Spermidine derivative capable of activating cancer immunity

Abstract

The present invention provides: a compound having a cancer immunity activating effect and represented by formula (I) [wherein the ring A is as defined in the description] or a pharmaceutically acceptable salt thereof (excluding a compound represeneted by [Chemical formula 2]); and a pharmaceutical composition which has higher biological stability than that of spermidine, can selectively bind to a mitochondrial protein to enhance the activation of FAO, and improves anti-tumor immunity in a cancer immunotherapy, the pharmaceutical composition containing the compound or the pharmaceutically acceptable salt.

Description

Spermidine derivatives that activate cancer immunity

[0001] The present invention relates to a spermine derivative that activates cancer immunity, particularly a compound of formula (I) or a salt thereof, a pharmaceutical composition containing the compound, and uses thereof.

[0002] Recent clinical trials have revealed that cancer immunotherapy, such as PD-1 blockade immunotherapy, is more effective than conventional standard treatments for various cancers (Non-Patent Documents 1 to 3). However, cancer immunotherapy, particularly PD-1 blockade immunotherapy, has the drawback of being less effective in elderly patients due to reduced T cell immunity. Therefore, methods to enhance the effectiveness of PD-1 blockade immunotherapy have been investigated, and a compound has been found that synergistically enhances antitumor immunity when used in combination with this method (Patent Document 1).

[0003] One such compound is the polyamine N-(3-aminopropyl)butane-1,4-diamine (hereinafter referred to as spermidine (SPD)). Spermidine has been shown to enhance fatty acid oxidation (FAO) by directly binding to and activating hydroxyl coenzyme A (CoA) dehydrogenase subunit α (HADHA), a subunit of mitochondrial trifunctional protein (mTFP) that oxidizes fatty acids to produce NADH (Non-Patent Document 4). Activation of FAO improves the mitochondrial activity and cytotoxicity of T cells and enhances the efficacy of PD-1 blockade immunotherapy in aging mice. However, the usefulness of spermidine as a therapeutic agent is hindered by its pleiotropic effects in addition to FAO activation and its limited biological stability.

[0004] As mentioned above, although compounds with cancer immunity activating effects have already been discovered, there is a need to explore alternative compounds to spermidine from the standpoint of compound stability and greater efficacy.

[0005] International Publication No. 2021 / 09599 Pamphlet

[0006] Bramer J, Reckamp K, et al: Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med, 373:1627-1639,2015.Hamanishi J, Mandai M, Ikeda T, et al: Safety and Antitumor Activity of Anti-PD-1 Antibody, Nivolumab, in Patients With Platinum-Resistant Ovarian Cancer. J Clin Oncol, 33:4015-4022, 2015.Motzer RJ, Escudier B, McDermott DF, et al: Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med, 373:1803-1813,2015.Al-Habsi, M., Chamoto, K., Matsumoto, K., et al.: Spermidine Activates Mitochondrial Trifunctional Protein and Improves Antitumor Immunity in Mice. Science 2022, 378 (6618), eabj3510.

[0007] The present invention aims to investigate novel spermidine derivatives that have cancer immunoactivating effects, and to provide a pharmaceutical composition (e.g., an antitumor immunity activator) that is more biologically stable than spermidine and can selectively bind to mitochondrial proteins to enhance FAO activation, thereby improving antitumor immunity in cancer immunotherapy.

[0008] In order to solve the above problems, the present inventors have found that spermidine activates FAO, thereby enhancing the efficacy of PD-1 blockade immunotherapy in aged mice. Based on this finding, they have used chemoproteomics techniques to profile proteins that bind to spermidine and screen for compounds structurally similar to spermidine, which activates mitochondrial FAO. As a result of extensive research into compounds that are more biologically stable than spermidine and can selectively bind to mitochondrial proteins to enhance FAO activation, they have found a compound with the following structural formula: The present inventors have found that compounds represented by the following formula (I) or salts thereof (hereinafter sometimes referred to as "compounds of the present invention"), including compounds represented by the following formula (I):

[0009] That is, the present invention provides the following aspects: [Item 1] Formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ or a pharmaceutically acceptable salt thereof, provided that: [Item 2] Compounds in which ring A is one or more R １ C optionally substituted with ５－６ a carbocyclic ring or one or more R １ is a 5- or 6-membered heterocycle optionally substituted with; and R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４acyl, carbocycle, and heterocycle, wherein said cycloalkyl and heterocycloalkyl are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ Item 3: The compound according to [Item 1], or a pharmaceutically acceptable salt thereof, wherein Ring A is optionally substituted with one or two or more of the same or different groups selected from the group consisting of acyl. (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ acyl, cycloalkyl, and heterocycloalkyl, wherein said cycloalkyl and heterocycloalkyl are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４Item 2], wherein the compound or a pharmaceutically acceptable salt thereof is a compound of formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ [Item 5] A pharmaceutical composition comprising a compound represented by the formula (I), wherein R is a carbon ring or a 5- or 6-membered non-aromatic heterocycle, or a pharmaceutically acceptable salt thereof. [Item 5] The pharmaceutical composition according to [Item 4], for activating anti-tumor immune responses. [Item 6] The pharmaceutical composition according to [Item 4] or [Item 5], which is used in combination with a PD-1 signaling inhibitor. [Item 7] The pharmaceutical composition according to [Item 6], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. [Item 8] The pharmaceutical composition according to [Item 6] or [Item 7], for treating a disease caused by decreased T-cell immunity. [Item 9] The pharmaceutical composition according to [Item 8], wherein the disease caused by decreased T-cell immunity is cancer or an infectious disease. [Item 10] An anti-tumor immune activator for use in cancer immunotherapy, comprising a compound represented by formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ [Item 11] The anti-tumor immunity activator according to [Item 10], wherein the drug used in cancer immunotherapy is a PD-1 signal inhibitor. [Item 12] The anti-tumor immunity activator according to [Item 11], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. [Item 13] The compound represented by formula (I) is selected from the group consisting of the following: The antitumor immune activator according to any one of [Item 10] to [Item 12], which is a compound of the formula:

[0010] The present invention further provides the following aspects. [Item 14] A method for treating a disease caused by decreased T-cell immunity, comprising administering to a patient in need of treatment a therapeutically effective amount of a PD-1 signal inhibitor and a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. [Item 15] The method of treatment according to [Item 14], wherein the PD-1 signal inhibitor is administered before, simultaneously with, or after the administration of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof. [Item 16] The method of treatment according to [Item 14] or [Item 15], wherein the disease caused by decreased T-cell immunity is cancer or an infectious disease. [Item 17] A compound represented by formula (I) or a pharmaceutically acceptable salt thereof for use in activating anti-tumor immunity. [Item 18] Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of an anti-tumor immunity activator. [Item 19] A method for activating anti-tumor immunity in cancer immunotherapy, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0011] The present invention also provides the following aspects: [Item 1'] Formula (I): [Wherein: Ring A is an optionally substituted non-aromatic C ４－１０ a carbocycle, an optionally substituted 4- to 10-membered non-aromatic heterocycle, or an optionally substituted 5- to 10-membered aromatic heterocycle, or a pharmaceutically acceptable salt thereof, [Item 2'] Compounds in which ring A is one or more R １ Non-aromatic C optionally substituted with ４－１０ Carbocyclic ring, one or more R １ a 4- to 10-membered non-aromatic heterocycle optionally substituted with one or more R １ is a 5- to 10-membered aromatic heterocycle optionally substituted with R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６Hydroxyalkoxy, oxo, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -NHCOR ３ , -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; and R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ The compound according to [Item 1'], or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from the group consisting of haloalkyl. (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C１－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ and n is an integer of 0 to 13. [Item 4'] The compound according to [Item 2'], or a pharmaceutically acceptable salt thereof, wherein R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, hydroxy, -NHCO-C １－６ The compound according to [Item 3'], wherein R is alkyl or phenyl; and n is 0 to 3, or a pharmaceutically acceptable salt thereof. [Item 5'] R ２ But hydrogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－４ Acyl, -SO ２ -C １－６ The compound according to [Item 4'], wherein the compound is alkyl or benzoyl, or a pharmaceutically acceptable salt thereof. [Item 6'] Formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ or a pharmaceutically acceptable salt thereof, provided that: [Item 7'] Compounds in which ring A is one or more R １ C optionally substituted with ５－６ a carbocyclic ring or one or more R １ is a 5- or 6-membered heterocycle optionally substituted with R １ each independently represents a halogen, C １－６ Alkyl, C１－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, oxo, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -NHCOR ３ , -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; wherein R １ When two R are bonded to the same atom or adjacent atoms on ring A, １ together with the atoms on ring A to form a non-aromatic C ３－５ may form a carbocyclic ring or a 5- or 6-membered non-aromatic heterocyclic ring; and R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ [Item 8'] The compound according to [Item 6'], or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from the group consisting of one or more R １ C optionally substituted with ５－６ a carbocyclic ring or one or more R １ is a 5- or 6-membered heterocycle optionally substituted with; and R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ acyl, carbocycle, and heterocycle, wherein said carbocycle and heterocycle are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ The compound according to [Item 6'], or a pharmaceutically acceptable salt thereof, wherein Ring A is optionally substituted with one or two or more of the same or different groups selected from the group consisting of acyl. [Item 9'] The compound according to [Item 6'], or a pharmaceutically acceptable salt thereof, wherein Ring A is optionally substituted with one or two or more of the same or different groups selected from the group consisting of acyl. (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ and n is an integer of 0 to 12. [Item 10] The compound according to [Item 7'], or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from the group consisting of: (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ acyl, cycloalkyl, heterocycloalkyl, and benzoyl, wherein said cycloalkyl and heterocycloalkyl are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ and n is an integer of 0 to 12. [Item 11'] The compound according to [Item 8'], or a pharmaceutically acceptable salt thereof, having the following structure: or a pharmaceutically acceptable salt thereof. [Item 12'] A pharmaceutical composition comprising a compound according to any one of [Item 1'] to [Item 11'] or a pharmaceutically acceptable salt thereof. [Item 13'] A compound of formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ [Item 14'] A pharmaceutical composition comprising a compound of the formula: wherein R is a carbon ring or a 5- or 6-membered non-aromatic heterocycle, or a pharmaceutically acceptable salt thereof. [Item 14'] The pharmaceutical composition according to [Item 12'] or [Item 13'], for activating anti-tumor immune responses. [Item 15'] The pharmaceutical composition according to [Item 12'] or [Item 13'], characterized by being used in combination with a PD-1 signaling inhibitor. [Item 16'] The pharmaceutical composition according to [Item 15'], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. [Item 17'] The pharmaceutical composition according to [Item 15'], for treating a disease caused by decreased T-cell immunity. [Item 18'] The pharmaceutical composition according to [Item 17'], wherein the disease caused by decreased T-cell immunity is cancer or an infectious disease. [Item 19'] An anti-tumor immune activator for use in cancer immunotherapy, comprising the compound according to any one of [Items 1'] to [Item 11'], or a pharmaceutically acceptable salt thereof. [Item 20'] An anti-tumor immune activator for use in cancer immunotherapy, comprising the compound of formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ or a pharmaceutically acceptable salt thereof, wherein R is a carbon ring or a 5- or 6-membered non-aromatic heterocycle.

[0012] According to the present invention, the antitumor effect can be synergistically enhanced by combining the compound with PD-1 blockade immunotherapy (e.g., a PD-1 signal inhibitor). Furthermore, the compound of the present invention is expected to serve as a small molecule antitumor immune activator.

[0013] Figure 1 shows fluorescent gel images (left) and silver-stained gel images (right). HEK293 cells were treated with probe 1 (0, 1, 10, or 100 μM) for 30 minutes, and the resulting cell lysates were subjected to click chemistry with TAMRA-azide. After separation by SDS-PAGE, the gels were analyzed using a fluorescent scanner or silver staining. Figure 2 shows fluorescent images of gels separated by SDS-PAGE. (A) HEK293 cells were treated with probe 2 (0, 1, 10, or 25 μM) for 20 minutes, followed by 20 minutes of UV irradiation. The resulting cell lysates were subjected to click chemistry with TAMRA-azide. After separation by SDS-PAGE, the gels were analyzed using a fluorescent scanner. (B) shows the results for probe 2 (25 μM) or probe 3 (25 μM) without UV irradiation (UV-) and probe 2 (25 μM) or probe 3 (25 μM) with UV irradiation (UV+). (C) shows the results for HEK293 cells cultured for 48 hours in the presence or absence of 2.5 mM difluoromethylornithine (DFMO), treated with probe 2 (25 μM), and then exposed to UV. Figure 3 shows the proteome labeling workflow and Western blot analysis of probe 2-HADHA binding interactions. (A) shows the proteome labeling workflow using a biotin-azide tag. HEK293 cells were treated with photoaffinity probe 2, followed by UV irradiation for 20 minutes. The resulting cell lysates were subjected to click chemistry with biotin-azide to purify biotin-labeled proteins using avidin beads, followed by Western blot analysis of the probe 2-HADHA binding interaction. (B) Western blot analysis of HADHA labeled with probe 2 (1, 10, or 25 μM). (C) Western blot analysis of the probe 2-HADHA binding interaction in the presence or absence of DFMO and in the presence of DFMO and spermidine or spermidine phosphate. HEK293 cells were treated with or without DFMO for 48 hours, followed by photoreaction with probe 2 (25 μM) for 20 minutes. The effect of adding 1 mM spermidine or spermidine phosphate was also evaluated.(D) Western blot analysis of probe 2-HADHA binding interaction in the presence of DFMO with or without UV irradiation. HEK293 cells preincubated in the presence of DFMO were treated with probe 2 (10 μM), lysed without or with UV irradiation, and the resulting cell lysates were subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay and subsequent HADHA Western blot analysis to confirm the effect of UV irradiation. (E) Western blot analysis of probe 2-HADHA binding interaction in the soluble and insoluble fractions in the presence or absence of DFMO. HEK293 cells were preincubated with or without DFMO, photoreaction with probe 2 (1 or 10 μM) was performed, the resulting cell lysates were fractionated into soluble and insoluble fractions, and an avidin pull-down assay was performed, followed by Western blot analysis for HADHA. (F) Western blot analysis of the probe 2-HADHA binding interaction in the soluble and insoluble fractions in the presence or absence of DFMO, probe 2, and spermidine. HEK293 cells were preincubated with or without DFMO, photoreaction with probe 2 (10 μM) in the presence of 1 mM spermidine, the cell lysates were fractionated into soluble and insoluble fractions, and an avidin pull-down assay was performed, followed by Western blot analysis for HADHA. Figure 4 shows the activity of HADHA (HADHA-HADHB complex) (UV absorbance at 340 nm indicating the production of NADH) upon treatment with DMSO, spermidine (20 μM), and probe 2 (20 μM). Data represent mean ± SEM (n=3). Figure 5 shows the workflow for compound screening of spermidine derivatives.(A) HEK293 cells were preincubated for 48 hours in the presence of 2.5 mM DFMO, and compounds 4 to 32 (1 mM) were added individually 60 minutes before photoreaction with probe 2. Photoreaction with probe 2 (10 μM) was then performed for 20 minutes. The resulting cell lysates were subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay and subsequent HADHA Western blot analysis to evaluate the competitiveness of each compound. (B) The structures of the spermidine derivatives used in this experiment are shown. Figure 6-1 shows the results of Western blot analysis of spermidine and compounds 4 to 18. The symbols (+ and ++) indicate the degree of competition, as assessed by a decrease in band intensity on Western blot. Figure 6-2 shows the results of Western blot analysis of spermidine and compounds 19 to 32. The symbols (+ and ++) indicate the degree of competition, as assessed by a decrease in band intensity on Western blot. Figure 7 shows the comparison of the HADHA activation effects of compound 13 and spermidine. (A) shows the results of Western blot analysis of spermidine (compound 13)-HADHA binding interaction. HEK293 cells were preincubated for 48 hours in the presence of 2.5 mM DFMO. Spermidine or compound 13 (10 μM, 100 μM, or 1000 μM) was added individually 60 minutes before photoreaction with probe 2. Photoreaction with probe 2 (10 μM) was then performed for 20 minutes. The resulting cell lysates were subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay and subsequent HADHA Western blot analysis to evaluate the competitiveness of each compound. (B) shows the quantification of band intensity at each concentration of spermidine or compound 13. The band intensity at each concentration is expressed as a percentage (%) of the band intensity without the addition of compound, which is set to 100%. Data represent the mean ± SD (n = 3). (C) shows the activity of HADHA (HADHA-HADHB complex) (UV absorbance at a wavelength of 340 nm indicating the production of NADH) when treated with spermidine (0-20 μM) and compound 13 (0-20 μM). Data represent the mean ± SEM (n = 2).Figure 8 shows the workflow and results of chemoproteomic profiling of spermidine-binding proteins, as well as the results of Western blot analysis of four representative spermidine-binding proteins. (A) shows the workflow of chemoproteomic profiling of spermidine-binding proteins. HEK293 cells were preincubated in the presence of 2.5 mM DFMO for 48 hours, followed by photoreaction with probe 2 (10 μM) for 20 minutes. To increase spermidine-specific proteins, 1 mM spermidine was added before the photoreaction to competitively displace probe 2. The resulting cell lysate was subjected to click chemistry with biotin-azide to perform avidin pulldown, followed by LC-MS / MS analysis. (B) shows the volcano plot (Log). ２ Ratio [probe 2 / DMSO] vs. Log １０ p-value) (left) and Volcano plot (Log ２ Ratio [probe 2 / spermidine] vs. Log １０ p-value) (right). In the Volcano plot, the p-value cutoff was set to 0.05, and the Log ２The ratio cutoff was set at 3. Proteins enclosed in squares fulfill these criteria. (C) shows the subcellular localization of 67 spermidine-binding proteins. Gene ontology (GO) enrichment analysis was performed on the 67 identified proteins (subcellular components). (D) shows Western blot analysis results for four representative spermidine-binding proteins (EPHX1: epoxide hydrolase; DLD: dihydrolipoamide dehydrogenase; IDH2: isocitrate dehydrogenase-2; NIPSNAP1: NipSnap homolog 1). Figure 9 shows the cytotoxicity of spermidine and compound 13 in the absence and presence of the amino oxidase inhibitor aminoguanidine (n = 3 (technical replicates), t-test for each point). (A) shows the percentage (%) of dead cells at each concentration of spermidine and Compound 13 in the absence of aminoguanidine, an amino oxidase inhibitor. ＋ T cells were stimulated for 22 hours, and cytotoxicity was assessed by propidium iodide staining. (B) shows the percentage of dead cells for spermidine and Compound 13 at various concentrations under the same conditions as (A), except for the addition of aminoguanidine, an amino oxidase inhibitor. Data are means ± SEM. ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001, ＊＊＊＊＊p<0.00001. Figure 10 shows the half-lives (hr) of spermidine and compound 13. Spermidine or compound 13 was incubated in DMEM containing 5% FBS for various times, and the amines were labeled with benzoyl chloride. The labeled products were then analyzed by HPLC. The half-life of each compound was calculated by curve fitting. Figure 11 shows the results of Western blot analysis of compound 13 for four representative spermidine-binding proteins (EPHX1, DLD, IDH2, and NIPSNAP1). Various concentrations of compound 13 were added to the cell culture medium before photoreaction with probe 2, and Western blot analysis was performed to evaluate the binding ability of compound 13 to each protein. Figure 12 shows the results of measuring the mitochondrial activity of spermidine and compound 13 using the Seahorse assay. (A) shows an illustration of the Seahorse XF mitochondrial stress test. (B) shows the oxygen consumption rate (OCR) measured by a Seahorse assay in the presence of spermidine or Compound 13, and the spare respiratory capacity (SRC) calculated from the measured OCR. ＋ T cells were stimulated with CD3 / CD28 beads for 1 hour in the presence of spermidine or Compound 13, and real-time OCR of the cells was measured by Seahorse assay. Data represent mean ± SD (n = 5 (technical replicates)) and mean ± SEM (n = 5 (technical replicates)). ns indicates no significant difference. ＊ p<0.05, ＊＊* indicates p<0.01. Figure 13 shows tumor growth curves for the control group, PD-L1 mAb group, PD-L1 mAb + Compound 13 group, and PD-L1 mAb + spermidine group. Data represent mean ± SEM (n=5 per group). MC38 tumor-bearing mice were administered PD-L1 mAb (20 μg / mouse), PD-L1 mAb + Compound 13 (4 mg / kg), or PD-L1 mAb + spermidine (4 mg / kg) on ​​days 7, 12, and 18 after cell inoculation. Figure 14 shows the results of measuring the mitochondrial activity of Compound 13 and Compounds 33-36 by Seahorse assay. (A) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (33, 34, 35, and 36). Figure 15 shows the results of measuring the mitochondrial activity of compound 13 and compounds 37-40 by seahorse assay. (A) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (37, 38, 39, and 40). Figure 16 shows the results of measuring the mitochondrial activity of compound 13 and compounds 41-44 by seahorse assay. (A) shows the results of oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the results of oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (41, 42, 43, and 44). Figure 17 shows the results of measuring the mitochondrial activity of compound 13 and compounds 45-47 and 56 by seahorse assay.(A) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (45, 46, 47, and 56). Figure 18 shows the results of measuring the mitochondrial activity of compound 13 and compounds 48-51 by seahorse assay. (A) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (48, 49, 50, and 51). Figure 19 shows the results of measuring the mitochondrial activity of compound 13 and compounds 52-55 by seahorse assay. (A) shows the results of oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the results of oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (52, 53, 54, and 55). Figure 20 shows the results of measuring the mitochondrial activity of compound 13 and compounds 57-60 by seahorse assay. (A) shows the results of oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the results of oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and each compound (57, 58, 59, and 60). Figure 21 shows the results of measuring the mitochondrial activity of compound 13 and compound 61 by seahorse assay.(A) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 13 (13), and (B) shows the oxygen consumption rate (OCR) measured by seahorse assay in the presence of control (Ctrl) and compound 61 (61). Figure 22 shows a comparison of the maximum OCR measured after FCCP administration in the seahorse assay. Figure 23 shows tumor growth curves for the control administration group, PD-L1 mAb administration group, PD-L1 mAb + compound 34 administration group, PD-L1 mAb + compound 36 administration group, PD-L1 mAb + compound 40 administration group, PD-L1 mAb + compound 44 administration group, PD-L1 mAb + compound 51 administration group, and PD-L1 mAb + compound 56 administration group. (A) Tumor growth curves for the control group, PD-L1 mAb group, PD-L1 mAb + Compound 34 group, PD-L1 mAb + Compound 36 group, and PD-L1 mAb + Compound 40 group are shown. (B) Tumor growth curves for the control group, PD-L1 mAb group, PD-L1 mAb + Compound 44 group, PD-L1 mAb + Compound 51 group, and PD-L1 mAb + Compound 56 group are shown. Data represent mean ± SEM (n = 5 per group). MC38 tumor-bearing mice were administered PD-L1 mAb (20 μg / mouse) or PD-L1 mAb + each compound (4 mg / kg) on ​​days 7, 12, and 18 after cell inoculation.

[0014] The terms used in this specification are explained below.

[0015] As used herein, "non-aromatic C ４－１０ "Carbocycle" means a monocyclic or bicyclic non-aromatic (saturated or partially unsaturated) hydrocarbon ring having 4 to 10 carbon atoms. ５－６"Carbocycle" means a monocyclic non-aromatic hydrocarbon ring having 5 to 6 carbon atoms. These may be substituted at substitutable positions with one or more substituents included in the present invention, if desired. Examples include, but are not limited to, cyclobutane, cyclopentane, cyclohexane, cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, etc. Also, "non-aromatic C ４－１０ The term "carbocycle" also includes a spiro ring that is partially spiro-modified and a bridged ring that is partially bridged. The spiro ring and the bridged ring may contain one heteroatom, or two or more (e.g., 2 to 4), which may be the same or different, selected from nitrogen, sulfur, or oxygen. Examples of the spiro ring and the bridged ring include, but are not limited to, the following structures. Furthermore, the "non-aromatic C ４－１０ "Carbocycle" includes monocyclic non-aromatic carbocycles (e.g., C ４－６ Also encompassed are fused rings of a carbon ring) with a benzene ring or a 5- or 6-membered ring containing one, or two or more (e.g., 2 to 4) identical or different heteroatoms selected from nitrogen, sulfur, or oxygen (for example, a 5- or 6-membered ring among the "4- to 10-membered non-aromatic heterocycles" and "5- to 10-membered aromatic heterocycles" described below). Examples of such fused rings include, but are not limited to, the following structures.

[0016] In addition, the "non-aromatic C ５－６ A "carbocyclic ring" is a ring that, when two substituents are attached to the same or adjacent carbon atoms on the ring, is taken together with the carbon atoms on the ring to form a "non-aromatic carbon ring." ４－１０ Spiro rings, bridged rings and fused rings can be formed within the term "carbocycle."

[0017] As used herein, a "4- to 10-membered non-aromatic heterocycle" refers to a monocyclic or bicyclic non-aromatic (saturated or partially unsaturated) heterocycle composed of 4 to 10 carbon atoms and heteroatoms, containing at least one heteroatom selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S). A "5- or 6-membered non-aromatic heterocycle" refers to a monocyclic non-aromatic heterocycle composed of 5 or 6 carbon atoms and heteroatoms, in which at least one ring carbon atom is replaced with a heteroatom selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S). These may be substituted at available positions with one or more substituents within the scope of the present invention, as desired. The nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. Examples of such heterocyclic rings include, but are not limited to, oxetane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, tetrahydropyran, tetrahydrothiopyran, piperazine, morpholine, and thiomorpholine. Furthermore, the term "4- to 10-membered non-aromatic heterocycle" also includes partially spiro-modified spiro rings and partially bridged rings. Furthermore, the spiro rings and bridged rings may contain one, or two or more (e.g., 2 to 4) heteroatoms, which may be the same or different, selected from nitrogen, sulfur, or oxygen. Examples of such spiro rings and bridged rings include, but are not limited to, the following structures: Furthermore, the "4- to 10-membered non-aromatic heterocycle" also encompasses a fused ring formed between a monocyclic non-aromatic heterocycle (e.g., a 4- to 6-membered heterocycle) and a benzene ring or a 5- or 6-membered ring containing one, or two or more (e.g., 2 to 4) identical or different heteroatoms selected from nitrogen, sulfur, or oxygen (e.g., a 5- or 6-membered ring among the "4- to 10-membered non-aromatic heterocycle" and "5- to 10-membered aromatic heterocycle" described below). Examples of such fused rings include, but are not limited to, the following structures.

[0018] In addition, when two substituents are bonded to the same atom or adjacent atoms on the ring, the "5- or 6-membered non-aromatic heterocycle" can form a spiro ring, a bridged ring, or a fused ring included in the "4- to 10-membered non-aromatic heterocycle" together with carbon atoms on the ring.

[0019] As used herein, the term "5- to 10-membered aromatic heterocycle" refers to a monocyclic or bicyclic aromatic heterocycle composed of 5 to 10 carbon atoms and heteroatoms, containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur atoms. These may be substituted at substitutable positions with one or more substituents included in the present invention, as desired. The "5- to 10-membered aromatic heterocycle" may be a 5- or 6-membered monocyclic aromatic heterocycle, preferably containing one or two nitrogen atoms. Specific examples of such compounds include pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiophene, pyrrole, thiazole, isothiazole, pyrazole, imidazole, furan, oxazole, isoxazole, oxadiazole, thiadiazole, triazole, tetrazole, quinoline, isoquinoline, naphthyridine, quinazoline, benzofuran, benzothiophene, indole, benzoxazole, benzisoxazole, 1H-indazole, 2H-indazole, benzimidazole, benzoxadiazole, benzothiadiazole, indolizine, benzofurazine, thienopyrimidine, pyrazolopyridine, imidazopyridine, imidazopyrazine, pyrazolopyrimidine, triazolopyrimidine, thienothiophene, and imidazothiazole.

[0020] As used herein, "halogen" means fluorine, chlorine, bromine or iodine.

[0021] As used herein, "C １－６"Alkyl" means a straight or branched chain saturated hydrocarbon having from 1 to 6 carbon atoms, which may be optionally substituted at any available position with one or more substituents included in the present invention. Examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, and the like.

[0022] As used herein, "C １－６ "Haloalkyl" means an alkyl group as defined above having 1 to 6 carbon atoms in which one or more hydrogen atoms have been replaced by a halogen atom(s). The number of replaced hydrogen atoms can range from 1 up to the total number of other hydrogen atoms present in the parent alkyl group. When there are multiple halogen atoms, they may be replaced with the same or different halogen atoms. Examples include, but are not limited to, fluoromethyl, chloromethyl, bromoethyl, trifluoromethyl, 2,2,2-trifluoroethyl, etc.

[0023] As used herein, "C １－６ "Hydroxyalkyl" means an alkyl group as defined above having from 1 to 6 carbon atoms in which one or more hydrogen atoms have been replaced by a hydroxy (OH) group(s). The number of replaced hydrogen atoms can range from 1 up to the total number of other hydrogen atoms present in the parent alkyl group. Examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl, and the like.

[0024] As used herein, "C １－６ "Alkoxy" means an alkyl group as defined above having 1 to 6 carbon atoms attached via an oxygen atom. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butyloxy, pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, and the like.

[0025] As used herein, "C１－６ "Haloalkoxy" means an alkoxy group as defined above having 1 to 6 carbon atoms in which one or more hydrogen atoms have been replaced by halogen atom(s). The number of replaced hydrogen atoms can range from 1 up to the total number of other hydrogen atoms present in the parent alkyl group. When there are multiple halogen atoms, they may be replaced with the same or different halogen atoms. Examples include, but are not limited to, chloromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, etc.

[0026] As used herein, "C １－６ "Hydroxyalkoxy" means an alkoxy group as defined above having from 1 to 6 carbon atoms in which one or more hydrogen atoms have been replaced by a hydroxy (OH) group(s). The number of replaced hydrogen atoms can range from 1 up to the total number of other hydrogen atoms present in the parent alkyl group. Examples include, but are not limited to, hydroxymethoxy, 2-hydroxyethoxy, 2-hydroxypropoxy, 3-hydroxypropoxy, 4-hydroxybutoxy, and the like.

[0027] As used herein, "mono- or di-C １－６ "Alkylamino" means an amino group in which one or two hydrogen atoms are replaced by the above alkyl group having 1 to 6 carbon atoms. When two alkyl groups are substituted, they may be the same or different alkyl groups. Examples thereof include, but are not limited to, methylamino, ethylamino, dimethylamino, diethylamino, etc.

[0028] As used herein, "C １－４ "Acyl" means a carbonyl group (-C(=O)) having attached thereto an alkyl group, as defined above, having one to three carbon atoms. Examples include, but are not limited to, formyl, acetyl, propionyl, and the like.

[0029] As used herein, the term "carbocyclic group" refers to a monocyclic or bicyclic non-aromatic or aromatic hydrocarbon ring group excluding one hydrogen atom bonded to the non-aromatic or aromatic carbon ring. A non-aromatic carbocyclic group is also referred to as a "cycloalkyl," and an aromatic carbocyclic group is also referred to as an "aryl." The "carbocyclic group" of the present invention may be a benzene ring, a spiro ring group containing a heteroatom, a bridged ring group, or a fused ring group. Furthermore, the "carbocyclic group" of the present invention is preferably a cyclic group consisting of 4 to 10 carbon atoms or a cyclic group consisting of 4 to 10 carbon atoms and at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. Examples of "carbocyclic groups" include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, etc.

[0030] As used herein, the term "heterocyclic group" refers to a monocyclic or bicyclic non-aromatic or aromatic heterocyclic group in which one hydrogen atom bonded to the non-aromatic or aromatic heterocyclic ring has been removed. A non-aromatic heterocyclic group is also referred to as a "heterocycloalkyl," and an aromatic heterocyclic group is also referred to as a "heteroaryl." The "heterocyclic group" in the present invention may be a spirocyclic group, a bridged ring group, or a fused ring group containing a benzene ring or a carbocyclic ring. Furthermore, the "heterocyclic group" in the present invention is preferably a cyclic group consisting of 4 to 10 carbon atoms and at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. Examples of "heterocyclic groups" include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, homopiperidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyridyl, pyridazinyl, isothiazolyl, pyrrolyl, furyl, thienyl, thiazolyl, imidazolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrazinyl, triazinyl, triazolyl, imidazolidinyl, oxadiazolyl, triazolyl, tetrazolyl, indolyl, indazolyl, chromenyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzotriazolyl, benzimidazolyl, and the like.

[0031] As used herein, "optionally substituted" includes cases where the substitutable positions of the group are not substituted (unsubstituted) and cases where they are substituted. "Unsubstituted" means that all substitutable positions of the group are hydrogen atoms. When substituted, if possible, the group may be substituted with multiple substituents, and the substituents may be the same or different. Examples of the substituted substituent include, but are not limited to, halogen, hydroxy, alkyl, alkenyl, alkynyl, cycloalkyl (non-aromatic carbocyclic group), heterocycloalkyl (non-aromatic heterocyclic group), haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, nitro, cyano, carbamoyl, carboxyl, formyl, acetyl, mesyl, ethylsulfonyl, isopropylsulfonyl, benzoyl, benzoyloxy, acylamino, methylamide, ethylamide, propylamide, aromatic carbocyclic group, aromatic heterocarbocyclic group, etc. Preferred substituents of the present invention include halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -NHCO-C １－６ Alkyl, —SO ２ -C １－６ Examples include alkyl, phenyl, and benzoyl.

[0032] Rings A and R in the compound represented by formula (I) １ ~R ３ Regarding n, the preferred ones are as follows, but the technical scope of the present invention is not limited to the range of the compounds listed below. ４－１０ Examples include a carbocycle, a 4- to 10-membered non-aromatic heterocycle, and a 5- to 10-membered aromatic heterocycle, and preferably the following structure: Examples of the ring include those represented by the following formula: １Examples of the halogen include C. １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, oxo, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -NHCOR ３ , -SO ２ R ３ , a carbocyclic group, a heterocyclic group and benzoyl, and preferably halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, hydroxy, -NHCO-C １－６ Carbocyclic and heterocyclic groups may be substituted with halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ R may be substituted with one or more identical or different groups selected from the group consisting of acyl, ２ Examples include hydrogen, halogen, and C. １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -SO ２ R ３ , a carbocyclic group, a heterocyclic group and benzoyl, and preferably hydrogen, C １－６ Alkyl, C１－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－４ Acyl, -SO ２ -C １－６ Carbocyclic and heterocyclic groups may be substituted with halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ R may be substituted with one or more identical or different groups selected from the group consisting of acyl, ３ Examples of the halogen include C. １－６ Alkyl and C １－６ haloalkyl, preferably C １－６ n is an integer of 0 to 13, preferably 0 to 4, and more preferably 0 to 3.

[0033] Specific examples of the compound of formula (I) include the following compounds: Examples include:

[0034] In the present invention, the compound of formula (I) may be in the form of a salt, and is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples of such salts include inorganic acid salts such as hydrochloride, sulfate, phosphate, and nitrate; organic acid salts such as lactate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, benzenesulfonate, and ascorbate; alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; and ammonium salts such as tetramethylammonium salt. The compound of the present invention may also be in the form of a hydrate and / or solvate, and these hydrates and / or solvates are also encompassed within the scope of the present invention. Furthermore, the compound of the present invention also includes all tautomers, all existing stereoisomers, and mixtures thereof.

[0035] In the present invention, the term "spermidine derivative" means a compound of formula (I) having a structure similar to spermidine or a pharmaceutically acceptable salt thereof.

[0036] The compound of the present invention can be used as a pharmaceutical composition. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

[0037] The compound or pharmaceutical composition of the present invention can be used in combination with cancer immunotherapy, for example, a PD-1 signal inhibitor.

[0038] As used herein, "PD-1 signaling" refers to a signaling mechanism mediated by PD-1. One example of this mechanism is the signaling mechanism in which PD-1 cooperates with its ligands, PD-L1 and PD-L2, to suppress T cell activation. PD-1 (Programmed Cell Death-1) is a membrane protein expressed on activated T cells and B cells, and its ligands, PD-L1 and PD-L2, are expressed on a variety of cells, including antigen-presenting cells such as monocytes and dendritic cells, and cancer cells. PD-1, PD-L1, and PD-L2 act as inhibitors that suppress T cell activation. Certain cancer cells and virus-infected cells express PD-1 ligands to suppress T cell activation and escape host immune surveillance.

[0039] PD-1 signal inhibitors include substances that specifically bind to PD-1, PD-L1, or PD-L2. Such substances may include proteins, polypeptides, oligopeptides, nucleic acids (including natural and artificial nucleic acids), low-molecular-weight organic compounds, inorganic compounds, cell extracts, extracts from animals, plants, soil, etc. The substances may be natural or synthetic. Preferred PD-1 signal inhibitors are antibodies, more preferably anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, etc. The antibodies may be any antibodies capable of inhibiting PD-1 signaling, and may be polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, or human antibodies. Methods for producing such antibodies are known. The antibodies may be derived from any organism, such as humans, mice, rats, rabbits, goats, or guinea pigs. In addition, as used herein, the term "antibody" refers to Fab, F(ab)', or the like. ２ , ScFv, Diabody, V Ｈ , V Ｌ , Sc(Fv) ２ , Bispecific sc (Fv) ２ This concept also includes low molecular weight substances such as minibodies, scFv-Fc monomers, and scFv-Fc dimers.

[0040] The compounds of the present invention can improve the mitochondrial activity and cytotoxicity of T cells by activating FAO, and therefore the pharmaceutical compositions of the present invention can treat diseases caused by impaired T cell immunity. Furthermore, the compounds of the present invention can enhance anti-tumor immunity in cancer immunotherapy, and therefore the compounds of the present invention can also be used as drugs used in cancer immunotherapy, for example, as anti-tumor immune response activators for PD-1 signal inhibitors.

[0041] As used herein, "compromised T-cell immunity" refers to a state in which T-cell function is suppressed and anti-tumor immune responses against cancer cells or pathogens are not induced. Furthermore, as used herein, "diseases caused by compromised T-cell immunity" refers to cancer, infectious diseases, or symptoms associated therewith.

[0042] "Cancer" as used herein includes leukemia, lymphoma (Hodgkin's disease, non-Hodgkin's lymphoma, etc.), multiple myeloma, brain tumor, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck cancer (larynx cancer, etc.), oral cancer (floor of the mouth cancer, etc.), gum cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, and the like. These include, but are not limited to, cancer, sinus cancer (maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, sphenoid sinus cancer, etc.), thyroid cancer, kidney cancer, lung cancer, osteosarcoma, prostate cancer, testicular tumor (testicular cancer), renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer (basal cell carcinoma, squamous cell carcinoma, malignant melanoma, actinic keratosis, Bowen's disease, Paget's disease, etc.), anal cancer, etc.

[0043] As used herein, the term "infectious disease" refers to various bacterial infections (such as streptococci (group A beta-hemolytic streptococci, pneumococci, etc.), Staphylococcus aureus (MSSA, MRSA), Staphylococcus epidermidis, enterococci, listeria, Streptococcus meningitidis, Neisseria gonorrhoeae, pathogenic Escherichia coli (O157:H7, etc.), Klebsiella (pneumoniae), Bacillus proteus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia marcescens, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, etc.) infectious diseases, tuberculosis, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, syphilis, tetanus, leprosy, Legionnaires' pneumonia (legionnaires' disease), leptospirosis, Lyme disease, tularemia, Q fever, etc.), rickettsial infections (typhus, scrub typhus, Japanese spotted fever, etc.), chlamydial infections (trachoma, genital chlamydia infection, psittacosis, etc.), fungal infections (aspergillosis, candidiasis, cryptococcosis, tinea, histoplasmosis, nephritis, pneumonia, etc.), parasitic protozoan infections (amebic dysentery, malaria, toxoplasmosis, leishmaniasis, cryptosporidium, etc.), parasitic helminth infections (echinococcosis, schistosomiasis japonicum, filariasis, ascariasis, diphyllobothriasis, etc.), viral infections (influenza, viral hepatitis, viral meningitis, acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, yellow fever, common cold syndrome, rabies, rhinoceros disease, etc.) These include, but are not limited to, tomegalovirus infection, severe acute respiratory syndrome (SARS), progressive multifocal leukoencephalopathy, chickenpox, shingles, hand, foot and mouth disease, dengue fever, erythema infectiosum, infectious mononucleosis, smallpox, rubella, polio, measles, pharyngoconjunctival fever (swimming pool fever), Marburg hemorrhagic fever, hantavirus renal hemorrhagic fever, Lassa fever, mumps, West Nile fever, herpangina, and chikungunya fever.

[0044] As used herein, "treatment" refers to any cure and / or amelioration of a disease or disorder and its associated symptoms in a mammal, particularly a human. For example, in the treatment of cancer, this includes eliminating cancer cells, inhibiting the proliferation of cancer cells, preventing the recurrence of cancer, and alleviating or reducing symptoms associated with cancer. In addition, in the treatment of infectious diseases, this includes inhibiting the proliferation of pathogens that cause infectious diseases, preventing the recurrence of infectious diseases, and alleviating or reducing symptoms associated with infectious diseases.

[0045] As used herein, the term "patient" refers to humans and other animals, such as dogs, cats, horses, etc. The patient is preferably a mammal, and more preferably a human.

[0046] As used herein, a "therapeutically effective amount" refers to an amount that results in a cure, amelioration, alleviation, and / or reduction of a disease or disorder and its associated symptoms compared to an untreated subject. Such an effective amount includes the amount of a compound of the present invention alone, the amount of a combination of compounds of the present invention, and / or the amount of a compound of the present invention in combination with other active ingredients useful in cancer immunotherapy.

[0047] The compound or pharmaceutical composition of the present invention may be used in combination with a PD-1 signal inhibitor, or may be used as a combination drug.

[0048] When the compound or pharmaceutical composition of the present invention is used in combination with a PD-1 signal inhibitor, the compound or pharmaceutical composition of the present invention may be administered before, simultaneously with, or after administration of the PD-1 signal inhibitor.

[0049] The compounds and pharmaceutical compositions of the present invention can be administered orally or parenterally by being formulated into appropriate dosage forms.

[0050] The dosage forms of the compound and pharmaceutical composition of the present invention include, for example, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets), capsules, granules (effervescent granules), powders, oral liquids (elixirs, suspensions, emulsions, lemonades), syrups (syrup preparations), oral jellies, oral tablets (troches, sublingual tablets, buccal tablets, adhesive tablets, gums), oral sprays, oral semisolid preparations, mouthwashes, injections (infusions, implantable injections, sustained-release injections), dialysis preparations ( Examples of the pharmaceutical composition include, but are not limited to, peritoneal dialysis agents, hemodialysis agents), inhalants (inhalation powders, inhalation liquids, inhalation aerosols), suppositories, rectal semisolid preparations, enemas, eye drops, eye ointments, ear drops, nasal preparations (nasal powders, nasal liquids), vaginal tablets, vaginal suppositories, solid preparations for external application (external powders), liquid preparations for external application (liniments, lotions), sprays (external aerosols, pump sprays), ointments, creams, gels, patches (tapes, poultices), etc. The formulations of the present invention can be produced by known methods using the compounds of the present invention and pharmaceutically acceptable additives.

[0051] Examples of pharmaceutically acceptable additives (carriers) include stabilizers, surfactants, solubilizers, buffers, suspending agents, antioxidants, coating agents, wetting agents, moisturizing agents, cooling agents, colorants, flavoring agents, isotonicity agents, emulsifiers, pH adjusters, skin protective agents, dispersants, propellants, fragrances, preservatives, solvents, etc., and these additives (carriers) can be used depending on the purpose.

[0052] The compound of the present invention may be used in combination with other drugs, for example, anticancer agents or anti-infective agents, as long as the activation of anti-tumor immunity is not hindered.

[0053] The dose of the compound of the present invention varies depending on the individual compound, and on the patient's disease, age, body weight, sex, symptoms, administration route, etc., but typically, for an adult (body weight 60 kg), the compound of the present invention can be administered at a dose of 0.001 to 1000 mg / day at least once a day at a frequency at which the desired effect can be confirmed.

[0054] When the compound of the present invention is used in combination with a PD-1 signal inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody), the ratio (by mass) of the compound of the present invention to the PD-1 signal inhibitor is, for example, 1:1 to 1:10.

[0055] A PD-1 signal inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody) may be dissolved in a buffer solution such as PBS, physiological saline, or sterile water, and, if necessary, sterilized by filtration using a filter or the like, and then administered to a subject or test animal by injection or infusion. This solution may also contain additives (e.g., colorants, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonicity agents, etc.). Possible routes of administration include intravenous, intramuscular, peritoneal, subcutaneous, and intradermal administration.

[0056] The dose of a PD-1 signal inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody) varies depending on the individual compound, as well as the patient's disease, age, weight, sex, symptoms, administration route, and the like. Generally, for an adult (body weight 60 kg), the compound of the present invention can be administered at a dose of 0.001 to 1000 mg / day at least once a day, at a frequency at which the desired effect can be confirmed.

[0057] The present invention will be further illustrated by the following synthesis examples and test examples, but these are not intended to limit the scope of the present invention.

[0058] (1) Materials and Methods Materials and methods used in the following synthesis examples and test examples are as follows, but are not limited thereto.

[0059] (1-1) Materials and Biological Reagents Dulbecco's Modified Eagle's Medium (DMEM) and phosphate-buffered saline (PBS, pH 7.4) were used. Dulbecco's Modified Eagle's Medium (DMEM) and phosphate-buffered saline (PBS, pH 7.4) were purchased from Thermo Fisher Scientific. A mixed solution of penicillin and streptomycin was purchased from Nacalai Tesque Inc. Fetal bovine serum (FBS) was purchased from Biowest. All DMEM used in this study was stored in a humidified 5% CO atmosphere. ２ The medium was incubated in an incubator at 37°C and supplemented with a 1% (v / v) mixed solution of penicillin and streptomycin and 10% (v / v) fetal bovine serum (FBS, Biowest, S1820-500).

[0060] Mice: C57BL / 6NCrSlc female mice purchased from Japan SLC were used. All mice were maintained under specific pathogen-free conditions. All mouse experiments were performed under the approval of the respective institutional review boards.

[0061] Antibodies The antibodies shown in the table below were used.

[0062] Compounds The compounds shown in the table below were used in Test Example 4 below.

[0063] (1-2) Method 1. In-gel Fluorescence Imaging (A) Labeling with Probe 1. HEK293 cells were washed with PBS and added to serum-free medium containing Probe 1. The cells were harvested and lysed in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail. The resulting cell lysate was centrifuged at 20,000 g for 20 minutes. Protein concentration was measured using a BCA protein assay kit. To bind the Probe 2-labeled proteome by quick chemistry, TAMRA-azide was added to the cell lysate to a final concentration of 100 μM, followed by the addition of a cocktail containing 2 mM tris(3-hydroxypropyltriazolylmethyl)amine, 1 mM copper sulfate, and 5 mM sodium ascorbate. The reaction mixture was shaken at room temperature for 2 hours using a tube mixer (Waken, Japan). The reaction was stopped by adding SDS-PAGE sample buffer (Nacalai Tesque), and the proteins were resolved on a 10% SDS-PAGE acrylamide gel, followed by fluorescent imaging using a FluoroImage Analyzer (Amersham Typhoon, GE Healthcare).

[0064] (B) Photoaffinity labeling. HEK293 cells were washed with PBS and added to serum-free medium containing probe 2. The cells were then incubated on ice in a BIO-LINK （登録商標）The cells were exposed to UV light (365 nm wavelength) for 20 minutes using a crosslinker (BLX-365, Vilber-Loumat). Cells were harvested and lysed in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail. The resulting cell lysate was centrifuged at 20,000 g for 20 minutes. Protein concentration was measured using a BCA protein assay kit. To bind the probe-labeled proteome via click chemistry, TAMRA-azide was added to the cell lysate to a final concentration of 100 μM, followed by the addition of a cocktail containing 2 mM tris(3-hydroxypropyltriazolylmethyl)amine, 1 mM copper sulfate, and 5 mM sodium ascorbate. The reaction mixture was shaken at room temperature for 2 hours in a tube mixer (Waken, Japan). The reaction was stopped by adding SDS-PAGE sample buffer (Nacalai Tesque), and the proteins were resolved on a 10% SDS-PAGE acrylamide gel, followed by fluorescent imaging using a FluoroImage Analyzer (Amersham Typhoon, GE Healthcare).

[0065] (C) Photoaffinity labeling in the presence of DFMO. HEK293 cells were treated with 2.5 mM difluoromethylornithine (DFMO) on days 1 and 2. On day 3, cells were washed with PBS and added to serum-free medium containing probe 2. The cells were then incubated on ice in a BIO-LINK （登録商標）The cells were exposed to UV light (365 nm wavelength) for 20 minutes using a crosslinker (BLX-365, Vilber-Loumat). Cells were harvested and lysed in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail. The resulting cell lysate was centrifuged at 20,000 g for 20 minutes. Protein concentration was measured using a BCA protein assay kit. To bind the probe-labeled proteome via click chemistry, TAMRA-azide was added to the cell lysate to a final concentration of 100 μM, followed by the addition of a cocktail containing 2 mM tris(3-hydroxypropyltriazolylmethyl)amine, 1 mM copper sulfate, and 5 mM sodium ascorbate. The reaction mixture was shaken at room temperature for 2 hours on a tube mixer (Waken, Japan). The reaction was stopped by adding SDS-PAGE sample buffer (Nacalai Tesque), and the proteins were resolved on a 10% SDS-PAGE acrylamide gel, followed by fluorescent imaging using a fluoroimage analyzer (Typhoon™ FLA 900).

[0066] 2. Pull-down assay (A) Photoaffinity labeling HEK293 cells were washed with PBS and added to serum-free medium containing probe 2. The cells were then incubated on ice in a BIO-LINK （登録商標）The cells were exposed to UV light (365 nm wavelength) for 20 minutes using a crosslinker (BLX-365, Vilber-Loumat). Cells were harvested and lysed in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail. The resulting cell lysate was centrifuged at 20,000 g for 20 minutes. Protein concentration was measured using a BCA protein assay kit. To bind the probe-labeled proteome via click chemistry, TAMRA-azide was added to the cell lysate to a final concentration of 100 μM, followed by the addition of a cocktail containing 2 mM tris(3-hydroxypropyltriazolylmethyl)amine, 1 mM copper sulfate, and 5 mM sodium ascorbate. The reaction mixture was shaken at room temperature for 2 hours in a tube mixer (Waken, Japan). Three volumes of cold acetone were then added to the reaction mixture, which was then maintained at -30°C for at least 30 minutes. After centrifugation at 20,000 g for 10 minutes, the precipitated proteins were washed with acetone (at least three times). The pellet was air-dried and dissolved in 100 μL of 1% SDS-PBS solution by gentle sonication. 900 μL of RIPA buffer was added, and the mixture was centrifuged at 20,000 g for 10 minutes. An aliquot from the supernatant was set aside as the "input sample" and incubated with SDS-PAGE sample buffer at room temperature for 1 hour. 20 μL of Softlink Soft-Release Avidin resin (Promega) was added to the supernatant, and the mixture was stirred overnight at 4°C in a rotary incubator (Taitec RT-50, Japan). The sample was centrifuged at 3,000 g for 1 minute. The supernatant was discarded, and the beads were washed with 1 mL of RIPA buffer. The beads were recovered by centrifugation at 3,000 g for 1 minute. The beads were washed three times. Sixty microliters of a 5 mM biotin solution in RIPA buffer (freshly prepared) was added, and the mixture was further shaken at room temperature for 15 minutes to elute the proteins adsorbed to the resin. The mixture was centrifuged at 15,000 g for 1 minute. The supernatant was removed, and SDS-PAGE sample buffer was added to resolve the proteins on a 10% SDS-PAGE acrylamide gel. Fluorescence imaging was performed using a FluoroImage Analyzer (Typhoon™ FLA 900).

[0067] (B) Photoaffinity labeling in the presence of DFMO. HEK293 cells were treated with 2.5 mM difluoromethylornithine (DFMO) on days 1 and 2. On day 3, cells were washed with PBS and added to serum-free medium containing probe 2. The cells were then incubated on ice in a BIO-LINK （登録商標）The cells were exposed to UV light (365 nm wavelength) for 20 minutes using a crosslinker (BLX-365, Vilber-Loumat). Cells were harvested and lysed in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail. The lysate was centrifuged at 20,000 g for 20 minutes. Protein concentration was measured using a BCA protein assay kit. To bind the probe-labeled proteome via click chemistry, TAMRA-azide was added to the cell lysate to a final concentration of 100 μM, followed by a cocktail containing 2 mM tris(3-hydroxypropyltriazolylmethyl)amine, 1 mM copper sulfate, and 5 mM sodium ascorbate. The reaction mixture was shaken at room temperature for 2 hours using a tube mixer (Waken, Japan). Three volumes of cold acetone were then added to the reaction mixture, which was then kept at -30°C for at least 30 minutes. After centrifugation at 20,000 g for 10 minutes, the precipitated protein was washed with acetone (at least three times). The pellet was air-dried and dissolved in 100 μL of 1% SDS-PBS solution by gentle sonication. 900 μL of RIPA buffer was added, and the mixture was centrifuged at 20,000 g for 10 minutes. An aliquot from the supernatant was set aside as the "input sample" and incubated with SDS-PAGE sample buffer at room temperature for 1 hour. 20 μL of Softlink Soft-Release Avidin resin (Promega) was added to the supernatant, and the mixture was agitated overnight at 4°C in a rotary incubator (Taitec RT-50, Japan). The sample was centrifuged at 3,000 g for 1 minute. The supernatant was discarded, and the beads were washed with 1 mL of RIPA buffer. The beads were recovered by centrifugation at 3,000 g for 1 minute. The beads were washed three times. 60 μL of a solution of 5 mM biotin in RIPA buffer (freshly prepared) was added, and the mixture was further shaken at room temperature for 15 minutes to elute the proteins adsorbed to the resin. The mixture was centrifuged at 15,000 g for 1 minute, and the supernatant was removed, and SDS-PAGE sample buffer was added to separate the proteins on a 10% SDS-PAGE acrylamide gel. Fluorescence imaging was performed using a fluoroimage analyzer (Typhoon™ FLA 900).

[0068] 3. Fractionation of the proteome in membrane-soluble and insoluble fractions. Harvested HEK293 cells were washed with a PBS solution containing a protease inhibitor cocktail and lysed by sonication. The mixture was centrifuged at 20,000 g for 15 minutes at 4°C, and the lysate was collected as the PBS-soluble fraction. The pellet was rehomogenized with PBS containing 1% NP-40 solution by sonication. The mixture was centrifuged at 20,000 g for 15 minutes at 4°C, and the lysate was collected as the PBS-insoluble fraction. The soluble and insoluble fractions were further collected for use in the click reaction in the pull-down assay described above.

[0069] 4. Western blot analysis Gels were prepared with 10% acrylamide using reagents (Nacalai Tesque). Gel electrophoresis and nitrocellulose blotting membranes (Amersham) were used. （登録商標） Protran （登録商標） ) was sequentially performed using a Mini Trans-Blot system (Bio-Rad). After protein electrophoresis, the membrane was incubated with skim milk solution in PBST (0.05% Tween / PBS) for 1 hour. The membrane was washed three times with PBST solution for 5 minutes and incubated overnight at 4°C with primary antibody, uniformly rocked on a Rocker 35 (Labnet International). The membrane was washed three times with PBST solution for 5 minutes and incubated with secondary antibody on a rocking mixer (AS ONE) for 1 hour at room temperature. The membrane was washed three times with PBST solution for 5 minutes and treated with ECL Prime Western blotting detection reagent (GE Healthcare) for 1 minute. Protein bands were visualized using an ImageQuant LAS 500 (GE Healthcare, MA, USA).

[0070] 5. Competition Assay HEK293 cells were treated with 2.5 mM difluoromethylornithine (DFMO) on days 1 and 2. On day 3, the cells were washed with PBS and added to serum-free medium containing probe 2 (0 or 10 μM). After 20 minutes, 1 mM spermidine or each screening compound (compounds 4 to 32) dissolved in type 1 water (polyamines were dissolved in DMSO) was added. The cells were then incubated on ice in a BIO-LINK （登録商標）The cells were exposed to UV light (wavelength: 365 nm) for 20 minutes using a crosslinker (BLX-365, Vilber-Loumat). Cells were harvested and lysed in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail by sonication. The resulting cell lysate was centrifuged at 20,000 g for 20 minutes. Protein concentration was measured using a BCA protein assay kit. To bind the probe-labeled proteome via click chemistry, biotin-azide was added to the cell lysate to a final concentration of 100 μM, followed by a cocktail containing 2 mM tris(3-hydroxypropyltriazolylmethyl)amine, 1 mM copper sulfate, and 5 mM sodium ascorbate. The reaction mixture was shaken at room temperature for 2 hours using a tube mixer (Waken, Japan). Three volumes of cold acetone were then added to the reaction mixture, which was then maintained at -30°C for at least 30 minutes. After centrifugation at 20,000 g for 10 minutes, the precipitated proteins were washed with acetone (three times). The pellet was air-dried and dissolved in 100 μL of 1% SDS-PBS solution by gentle sonication. 900 μL of RIPA buffer was added, and the mixture was centrifuged at 20,000 g for 10 minutes. An aliquot from the supernatant was set aside as the "input sample" and incubated with SDS-PAGE sample buffer at room temperature for 1 hour. 20 μL of Softlink Soft-Release Avidin resin (Promega) was added to the supernatant, and the mixture was stirred overnight at 4°C in a rotary incubator (Taitec RT-50, Japan). The sample was centrifuged at 3,000 g for 1 minute. The supernatant was discarded, and the beads were washed with 1 mL of RIPA buffer. The beads were collected by centrifugation at 3,000 g for 1 minute. The beads were washed three times. 60 μL of a 5 mM biotin solution in RIPA buffer (freshly prepared) was added and the mixture was further shaken at room temperature for 15 minutes to elute the proteins adsorbed to the resin. The mixture was centrifuged at 15,000 g for 1 minute. The supernatant was removed, and SDS-PAGE sample buffer was added and incubated for 1 hour. The samples were analyzed by Western blot analysis.

[0071] 6. Purification of the Mitochondrial Trifunctional Protein Expression plasmid DNA encoding tag-free HADHA and His8-tagged HADHB was co-transformed into Escherichia coli BL21(DE3) cells (Agilent Technologies). The co-transformed E. coli cells were grown in 1 L of LB medium at an OD ６００ The cells were cultured at 37°C until the pH reached 0.6. After lowering the temperature to 23°C, 0.1 mM IPTG was added and the cells were cultured overnight with constant shaking to slowly express both proteins. The cells were harvested and resuspended in Buffer A (20 mM monobasic potassium phosphate pH = 7.7, 100 mM NaCl, 0.02% DDM, 10 mM imidazole, 5% glycerol, protease inhibitor cocktail (Nacalai Tesque), and 50 μg / mL lysozyme (Wako). After incubation on ice for 10 minutes, the cells were lysed by sonication and then incubated at 4°C for 1 hour. The mixture was centrifuged at 20,000 g for 30 minutes at 4°C to remove cell debris. The supernatant was removed and loaded onto a Ni column equilibrated with Buffer A, and passed through the column under gentle pressure. The column was washed with buffer B (20 mM monobasic potassium phosphate pH = 7.7, 100 mM NaCl, 0.02% DDM, 20 mM imidazole, 5% glycerol) followed by buffer C (20 mM monobasic potassium phosphate pH = 7.7, 100 mM NaCl, 0.02% DDM, 40 mM imidazole, 5% glycerol) to remove nonspecifically bound proteins. The purified protein was eluted with buffer D (20 mM monobasic potassium phosphate pH = 7.7, 100 mM NaCl, 0.02% DDM, 400 mM imidazole, 5% glycerol). The eluate was concentrated and buffer-exchanged into buffer E (20 mM HEPES-NaOH pH = 7.5, 100 mM NaCl, 0.02% DDM) using a 100-kDa MWCO Amicon Ultra centrifugal filter (Merck Millipore). Aliquots of the pass-through fractions from buffers B, C, and D were saved in SDS-PAGE sample buffer. The purified trifunctional protein complex was aliquoted into multiple fractions and stored at -80°C until use. Each fraction sample was analyzed by gel electrophoresis followed by Coomassie brilliant blue staining.

[0072] 7. In vitro mTFP enzyme assay. To 88.5 μL of buffer solution (50 mM Tris-HCl (pH = 7.5) and 1 mM EDTA), 10 μL of 10 mM NAD solution was added, followed by 2.5 μL of 1 mM 2-trans-dodecenoyl-CoA (TCI, Japan). The reaction was initiated by adding 0.5 μg of trifunctional protein. NADH production was monitored for 20 minutes by observing the increase in absorbance at a wavelength of 340 nm using a Hitachi U-3010 spectrophotometer. The data after 10 minutes were used to evaluate enzyme activity.

[0073] 8. LC-MS / MS Proteome and GO Enrichment Analysis. The protein solution was subjected to the SP3 method to prepare peptides for mass spectrometry analysis. Mass spectra were obtained using a nanoflow UHPLC system (ADVANCE UHPLC; AMR Inc.) equipped with an LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific) and an Advanced Captive Spray SOURCE (AMR Inc.). The enriched peptide mixture was loaded onto a C18 0.1 mm i.d. x 20 mm, 5 μm particle size trap column (Acclaim PepMap 100 C18, Thermo Fisher Scientific) and fractionated on a C18 0.075 mm i.d. x 150 mm, 3 μm particle size column (CELI). Peptides from in-gel digestion and SP3 were eluted over 46 and 160 minutes, respectively, with a linear gradient of 5-35% solvent B at a flow rate of 300 nL / min. The solvent compositions of buffers A and B were 100% H, respectively. ２The solvent was 0, 0.1% formic acid, and 100% acetonitrile. The mass spectrometer was programmed to perform 13 consecutive scans, consisting of a full-scan MS in the m / z range of 350–1,600 using the Orbitrap at 60,000 resolution and an automated data-dependent MS / MS scan of the 12 most intense ion signals from the initial precursor scan using the ion trap detector. MS / MS spectra were acquired for molecules in the same m / z range, using an isolation width of 2 m / z, an exclusion time of 90 seconds, and a normalized collision energy of 35% CID. Raw data files were searched using Proteome Discoverer 2.5 software (Thermo Fisher Scientific) with the MASCOT version 2.6 search engine against the Homo sapiens dataset (Uniprot Proteome UP000005640, 2022_01downloaded) and the Common Repository of Adventitious Proteins (cRAP, ftp: / / ftp.thegpm.org / fasta / cRAP) and avidin sequences for recognizing contaminating proteins, with a false discovery rate (FDR) set to 0.01. Carbamidomethylation of cysteines was a fixed modification, while methionine oxidation and acetylation of protein N-termini were variable modifications. Label-free quantification of proteins was also performed using Proteome Discoverer 2.5.The S7 parameters were as follows: Peptides to Use: Unique + Razor, Consider Protein Groups for Peptide Uniqueness: True, Use Shared Quan Results: True, Reject Quan Results with Missing Channels: False, Precursor Abundance Based On: Intensity, Min. #Replicate Features [%]: 0, Normalization Mode: None, Scaling Mode: On All Average, Protein Abundance Calculation: Summed Abundances, N for Top N: 3, Protein Ratio Calculation: Pairwise Ratio Based, Maximum Allowed Fold Change: 100, Imputation Mode: None, Hypothesis Test: t-test (background-based). Volcano plots were created using GraphPad Prism, and statistical significance was determined using a t-test. Significant proteins were used for GO enrichment analysis using DAVID.

[0074] 9. Stability test: 100 μM spermidine or Compound 13 was added to DMEM supplemented with 5% FBS, and the cells were incubated in a humidified atmosphere of 5% CO ２The mixture was incubated at 37°C in an incubator, and the medium was transferred to a 2 mL Eppendorf tube. 1 mL of acetonitrile was added, followed by shaking until homogenous. The sample was centrifuged at 15,000 g for 5 minutes. The supernatant was removed and filtered through a 0.22 μm filter. 5 μL of benzoyl chloride (Wako) was added, followed by 10 μL of 10 M sodium hydroxide solution. The reaction mixture was mixed in a tube mixer for 10 minutes, and the benzoylated derivative was extracted with dichloromethane. The organic layer was dried over sodium sulfate. 100 μL of methanol was added to the organic residue, and 10 μL of the sample was subjected to HPLC analysis. Graph-pad Prism was used for curve fitting and half-life calculation.

[0075] 10. Isolation of T cells for in vitro analysis. Spleen cells collected from 6- to 8-week-old female mice were treated with ammonium chloride-potassium (ACK) buffer for 2 minutes to lyse red blood cells. The stored cells were purified using the MojoSort Mouse CD8 (naive) T cell isolation kit (BioLegend, CD8 + Naive T cells (480044; CD8+ T cells (480035)) were used and treated according to their manufacturer's instructions. Briefly, cells were incubated with the appropriate antibody cocktail and magnetic nanobeads, then added to a magnetic mug. Negatively selected CD8 ＋ Naive cells or total CD8 ＋ T cells were subjected to mitochondrial activity measurement or spermidine toxicity test, respectively.

[0076] 11. Measurement of Mitochondrial Activity by Seahorse (Seahorse Assay) The oxygen consumption rate (OCR) of T cells was measured using a Seahorse XFe96 extracellular flux analyzer (Agilent Technologies) according to a known procedure. Briefly, naive mouse CD8 T cells were stimulated with Dynabeads Mouse T-Activator CD3 / CD28 (beads:cells = 1:2.5) (Thermo Fisher Fisher, 11453D) in the presence of spermidine (0.2 μM) (Nacalai Tesque, 3210891) or compound 13 (0.2 μM) for 1 hour. Cells were cultured on XFe96 plates coated with Cell-Tak (Corning, 354240) in XF DMEM medium (Agilent Technologies, 103575-100) supplemented with 10 mM glucose (Wako, 041-00595), 1 mM pyruvate (Thermo Fisher, 11360-070), and 2 mM glutamine (Agilent, 103579-100). Three compounds included in the XF Cell Mito Stress Test Kit (Agilent Technologies): oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (1 μM), and rotenone / antimycin A (0.5 μM) were sequentially added. Mitochondrial parameters are shown in FIG. 12A, and spare respiratory capacity (SRC) can be calculated by the following formula with reference to FIG. 12A: SRC (pmol / min) = OCR ｍａｘｉｍａｌ -OCR ｂａｓａｌ

[0077] 12. Tumor therapy model MC38 cells (5 × 10 ５ Mice were intradermally inoculated with Compound 13 (4 mg / kg) into one flank (day 0). PD-L1 monoclonal antibody (20 μg / mouse) (clone 1-111A, housemade) was intraperitoneally injected, and spermidine (4 mg / kg) or Compound 13 (4 mg / kg) was orally administered on days 7, 12, and 18 after tumor injection. Tumor volume was calculated using the following formula:

[0078] 13. Toxicity test on T cells Mouse total CD8 ＋ T cells (1.5 x 10 ５ ) were stimulated for 22 hours with Dynabeads Mouse T-Activator CD3 / CD28 (beads:cells = 1:2.5) in the presence of spermidine (0-2 μM) or a compound of the present invention (compound 13) (0-2 μM) with or without aminoguanidine (1 mM) (Sigma Aldrich, 396494). The cells were separated from the beads using a magnetic stand and incubated with 10 μL of CD16 / CD32 antibody (Invitrogen, 16-0161-82) (×100 dilution in 2% FCS / PBS) for 5 minutes at room temperature, followed by the addition of a mixture of 10 μL of APC-conjugated CD3 antibody (eBioscience, 17-0031-82) (×100 dilution in 2% FCS / PBS) and Brilliant Violet 421-conjugated CD8 antibody (Biolegend, 100738) (×50 dilution in 2% FCS / PBS) for 12 minutes at 4°C. Cells were washed with 2% FCS / PBS and stained with 50 μL propidium iodide (PI) (Sigma Aldrich, p-4864) (×1000 dilution in Annexin buffer) for 1 minute at 4°C. After one wash, they were subjected to flow cytometry analysis (BD Biosciences, LSR Fortessa X-20). FACS data were analyzed using FlowJo software (BD Biosciences). PI-positive cells were defined as dead cells.

[0079] 14. Quantification and Statistical Analysis Student's t-test was used to calculate p-values. All biochemical curves and associated statistical analyses were performed using GraphPad Prism (GraphPad Software).

[0080] (2) Synthesis Example <2-1. Materials> Solvents and chemicals used in the chemical synthesis were purchased commercially and used without further purification unless otherwise specified. Solvents and chemicals were purchased from Wako Pure Chemicals, TCI, or Sigma-Aldrich. Analytical thin-layer chromatography (TLC) was performed using the fluorescent indicator F 254 Merck 60 silica gel glass plates coated with fluorescent indicator F 254 Silica gel column chromatography was performed using Wakosil® C-200 (Wako) silica gel and chromatorex NH-DM1020 silica. 1 H and 13 C NMR spectra were recorded using Bruker (Avance III 600) 600-MHz and (Avance III 800) 800-MHz NMR spectrometers. High-resolution mass spectra (HRMS) were recorded using a Bruker timsTOF mass spectrometer.

[0081] <Synthesis of Probe 1>

[0082] Synthesis of probe 1 N1,N4-Bis-Boc-spermidine (200 mg, 0.57 mmol) was dissolved in acetonitrile, followed by the addition of DIPEA (149 mg, 1.16 mmol). Propargyl bromide (68 mg, 0.57 mmol) was added, and the reaction mixture was stirred for 1 hour. The reaction was monitored by TLC and LC-MS. The solvent was removed under reduced pressure to give an oil containing the mono- and bis-alkylated products. The mono-alkylated product was purified by flash chromatography. The purified compound was dissolved in dichloromethane, and HCl / 1,4-dioxane (4N, 200 μL) was added dropwise. After stirring overnight, the solvent was concentrated under reduced pressure. The resulting salt was washed multiple times with dichloromethane and ether. The title compound was obtained as a white solid (50 mg, 0.27 mmol).

[0083] 1 H NMR (800 MHz, CDCl3) δ: 1.79-1.81(m, 4H), 2.08-2.12(m, 2H), 3.00-3.01(m, 1H), 3.10-3.21(m, 10H), 3.94(m, 2H); 13 C NMR (201 MHz, CDCl3) δ: 22.63, 22.75, 23.72, 36.33, 36.52, 44.51, 45.85, 46.97, 73.13, 77.99. HRMS (ESI) m / z calcd for [C 10 H 22 N3] + [M+H] + : 184.1808, found 184.1806.

[0084] <Synthesis of Probe 2>

[0085] Synthesis of Compound S1 To a solution of N-(tert-butoxycarbonyl)-1,4-diaminobutane (480 mg, 2.55 mmol) in acetonitrile (5 ml) at 0° C. was added DIPEA (658 mg, 5.11 mmol) and stirred at room temperature for 10 minutes. To the solution was added propargyl bromide (303 mg, 2.55 mmol) dropwise, and the reaction mixture was stirred for 30 minutes. The reaction was quenched by the addition of water, followed by the addition of excess ethyl acetate. The title compound was extracted into ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to give an oil containing a mixture of mono-, bis-, and tris-alkylated products. The resulting oil was purified by flash chromatography (1-4% methanol:dichloromethane) to give the title compound (250 mg, 1.11 mmol) as a colorless oil.

[0086] 1 H NMR (600 MHz, CDCl3) δ: 1.43(s, 9H), 1.50-1.55(m, 4H), 2.21(t, J=2.4Hz, 2H), 2.70-2.72(m, 2H), 3.13-3.14(m, 2H), 3.42(d, J=2.4Hz,2H), 4.86(brs,1H); 13 C NMR (151 MHz, CDCl3) δ: 27.08, 27.82, 28.43, 38.1, 40.42, 48.18, 71.28, 79.04, 82.18, 155.98. HRMS (ESI) m / z calcd for [C 12 H 23 N2O2] + [M+H] + : 227.1754, found 227.1753.

[0087] Synthesis of Compound S2 To a solution of compound S1 (129 mg, 0.57 mmol) in acetonitrile under an argon atmosphere was added DIPEA (149 mg, 1.16 mmol), followed by 3-(4-(bromomethyl)phenyl)-3-(trifluoromethyl)-3H-diazirine (119 mg, 0.43 mmol) at room temperature. The reaction mixture was stirred overnight, the solvent removed under reduced pressure, and then purified by flash chromatography (10-30% ethyl acetate:hexanes). The title compound was obtained as a colorless oil (120 mg, 0.28 mmol).

[0088] 1 H NMR (600 MHz, CDCl3) δ: 1.43(s, 9H), 1.52(m, 4H), 2.22(brs, 1H), 2.53(brs, 2H), 3.12(brs, 2H), 3.27(br s, 2H), 3.62(s, 2H), 4.68(brs, 1H), 7.13(d, J=8.4Hz, 2H), 7.38 (d, J=8.4Hz, 2H); 13 C NMR (151 MHz, CDCl3) δ: 24.76, 27.73, 28.43, 28.37(q, J= 40.8Hz), 40.45, 41.19, 52.82, 57.26, 73.37, 78.04, 79.08, 122.15(q, J= 274.8Hz), 126.441, 127.92, 129.36, 140.72, 155.97. 21 H 28 F3N4O2] + [M+H] + : 425.2159, found 425.2160.

[0089] Synthesis of Compound S3 Compound S2 (100 mg, 0.24 mmol) was dissolved in dichloromethane and the solution was stirred at 0° C. To the reaction mixture was added HCl / 1,4-dioxane (4N, 500 μL) dropwise. After stirring overnight, the solvent was removed under reduced pressure. The residue was washed with saturated sodium bicarbonate solution and the crude compound was extracted into ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure and purified by flash chromatography on aminosilica (10-30% ethyl acetate:hexane) to give the title compound (50 mg, 0.15 mmol) as a pale yellow oil.

[0090] 1 H NMR (600 MHz, CDCl3) δ: 1.46-1.55(m, 6H), 2.21(t, J=2.4Hz, 1H), 2.52-2.54(m, 2H), 2.69-2.71(m, 2H), 3.28(d, J=2.4Hz, 2H), 3.62(s, 2H), 7.13(d, J=8.4Hz, 2H), 7.38 (d, J=9.0Hz, 2H); 13 C NMR (151 MHz, CDCl3) δ: 24.84, 28.37(q, J= 40.8Hz), 31.31, 41.33, 42.04, 53.11, 57.28, 73.27, 78.18, 122.15(q, J= 274.8Hz),126.42, 127.88, 129.34, 140.85. HRMS (ESI) m / z calcd for [C 16 H 20 F3N4] + [M+H] + : 325.1635, found 325.1627.

[0091] Synthesis of probe 2 (compound S4) To a solution of compound S3 (50 mg, 0.15 mmol) in acetonitrile, DIPEA (39 mg, 0.31 mmol) was added and stirred for 15 minutes. 3-(tert-Butoxycarbonylamino)propyl bromide (36 mg, 0.15 mmol) was added to the reaction mixture, and the reaction mixture was stirred overnight. Mono- and di-substituted products were observed by TLC and LC-MS. The mono-substituted product was purified by flash chromatography on amino silica gel. The purified compound was dissolved in dichloromethane, and HCl / 1,4-dioxane (4N, 200 μL) was added dropwise. After stirring overnight, the solvent was concentrated under reduced pressure, and the residue was washed with saturated sodium bicarbonate solution, and the crude compound was then extracted into ethyl acetate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography on amino silica gel (1-4% methanol:dichloromethane) afforded the title compound (30 mg, 0.078 mmol) as a pale yellow oil.

[0092] 1 H NMR (600 MHz, CDCl3) δ: 1.48-1.50(m, 4H), 1.59-1.64(m, 2H), 2.19(t, J=2.4Hz, 1H), 2.49-2.51(m, 2H), 2.57-2.59(m, 2H), 2.63-2.66(m, 2H), 2.74-2.76(m, 2H), 3.25(d, J=1.8Hz, 2H), 3.59(s, 2H), 7.11(d, J=7.8Hz, 2H), 7.35(d, J=8.4Hz, 2H); 13 C NMR (151 MHz, CDCl3) δ: 25.27, 27.75, 28.36(q, J= 40.8Hz), 33.73, 40.63, 41.32, 47.99, 49.91, 53.08, 57.27, 73.22, 78.19, 122.13(q, J= 274.8Hz), 126.38, 127.83, 129.31, 140.88. HRMS (ESI) m / z calcd for [C 19 H 27 F3N5] + [M+H] +: 382.2213, found 382.2214.

[0093] <Synthesis of Probe 3>

[0094] Synthesis of Compound S5 To a solution of compound S1 (260 mg, 1.14 mmol) in acetonitrile under an argon atmosphere, DIPEA (294 mg, 2.28 mmol) was added, followed by benzyl bromide (196 mg, 1.14 mmol) at room temperature. The reaction mixture was stirred overnight. The solvent was removed under reduced pressure and purified by flash chromatography (10-30% ethyl acetate:hexanes) to give the title compound (250 mg, 0.79 mmol) as a colorless oil.

[0095] 1 H NMR (600 MHz, CDCL3) δ: 1.42 (s, 9H), 1.50-1.52(m, 4H), 2.20(t, J=2.4Hz,1H), 2.52-2.54(m, 2H), 3.09-3.10(m, 2H), 3.27-3.28(m, 2H), 3.59(s, 2H), 4.72(brs, 1H), 7.21-7.23(m, 1H), 7.27-7.32 (m, 4H); 13 C NMR (151 MHz, CDCl3) δ: 24.79, 27.68, 28.41, 40.44, 41.14, 52.74, 57.77, 73.14, 78.37, 78.98, 127.11, 128.27, 129.07, 138.62, 155.96. HRMS (ESI) m / z calcd for [C 19 H 29 N2O2] + [M+H] + : 317.2224, found 317.2222.

[0096] Synthesis of Compound S6 Compound S5 (250 mg, 0.79 mmol) was dissolved in dichloromethane, and the solution was stirred at 0° C. To the solution was added HCl / 1,4-dioxane (4 N, 500 μL) dropwise. After stirring overnight, the solvent was removed under reduced pressure. The residue was washed with saturated sodium bicarbonate solution, and the crude compound was extracted into ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by flash chromatography on aminosilica (10-30% ethyl acetate:hexanes) to give the title compound (140 mg, 0.65 mmol) as an oil.

[0097] 1 H NMR (600 MHz, CDCl3) δ: 1.45-1.54(m, 6H), 2.20(t, J=2.4Hz,1H), 2.52-2.54(m, 2H), 2.66-2.68(m, 2H), 3.27-3.29(m, 2H), 3.59(s, 2H), 7.20-7.23(m, 1H), 7.27-7.33(m, 4H); 13 C NMR (151 MHz, CDCl3) δ: 24.81, 31.39, 41.25, 42.01, 53.03, 57.76, 73.01, 78.51, 127.04, 128.23, 129.01,138.76. HRMS (ESI) m / z calcd for [C 14 H 21 N2] + [M+H] + : 217.1699, found 217.1698.

[0098] Synthesis of probe 3 (compound S7) To a solution of compound S6 (140 mg, 0.65 mmol) in acetonitrile, DIPEA (167 mg, 1.30 mmol) was added and stirred for 15 minutes. 3-(tert-Butoxycarbonylamino)propyl bromide (154 mg, 0.65 mmol) was added to the reaction mixture, and the reaction mixture was stirred overnight. The mono- and di-substituted products were monitored by TLC and LC-MS. The mono-substituted product was purified by flash chromatography. The purified compound was dissolved in dichloromethane, and HCl / 1,4-dioxane (4 N, 200 μL) was added dropwise. After stirring overnight, the solvent was removed under reduced pressure. The residue was washed with saturated sodium bicarbonate solution, and the crude compound was extracted into ethyl acetate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The crude mixture contained mono- and bis-alkylated products. The crude mixture was purified by flash chromatography on aminosilica (1-4% methanol:dichloromethane) to give the title compound as a pale yellow oil (80 mg, 0.29 mmol).

[0099] 1 H NMR (600 MHz, CDCl3) δ: 1.52(m, 4H), 1.59-1.64(m, 2H), 1.89(brs, 1H), 2.19(t, J=2.4Hz, 1H), 2.51-2.53(m, 2H), 2.59(brs, 2H), 2.64-2.66(m, 2H), 2.73-2.76(m,2H), 3.28(d, J=2.4Hz, 2H), 3.59(s, 2H), 7.20-7.22(m, 1H), 7.26-7.32(m, 4H); 13 C NMR (151 MHz, CDCl3) δ: 25.28, 27.71, 33.58, 40.62, 41.27, 47.94, 49.87, 53.06, 57.78, 73.02, 78.54, 127.05, 128.25, 129.04, 138.79. HRMS (ESI) m / z calcd for [C 17 H 28 N3] + [M+H] + : 274.2278, found 274.2278.

[0100] <2-2. Synthesis of Compounds of the Present Invention> Synthesis Methods The compounds of the present invention may be synthesized by a method combining the production methods shown below and known synthesis methods. The compounds in the reaction schemes may each form a salt, and examples of such salts include the same as the salt of the compound (I) above. Note that these reactions are merely illustrative, and the compounds of the present invention may also be produced by other appropriate methods based on the knowledge of those skilled in organic synthetic chemistry.

[0101] The compounds were identified by proton nuclear magnetic resonance spectroscopy ( １ The LC-MS analysis conditions for the compound of the present invention are as follows: HPLC conditions Column: L-Column 2 C １８ (3.0 mm ID x 50 mm, 3 μm) Mobile phase: A: 0.05% TFA in water B: 0.05% TFA in acetonitrile Gradient program: B% 5% to 55% 0.01 to 1.67 min, B% 55% to 90% 1.67 to 1.68 min, B% 90% 1.68 to 2.0 min Flow rate: 1.5 ml / min Detection: PDA 220 nm Temperature: 40 ° C Injection volume: 0.002 ml MS conditions Ionization: ES / APCI (Dual Ion Source), Positive mode Mode: Scan (m / z 100-800)

[0102] Scheme 1 The compound of formula (I) or a pharmaceutically acceptable salt thereof can be prepared, for example, by the following synthetic method.

[0103] Step 1 (Condition 1): Benzyl (3-oxopropyl)carbamate A (41 mg, 0.20 mmol) was added to a solution of the corresponding amine compound (0.24 mmol) in acetic acid (100 μL) and methanol (500 μL) at room temperature. After stirring at room temperature for 10 minutes, borane-2-methylpyridine complex (Pic-BH ３) (43 mg, 0.40 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 3 hours. The mixture was poured into water at room temperature and evaporated by blowing with air at 60°C. The residue was purified by preparative HPLC (YMC-Triart C18, H in acetonitrile (MeCN) containing 0.1% triacetoxyacetic acid (TFA)). ２ The resulting fractions were evaporated by blowing with air at 60° C. to give the desired product C.

[0104] Step 1 (Condition 2): Benzyl (3-oxopropyl)carbamate A (41 mg, 0.20 mmol) was added to a solution of the corresponding amine compound (0.24 mmol) in acetic acid (100 μL) and methanol (500 μL) at room temperature. After stirring at room temperature for 10 minutes, Pic-BH ３ (43 mg, 0.40 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 3 hours. The mixture was poured into water at room temperature and evaporated by blowing with air at 60°C. The residue was purified by preparative HPLC (YMC-Triart C18, 10 mM ammonium bicarbonate (NH ４ HCO ３ ) containing H in MeCN ２ The resulting fractions were evaporated by blowing with air at 60° C. to give the desired product C.

[0105] Step 1 (Condition 3): The corresponding amine compound (0.24 mmol) and acetic acid (100 μL, 1.75 mmol) were added to a solution of benzyl (3-oxopropyl)carbamate A (0.20 mmol) in TFA (500 μL). After stirring at room temperature for 1 hour, sodium triacetoxyborohydride (NaBH(OAc) ３ ) (85 mg, 0.40 mmol) was added to the reaction mixture. The mixture was stirred at room temperature overnight. The mixture was poured into water at room temperature and evaporated by blowing off air at 60°C. The residue was purified by preparative HPLC (YMC-Triart C18, H in MeCN containing 0.1% TFA). ２ The resulting fractions were lyophilized to give the desired product C.

[0106] Step 1 (Condition 4): The corresponding amine compound (0.24 mmol) and acetic acid (100 μL, 1.75 mmol) were added to a solution of benzyl (3-oxopropyl)carbamate A (0.20 mmol) in TFA (500 μL). After stirring at room temperature for 1 hour, sodium triacetoxyborohydride (NaBH(OAc) ３ ) (85 mg, 0.40 mmol) was added to the reaction mixture. The mixture was stirred at room temperature overnight. The mixture was poured into water at room temperature and evaporated by blowing off air at 60 °C. The residue was purified by preparative HPLC (YMC-Triart C18, 10 mM NH ４ HCO ３ H in MeCN containing ２ The resulting fractions were lyophilized to give the desired product C.

[0107] Step 2 (Condition 1): A mixture of product C (0.20 mmol) obtained in Step 1 and Pd / C (20 mg, 10 wt%) in ethanol (1 mL) was hydrogenated at room temperature for 2 hours (using a parallel gas reactor) under 0.1-0.3 MPa. The catalyst was filtered off. 1N HCl (0.50 mL) was added to the filtrate, which was then evaporated by blowing with air at 60°C. The residue was dissolved in water / MeCN and lyophilized to give the desired product E (compounds 33-35, 38-45, 48-49, 51, 53-55) as a solid.

[0108] Step 2 (Condition 2): A mixture of benzyl (3-((2-oxopiperidin-4-yl)amino)propyl)carbamate obtained in Step 1, trifluoroacetic acid (75.1 mg, 179 μmmol) and Pd / C (20 mg, 10 wt %) in ethanol (4 mL) was hydrogenated under balloon pressure at room temperature for 2 hours. The catalyst was filtered off. The filtrate was concentrated in vacuo. The residue was dissolved in water / MeCN (1 mL) and lyophilized to give 4-((3-aminopropyl)amino)piperidin-2-one (Compound 37) as a pale orange oil (28.3 mg, 92.3%).

[0109] Step 2 (Condition 3): A mixture of benzyl (3-(((5s,8s)-2-oxo-1-azaspiro[4,5]decan-8-yl)amino)propyl)carbamate obtained in Step 1, trifluoroacetic acid (58 mg, 0.12 mmol), and Pd / C (13 mg, 10 wt %) in ethanol (4 mL) was hydrogenated under balloon pressure at room temperature for 2 hours. The catalyst was removed by filtration. The filtrate was concentrated in vacuo to give (5s,8s)-8-((3-aminopropyl)amino)-1-azaspiro[4,5]decan-2-one (Compound 50) as a pale orange powder (25.9 mg, 94%).

[0110] Step 2 (Condition 4): A mixture of product C (0.20 mmol) obtained in Step 1 and Pd / C (20 mg, 10 wt%) in ethanol (1 mL) was hydrogenated at room temperature for 2 hours (using a parallel gas reactor) under 0.1-0.3 MPa. The catalyst was filtered off. The filtrate was concentrated in vacuo. A mixture of the residue and TFA (300 μL) was stirred at room temperature for 0.5 hours. The reaction mixture was concentrated in vacuo. The residue was dissolved in water / MeCN (1 mL) and lyophilized to give the desired products (compounds 36 and 46) as solids.

[0111] Step 2 (Condition 5): A mixture of product C (0.20 mmol) obtained in Step 1 and Pd / C (20 mg, 10 wt%) in ethanol (1 mL) was hydrogenated at room temperature for 2 hours (using a parallel gas reactor) under 0.1-0.3 MPa. The catalyst was filtered off. 1N HCl (0.50 mL) was added to the filtrate, which was then concentrated under vacuum at room temperature. The residue was dissolved in water / MeCN and lyophilized to give the desired products (compounds 47 and 52) as solids.

[0112] Scheme 2 The compound of formula (I) or a pharmaceutically acceptable salt thereof can also be prepared, for example, by the following synthetic method.

[0113] Step 1: Benzyl (3-aminopropyl)carbamate hydrochloride B (59 mg, 0.24 mmol), DIEA (42 μL, 0.24 mmol) and Ti(OEt) at room temperature ４(50 μL, 0.24 mmol) was added to a solution of the corresponding ketone (0.20 mmol) in THF (1 mL). After stirring at 70 °C for 3 h, the THF was evaporated by nitrogen blowing. Methanol (500 μL) and NaBH(OAc) ３ (85 mg, 0.40 mmol) was added to the reaction mixture. The mixture was stirred at room temperature overnight and poured into water at room temperature. The insoluble material was removed by filtration, and the filtrate was evaporated with air at 60°C. The residue was purified by preparative HPLC (YMC-Triart C18, 10 mM NH ４ HCO ３ H in MeCN containing ２ The resulting fractions were evaporated by air blowing at 60° C. to give the desired product C.

[0114] Step 2: A mixture of product C (0.20 mmol) obtained in Step 1 and Pd / C (21 mg, 10 wt%) in ethanol (1 mL) was hydrogenated at room temperature for 2 hours (using a parallel gas reactor) under 0.1-0.3 MPa. The catalyst was filtered off. 1N HCl (0.20 mL) was added to the filtrate, which was then evaporated with air at 60°C. The residue was dissolved in water / MeCN and lyophilized to give the desired products (compounds 57-61) as solids.

[0115] Synthesis of Compounds 33 to 55 Compounds 33 to 55 shown in the table below were synthesized using benzyl(3-oxopropyl)carbamate and the corresponding amine compound (R-amine) according to the method described in Scheme 1 above.

[0116] The instrumental analysis data for compounds 33 to 55 are as follows: Compound 33: 1H NMR (400 MHz, DMSO-d6, 298K) δ 1.35-1.58 (3H, m), 1.63-1.91 (3H, m), 2.00 (2H, quin, J = 7.5 Hz), 2.05-2.15 (1H, m), 2.31-2.44 (1H, m), 2.85-2.96 (2H, m), 2.97-3.08 (2H, m), 3.11-3.23 (1H, m), 5.05 (1H, br d, J = 47.4 Hz), 8.17 (3H, br s), 9.36 (2H, br s). LCMS (ESI) m / z calcd for [C9H 19 FN2] + [M+H] + : 174.259, found 174.15. Compound 34: LCMS (ESI) m / z calcd for [C 10 H 19 F3N 12 O] + [M+H] + : 240.266, found 240.14. Compound 35: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 0.97 (3H, t, J = 7.6 Hz), 1.10-1.27 (2H, m), 1.37-1.53 ​​(2H, m), 1.84 (2H, br d, J = 11.0 Hz), 1.94-2.12 LCMS (ESI) m / z calcd for [C 12 H 25 N3O] + [M+H] + : 227.346, found 227.2. Compound 36: 1H NMR (400 MHz, DMSO-d6, 298K) δ 0.64-0.78 (3H, m), 0.92-1.03 (1H, m), 1.83-2.05 (2H, m), 2.06-2.21 (2H, m), 2.61 (1H, br d, J = 13.4 Hz), 2.79-2.94 (3H, m), 2.96-3.17 (2H, m), 3.19-3.35 (2H, m), 3.49 (1H, br t, J = 11.5 Hz), 7.94 (3H, br s), 8.61-9.36 (4H, m). LCMS (ESI) m / z calcd for [C 10 H 21 N3] + [M+H] + : 183.294, found 183.17. Compound 37: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.41-1.53 ​​(1H, m), 1.66 (2H, quin, J = 7.0 Hz), 1.82-1.91 (1H, m), 1.96 (1H, dd, J = 17.0, 8.3 Hz), 2.37 (1H, dd, J = 17.0, 4.6 Hz), 2.56-2.68 (2H, m), 2.80-2.93 (3H, m), 2.99-3.08 (1H, m), 3.15-3.22 (1H, m), 5.81-7.11 (3H, m), 7.42 (1H, br s). LCMS (ESI) m / z calcd for [C8H 17 N3O] + [M+H] + : 171.24, found 171.14. Compound 38: LCMS (ESI) m / z calcd for [C 11 H 22 N2] + [M+H] + : 182.306, found 182.18. Compound 39: 1H NMR (400 MHz, DMSO-d6, 298K) δ 0.48-0.56 (2H, m), 0.56-0.64 (2H, m), 2.03-2.09 (2H, m), 2.31-2.39 (2H, m), 2.46-2.54 (2H, m), 3.03-3.08 (4H, m), 3.97 (1H, quin, J = 7.8 Hz). LCMS (ESI) m / z calcd for [C9H 18 N2] + [M+H] + : 154.253, found 154.15. Compound 40: LCMS (ESI) m / z calcd for [C 12 H 25 N3O] + [M+H] + : 227.346, found 227.2. Compound 41: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.48-1.68 (2H, m), 1.94-2.19 (4H, m), 2.91 (6H, br d, J = 6.5 Hz), 3.21-3.41 (2H, m), 4.30-4.72 (1H, m), 7.34-7.40 (2H, m), 7.44-7.49 (3H, m), 8.08 (3H, br s), 9.35 (2H, br s). LCMS (ESI) m / z calcd for [C 15 H 23 N3O] + [M+H] + : 261.363, found 261.18. Compound 42: LCMS (ESI) m / z calcd for [C 11 H 25 N3O2S] + [M+H] + : 263.4, found 263.17. Compound 43: LCMS (ESI) m / z calcd for [C 10 H 22 N2O] + [M+H] + : 186.294, found 186.17. Compound 44:1 H NMR (600 MHz, DMSO-d6, 298K) δ 1.35 (3H, s), 1.80-1.88 (4H, m), 1.90-2.10 (6H, m), 2.87-2.92 (2H, m), 2.96-3.01 (2H, m), 7.88 (3H, br s), 8.82 (2H, br s). LCMS (ESI) m / z calcd for [C 10 H 20 F2N2] + [M+H] + : 206.276, found 206.16. Compound 45: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.93-2.01 (2H, m), 2.02 (6H, s), 2.66 (1H, s), 2.82-3.03 (4H, m), 8.15 (3H, br s), 10.02 (2H, br s). LCMS (ESI) m / z calcd for [C8H 16 N2] + [M+H] + : 140.226, found 140.13. Compound 46: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.58 (3H, s), 1.92 (2H, quin, J = 7.6 Hz), 2.83-2.95 (2H, m), 2.97-3.09 (2H, m), 4.41 (2H, d, J = 7.5 Hz), 4.71 (2H, d, J = 7.5 Hz), 7.96 (3H, br s), 9.63 (2H, br s). LCMS (ESI) m / z calcd for [C7H 16 N2O] + [M+H] + : 144.215, found 144.13. Compound 47: LCMS (ESI) m / z calcd for [C8H 18 N2O] + [M+H] +: 158.241, found 158.14. Compound 48: LCMS (ESI) m / z calcd for [C 11 H 17 N3O] + [M+H] + : 207.272, found 207.14. Compound 49: LCMS (ESI) m / z calcd for [C9H 17 N5] + [M+H] + : 195.265, found 195.15. Compound 50: LCMS (ESI) m / z calcd for [C 12 H 23 N3O] + [M+H] + : 225.331, found 225.18. Compound 51: LCMS (ESI) m / z calcd for [C 11 H 22 F2N2] + [M+H] + : 220.303, found 220.18. Compound 52: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.09 (3H, s), 1.82 (2H, br t, J = 9.6 Hz), 1.88-2.00 (2H, m), 2.12 (2H, br t, J = 9.9 Hz), 2.77-2.96 (4H, m), 3.23 (2H, s), 3.61-3.81 (2H, m), 8.13 (3H, br s), 9.29 (2H, br s). LCMS (ESI) m / z calcd for [C9H 20 N2O] + [M+H] + : 172.268, found 172.16. Compound 53: LCMS (ESI) m / z calcd for [C9H 18 F2N2] + [M+H] + : 192.249, found 192.14. Compound 54: LCMS (ESI) m / z calcd for [C 14 H22 N2] + [M+H] + : 218.338, found 218.18. Compound 55: 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.87-1.91 (2H, m), 2.28-2.39 (2H, m), 2.60-2.68 (2H, m), 2.70-2.79 (2H, m), 2.86-2.91 (2H, m), 3.13 (2H, br t, J = 11.6 Hz), 3.89 (2H, br d, J = 11.3 Hz), 7.44-7.55 (3H, m), 7.70 (2H, br d, J = 7.6 Hz), 7.98 (3H, br s), 9.70 (2H, br s). LCMS (ESI) m / z calcd for [C 14 H 22 N2O] + [M+H] + : 234.337, found 234.17.

[0117] Synthesis of Compound 56 Compound 56:

[0118] Compound 56 was prepared according to the following synthetic method.

[0119] Step 1: A mixture of N-(5-aminopyridin-2-yl)acetamide (36 mg, 0.24 mmol), tert-butyl(3-bromopropyl)carbamate (48 mg, 0.20 mmol), and DMF (500 μL) was stirred at 65° C. overnight. Insoluble matter was removed by filtration. The filtrate was purified by preparative HPLC (YMC-Triart C18, 10 mM NH ４ HCO ３ H in MeCN containing ２ The resulting fraction was evaporated by air blowing at 60° C. to give tert-butyl (3-((6-acetamidopyridin-3-yl)amino)propyl)carbamate (8.0 mg, 13%) as a pale yellow oil.

[0120] Step 2: A mixture of tert-butyl (3-((6-acetamidopyridin-3-yl)amino)propyl)carbamate (8.0 mg, 26 μmol) and TFA (500 μL) was stirred at 0° C. for 10 minutes. The reaction mixture was concentrated in vacuo. The residue was dissolved in water / MeCN and lyophilized to give N-(5-((3-aminopropyl)amino)pyridin-2-yl)acetamide, di-TFA salt (Compound 56) (8.3 mg, 73%) as a light brown oil. 1 H NMR (400 MHz, DMSO-d6, 298K) δ 1.64-1.74 (2H, m), 1.75 (3H, br s), 2.72-2.86 (2H, m), 3.64 (2H, t, J = 6.9 Hz), 7.02 (1H, dd, J = 8.4, LCMS (ESI) m / z calcd for [C 10 H 16 N4O] + [M+H] + : 208.26, found 208.13.

[0121] Synthesis of Compounds 57 to 61 Compounds 57 to 61 shown in the table below were synthesized using benzyl (3-aminopropyl)carbamate hydrochloride and the corresponding ketone compound (R-ketone) according to the method described in Scheme 2 above.

[0122] The instrumental analysis data for Compounds 57 to 61 are as follows: Compound 57: LCMS (ESI) m / z calcd for [C 13 H20N2] + [M+H] + : 204.311, found 204.16. Compound 58: LCMS (ESI) m / z calcd for [C 10 H 18 N4] + [M+H] +: 194.277, found 194.15. Compound 59: LCMS (ESI) m / z calcd for [C 12 H 17 FN 2O ] + [M+H] + : 224.275, found 224.13. Compound 60: LCMS (ESI) m / z calcd for [C 13 H 20 N2O] + [M+H] + : 220.16, found 220.311. Compound 61: LCMS (ESI) m / z calcd for [C 10 H 18 N4] + [M+H] + : 194.277, found 194.15.

[0123] (3) Test Examples Test Example 1: Evaluation of Spermidine Probe (A) Labeling with Probe 1 Probe 1 was used to confirm whether spermidine covalently binds to intracellular proteins. Specifically, HEK293 cells were treated with Probe 1 (0, 1, 10, or 100 μM) for 30 minutes, and the resulting cell lysate was subjected to click chemistry with TAMRA-azide. After separation by SDS-PAGE, the gel was analyzed by a fluorescent scanner or silver staining. The results are shown in Figure 1. Figure 1 does not confirm the covalent binding of Probe 1 to intracellular proteins.

[0124] (B) Labeling with Probes 2 and 3. Proteins involved in noncovalent interactions with spermidine were analyzed using Probe 2, which has a photoactive group, and Probe 3, which does not. First, HEK293 cells were treated with Probe 2 (0, 1, 10, or 25 μM) for 20 minutes, followed by 20 minutes of UV irradiation. The resulting cell lysates were subjected to click chemistry with TAMRA-azide. After separation by SDS-PAGE, the gels were analyzed with a fluorescent scanner. Furthermore, HEK293 cells were treated with Probe 2 (25 μM) or Probe 3 (25 μM) for 20 minutes, followed by 20 minutes of UV irradiation with or without UV irradiation. The resulting cell lysates were subjected to click chemistry with TAMRA-azide. After separation by SDS-PAGE, the gels were analyzed with a fluorescent scanner to confirm the effect of UV irradiation. The results are shown in Figures 2A and 2B. From the test results, it was confirmed that probe 2 having a photoactive group was covalently bound to an intracellular protein.

[0125] (C) Labeling with Probe 2 in the Presence of DFMO. The results of (B) suggest that endogenous intracellular spermidine competes with the probe, preventing it from binding to the protein. Therefore, labeling with Probe 2 was performed in the absence and presence of DFMO, a polyamine synthase, to examine the effect of endogenous intracellular spermidine. The results are shown in Figure 3C. As shown in Figure 3C, no band was observed in the absence of DFMO, but a band was observed in the presence of DFMO. These test results confirmed that endogenous intracellular spermidine affects probe binding.

[0126] Test Example 2: Examination of Probe 2-HADHA Binding Interaction Biotin-azide was used instead of TAMPA-azide in the click reaction to verify the presence or absence of HADHA in spermidine-binding proteins. Specifically, as shown in Figure 3A, HEK293 cells were treated with photoaffinity probe 2 and then irradiated with UV light for 20 minutes. The resulting cell lysate was subjected to click chemistry with biotin-azide. The biotin-labeled protein was purified using avidin beads, followed by Western blot analysis of the probe 2-HADHA binding interaction. In addition, given that HADHA is a membrane-bound protein, the cell lysate was fractionated into soluble and insoluble fractions. These fractions were then subjected to click chemistry with biotin-azide. The biotin-labeled protein was purified using avidin beads, followed by Western blot analysis of the probe 2-HADHA binding interaction.

[0127] The results are shown in Figure 3. Figure 3B shows the results of Western blot analysis of HADHA labeled with probe 2 (1, 10, or 25 μM), Figure 3C shows the results of Western blot analysis of probe 2-HADHA binding interaction in the presence or absence of DFMO and in the presence of DFMO and spermidine or spermidine phosphate, Figure 3D shows the results of Western blot analysis of probe 2-HADHA binding interaction in the presence of DFMO with or without UV irradiation, Figure 3E shows the results of Western blot analysis of probe 2-HADHA binding interaction in the soluble and insoluble fractions in the presence or absence of DFMO, probe 2, and spermidine, and Figure 3F shows the results of Western blot analysis of probe 2-HADHA binding interaction in the soluble and insoluble fractions in the presence or absence of DFMO, probe 2, and spermidine. The results of this study demonstrated that HADHA could be labeled with Probe 2 in a dose-dependent manner (Fig. 3B). Furthermore, labeling HADHA with Probe 2 in the presence of DFMO enhanced the Probe 2-HADHA binding interaction. Furthermore, the addition of excess spermidine or spermidine phosphate weakened the Probe 2-HADHA binding interaction by competing with it (Fig. 3C). Furthermore, in the presence of DFMO, most of the biotin-labeled HADHA was found in the insoluble fraction (Fig. 3D), and the labeling level was reduced by the addition of excess spermidine (Fig. 3E). Therefore, probe 2 primarily labeled HADHA localized in the mitochondrial membrane. The increased labeling efficiency of probe 2 in the presence of DFMO and the decreased labeling efficiency of probe 2 with the addition of excess spermidine suggested that the presence of endogenous intracellular spermidine competes for binding to HADHA.

[0128] Test Example 3: Examination of the usefulness of probe 2 To examine the usefulness of probe 2 as a functional spermidine probe, the above-described in vitro enzyme assay was carried out using purified mTFP reconstituted with recombinant HADHA and HADHB.

[0129] The results are shown in Figure 4. Figure 4 shows that probe 2 promoted the enzymatic activity of HADHA in producing NADH by oxidation of 2-trans-dodecenoyl-CoA, and that the activity was equivalent to that of spermidine. Therefore, it was demonstrated that probe 2 is useful as a functional spermidine probe.

[0130] Test Example 4: Screening for Compounds with Similar Effects to Spermidine The 29 selected compounds (Figure 5B) were used to search for compounds with similar effects to spermidine. Specifically, as shown in Figure 5A, HEK293 cells were preincubated for 48 hours in the presence of 2.5 mM DFMO, and compounds 4 to 32 (1 mM) were added individually 60 minutes before photoreaction with probe 2. Photoreaction with probe 2 (10 μM) was then carried out for 20 minutes. The resulting cell lysates were subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay and subsequent HADHA Western blot analysis to evaluate the competitiveness of each compound. Spermidine (SPD) was used as a control.

[0131] The results are shown in Figures 6-1 and 6-2. Figures 6-1 and 6-2 indicate that compounds 8, 13, 18, and 25 have high levels of competitiveness. Furthermore, it was confirmed that these compounds share a primary amine and a secondary amine structure separated by a three-carbon linker. Therefore, these test results suggest that these groups are important for binding to HADHA.

[0132] Test Example 5: Examination of HADHA Activation by Compounds of the Present Invention Spermidine is known to enhance HADHA activation. Therefore, the HADHA activation effect of compounds of the present invention was examined. In this test, compound 13, which showed high competitiveness, was used as the compound of the present invention. HEK293 cells were preincubated for 48 hours in the presence of 2.5 mM DFMO, and spermidine or compound 13 (10 μM, 100 μM, or 1000 μM) was added individually 60 minutes before photoreaction with probe 2. Photoreaction with probe 2 (10 μM) was then carried out for 20 minutes. The resulting cell lysates were subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay and subsequent HADHA Western blot analysis to evaluate the competitiveness of each compound.

[0133] The results are shown in Figure 7. Figure 7A shows the results of Western blot analysis of spermidine (compound 13)-HADHA binding interaction, demonstrating that competition with probe 2 increases in a concentration-dependent manner. Figure 7B shows the quantification of band intensity at each concentration of spermidine or compound 13. Band intensity at each concentration is expressed as a percentage (%) of the band intensity without compound added, which is set at 100%. Data represent mean ± SD (n = 3). Figure 7C shows the activity of HADHA (HADHA-HADHB complex) (UV absorbance at 340 nm indicating NADH production) upon treatment with spermidine (0-20 μM) and compound 13 (0-20 μM). Data represent mean ± SEM (n = 2). The test results showed that compound 13 competed with probe 2 at a concentration less than 1 / 10 that of spermidine, and that compound 13 enhanced the enzymatic activity of HADHA more than spermidine. Therefore, it was suggested that compound 13 is a compound that provides a more effective HADHA activation effect than spermidine.

[0134] Test Example 6: Chemoproteome Analysis of Spermidine-Binding Proteins To comprehensively analyze spermidine-binding proteins, chemoproteomic profiling of spermidine-binding proteins was performed using Probe 2. Specifically, HEK293 cells were preincubated in the presence of 2.5 mM DFMO for 48 hours, followed by photoreaction with Probe 2 (10 μM) for 20 minutes. To compete with Probe 2 and increase spermidine-specific proteins, 1 mM spermidine was added prior to photoreaction with Probe 2. The resulting cell lysates were subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay, followed by LC-MS / MS analysis. Gene ontology (GO) enrichment analysis was also performed to examine the localization of proteins identified by GO enrichment analysis. Furthermore, from the identified mitochondrial proteins, three proteins (epoxide hydrolase (EPHX1), dihydrolipoyl dehydrogenase (DLD), and isocitrate dehydrogenase-2 (IDH2)) that are representative proteins contributing to lipid metabolism, and NipSnap homolog 1 (NIPSNAP1), a mitochondrial protein that does not contribute to lipid metabolism but has the highest -log P value and spermidine competition rate, were selected and analyzed by Western blot analysis for labeling with probe 2 and competition with spermidine.

[0135] The results are shown in Figure 8. Figure 8B shows that a set of 239 proteins was identified as proteins labeled with Probe 2 (left panel), and that of these 239 proteins, 67 proteins were significantly competed for by preincubation with 1 mM spermidine prior to photoreaction with Probe 2 (right panel). HADHA was included in the set of 67 identified proteins. Figure 8C also revealed that the majority of spermidine-binding proteins were mitochondrial proteins. Furthermore, it was confirmed that mitochondrial proteins were labeled with Probe 2 and that competition with Probe 2 was observed upon addition of spermidine.

[0136] Test Example 7: Examination of the biological stability of the compound of the present invention Spermidine is known to be rapidly degraded by amine oxidase in serum, producing cytotoxic acrolein metabolites and inducing cell death. Furthermore, the inherent low biological stability of spermidine is believed to hinder its potential for therapeutic application. In this test, total CD8 was isolated using CD3 / CD28 beads in the presence of spermidine or compound 13. ＋ T cells were stimulated for 22 hours, and cytotoxicity was assessed by propidium iodide staining. Furthermore, spermidine or compound 13 was incubated in DMEM containing 5% FBS for various times to label the amines with benzoyl chloride. The labeled products were analyzed by HPLC, and the half-life of each compound was calculated by curve fitting.

[0137] The results are shown in Figures 9 and 10. Figure 9 shows that increasing the concentration of spermidine in a medium containing 10% fetal bovine serum (FBS) increased the CD8 ＋ It was shown that the death rate of CD8 T cells was induced, and the cell death was alleviated by the simultaneous addition of the amine oxidase inhibitor aminoguanidine. On the other hand, Compound 13, which is a compound of the present invention, showed a low rate of dead cells regardless of the presence or absence of the addition of aminoguanidine, indicating that the compound 13 is a CD8 T cell-specific inhibitor. ＋ It was shown that the compound of the present invention has no cytotoxicity to cells. Therefore, it was shown that the compound of the present invention is not decomposed by aminooxidase and is chemically stable. Figure 10 shows that the half-life of spermidine is 2.8 hours, while the half-life of compound 13 is 501.6 hours. Therefore, it was shown that the compound of the present invention is a compound with much higher stability than spermidine. Therefore, it was suggested that the compound of the present invention exhibits excellent biological stability.

[0138] Test Example 8: Examination of target selectivity of compounds of the present invention The binding ability of compound 13 to four spermidine-binding proteins (EPHX1, DLD, IDH2, and NIPSNAP1) was evaluated. Specifically, HEK293 cells were preincubated for 48 hours in the presence of 2.5 mM DFMO, and compound 13 (10 μM, 100 μM, or 1000 μM) was added 60 minutes before photoreaction with probe 2. Photoreaction with probe 2 (10 μM) was then carried out for 20 minutes. The resulting cell lysate was subjected to click chemistry with biotin-azide, followed by an avidin pull-down assay, followed by Western blot analysis.

[0139] The results are shown in Figure 11. As shown in Figure 11, compound 13 competed with probe 2 at 10 µM for three lipid metabolism-related proteins (EPHX1, DLD, and IDH2), but did not compete with probe 2 for NIPSNAP1, which is not a lipid metabolism-related protein, even at 1000 µM. On the other hand, spermidine competed with probe 2 for four proteins (Figure 8D). This suggests that the compounds of the present invention exhibit higher target selectivity for lipid metabolism-related proteins than spermidine.

[0140] Test Example 9: Study of mitochondrial activity of the compound of the present invention (1) Since the target HADHA is involved in the activation of FAO, the mitochondrial activity of the compound of the present invention was studied by evaluating the effect of compound 13 on the bioenergetics of CD8+ T cells in vitro. ＋T cells were stimulated with CD3 / CD28 beads for 1 hour in the presence of spermidine or compound 13, and the real-time oxygen consumption rate (OCR) of the cells was measured using a Seahorse assay. Spare respiratory capacity (SRC) was calculated from the measured OCR. OCR was measured under basal conditions and after the sequential addition of oligomycin (which inhibits oxidative phosphorylation-dependent ATP synthesis), carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP, which uncouples ATP synthesis from the electron transport chain), rotenone, and antimycin A (which inhibit electron transport complexes I and III, respectively). OCR assessment was performed in the presence of the amine oxidase inhibitor aminoguanidine to eliminate the influence of enzymatic degradation of spermidine. SRC is an indicator of mitochondrial metabolic capacity, represented by OCR uncoupled from ATP production.

[0141] The results are shown in Figure 12. Figure 12 shows that compound 13 enhanced SRC in a similar manner to spermidine. Therefore, it was revealed that compound 13 retains the ability of spermidine to enhance mitochondrial bioenergetics.

[0142] Test Example 10: Examination of the combined effect of the compound of the present invention in PD-1 blockade immunotherapy (1) It has already been reported that oral supplementation with spermidine enhances the antitumor activity of PD-L1 monoclonal antibody (PD-L1 mAb) in both young and aged mice. Therefore, the combined effect of the compound of the present invention in PD-1 blockade immunotherapy was examined using Compound 13 of the present invention. According to the above "12. Tumor therapy model," MC38 tumor-bearing mice were administered Control, PD-L1 mAb, PD-L1 mAb + Compound 13, or PD-L1 mAb + spermidine, and tumor volume (mm ３ ) was measured to evaluate the antitumor effect of PD-L1 mAb. Data represent the mean ± SEM (n = 5 per group).

[0143] The results are shown in Figure 13. As shown in Figure 13, administration of the PD-L1 antibody alone was ineffective in suppressing tumor growth, but compound 13, like spermidine, suppressed tumor growth when used in combination with the PD-L1 antibody. In other words, oral supplementation with compound 13 was shown to enhance the antitumor activity of PD-L1 mAb. Therefore, it was suggested that the compounds of the present invention, like spermidine, activate cancer immunity when used in combination with cancer immunotherapy.

[0144] Test Example 11: Study of Mitochondrial Activity of Compounds of the Present Invention (2) The effects of Compounds 33 to 61 on the bioenergetics of CD8+ T cells in vitro were evaluated according to the method described in Test Example 9 to study the mitochondrial activity of the compounds of the present invention. Specifically, the OCR of each compound was measured according to the following procedure. Naive CD8 cells were isolated from the spleen and lymph nodes of B6N mice using Mojo sort. 3 x 10 ５ Cells / well, CD3 / 28 beads, and compound 13 or compounds 33-61 (final concentration 0.2 μM) were mixed and plated on a 96-well U-plate (12 wells / compound) and incubated at 37°C for 90 minutes. The magnetic beads were removed using a magnetic stand, and the cells were collected on a V-plate. The wells of the U-plate were then washed with medium and combined with the previously collected cells (both washed). After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded by decantation, 150 μl of PBS was added, centrifuged, and the supernatant was discarded by decantation. 50 μl of FAO medium was added to each well, and the mixture was transferred to a Seahorse medium plate. After centrifugation at 800 rpm for 2 minutes, the cells were incubated at 37°C for 27 minutes, and then etomoxir (Et) (final concentration: 5 μM) or FAO medium was added. After further incubation for 20 minutes, the cells were placed in a Seahorse. The OCR of each compound was measured according to the Seahorse assay described above. 2-mercaptoethanol (4 μL) and aminoguanidine (final concentration: 1 mM) were added to the RPMI medium used for incubation. FAO medium containing carnitine was used.

[0145] The results are shown in Figures 14 to 21. Figures 14 to 21 show that compounds 33 to 61, like compound 13, enhanced OCR. Figure 22 shows a comparison of the maximum OCR values ​​after administration of FCCP to seahorses. These results demonstrate that compounds 33 to 61, like compound 13, retain the ability to enhance mitochondrial bioenergetics.

[0146] Test Example 12: Examination of the combined effect of the compound of the present invention in PD-1 blockade immunotherapy (2) In accordance with the method described in Test Example 10, the combined effect of the compound of the present invention in PD-1 blockade immunotherapy was examined using compounds with a maximum OCR value equal to or greater than that of Compound 13. According to the above "12. Tumor therapy model," MC38 tumor-bearing mice were administered Control, PD-L1 mAb, PD-L1 mAb + Compound 34, PD-L1 mAb + Compound 36, PD-L1 mAb + Compound 40, PD-L1 mAb + Compound 44, PD-L1 mAb + Compound 51, and PD-L1 mAb + Compound 56, and tumor volume (mm ３ ) was measured to evaluate the antitumor effect of PD-L1 mAb. Data represent the mean ± SEM (n = 5 per group).

[0147] The results are shown in Figure 23. As shown in Figure 23, administration of the PD-L1 antibody alone had no effect on tumor growth inhibition, but compounds 34, 36, 40, 44, 51, and 56, when administered in combination with the PD-L1 antibody, inhibited tumor growth. This indicates that oral supplementation of these compounds enhances the antitumor activity of PD-L1 mAb. Therefore, it is suggested that the compounds of the present invention activate cancer immunity when used in combination with cancer immunotherapy.

[0148] The compounds of the present invention can synergistically enhance the antitumor effect when used in combination with PD-1 blockade immunotherapy (e.g., PD-1 signal inhibitors). Furthermore, the compounds of the present invention are expected to serve as small molecule antitumor immunoactivators.

Claims

1. Formula (I): [Wherein: Ring A is an optionally substituted non-aromatic C ４－１０ a carbocycle, an optionally substituted 4- to 10-membered non-aromatic heterocycle, or an optionally substituted 5- to 10-membered aromatic heterocycle, or a pharmaceutically acceptable salt thereof, Compounds of are excluded.

2. Ring A is one or more R １ Non-aromatic C optionally substituted with ４－１０ Carbocyclic ring, one or more R １ a 4- to 10-membered non-aromatic heterocycle optionally substituted with one or more R １ is a 5- to 10-membered aromatic heterocycle optionally substituted with R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, oxo, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -NHCOR ３ , -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; and R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R is selected from the group consisting of haloalkyl; 3. Ring A is (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein n is selected from the group consisting of haloalkyl; and n is an integer from 0 to 13.

4. R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, hydroxy, -NHCO-C １－６ 4. The compound of claim 3, wherein n is alkyl or phenyl; and n is 0 to 3, or a pharmaceutically acceptable salt thereof.

5. R ２ But hydrogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－４ Acyl, -SO ２ -C １－６ 5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, which is alkyl or benzoyl.

6. Formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ a carbocyclic ring or a 5- or 6-membered non-aromatic heterocyclic ring; or a compound represented by the formula: Compounds of are excluded.

7. Ring A is one or more R １ C optionally substituted with ５－６ a carbocyclic ring or one or more R １ is a 5- or 6-membered heterocycle optionally substituted with R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, oxo, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -NHCOR ３ , -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; wherein R １ When two R are bonded to the same atom or adjacent atoms on ring A, １ together with the atoms on ring A to form a non-aromatic C ３－５ may form a carbocyclic ring or a 5- or 6-membered non-aromatic heterocyclic ring; and R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein: R is selected from the group consisting of haloalkyl; 8. Ring A is one or more R １ C optionally substituted with ５－６ a carbocyclic ring or one or more R １ is a 5- or 6-membered heterocycle optionally substituted with; and R １ each independently represents a halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ acyl, carbocycle, and heterocycle, wherein said carbocycle and heterocycle are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ 7. The compound according to claim 6, or a pharmaceutically acceptable salt thereof, wherein the compound is optionally substituted with one or two or more of the same or different groups selected from the group consisting of acyl.

9. Ring A is (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ Acyl, -SO ２ R ３ , carbocyclic groups, heterocyclic groups, and benzoyl, wherein said carbocyclic groups and heterocyclic groups are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ acyl; R ３ each independently represents a halogen, C １－６ Alkyl and C １－６ 8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein n is selected from the group consisting of haloalkyl; and n is an integer from 0 to 12.

10. Ring A is (wherein each bond marked with * is bonded to —NH—); R ２ But hydrogen, halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, carboxy, nitro, amino, mono- or di-C １－６ Alkylamino, C １－４ acyl, cycloalkyl, heterocycloalkyl, and benzoyl, wherein said cycloalkyl and heterocycloalkyl are selected from the group consisting of halogen, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Hydroxyalkyl, C １－６ Alkoxy, C １－６ Haloalkoxy, C １－６ Hydroxyalkoxy, hydroxy, cyano, nitro, amino, mono- or di-C １－６ alkylamino, and C １－４ 10. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein:

11. The structure: or a pharmaceutically acceptable salt thereof.

12. Formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ a carbocyclic ring or a 5- or 6-membered non-aromatic heterocyclic ring; or a pharmaceutically acceptable salt thereof.

13. The pharmaceutical composition according to claim 12 for activating anti-tumor immune responses.

14. The pharmaceutical composition according to claim 12, which is used in combination with a PD-1 signal inhibitor.

15. The pharmaceutical composition according to claim 14, wherein the PD-1 signal inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

16. The pharmaceutical composition according to claim 14 for treating a disease caused by decreased T-cell immunity.

17. The pharmaceutical composition according to claim 16, wherein the disease caused by decreased T-cell immunity is cancer or an infectious disease.

18. An antitumor immune activator used in cancer immunotherapy, comprising the compound of formula (I): wherein ring A is an optionally substituted non-aromatic C ５－６ or a pharmaceutically acceptable salt thereof, wherein R is a carbon ring or a 5- or 6-membered non-aromatic heterocycle.

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