoacetate and 4-dimethylaminopyridine.

[0033] (18) The preparation method of any one of (9) to (17), wherein the coupling agent is 1-hydroxybenzotriazole.

[0034] (19) The preparation method of any one of (9) to (18), wherein the solvent is selected from N,N-dimethylformamide, dichloromethane, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N-methylpyrrolidone.

[0035] (20) The preparation method of any one of (9) to (19), wherein the solvent is N,N-dimethylformamide.

[0036] (21) The preparation method of any one of (9) to (20), wherein the solvent is an anhydrous solvent.

[0037] (22) The preparation method of any one of (9) to (21) has a reaction temperature of 5℃ to 80℃.

[0038] (23) The preparation method of any one of (9) to (22) has a reaction temperature of 10℃ to 40℃.

[0039] (24) The preparation method of any one of (9) to (23) has a reaction temperature of 20℃ to 30℃.

[0040] (25) The preparation method of any one of (9) to (24) has a reaction time of more than 1 hour.

[0041] (26) The preparation method of any one of (9) to (25) has a reaction time of more than 10 hours.

[0042] (27) The preparation method of any one of (9) to (26) has a reaction time of less than 48 hours.

[0043] (28) The preparation method of any one of (9) to (27) has a reaction time of 16 hours to 28 hours.

[0044] Application of compounds (29)(1) in the preparation of antitumor drugs.

[0045] (30) As in (29), wherein the tumor involved in the antitumor drug is selected from colon cancer, melanoma and prostate cancer.

[0046] The combined use of (31)(1) compounds with immune checkpoint blockade therapy.

[0047] The combined use of any one of the drug compositions in (32)(2) to (4) with immune checkpoint blockade therapy.

[0048] Technical effects of the invention

[0049] The mitochondrial-targeting compound, triphenylphosphine GW7647 conjugate (TPP-GW), efficiently induces ICD in tumor cells and activates the maturation of BMDCs (miceolecularly derived dendritic cells) and T cells. In vitro studies have shown that TPP-GW has nearly 10 times the cytotoxicity of GW7647 and exhibits specific tumor cell killing. Furthermore, TPP-GW demonstrates excellent in vivo tumor-suppressing effects. Attached Figure Description

[0050] Figure 1 This is a synthesis route diagram for the TPP-GW of the present invention.

[0051] Figure 2 This is the mass spectrum of the TPP-GW of the present invention.

[0052] Figure 3 This is the hydrogen NMR spectrum of the TPP-GW of this invention.

[0053] Figure 4a These are electron microscopy images showing the effects of TPP-GW, GW7647, and PBS on mitochondrial structure in tumor cells according to the present invention.

[0054] Figure 4b Fluorescence images showing the effects of TPP-GW, GW7647, TPP, and PBS of the present invention on Mitotracker, a fluorescent dye that indicates mitochondrial function in tumor cells.

[0055] Figure 4c This is a dose-toxicity curve of TPP-GW, GW7647 and TPP in mouse melanoma B16 cells of the present invention; data are expressed as mean ± SD (n = 3).

[0056] Figure 4d This is a dose-toxicity curve of TPP-GW, GW7647 and TPP in mouse colon cancer MC38 cells of the present invention; data are expressed as mean ± SD (n = 3).

[0057] Figure 4e This is a dose-toxicity curve of TPP-GW, GW7647 and TPP in mouse prostate cancer RM-1 cells; data are expressed as mean ± SD (n = 3).

[0058] Figure 4f This is a dose-toxicity curve of TPP-GW, GW7647 and TPP in mouse liver AML12 cells; data are expressed as mean ± SD (n = 3).

[0059] Figure 5aThis is a statistical graph showing the effects of TPP-GW, GW7647, TPP, and PBS on HMGB1-Gluc release according to the present invention; data are expressed as mean ± SD (n = 3).

[0060] Figure 5b This is a statistical graph showing the effects of TPP-GW, GW7647, TPP, and PBS on ATP secretion according to the present invention; data are expressed as mean ± SD (n = 3).

[0061] Figure 5c Representative flow cytometry images and average fluorescence intensities show the effects of TPP-GW, GW7647, TPP, and PBS on CRT release in this invention.

[0062] Figure 6a Representative flow cytometry images and average fluorescence intensities of CD80 and CD86 expression on the surface of BMDC cells co-cultured with TPP-GW, GW7647, TPP, and PBS as promoted by this invention.

[0063] Figure 6b Representative flow cytometry images and average fluorescence intensities of B3Z T cells co-cultured with TPP-GW, GW7647, TPP, and PBS to promote maturation (expression of CD69 and CD8 on the cell surface) according to this invention.

[0064] Figure 6c These are representative flow cytometry images and average fluorescence intensities of the effector cytokine GzmB secreted by co-cultured B3Z T cells using TPP-GW, GW7647, TPP, and PBS, as described in this invention.

[0065] Figure 6d These are representative flow cytometry images and average fluorescence intensities of IFN-γ secretion from co-cultured B3Z T cells promoted by TPP-GW, GW7647, TPP, and PBS according to the present invention.

[0066] Figure 7a This is a schematic diagram of the intratumoral drug delivery process in animal experiments according to the present invention.

[0067] Figure 7b Images of MC38 xenografts in animal experiments for this invention.

[0068] Figure 7c The figures show the tumor volume change curves for each group in the animal experiments of this invention.

[0069] Figure 7d This is a statistical chart of tumor weight in each group of animal experiments according to the present invention. The data are expressed as mean ± SD (n = 6).

[0070] Figure 7eImages of CD8 and TUNEL staining in animal experiments according to this invention.

[0071] Figure 7f The figures show the weight change curves of mice in each group during the animal experiments of this invention.

[0072] Figure 7g This is a schematic diagram of the flow cytometry experimental procedure for animal experiments according to the present invention.

[0073] Figure 7h CD8 infiltrating tumor tissue in animal experiments of this invention + T cell percentage, data are expressed as mean ± SD (n = 6).

[0074] Figure 7i CD8+ in tumor tissue flow cytometry for animal experiments according to this invention + GzmB released by T cells, data are presented as mean ± SD (n = 6).

[0075] Figure 7j CD8+ in tumor tissue flow cytometry for animal experiments according to this invention + IFN-γ released by T cells, data are presented as mean ± SD (n = 6).

[0076] Figure 7k The number of NK cells infiltrating tumor tissue in animal experiments of this invention is given, and the data are expressed as mean ± SD (n = 6).

[0077] Figure 7l The data represents GzmB released by NK cells in tumor tissue during flow cytometry experiments in the animal experiments of this invention. The data are expressed as mean ± SD (n = 6).

[0078] Figure 7m The data represents the IFN-γ released by NK cells in tumor tissue during flow cytometry experiments in the animal experiments of this invention. The data are expressed as mean ± SD (n = 6).

[0079] Figure 7n The percentage of Treg cells infiltrating tumor tissue in animal experiments of this invention is given, and the data are expressed as mean ± SD (n = 6).

[0080] Figure 7o Macrophages infiltrating tumor tissue in animal experiments of this invention CD86 expression in cells, data are presented as mean ± SD (n = 6).

[0081] Figure 7p Macrophages infiltrating tumor tissue in animal experiments of this invention CD80 expression in cells, data are presented as mean ± SD (n = 6).

[0082] Figure 7q Macrophages infiltrating tumor tissue in animal experiments of this invention CD206 expression in cells, data are presented as mean ± SD (n = 6).

[0083] Figure 7r Macrophages infiltrating tumor tissue in animal experiments of this invention MHC II expression in cells, data are presented as mean ± SD (n = 6).

[0084] Figure 7s The data represents CD86 expressed by DC cells infiltrating tumor tissue in animal experiments of this invention. Data are expressed as mean ± SD (n = 6).

[0085] Figure 7t The data represents CD80 expressed by DC cells infiltrating tumor tissue in animal experiments of this invention. The data are expressed as mean ± SD (n = 6).

[0086] Figure 7u The data represents CD40 expressed by DC cells infiltrating tumor tissue in animal experiments of this invention. The data are expressed as mean ± SD (n = 6).

[0087] Figure 8a This is a schematic diagram of the animal experiment drug delivery process for the combined application of the present invention and immunoblocking therapy.

[0088] Figure 8b Images of B16 xenografts in animal experiments using the combination of immunoblockade therapy in this invention.

[0089] Figure 8c The figures show the tumor volume change curves for each group in the animal experiments of this invention.

[0090] Figure 8d This is a statistical chart of tumor weight in each group of animal experiments using the combined immunoblocking therapy of this invention. The data are expressed as mean ± SD (n = 6).

[0091] Figure 8e The figures show the weight change curves of mice in each group during animal experiments using the combined immunoblocking therapy of this invention. Detailed Implementation

[0092] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. In this application, the use of "~" to indicate a numerical range, unless otherwise specified, means that the numerical values ​​before and after it are included as the lower and upper limits.

[0093] The first aspect of the present invention relates to a mitochondrial-targeting compound, which is a triphenylphosphine GW7647 conjugate, the chemical formula of which is shown below:

[0094] Its molecular weight is 804.4685.

[0095] The second aspect of the present invention relates to a method for preparing the above-mentioned compound, as detailed in [reference needed]. Figure 1 The synthesis route map.

[0096] The preparation method includes the step of coupling (3-aminopropyl)triphenylphosphine with GW7647. The ratio of GW7647 to (3-aminopropyl)triphenylphosphine, in molar ratio, is 1:1 to 1:2, preferably 1:1 to 1:1.5, and more preferably 1:1 to 1:1.2.

[0097] The specific steps include: dissolving GW7647, an organic base, a condensing agent, and a coupling agent in a solvent, stirring, and then adding (3-aminopropyl)triphenylphosphine to the reaction system to continue the reaction. After the reaction is complete, the solvent in the reaction system is evaporated to dryness, then reconstituted with a suitable solvent, such as dichloromethane, and the dichloromethane layer is washed with saturated NaCl solution. After drying the dichloromethane layer overnight with anhydrous sodium sulfate, the mitochondrial-targeted (3-aminopropyl)triphenylphosphine GW7647 conjugate is obtained by rotary evaporation.

[0098] Regarding the proportions of the components participating in the reaction, in molar ratio, GW7647:organic base:condensing agent:coupling agent:(3-aminopropyl)triphenylphosphine is 1:1:1:1:1 to 1:2:2:2:2, preferably 1:1:1:1:1 to 1:1.5:1.5:1.5:1.5, more preferably 1:1:1:1:1 to 1:1.1:1.1:1.1:1.2.

[0099] The organic base is selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine (NMM), 1,8-diazabicycloundec-7-ene (DBU), pyridine, 2,6-dimethylpyridine, and tetramethylethylenediamine (TMEDA), etc., preferably N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, 1,8-diazabicycloundec-7-ene, and more preferably N,N-diisopropylethylamine.

[0100] The condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and benzotriazole-1-yl-oxytripyrrolidine hexafluorophosphate. Phosphorus (pyBOP), propylphosphonic anhydride (T3P), and N,N'-carbonyldiimidazole (CDI), preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, more preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0101] The coupling agent is selected from 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azobenzotriazole (HOAT), N-hydroxysuccinimide (HOSu), ethyl 2-oxime cyanoacetate (Oxyma Pure), and 4-dimethylaminopyridine (DMAP), etc., preferably 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, N-hydroxysuccinimide, and more preferably 1-hydroxybenzotriazole.

[0102] The solvent is selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), and N-methylpyrrolidone (NMP), etc., preferably N,N-dimethylformamide, dichloromethane, or tetrahydrofuran, and more preferably N,N-dimethylformamide. The solvent is preferably an anhydrous solvent.

[0103] In the preparation method of the present invention, the reaction temperature is 5℃~80℃, preferably 10℃~40℃, more preferably 20℃~30℃, and the reaction time is more than 1 hour, preferably more than 10 hours, more preferably less than 48 hours, and more preferably 16 hours~28 hours.

[0104] The advantages of TPP-GW, prepared according to the above method, in inducing tumors to exert anti-tumor immune effects are: (1) Simple synthesis route: The carboxyl group of GW7647 is used to perform an amide bond reaction with the amino group of the small molecule (3-aminopropyl)triphenylphosphine with mitochondrial targeting, which increases the accumulation of GW7647 in mitochondria and achieves targeting of tumor cells; (2) Excellent anti-tumor function: TPP-GW is a compound that strongly induces ICD in tumor cells, which can destroy the structure and function of mitochondria in tumor cells, promote the maturation of BMDCs co-cultured with tumor cells and the activation of T cells; (3) No safety risk: GW7647 has high in vivo safety. The animal experiment was administered via intratumoral injection, and there was no systemic toxicity; (4) Synergistic effect with immune checkpoint blockade therapy: TPP-GW can exert anti-tumor immune effects by activating a variety of immune cells in the tumor immune microenvironment. The combined application with αPD-1 monoclonal antibody showed good efficacy against low immunogenic melanoma, etc.

[0105] A third aspect of the present invention relates to a pharmaceutical composition comprising the triphenylphosphine GW7647 conjugate obtained above, the pharmaceutical composition being used for antitumor purposes. The tumors referred to here include colon cancer, melanoma, and prostate cancer, among others.

[0106] The fourth aspect of this invention relates to the use of the above-mentioned compounds in the preparation of antitumor drugs. The tumors referred to here include colon cancer, melanoma, and prostate cancer, etc. The fifth aspect of this invention relates to the combined use of the above-mentioned compounds with immune checkpoint blockade therapy, and the sixth aspect of this invention relates to the combined use of a pharmaceutical composition with immune checkpoint blockade therapy. As described above, when TPP-GW or a pharmaceutical composition containing TPP-GW is used in combination with immune checkpoint blockade therapy, TPP-GW can exert antitumor immune effects by activating various immune cells in the tumor immune microenvironment, and its combined use with αPD-1 monoclonal antibody shows good efficacy against low-immunogenic melanoma.

[0107] Example

[0108] GW7647 (9 mg, 0.0179 mmol, purchased from MCE), N,N-diisopropylethylamine (2.7 mg, 0.021 mmol, purchased from Innochem), EDCI (3.8 mg, 0.01982 mmol, purchased from Innochem), and 1-hydroxybenzotriazole (2.6 mg, 0.01924 mmol, purchased from Innochem) were dissolved in anhydrous DMF (purchased from Innochem) and stirred at room temperature for 3 hours. (3-aminopropyl)triphenylphosphine (8 mg, 0.02497 mmol, purchased from Innochem) was added to the reaction system, and the reaction was continued at room temperature for 12 hours. After the reaction was complete, the solvent DMF in the reaction system was evaporated to dryness, redissolved in dichloromethane, and the dichloromethane layer was washed twice with saturated NaCl solution. After the dichloromethane layer was dried overnight with anhydrous sodium sulfate, mitochondrial-targeting GW7647 (15 mg) was obtained by rotary evaporation, with a yield of 88%.

[0109] like Figure 2 The mass spectrum of TPP-GW shown is MS:C 50 H 67 N3O2PS + Cal. 804.46, found 804.4685(M + Its molecular weight is 804.4685.

[0110] like Figure 3 The TPP-GW is shown in the 1H NMR spectrum. 1 H-NMR (600MHz, CDCl3-d6): δ8.646(t,J=6.0Hz,1H),7.797-7.764(m,9H),7.685-7.653(m,6H),7.420(d,J =8.4Hz,2H),7.061(d,J=8.4Hz,2H),4.069(d,J=7.8Hz,1H),3.940-3.891(m,2H),3.627-3.609(m,3H),3. 375(t,J=7.8Hz,2H),3.036(t,J=7.2Hz,2H),2.777(t,J=7.8Hz,2H),1.913-1.889(m,4H),1.683-1.666(m ,7H),1.558(s,6H),1.494-1.444(m,2H),1.390-1.313(m,4H),1.212-1.028(m,10H),0.878-0.844(m,2H).

[0111] Test case

[0112] 1. TPP-GW inhibits tumor cell growth by disrupting mitochondrial structure and function. To determine whether TPP-GW targets mitochondria, after 12 hours of administration of GW7647 (10 μM), TPP-GW (6 μM), and PBS (Thermo Biotech), respectively, the mitochondrial structure of mouse colon cancer MC38 cells (purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) was scanned and photographed using a transmission electron microscope (HITACHIH, T7800). The results are as follows: Figure 4a As shown, TPP ((3-aminopropyl)triphenylphosphine) mediated and enhanced the destructive effect of the compound on mitochondria. In TPP-GW treated cells, the mitochondrial membrane cristae disappeared, and contents flowed out. Six hours after administration (GW7647 (10 μM), TPP-GW (6 μM), TPP (10 μM)), mitochondria were labeled with the mitochondrial dye MitoTracker Red (red fluorescence, purchased from Thermo Fisher Scientific), and photographed using a laser confocal microscope (OLYMPUS, FV3000). The results are as follows. Figure 4b As shown, cells treated with TPP-GW showed almost no red fluorescence, while cells treated with TPP or GW7647 showed no difference in red fluorescence compared to the control group. These results indicate that TPP-GW can more effectively disrupt mitochondrial structure and function.

[0113] The inventors also studied the growth inhibition effects of GW7647 and TPP-GW on tumor cells in different cell lines.

[0114] like Figures 4c to 4e As shown, compared with GW7647, TPP-GW enhanced its cytotoxicity in mouse melanoma B16 cells (purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), mouse colon cancer MC38 cells, and mouse prostate cancer RM-1 cells (purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), exhibiting an IC50 value. 50 The value decreased; in addition, the cytotoxicity of TPP-GW was tested using normal mouse liver cells AML12 (purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) to determine whether it was tumor-specific. Figure 4f As shown, IC in AML12 50 The value is much higher than the above three tumor cell lines, which indicates that TPP-GW has a certain degree of tumor cell specificity.

[0115] 2. TPP-GW induces ICD in tumor cells (compound concentrations: TPP (10 μM), GW7647 (10 μM), TPP-GW (6 μM))

[0116] HMGB1 release, ATP secretion, and CRT exposure on the cell membrane surface are markers of ICD in tumor cells. Experiments have shown that TPP-GW can induce the upregulation of the expression of several markers.

[0117] In MC38-HMGB1-Gluc reporter gene stable transfected cells (obtained by transfecting HMGB1-Gluc plasmid (purchased from Addgene) into MC38 cells and selecting single clones), Renilla- Renilla- The Luciferase Assay System (Promega) was used to detect the activity of HMGB1-Gluc secreted by cells into the culture supernatant. Results are as follows: Figure 5a As shown, TPP-GW significantly upregulated HMGB1-Gluc activity after 12 hours of treatment, and this upregulation was dose-dependent.

[0118] use ATP detection system The ATP Assay System (Promega) detects ATP secretion, and the results are as follows: Figure 5b As shown, TPP-GW significantly promoted ATP secretion after 12 hours of treatment.

[0119] The expression of CRT on the surface of tumor cells was detected by flow cytometry (CRT antibody was purchased from Cell Signaling Technology). The results are as follows: Figure 5c As shown, TPP-GW can significantly induce CRT exposure after 6 hours of application.

[0120] The above results indicate that TPP-GW can induce the production of ICD markers in tumor cells.

[0121] 3. TPP-GW induces DC maturation and T cell activation (compound concentrations: TPP (10 μM), GW7647 (10 μM), TPP-GW (6 μM))

[0122] The HMGB1, CRT, and ATP released by ICDs can promote the maturation of dendritic cells (DCs), and mature DCs can initiate and activate T cells.

[0123] MC38 tumor cells that had undergone ICD after treatment with TPP-GW (6 μM) for 6 h were co-cultured with bone marrow-derived dendritic cells (BMDCs) (isolated from mouse leg bones and induced to differentiate for 7 days with GM-CSF (Peprotech) and IL-4 (Peprotech)) for 24 h. The maturation status of BMDCs was detected by flow cytometry. Results are as follows: Figure 6a As shown, TPP-GW can induce an increase in CD80 and CD86 on the BMDC surface.

[0124] T cell activation requires the participation of dendritic cells (DCs). The inventors utilized a three-cell co-culture system (B16-OVA (mouse melanoma cells - OVA), BMDCs, and B3Z T cells (mouse T cell hybridoma cells) (a gift from Professor Xia Xiaojun of Sun Yat-sen University)). B16-OVA cells were first treated with TPP-GW (6 μM) for 6 hours, then co-cultured with BMDCs and B3Z T cells for 24 hours. Flow cytometry was used to detect the expression of CD69, a marker of T cell activation, as well as the T cell effector molecules IFN-γ and GzmB. The results are as follows: Figure 6b As shown, TPP-GW can promote the activation of T cells in a three-cell co-culture system, such as... Figure 6c As shown, TPP-GW can promote the secretion of GzmB by T cells, such as Figure 6d As shown, TPP-GW can promote the secretion of IFN-γ by T cells.

[0125] 4. TPP-GW induces antitumor effects in vivo

[0126] To investigate the antitumor effect of TPP-GW in a mouse MC38 colon cancer xenograft model. Six- to eight-week-old C57BL / 6J mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were subcutaneously inoculated with MC38 cells on the right side. 6 One per tumor. When the tumor volume is approximately 100 mm. 3 Mice were randomly divided into four groups, and intratumoral injections were administered of PBS, GW7647 5 mg / kg, TPP-GW 5 mg / kg, and TPP-GW 10 mg / kg, respectively. The experimental procedure is as follows: Figure 7a As shown. On day 15 of the experiment, mice were euthanized by CO2 asphyxiation, and tumor tissue was removed. Figure 7b As shown, both the TPP-GW 5 mg / kg and TPP-GW 10 mg / kg groups inhibited the growth of colon cancer xenografts in mice. Figure 7c and Figure 7d As shown, there was no statistically significant difference in tumor volume and weight between the GW7647 5 mg / kg group and the PBS control group. Immunohistochemical experiments were performed on the tumor tissue, and the results are as follows: Figure 7e As shown, TPP-GW can increase the number of infiltrating CD8 cells in tumor tissue and increase the number of apoptotic cells in tumor tissue (TUNEL). + During the experiment, the weight of the mice was measured and recorded, and the results are as follows: Figure 7f As shown, there was no statistically significant difference in body weight among the experimental groups, indicating that intratumoral administration of GW7647 or TPP-GW did not produce systemic toxicity.

[0127] Next, we analyzed the immune cells infiltrating the tumor tissue. The flow cytometry flowchart is shown below. Figure 7g As shown. When the tumor volume is approximately 100 mm... 3 Mice were randomly divided into four groups, and their tumors were injected intratumorally with PBS, GW7647 5 mg / kg, TPP-GW 5 mg / kg, and TPP-GW 10 mg / kg, respectively. On day 6, mice were sacrificed by CO2 asphyxiation, and tumor tissue was dissected. The tumor tissue was digested, ground, filtered, washed, and stained with antibodies by flow cytometry before being analyzed. The results are as follows: Figure 7h As shown, TPP-GW can increase the number of CD8 cells infiltrating tumor tissue. + The number of T cells; such as Figure 7i and 7j As shown, it can also increase the secretion of effector molecules GzmB and IFN-γ in CD8 cells; at the same time, as Figure 7k As shown, it can increase the number of NK cells; such as Figure 7l and Figure 7m As shown, it can increase the secretion of effector molecules GzmB and IFN-γ in NK cells; such as Figure 7n As shown, it can reduce the infiltration of suppressive immune cells (Tregs); such as Figure 7o , 7p As shown in 7q and 7r, it can increase the conversion of macrophages from M2 type alternative activation type to M1 type classical activation type, and promote anti-tumor response; Figure 7s , 7t As shown in 7u, it can promote the maturation of DC cells.

[0128] 5. TPP-GW synergistically enhances in vivo antitumor effects with αPD-1 monoclonal antibody.

[0129] In melanomas with low immunogenicity, ICD therapy is not very effective; however, combining it with immune checkpoint blockade therapy (ICB) achieves better in vivo tumor suppression. 6-8 week old C57BL / 6J mice were subcutaneously inoculated with 2.5 × 10⁻⁶ B16 cells on the right side. 5 One per tumor. When the tumor volume is approximately 100 mm. 3 Mice were randomly divided into 4 groups. TPP-GW 5 mg / kg was injected intratumorally, and αPD-1 monoclonal antibody (purchased from BioCell) 200 μg / mouse was injected intraperitoneally. The flowchart is shown in Figure 8a. On day 12, mice were sacrificed by CO2 asphyxiation, and tumor tissue was dissected and photographed. The results are as follows: Figure 8b and 8c As shown, there was little difference in tumor volume between the αPD-1 monoclonal antibody group and the PBS control group. The tumor volume in the TPP-GW group was slightly smaller than that in the PBS group. The tumor volume was smallest in the group using the combination of αPD-1 monoclonal antibody and TPP-GW. Figure 8dAs shown, the group receiving the combined use of αPD-1 monoclonal antibody and TPP-GW had the lightest tumor weight. This indicates that the tumor-suppressing effect of the combined αPD-1 monoclonal antibody and TPP-GW group was more significant than that of either drug alone. Mouse body weight was measured and recorded during the experiment. Figure 8e As shown, there was no statistically significant difference in body weight among the experimental groups.

[0130] Industrial availability

[0131] In summary, this application has developed a mitochondrial-targeted PPARα agonist, TPP-GW, which can act as an ICD inducer, triggering ICD in tumor cells and thereby reshaping the tumor immune microenvironment and enhancing in vivo anti-tumor immune effects. The inventors' experimental results show that TPP-GW alone can exhibit a certain in vivo tumor-suppressing effect, and combining it with αPD-1 monoclonal antibody blockade therapy can achieve a stronger in vivo tumor-suppressing effect. These results indicate that TPP-GW shows promise as a good partner for ICB therapy.

Claims

1. The use of a mitochondrial-targeting compound in the preparation of an antitumor drug, said compound being a triphenylphosphine GW7647 conjugate, with the following structural formula: The tumor targeted by the anti-tumor drug is prostate cancer.

2. The use of the mitochondrial-targeting compound according to claim 1 in the preparation of antitumor drugs, wherein, The preparation method of the triphenylphosphine GW7647 conjugate includes coupling NH2-TPP with GW7647. The structural formula of the NH2-TPP is as follows: The structural formula of GW7647 is as follows:

3. The use of the mitochondrial-targeting compound according to claim 2 in the preparation of antitumor drugs, wherein, The molar ratio of GW7647 to NH2-TPP is 1:1 to 1:

2.

4. The use of the mitochondrial-targeting compound according to claim 2 in the preparation of antitumor drugs, wherein, The molar ratio of GW7647 to NH2-TPP is 1:1 to 1:1.

5.

5. The use of the mitochondrial-targeting compound according to claim 2 in the preparation of antitumor drugs, wherein, The molar ratio of GW7647 to NH2-TPP is 1:1 to 1:1.2.

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