PPARδ Activators

Abstract

To provide a PPARδ activator having novel agonist of PPARδ (Peroxisome proliferator-activated receptor δ) as an active ingredient, and an exercise tolerant improver having the activator as an active ingredient.SOLUTION: The present invention relates to: a PPARδ activator that has a guanidine derivative or a biguanidine derivative as an active ingredient, and activates transcription activity of PPARδ; the PPARδ activator in which the guanidine derivative and the biguanidine derivative can be fitted inside a ligand binding pocket of PPARδ in the state where a guanidino group or a biguanidino group forms hydrogen bond with amino acid residues severally corresponding to His413, His287, Thr253, and Tyr437 of human PPARδ out of amino acid residues constituting an inner surface of the ligand binding pocket; and an exercise tolerant improver having any of the PPARδ activators as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an activator of peroxisome proliferator-activated receptor δ (PPARδ). This application claims priority based on Japanese Patent Application No. 2018-199523, filed on October 23, 2018, the contents of which are incorporated herein by reference. [Background technology]

[0002] PPAR is a member of the nuclear hormone receptor superfamily and is a ligand-inducible transcription factor. In mammals, PPAR has family members α, γ, and δ. PPARα regulates the expression of various genes related to lipolysis with free fatty acids as endogenous ligands, and is therefore a target of hyperlipidemia improving drugs. PPARγ promotes the differentiation of adipocytes with long-chain fatty acids and prostaglandins as endogenous ligands, and is a target substance of thiazolidinedione, an insulin resistance improving drug. PPARδ is widely expressed in various tissues, but its endogenous ligand with proven physiological activity is unknown, and therefore it is considered to be an orphan receptor (see, for example, Non-Patent Document 1). PPARδ is composed of an N-terminal domain with ligand-independent transcription activation ability, a zinc finger domain with a DNA binding region, and a ligand binding domain (LBD) with ligand-dependent transcription activation ability. The amino acid sequence of the LBD of PPARδ is 70% homologous (sequence identity) to the LBD of PPARα and 68% to the LBD of PPARγ (see, for example, Non-Patent Document 2).

[0003] In recent years, it has been reported that PPARδ is an important transcriptional regulator in the control of lipid catabolism, transport, accumulation, etc. (see, for example, Non-Patent Document 3), and has attracted attention as a molecular target for the development of therapeutic drugs for metabolic disorders, etc. For example, it has been reported that the ingestion of a PPARδ agonist can enhance exercise-induced effects such as exercise endurance, and that a PPARδ agonist can be used as an exercise performance enhancer (see, for example, Patent Document 1 and Non-Patent Document 4). In addition, the exercise effect of a subject is further improved by combining an agonist of AMPK (5' AMP-activated protein kinase) with an agonist of PPARδ (see, for example, Patent Document 2 and Non-Patent Document 4). In addition, it has been reported that the administration of a PPARδ agonist can suppress lipid-induced endoplasmic reticulum (ER) stress, which plays an important role in the development of cardiomyopathy (see, for example, Non-Patent Document 5), and that it has a hepatoprotective effect and an anti-fibrotic effect against liver fibrosis induced by carbon tetrachloride administration (see, for example, Non-Patent Document 6).

[0004] PPARδ agonists include, for example, phenoxyacetic acid derivatives, such as GW501516 (CAS No.: 317318-70-0). The basic skeleton of phenoxyacetic acid derivatives is a chemical structure in which a long-chain hydrophobic group such as a non-polar hydrocarbon is bound to a carboxyl group (-COOH). From the results of structural analysis of a complex between PPARδ and its agonist phenoxyacetic acid derivative, it has been revealed that the ligand binding pocket of PPARδ is a Y-shaped pocket consisting of three tunnel-like cavities called arms I, II, and III (see, for example, Non-Patent Documents 7 and 8). As PPARδ agonists, drugs with a skeleton with branched hydrophobic groups, such as GW2331 (CAS No.: 190844-95-2), have also been developed.

[0005] On the other hand, metformin is a biguanide drug and is widely used as an oral antidiabetic drug. It is known that metformin activates AMPK, thereby enhancing glucose uptake in skeletal muscles and controlling fatty acid β-oxidation in the liver (e.g., Non-Patent Document 9). It has also been reported that metformin inhibits mitochondrial glycerophosphate dehydrogenase in the liver, suppressing gluconeogenesis, and lowering blood glucose (e.g., Non-Patent Document 10). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2010-514804 [Patent Document 2] Special Publication No. 2011-507970 [Non-patent literature]

[0007] [Non-Patent Document 1] Evans et al., Nature Medicine 2004, vol. 10(4), p.355-361. [Non-Patent Document 2] Willson et al., Journal of Medicinal Chemistry, 2000, vol. 43(4), p.527-550. [Non-Patent Document 3] Barish et al., The Journal of Clinical Investigation, 2006, vol.116(3), p.590-597. [Non-Patent Document 4] Narkar et al., Cell, 2008, vol.134(3), p.405-415. [Non-Patent Document 5] Palomer et al., International Journal of Cardiology, 2014, vol.174, p.110-118.

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Summary of the Invention

[0008] A primary object of the present invention is to provide a PPARδ activator containing a novel PPARδ agonist as an active ingredient, and an exercise tolerance improving agent containing the same as an active ingredient. [Means for solving the problem]

[0009] As a result of intensive research, the present inventors have found that metformin is an agonist of PPARδ and activates the transcriptional activity of PPARδ. Furthermore, from the results of co-crystal structure analysis of metformin and the LBD of human PPARδ (hPPARδ), it was found that metformin enters the ligand-binding pocket of PPARδ, and two amino groups of the biguanide skeleton of metformin interact with and bind to amino acid residues near the opening of the ligand-binding pocket, thereby causing the LBD of PPARδ to take an active conformation, thus completing the present invention.

[0010] That is, the PPARδ activator and exercise tolerance improving agent according to the present invention are the following [1] to [8]. [1] A PPARδ activator that contains a guanidine derivative or biguanidine derivative as an active ingredient and activates the transcriptional activity of PPARδ (Peroxisome proliferator-activated receptor δ). [2] The PPARδ activator according to [1], wherein the guanidine derivative and the biguanidine derivative are capable of fitting into the inside of the ligand-binding pocket of PPARδ by forming hydrogen bonds with amino acid residues corresponding to histidine at position 413, histidine at position 287, threonine at position 253, and tyrosine at position 437 of hPPARδ, among the amino acid residues constituting the inner surface of the ligand-binding pocket of PPARδ. [3] The guanidine derivative is represented by the following general formula (1): [In formula (1), R 1 represents a monovalent organic group. The PPARδ activator according to [1] or [2] above, which is a compound represented by the following formula (excluding biguanidine derivatives):

[0011] [ka]

[0012] [4] The guanidine derivative is represented by the following general formulas (1-1-1) to (1-1-4): [In the formulas (1-1-1) and (1-1-2), Z 11 represents an oxygen atom or a sulfur atom, n1 represents 0 or 1, R 12 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, n12 represents an integer of 0 to 2, R 13 represents a hydrogen atom or an aliphatic hydrocarbon group having 1 to 6 carbon atoms, R 14 represents an aromatic hydrocarbon group which may have a substituent, and p1 represents an integer of 1 or more. In formulas (1-1-3) and (1-1-4), Z 11 represents an oxygen atom or a sulfur atom, n1 represents 0 or 1, R 15 represents an aliphatic hydrocarbon group or an alkoxy group having 1 to 6 carbon atoms, n15 represents an integer of 0 to 2, Z 2 represents a divalent linking group, R 5 represents an aromatic hydrocarbon group which may have a substituent, or a cyclic hydrocarbon group which may have a substituent, and p2 represents an integer of 1 or greater. The PPARδ activator according to [3] above, which is a compound represented by any one of the following:

[0013] [ka]

[0014] [ka]

[0015] [5] The PPARδ activator according to the above [1], wherein the guanidine derivative is a compound represented by any one of the following formulas (A-1) to (A-4) and (B-1) to (B-3):

[0016] [ka]

[0017] [ka]

[0018] [6] The biguanidine derivative is represented by the following general formula (2): [In formula (2), R 2 and R 3 R each independently represents a hydrogen atom or a monovalent organic group. 2 and R 3 When both are monovalent organic groups, they may be linked to form a ring structure. The PPARδ activator according to any one of the above [1] to [3], which is a compound represented by the following formula:

[0019] [ka]

[0020] [7] The PPARδ activator according to [1] above, which contains as an active ingredient one or more selected from the group consisting of metformin, phenformin, and buformin. [8] An exercise tolerance improving agent comprising any one of the PPARδ activators [1] to [7] above as an active ingredient. Effect of the Invention

[0021] The PPARδ activator according to the present invention can activate the transcriptional activity of PPARδ and affect various physiological actions transcriptionally regulated by PPARδ. Therefore, the PPARδ activator is useful as an active ingredient of an exercise tolerance improving agent, and is expected to be useful as an active ingredient of a pharmaceutical composition for treating or preventing metabolic disorders and the like. [Brief description of the drawings]

[0022] [Figure 1] The amino acid sequence of the ligand-binding pocket of hPPARδ. In the sequence, the amino acid residues with a black circle on the top indicate the amino acid residues that constitute the inner surface of the ligand-binding pocket of hPPARδ, and the amino acid residues enclosed in a box are the amino acid residues that form hydrogen bonds with the guanidino group in the guanidine derivative. [Diagram 2] FIG. 2 is a schematic diagram showing the structure of a complex between a guanidine derivative represented by general formula (1) and a compound in which R1 is an organic group branched in the direction of the arrow, and hPPARδ. [Diagram 3] FIG. 2 is a schematic diagram showing the immobilization reaction of metformin onto COOH FG beads in Example 1. [Figure 4] 1 shows silver-stained gel images after two-dimensional electrophoresis of the supernatant eluted from Met-beads (right panel) and the supernatant eluted from NC-beads (left panel) in affinity purification using Met-beads in Example 1. [Diagram 5] FIG. 2 shows the amino acid sequence of PPARδ and portions of the amino acid sequences of peptides identified by TOF-MS in Example 1. [Figure 6] FIG. 1 shows the results of Western blotting, using anti-PPARδ antibody, of immunoprecipitates obtained by adding Met-beads to purified Myc-hPPARδ and mouse liver extract in Example 1. [Figure 7] FIG. 1 shows the binding-dissociation curves of metformin immobilized on a biosensor and serially diluted purified Myc-hPPARδ in Example 1. [Figure 8]FIG. 1 is a schematic diagram of the full-length PPARδ and each mutant used in Example 1. [Figure 9] This figure shows the quantitative results of the relative immunoprecipitation amounts (relative amounts, with the amount of Myc-tagged protein in the cell extract taken as 100%) obtained using Met-beads from cell extracts of cells strongly expressing Myc-hPPARδ, Myc-ΔN-term, and Myc-ΔLBD in Example 1. [Figure 10A] FIG. 1 is a schematic diagram showing the mechanism by which the PPARδ complex (hPPARδ, PGC1α, RXRα) activates transcription via PPRE and the relationship with metformin. [Figure 10B] FIG. 13 shows the results of a luciferase assay using PPRE×2-tk-luciferase in cells treated with metformin or GW501516 in Example 2. [Figure 11] In Example 2, this figure shows the results of measuring the amount of PGC1α immunoprecipitated by immunoprecipitation using anti-Myc antibody on cell extracts from cells that strongly express Myc-hPPARδ, RXRα, and PGC1α and were treated with metformin, GW501516, or DMSO (PGC1α amount relative to Myc-hPPARδ amount in the immunoprecipitate: [PGC1α amount] / [Myc-hPPARδ amount]). [Figure 12] In Example 3, this figure shows the results of measuring the relative expression levels (expression level in DMSO-treated cells is set to 1) of PPARδ target genes in C2C12 cells on day 6 of differentiation, which were induced to differentiate into muscle by low serum stimulation, when treated with metformin, GW501516, or DMSO. [Figure 13] FIG. 13 shows the results of chromatin immunoprecipitation using an anti-FLAG antibody performed on C2C12-PPARδ cells treated with metformin, GW501516, or DMSO on day 6 of muscle differentiation induction by low serum stimulation in Example 3. [Figure 14] FIG. 1 shows the results of measuring mitochondrial activity in C2C12 cells treated with metformin or GW501516 in Example 4. [Figure 15]1 shows micrographs of a complex crystal of PPARδ-LBD and metformin (A) and a complex crystal of PPARδ-LBD and phenformin (B) in Example 5. [Figure 16] FIG. 1 shows the structure of the complex crystal of PPARδ-LBD and metformin in Example 5. [Figure 17] FIG. 13 shows a metformin molecule in the ligand-binding pocket of PPARδ-LBD in the complex crystal of PPARδ-LBD and metformin in Example 5. [Figure 18] FIG. 1 shows a comparison of the structures of metformin and phenformin bound to PPARδ-LBD in Example 5. [Figure 19] This figure shows the measurement results of the number of electrode contacts (NOS) for each group at each elapsed time from the start of training in Example 6. Figure 19(A) shows the results for the control group (in the figure, "cont") and the metformin administration group (in the figure, "met"), and Figure 19(B) shows the results for the training group (in the figure, "cont+train") and the training + metformin administration group (in the figure, "met+train"). [Figure 20] FIG. 13 is a diagram showing interactions between the guanidino group of metformin and amino acid residues on the inner surface of the ligand-binding pocket of PPARδ in the crystal structure of the complex of PPARδ and metformin in Example 7. [Figure 21] FIG. 13 shows the results of calculating the interaction energies between metformin, GW0742, or GW501516 and seven amino acid residues (Tyr437, Leu433, Met417, His287, Thr253, Gln250, Phe246) in the ligand-binding pocket of hPPARδ in Example 7. [Figure 22] FIG. 13 shows the results of a luciferase assay using PPRE×2-tk-luciferase in cells treated with compound (A-4) in Example 9. [Figure 23A]FIG. 13 shows the results of measuring the relative expression level of angptl4 gene in cells treated with compound (A-4) or GW0742 in Example 10. [Figure 23B] FIG. 13 shows the results of measuring the relative expression level of pdk4 gene in cells treated with compound (A-4) or GW0742 in Example 10. [Figure 23C] FIG. 13 shows the results of measuring the relative expression level of the cpt1a gene in cells treated with compound (A-4) or GW0742 in Example 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] PPARδ is composed of an N-terminal domain (N-term domain) with ligand-independent transcriptional activation, a zinc finger domain with a DNA-binding region, and a ligand-binding domain (LBD) with ligand-dependent transcriptional activation. The LBD consists of 12 α-helices and 3 β-sheets, and the third to eighth α-helices form a ligand-binding pocket. Figure 1 shows the amino acid sequence of the ligand-binding pocket of hPPARδ. In the amino acid sequence shown in Figure 1, the following 33 amino acid residues constitute the internal surface of the ligand-binding pocket: Asn191, Met192, Ile213, Leu219, Glu223, Trp228, Val245, Phe246, Arg248, Cys249, Gln250, Thr252, Thr253, Glu255, Thr256, Glu259, His287, Ile290, Phe291, Leu294, Ile297, Leu303, Val305, Ala306, Asn307, Val312, Phe316, Leu317, Ile327, Ile328, His413, Met417, Leu433, and Tyr437.

[0024] Structural studies of the complex between hPPARδ and GW501516 have revealed the following: Phenoxyacetic acid derivatives such as GW501516 specifically bind to amino acid residues at the entrance (arm I) of the ligand-binding pocket of PPARδ by forming multiple hydrogen bonds with their carboxyl groups. This binding causes the 12th α-helix (helix-12) of PPARδ to collapse so as to cover the ligand-binding pocket, and the remaining hydrophobic group fits into the non-polar pocket. This collapsed helix-12 becomes the lid of the ligand-binding pocket, activating the transcriptional activity of PPARδ (Non-Patent Documents 7 and 8).

[0025] The PPARδ activator according to the present invention is a drug that activates the transcription activity of PPARδ, and contains a guanidine derivative or a biguanidine derivative as an active ingredient. The guanidine derivative means a compound having a guanidino group. The biguanidine derivative means a compound having a biguanidino group. The active ingredient of the PPARδ activator according to the present invention may be a guanidine derivative having a biguanidino group, or a biguanidine derivative having a guanidino group.

[0026] The PPARδ activator according to the present invention fits into the ligand-binding pocket and forms multiple hydrogen bonds with amino acid residues at the entrance (arm I) of the ligand-binding pocket of PPARδ via the guanidino or biguanidino groups, thereby folding helix-12 over the ligand-binding pocket so as to cover it. The PPARδ activator according to the present invention can achieve the structural change (folding and immobilization of helix-12) required for activating the transcriptional activity of PPARδ solely through the guanidino or biguanidino groups.

[0027] Specifically, the guanidino group or biguanidino group of the PPARδ activator according to the present invention forms hydrogen bonds with amino acid residues corresponding to histidine at position 413 (His413), histidine at position 287 (His287), threonine at position 253 (Thr253), and tyrosine at position 437 (Tyr437) of hPPARδ, among the amino acid residues constituting the inner surface of the ligand-binding pocket of PPARδ. These hydrogen bonds position the PPARδ activator according to the present invention at the entrance of the ligand-binding pocket of PPARδ. As a result, helix-12 is inverted and fixed in a state in which the ligand-binding pocket is covered, activating PPARδ.

[0028] The guanidino or biguanidino group of the PPARδ activator according to the present invention also interacts with the amino acid residues corresponding to phenylalanine (Phe246) at position 246, methionine (Met417) at position 417, leucine (Leu433) at position 433, and glutamine (Gln250) at position 250 of hPPARδ, among the amino acid residues constituting the inner surface of the ligand-binding pocket of PPARδ. The interaction between the guanidino or biguanidino group and the entrance (arm I) of the ligand-binding pocket of PPARδ is more stable than that of phenoxyacetic acid derivatives that bind via a carboxy group, and can efficiently invert and fix helix-12, so that it is expected to have a greater activation effect on PPARδ than phenoxyacetic acid derivatives.

[0029] The guanidine derivative, which is an active ingredient of the PPARδ activator according to the present invention, is not particularly limited as long as it is a compound that can fit into the ligand binding pocket of PPARδ in a state where the guanidino group forms hydrogen bonds with His413, His287, Thr253, and Tyr437 of hPPARδ. Similarly, the biguanidine derivative, which is an active ingredient of the PPARδ activator according to the present invention, is not particularly limited as long as it is a compound that can fit into the ligand binding pocket of PPARδ in a state where the biguanidino group forms hydrogen bonds with His413, His287, Thr253, and Tyr437 of hPPARδ.

[0030] The phrase "a compound fits inside a ligand-binding pocket" means that the compound is contained within the ligand-binding pocket, and may occupy the entire pocket or only a part of the pocket. The active ingredient of the PPARδ activator according to the present invention may be a compound that occupies only a part or all of arm I of PPARδ, a compound that occupies only a part or all of arm I and arm II of PPARδ, or a compound that occupies only a part or all of arm I and arm III of PPARδ. In addition, the active ingredient may be a compound having a branched structure that occupies only a part or all of arm I and arm II and a part or all of arm III of PPARδ.

[0031] Among the active ingredients of the PPARδ activator according to the present invention, the guanidine derivatives include, for example, a compound represented by the following general formula (1) (excluding biguanidine derivatives) (hereinafter referred to as "compound (1)"). Among the active ingredients of the PPARδ activator according to the present invention, the biguanidine derivatives include, for example, a compound represented by the following general formula (2) (hereinafter referred to as "compound (2)"). In general formula (1), R 1 represents a monovalent organic group. 2 and R 3 Each independently represents a hydrogen atom or a monovalent organic group. 2 and R 3 The compound in which all of these are hydrogen atoms is metformin.

[0032] [ka]

[0033] R 1 , R 2 , and R 3The monovalent organic group is not particularly limited as long as it is an organic group of a size and shape that allows the entire compound to fit into the inside of the ligand binding pocket of PPARδ in a state in which the guanidino group or biguanidino group forms a hydrogen bond with the amino acid residues corresponding to His413, His287, Thr253, and Tyr437 of hPPARδ among the amino acid residues that constitute the inner surface of the ligand binding pocket of PPARδ. For example, the monovalent organic group may be an acidic group or a basic group. It may also be a hydrophilic group or a hydrophobic group.

[0034] The monovalent organic group may be linear, branched, or have a cyclic structure. 1 This is a schematic diagram showing the structure of a compound that is a branched organic group branched in the direction of the arrow and PPARδ. The branched organic group extends to arms II and III.

[0035] The monovalent organic group is, for example, -(Z 1 )-R 4 , (Z 1 represents a single bond, an oxygen atom, a sulfur atom, -NH-, -N=CH-, -CO-, -CO-O-, -O-CO-, -CO-NH-, or -NH-CO-. 4 represents an aliphatic hydrocarbon group, an aromatic hydrocarbon group, or a heterocyclic group.) Examples of the alkyl group include a carboxy group, a nitro group, a cyano group, a hydroxy group, and an amino group.

[0036] R 4When is an aliphatic hydrocarbon group, the aliphatic hydrocarbon group is not particularly limited, and examples thereof include saturated or unsaturated hydrocarbon groups having 1 to 20 carbon atoms. The monovalent aliphatic hydrocarbon group may be a chain hydrocarbon group or a cyclic hydrocarbon group. Specific examples of the monovalent chain hydrocarbon group include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, 2-ethylhexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl groups. Examples of the monovalent cyclic hydrocarbon group include groups obtained by removing one hydrogen atom from an alicyclic compound such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, adamantane, and dicyclopentadiene. These aliphatic hydrocarbon groups may have one or more substituents. Examples of the substituent include a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom), a trifluoromethyl group, a carboxy group, a nitro group, a cyano group, a hydroxy group, an amino group, a ketone group, an alkoxy group, an aromatic hydrocarbon group, a heterocyclic group, a multi-ring linking group, etc. Examples of the aromatic hydrocarbon group and the heterocyclic group include the R 4 Examples of the polycyclic linking group include the same as the polycyclic linking group that can be taken as the monovalent organic group described below. Specific examples of the alkoxy group include a methoxy group, an ethoxy group, a propyloxy group, an isopropyloxy group, an n-butoxy group, a t-butoxy group, an n-pentyloxy group, an isopentyloxy group, a neopentyloxy group, a tert-pentyloxy group, an n-hexyloxy group, and an isohexyloxy group. When the aliphatic hydrocarbon group has an aromatic hydrocarbon group, a heterocyclic group, or a polycyclic linking group as a substituent, the aliphatic hydrocarbon group preferably has 1 to 6 carbon atoms.

[0037] R 4When is an aromatic hydrocarbon group, the aromatic hydrocarbon group is not particularly limited, and examples thereof include groups in which one hydrogen atom has been removed from an aromatic cyclic compound such as benzene, naphthalene, anthracene, or phenanthrene. These aromatic hydrocarbon groups may have one or more substituents. Examples of the substituents include halogen atoms, trifluoromethyl groups, carboxy groups, nitro groups, cyano groups, hydroxy groups, amino groups, ketone groups, alkoxy groups, and aliphatic hydrocarbon groups. Examples of the aliphatic hydrocarbon group include the above-mentioned R 4 The alkoxy group includes the same groups as those exemplified as the substituents of the aliphatic hydrocarbon group.

[0038] R 4 When is a heterocyclic group, the heterocyclic group is not particularly limited, and examples thereof include 5-membered heterocyclic compounds such as pyrrolidine, pyrrole, imidazole, pyrazole, imidazoline, triazole, tetrazole, oxazole, thiazole, tetrahydrofuran, furan, dioxolane, tetrahydrothiophene, and thiophene; 6-membered heterocyclic compounds such as piperidine, pyridine, pyrimidine, pyrazine, pyridazine, morpholine, thiazine, oxane, pyrylium ion, dioxane, thiane, and thiapyran; and condensed heterocyclic compounds such as indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, xanthene, carbazole, and benzo-C-cinnoline, from which one hydrogen atom has been removed. These heterocyclic groups may have one or more substituents. Examples of the substituent include a halogen atom, a trifluoromethyl group, a carboxy group, a nitro group, a cyano group, a hydroxy group, an amino group, a ketone group, an alkoxy group, and an aliphatic hydrocarbon group. 4 The alkoxy group includes the same groups as those exemplified as the substituents of the aliphatic hydrocarbon group.

[0039] The monovalent organic group may be a group (multiple ring linking group) in which two or more rings are linked by a single bond or a divalent linking group. The ring is not particularly limited, and may be an alicyclic compound, an aromatic cyclic compound, or a heterocyclic compound. The linked rings may be the same type of ring or different types of rings. Specifically, the same alicyclic compounds, aromatic cyclic compounds, or heterocyclic compounds as described above may be used.

[0040] In the multi-ring linking group, examples of the divalent linking group linking rings together include divalent chain aliphatic hydrocarbon groups, -O-, -S-, -NH-, -N=CH-, -CO-, and groups in which two or more of these are bonded. The divalent chain aliphatic hydrocarbon group may be linear or branched. In addition, it may be a divalent aliphatic hydrocarbon group consisting of only saturated bonds, or a divalent aliphatic hydrocarbon group having one or more unsaturated bonds. As the divalent chain aliphatic hydrocarbon group, for example, an alkylene group or an alkenylene group having 1 to 10 carbon atoms can be used.

[0041] Examples of the divalent chain aliphatic hydrocarbon group, the group in which two or more of -O-, -S-, -NH-, -N=CH-, and -CO- are bonded include -CO-O-, -O-CO-, -CO-NH-, -NH-CO-, -(CH 2 ) n -O-(CH 2 ) m -, -(CH 2 ) n -S-(CH 2 ) m -(n and m are each independently an integer of 0 or more and satisfying n+m≧1. Note that -(CH 2 ) 0 - means a single bond.)

[0042] In the general formula (2), R 2 and R 3 When both are monovalent organic groups, they may be linked to form a ring structure. 2 and R3 The ring formed by linking may be an alicyclic compound, an aromatic ring compound, or a heterocyclic compound. The alicyclic compound, the aromatic ring compound, and the heterocyclic compound may each be the same as those described above for R 4 The cyclic compounds may have a substituent. Examples of the substituent include the same groups as those described above for R 1 , R 2 , and R 3 The monovalent organic group may be the same as that which may be taken as the monovalent organic group in the above formula (I).

[0043] An example of the compound (1) is a compound represented by the following general formula (1-1) (compound (1-1)).

[0044] [ka]

[0045] In general formula (1-1), Z 11 represents an oxygen atom or a sulfur atom, and n1 represents 0 or 1. In general formula (1-1), R 11 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, an aromatic hydrocarbon group which may have a substituent, or a polycyclic linking group which may have a substituent. The aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent and the aromatic hydrocarbon group which may have a substituent are each the same as those described above for R 4 Examples of the optionally substituted polycyclic linking group include the same groups as those which may be taken as the monovalent organic group.

[0046] Examples of compound (1-1) include a compound represented by the following general formula (1-1-1) (compound (1-1-1)) and a compound represented by the following general formula (1-1-2) (compound (1-1-2)).

[0047] [ka]

[0048] In general formula (1-1-1), Z 11 and n1 are the same as in general formula (1-1). In formula (1-1-2), p1 is an integer of 1 or more, preferably an integer of 1 to 6, more preferably an integer of 1 to 3, and particularly preferably 1.

[0049] In the general formulae (1-1-1) and (1-1-2), n12 represents an integer of 0 to 2. In the general formula (1-1-1) and the general formula (1-1-2), R 12 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms. The aliphatic hydrocarbon group may be any of the above-mentioned R 4 The examples of the groups include the same as those which can be taken as the group. In the general formula (1-1-1) and the general formula (1-1-2), R 13 represents a hydrogen atom or an aliphatic hydrocarbon group having 1 to 6 carbon atoms. The aliphatic hydrocarbon group may be any of the above-mentioned R 4 The examples of the groups include the same as those which can be taken as the group. In the general formula (1-1-1) and the general formula (1-1-2), R 14 represents an aromatic hydrocarbon group which may have a substituent. The aromatic hydrocarbon group may be any of the above-mentioned R 4 The examples of the groups include the same as those which can be taken as the group.

[0050] The compound (1-1-1) is, for example, Z 11 is an oxygen atom or a sulfur atom, n1 is 1, n12 is 0 or 1, R 12 is a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, an n-hexyl group, or an isohexyl group; R 13 is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a t-butyl group, a n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a n-hexyl group, or an isohexyl group, and R 14In particular, the compound in which Z is a phenyl group which may have a substituent is preferable. 11 is an oxygen atom or a sulfur atom, n1 is 1, n12 is 0 or 1, R 12 is a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, or a t-butyl group, and R 13 is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, or a t-butyl group, and R 14 is a phenyl group having no substituent, or a phenyl group having one or two substituents selected from the group consisting of a halogen atom, a trifluoromethyl group, and a methyl group, is preferred, 11 is an oxygen atom or a sulfur atom, n1 is 1, n12 is 0 or 1, R 12 is a methyl group, and R 13 is a hydrogen atom or a methyl group, and R 14 is a phenyl group having no substituent, or a phenyl group having one or two substituents selected from the group consisting of a halogen atom, a trifluoromethyl group, and a methyl group.

[0051] In the compound (1-1-2), p1 is 1, n12 is 0 or 1, and R 12 is a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, an n-hexyl group, or an isohexyl group; R 13 is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a t-butyl group, a n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a n-hexyl group, or an isohexyl group, and R 14 is a phenyl group which may have a substituent. 12 is a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, or a t-butyl group, and R 13is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, or a t-butyl group, and R 14 is a phenyl group having no substituent, or a phenyl group having one or two substituents selected from the group consisting of a halogen atom, a trifluoromethyl group, and a methyl group, p1 is 1, n12 is 0 or 1, and R 12 is a methyl group, and R 13 is a hydrogen atom or a methyl group, and R 14 is a phenyl group having no substituent, or a phenyl group having one or two substituents selected from the group consisting of a halogen atom, a trifluoromethyl group, and a methyl group.

[0052] Examples of the compound (1-1-1) include compounds of the following formulae (A-1) to (A-2): Examples of the compound (1-1-2) include compounds of the following formulae (A-3) to (A-4):

[0053] [ka]

[0054] Examples of compound (1-1) include a compound represented by the following general formula (1-1-3) (compound (1-1-3)) and a compound represented by the following general formula (1-1-4) (compound (1-1-4)).

[0055] [ka]

[0056] In general formula (1-1-3), Z 11 and n1 are the same as in general formula (1-1). In general formula (1-1-4), p2 is an integer of 1 or more, preferably an integer of 1 to 6, more preferably an integer of 1 to 3, and particularly preferably 1.

[0057] In the general formulae (1-1-3) and (1-1-4), n15 represents an integer of 0 to 2. In the general formula (1-1-3) and the general formula (1-1-4), R 15 represents an aliphatic hydrocarbon group or an alkoxy group having 1 to 6 carbon atoms. The aliphatic hydrocarbon group may be any of the above-mentioned R 4 The alkoxy group includes the same groups as those exemplified as the substituents of the aliphatic hydrocarbon group.

[0058] In the general formula (1-1-3) and the general formula (1-1-4), Z 2 is a divalent linking group. Examples of the divalent linking group include the same groups as those exemplified as the divalent linking group linking rings together in the multi-ring linking group. Compound (1-1-3) and compound (1-1-4) each include a divalent linking group. 2 But -(CH 2 ) 2 -, -(CH 2 ) 3 -, -O-CH 2 -, -CH 2 -O-, -CH 2 -O-CH 2 -,-S-CH 2 -, -CH 2 -S-, -CH 2 -S-CH 2 -, -NH-CH 2 -, -CH 2 -NH- or -CH 2 -NH-CH 2 Preferred are compounds in which -

[0059] In the general formula (1-1-3) and the general formula (1-1-4), R 5 is an aromatic hydrocarbon group which may have a substituent, or a cyclic hydrocarbon group which may have a substituent. The aromatic hydrocarbon group which may have a substituent is the same as that described above for R 4 The optionally substituted polycyclic linking group may be the same as the polycyclic linking group that may be the monovalent organic group. Compound (1-1-3) and compound (1-1-4) each include a polycyclic linking group that may be the monovalent organic group.5 is preferably a phenyl group which may have a substituent, or a cyclohexyl group which may have a substituent.

[0060] The compound (1-1-3) is, for example, Z 11 is an oxygen atom or a sulfur atom, n1 is 1, n15 is 0 or 1, R 15 is a methyl group, an ethyl group, a propyl group, an isopropyl group, a methoxy group, an ethoxy group, or a propyloxy group; Z 2 Ga-(CH 2 ) 2 -, -(CH 2 ) 3 -, -O-CH 2 -, -CH 2 -O-, -CH 2 -O-CH 2 -,-S-CH 2 -, -CH 2 -S-, -CH 2 -S-CH 2 -, -NH-CH 2 -, -CH 2 -NH- or -CH 2 -NH-CH 2 - and R 5 is preferably a phenyl group which may have a substituent, or a cyclohexyl group which may have a substituent, 11 is an oxygen atom or a sulfur atom, n1 is 1, n15 is 0 or 1, R 15 is a methyl group, an ethyl group, a propyl group, an isopropyl group, a methoxy group, an ethoxy group, or a propyloxy group; Z 2 Ga-(CH 2 ) 2 -, -(CH 2 ) 3 -, -O-CH 2 -, -CH 2 -O-, -CH 2 -O-CH 2 -,-S-CH 2 -, -CH 2 -S- or -CH 2 -S-CH 2 - and R 5More preferred is a compound in which Z is an unsubstituted cyclohexyl group, an unsubstituted phenyl group, or a phenyl group having one or two substituents selected from the group consisting of a halogen atom, a trifluoromethyl group, and a methyl group. 11 is a sulfur atom, n1 is 1, n15 is 0 or 1, R 15 is a methyl group or a methoxy group, and Z 2 -O-CH 2 -, -CH 2 -O- or -CH 2 -O-CH 2 - and R 5 Preferred are compounds in which is an unsubstituted cyclohexyl group or an unsubstituted phenyl group.

[0061] In the compound (1-1-4), p2 is 1, n15 is 0 or 1, and R 15 is a methyl group, an ethyl group, a propyl group, an isopropyl group, a methoxy group, an ethoxy group, or a propyloxy group; Z 2 Ga-(CH 2 ) 2 -, -(CH 2 ) 3 -, -O-CH 2 -, -CH 2 -O-, -CH 2 -O-CH 2 -,-S-CH 2 -, -CH 2 -S-, -CH 2 -S-CH 2 -, -NH-CH 2 -, -CH 2 -NH- or -CH 2 -NH-CH 2 - and R 5 is a phenyl group which may have a substituent, or a cyclohexyl group which may have a substituent, p2 is 1, n15 is 0 or 1, and R 15 is a methyl group, an ethyl group, a propyl group, an isopropyl group, a methoxy group, an ethoxy group, or a propyloxy group; Z 2 Ga-(CH2 ) 2 -, -(CH 2 ) 3 -, -O-CH 2 -, -CH 2 -O-, -CH 2 -O-CH 2 -,-S-CH 2 -, -CH 2 -S- or -CH 2 -S-CH 2 - and R 5 is an unsubstituted cyclohexyl group, an unsubstituted phenyl group, or a phenyl group having one or two substituents selected from the group consisting of a halogen atom, a trifluoromethyl group, and a methyl group. In particular, compound (1-1-3) is a compound in which p2 is 1, n15 is 0 or 1, and R 15 is a methyl group or a methoxy group, and Z 2 -O-CH 2 -, -CH 2 -O- or -CH 2 -O-CH 2 - and R 5 Preferred are compounds in which is an unsubstituted cyclohexyl group or an unsubstituted phenyl group.

[0062] Examples of compound (1-1-4) include N-({4-[(benzyloxy)methyl]phenyl}methyl)guanidine hydrobromide (PubChem CID: 51131487) (hereinafter, compound (B-1)), N-({3-[(cyclohexyloxy)methyl]phenyl}methyl)guanidine hydrogen iodide (PubChem CID: 53598567) (hereinafter, compound (B-2)), and N-{[4-(benzyloxy)-3-methoxyphenyl]methyl}guanidine hydrogen iodide (PubChem CID: 16261695) (hereinafter, compound (B-3)).

[0063] [ka]

[0064] An example of the compound (2) is a compound represented by the following general formula (2-1) (compound (2-1)).

[0065] [ka]

[0066] In general formula (2-1), R 21 and R 22 each independently represents a hydrogen atom or an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent. The aliphatic hydrocarbon group may be any of the above-mentioned R 4 The examples of the groups include the same as those which can be taken as the group.

[0067] Examples of the compound (2-1) include metformin, buformin, phenformin, and the like.

[0068] The results of crystal structure analysis of the ligand binding pocket of PPARδ have been published (Non-Patent Documents 7 and 8). Therefore, whether compound (1) or compound (2) has a size and shape that can be fitted into the ligand binding pocket of PPARδ can be determined by using the structural data of the ligand binding pocket of PPARδ. For example, the shape of the ligand binding pocket of hPPARδ is the shape of a three-dimensional structure drawn by incorporating the data of Tables 1 to 63 in Example 5 described later into the molecular graphics software PyMOL (https: / / www.pymol.org). In other words, a compound that has at least one of a guanidino group and a biguanidino group and has a shape that can be included in the three-dimensional structure is included in the active ingredient of the PPARδ activator according to the present invention.

[0069] The PPARδ activator according to the present invention can be introduced into cells expressing PPARδ to activate the transcriptional activity of PPARδ in the cells. The cells to be treated with the PPARδ activator according to the present invention may be cells present in a living body or cells cultured in a culture vessel. When the cells to be treated are cells cultured in a culture vessel, the PPARδ activator can be taken up into the cells by utilizing endocytosis by culturing the cells in a medium containing the PPARδ activator. The PPARδ activator may also be introduced into the cells by known introduction methods such as lipofection, calcium phosphate precipitation, lithium acetate, and electroporation.

[0070] When the cells to be treated with the PPARδ activator are cells in the living body of an animal, the administration route of the PPARδ activator to the animal is not particularly limited. For example, the administration route of the PPARδ activator according to the present invention includes oral administration, intravenous administration, intraperitoneal administration, enema administration, etc.

[0071] The animal to which the PPARδ activator according to the present invention is administered is not particularly limited, and may be a human or a non-human animal. As the non-human animal, preferred are livestock animals and laboratory animals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs.

[0072] The PPARδ activator according to the present invention can be formulated by a conventional method into oral solids such as powders, granules, capsules, tablets, chewables, and sustained-release agents, oral liquids such as solutions and syrups, injections, enemas, sprays, patches, ointments, etc. The formulation can be carried out by a conventional method by adding excipients, binders, lubricants, disintegrants, fluidizers, solvents, solubilizers, buffers, suspending agents, emulsifiers, isotonic agents, stabilizers, preservatives, antioxidants, flavoring agents, colorants, etc. according to the needs of the formulation.

[0073] The dosage of the PPARδ activator according to the present invention may be sufficient to enhance the transcriptional activity of PPARδ in the administered cells more than before administration, and is appropriately determined taking into consideration the species, sex, age, body weight, and dosage (administration route, dosage form, number of administrations per day, etc.) of the animal to be treated. For example, the daily dosage of the active ingredient for an adult (body weight 60 kg) is preferably 0.01 mg to 10 g, more preferably 1 mg to 5 mg, and even more preferably 100 mg to 1 g of the active ingredient (guanidine derivative or biguanidine derivative) of the PPARδ activator. Such a dosage can be administered once or in several divided doses.

[0074] The PPARδ activator according to the present invention is suitable as an active ingredient of a pharmaceutical composition for treating or preventing various diseases that can be expected to be treated by activating the transcriptional activity of PPARδ, such as various metabolic disorders including diabetes and obesity, diseases caused by ER stress including cardiomyopathy, and hepatic fibrosis.

[0075] The PPARδ activator according to the present invention is useful as an active ingredient of an exercise tolerance improving agent, similar to other PPARδ agonists. The exercise tolerance improvement by PPARδ activation means improving exercise tolerance, suppressing fatigue during exercise, increasing the amount of exercise possible, and increasing the effect of a certain amount of exercise. By taking an exercise tolerance improving agent, it is possible to exercise for a longer period of time with the same load than in the case of not taking the drug, and the effect of the exercise is enhanced. As a result, it is expected that lifestyle-related diseases such as obesity and diabetes will be improved. In addition, it is expected that improving the effect of exercise will lead to health promotion. It is preferable that the exercise tolerance improving agent containing the PPARδ activator according to the present invention also contains AMPK or is used in combination with AMPK.

[0076] When PPARδ activity is improved, mitochondrial activity is also improved. Therefore, the PPARδ activator according to the present invention is preferable as an active ingredient of a pharmaceutical composition for treating or preventing various diseases for which a therapeutic effect can be expected by improving mitochondrial activity. For example, when mitochondrial activity is improved, immune cells are also activated. Therefore, the PPARδ activator according to the present invention is effective as an immunopotentiator and can be used as an active ingredient of a pharmaceutical composition for immunotherapy. In addition, the PPARδ activator according to the present invention is also effective in combination with a cancer immunotherapeutic agent such as an immune checkpoint inhibitor, similar to metformin (Non-Patent Document 11).

Example

[0077] Next, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited by these examples.

[0078] [Example 1] Using an affinity purification method with FG beads (registered trademark) immobilized with metformin, the in vivo target molecule of metformin was searched for. For the search for the target of metformin, a cell extract of the human liver cancer-derived cell line HepG2 cells was used.

[0079] [Preparation of Met-beads] FG beads immobilized with metformin (hereinafter referred to as "Met-beads") were prepared. The immobilization method followed the protocol provided by the manufacturer of COOH FG beads. Specifically, first, an NHS body was obtained by dehydrative condensation of the carboxylic acid at the linker end of COOH FG beads (manufactured by Tama River Seiki Co., Ltd.) equilibrated with N,N'-dimethylformamide and N-hydroxysuccinimide (NHS). Next, metformin was added to the obtained NHS body, and the NH 2 group of metformin was reacted with the NHS ester to react the COOH group of the COOH FG beads with the NH 2It was immobilized by forming an amide bond with the base. A schematic diagram of the metformin immobilization reaction on COOH FG beads is shown in Figure 3. Among the COOH FG beads, the COOH groups unbound to metformin were masked with aminoethanol. After masking, the Met-beads were washed and then suspended in 50% by volume methanol-water for use in subsequent experiments.

[0080] Also, as a control, COOH FG beads masked with aminoethanol, washed, and then suspended in 50% by volume methanol-water were used as beads without immobilized metformin (hereinafter referred to as "NC-beads") in subsequent experiments.

[0081] <Affinity Purification Using Met-beads> The prepared Met-beads were suspended in a KCl buffer (100 mM KCl, 0.126 g / mL glycerol, 20 mM HEPES (pH 7.9), 1 mM MgCl 2 、200 μM CaCl 2 、0.2 mM EDTA, 0.1% NP-40), and then magnetic separation was performed to discard the supernatant. Next, 200 μL of the KCl buffer was added to these Met-beads, the beads were dispersed with an ultrasonic disperser, and then the supernatant was discarded by magnetic separation. This washing process was repeated three times.

[0082] The cell extract of human liver cancer cell line HepG2 cells was diluted with the KCl buffer to a protein concentration of 3 mg / mL, and the resulting diluted solution was centrifuged (5 krpm / 4°C / 30 minutes) to separate the supernatant, thereby removing the insoluble fraction. The washed Met-beads were added to 400 μL of this supernatant, dispersed, and reacted by inversion stirring on a rotator at 4°C for 4 hours. After the reaction, the supernatant was discarded by magnetic separation, and the collected Met-beads were washed four times by adding 200 μL of KCl buffer, dispersing the beads with an ultrasonic dispersion device, and then discarding the supernatant by magnetic separation. 40 μL of 2D sample buffer (60 mM Tris-HCl (pH 8.8), 7 M Urea, 2 M Thiourea, 1% CHAPS, 1% Triton X-100, 1 × protease inhibitor, 10 mM DTT, 1 × BPB) was added to the washed Met-beads, stirred, and left to stand at room temperature for 10 minutes. After that, 4 μL of an aqueous acrylamide solution (71 mg / mL) was added and suspended, and the mixture was left to stand at room temperature for 10 minutes, after which the supernatant was magnetically separated and collected. The collected supernatant was subjected to two-dimensional electrophoresis using an agar gel (PI: pH 3-10, e-PAGEL, manufactured by ATTO), and the gel after electrophoresis was silver stained to detect the proteins eluted from the Met-beads. The same experiment was also performed for NC-beads not immobilized with metformin, and the gel after silver staining was compared to visually confirm the spots specific to Met-beads. Figure 4 shows the silver-stained images of the gel after two-dimensional electrophoresis of the supernatant eluted from Met-beads (right) and the supernatant eluted from NC-beads (left).

[0083] In the silver stained image of the supernatant eluted from Met-beads, two specific spots (spots indicated by arrowheads in Figure 4) were observed, which were not seen in the silver stained image of the supernatant eluted from NC-beads. The gels of these two spots were cut out and collected, and analyzed by TOF-MS (time-of-flight mass spectrometry). As a result, the proteins of both spots were identified as PPARδ (peroxisome proliferator-activated receptor δ). Figure 5 shows the amino acid sequence of hPPARδ (SEQ ID NO: 1) and the amino acid sequence of the peptide identified by TOF-MS. In Figure 5, the underlined region is the peptide identified by TOF-MS. The peptide identified by TOF-MS covered 26% of the full length of hPPARδ.

[0084] <Metformin and PPARδ binding experiment by immunoprecipitation method> The binding of PPARδ to metformin was confirmed by co-immunoprecipitation. Myc-tagged hPPARδ (Myc-hPPARδ) was strongly expressed in human embryonic kidney-derived cell line HEK293 cells, and Myc-hPPARδ was purified by immunoprecipitation using anti-Myc antibody (sc-40, SantaCruz) from the cell extract of the cells that strongly express PPARδ. Immunoprecipitation was performed using Met-beads with Myc-hPPARδ and mouse liver extract.

[0085] Specifically, Met-beads were added to purified Myc-hPPARδ and mouse liver extract, respectively, and the mixture was stirred by inversion. The Met-beads were then collected by magnetic separation. Western blotting was performed on the proteins eluted from the collected Met-beads using an anti-PPARδ antibody (sc-74517, SantaCruz) that recognizes endogenous PPARδ. The results of the Western blotting are shown in FIG. 6. As a result, it was confirmed that both Myc-hPPARδ and endogenous PPARδ in the mouse liver extract bind to metformin.

[0086] <Verification of the binding of metformin to PPARα> PPARδ is a member of the nuclear receptor superfamily, which also includes α and γ family members. We performed affinity purification using Metformin-beads and HepG2 cell extracts to verify the binding of endogenous PPARα. As a result, we could not confirm the binding of metformin to PPARα.

[0087] <Verification of the binding between metformin and AMPK> Metformin is known to activate AMPK (5' AMP-activated protein kinase) to enhance glucose uptake in skeletal muscles and regulate fatty acid β-oxidation in the liver. We therefore performed affinity purification using Met-beads and HepG2 cell extracts to verify the binding of endogenous AMPK to metformin. As a result, no binding between metformin and AMPK was confirmed.

[0088] <Measurement of affinity between metformin and PPARδ> The affinity of metformin to PPARδ was measured using a molecular interaction measurement device (product name: BLItz (registered trademark), manufactured by Pall ForteBio). The molecular interaction between metformin and Myc-hPPARδ was measured using a biosensor immobilized with metformin and purified Myc-hPPARδ. Metformin was immobilized on the biosensor of the molecular interaction measurement device via the NH 2 The binding rate constant (ka) was calculated to be 3.40×10 4 M -1 S -1 The dissociation rate constant (kd) is 1.03 × 10 -6 S -1Dissociation constant (KD) 3.30×10 -10 was calculated to be M, and it was confirmed that PPARδ strongly binds to metformin.

[0089] <Identification of the Binding Site of Metformin in PPARδ> To determine the binding site of metformin in PPARδ, a mutant lacking the N-term domain of PPARδ (ΔN-term: a mutant lacking the amino acid region from positions 1 to 70) and a mutant lacking only the first three α-helices of the LBD and completely lacking the ligand-binding pocket (ΔLBD: a mutant lacking the amino acid region from positions 237 to 441) were prepared. A schematic diagram of the structures of the full-length PPARδ and each mutant is shown in Figure 8.

[0090] Similar to the PPARδ overexpressing cells that strongly express Myc-hPPARδ, using HEK293 cells, ΔN-term overexpressing cells that strongly express Myc-tagged ΔN-term (Myc-ΔN-term) and ΔLBD overexpressing cells that strongly express Myc-tagged ΔLBD (Myc-ΔLBD) were produced. Met-beads were reacted with the cell extracts (Lysate) of these overexpressing cells for affinity purification, and Western blotting was performed using an anti-Myc antibody on the proteins eluted from the recovered Met-beads to quantify the amount of immunoprecipitation. The quantification results are shown in Figure 9. As a result, when the amount of the Myc-tagged protein in the cell extract was set to 100%, the relative amount of the Myc-tagged protein bound to the Met-beads (relative immunoprecipitation amount) (%) was approximately 58% for Myc-hPPARδ, approximately 34% for Myc-ΔN-term, and 0% (no band was confirmed by Western blotting) for Myc-ΔLBD. From these results, it was found that metformin binds to the LBD of PPARδ.

[0091] [Example 2] PPARδ forms a heterodimer with the nuclear receptor RXR (Retinoid X receptor) in the nucleus and binds to the transcriptional activator PGC1α (peroxisome proliferative activated receptor gamma coactivator-1) in the presence of a ligand for PPARδ, acting as a positive transcriptional regulator. Therefore, a luciferase assay was performed to verify the effect of metformin on the transcriptional regulation of PPARδ.

[0092] <Luciferase assay using <PPRE×2-tk-luciferase> To examine the transcriptional activation of PPARδ by metformin, human PPARδ was strongly expressed in the human embryonic kidney-derived cultured cell line HEK293 cells together with RXRα and PGC1α. As the reporter, a thymidine kinase promoter (tk) / luciferase gene reporter plasmid having two DNA sequences (PPAR-Response Element) to which the PPARδ / RXRα complex binds was used, and a luciferase assay was performed. As a positive control for PPARδ activation, GW501516 (manufactured by GlaxoSmithKline), an agonist of PPARδ, was used. The ratio of the luminescence amount of the reaction solution to the luminescence amount of the reaction solution added with an equal amount of DMSO (control) was defined as the relative activity value.

[0093] The results of the luciferase assay are shown in Fig. 10B. In the presence of the PPARδ complex (hPPARδ, PGC1α, RXRα), metformin could activate transcription in a concentration-dependent manner via the PPRE (PPAR-Response Element, a PPAR response sequence having a PPARδ binding sequence) (Figs. 10A and B). Also, this activation ability was comparable to that of GW501516. From these results, it was confirmed that metformin activates transcription by hPPARδ.

[0094] <Measurement of PPARδ / PGC1α transcriptional complex formation by co-immunoprecipitation method> It is known that the transcriptional activation by an agonist of PPARδ occurs by the agonist binding to the ligand-binding pocket of PPARδ and inducing the formation of a transcriptional complex with PGC1α, which is a transcriptional cofactor. Therefore, the amount of PPARδ / PGC1α transcriptional complex in cells treated with metformin or GW501516 was quantified. As a control, DMSO (dimethyl sulfoxide) treatment was performed.

[0095] First, using HEK293 cells, strongly expressing cells that strongly express Myc-hPPARδ, RXRα, and PGC1α were produced. After treating these strongly expressing cells with metformin, GW501516, or DMSO, cell extracts were prepared. Immunoprecipitation was performed on the obtained cell extracts using an anti-Myc antibody, and Western blotting was performed on the obtained immunoprecipitates using an anti-PGC1α antibody (ab54481, manufactured by Abcam) to measure the relative amount of immunoprecipitated PGC1α (the amount of PGC1α relative to the amount of Myc-hPPARδ in the immunoprecipitate: [amount of PGC1α in the immunoprecipitate] / [amount of Myc-hPPARδ in the immunoprecipitate]). The results are shown in Fig. 11. In metformin-treated cells and GW501516-treated cells, an increase in the amount of immunoprecipitated PGC1α was observed compared to DMSO-treated cells. This indicates that metformin binds to the ligand-binding pocket of PPARδ and induces the formation of a transcriptional complex with PGC1α.

[0096] [Example 3] The effect of metformin on transcriptional activation by PPARδ in muscle differentiation was examined.

[0097] <qPCR analysis of target genes of PPARδ> Mouse skeletal muscle-derived myoblast cell line C2C12 cells were stimulated with low serum to induce muscle differentiation, and on the 6th day of differentiation, they were treated with 100 μM metformin, 1 μM GW501516, or DMSO. For the treated cells, the expression of target genes of PPARδ was analyzed by qPCR. Four target genes, the angptl4 gene, the pdk4 gene, the plin gene, and the ucp3 gene, were measured.

[0098] The measurement results of the relative expression levels when the expression level of DMSO-treated cells was set to 1 are shown in Fig. 12. As a result, although metformin was inferior to GW501516 in terms of the target gene induction efficiency in the case of GW501516 treatment, metformin was able to induce the expression of PPARδ target genes.

[0099] <Measurement of the effect of metformin on the recruitment of PPARδ to PPRE> It was examined whether the transcription factor PPARδ was recruited to the promoter of the target gene by metformin treatment. First, C2C12-PPARδ cells were prepared by constitutively expressing FLAG-tagged PPARδ (FLAG-PPARδ) in C2C12 cells. C2C12-PPARδ cells were stimulated with low serum to induce muscle differentiation, and on the 6th day of differentiation, they were treated with 100 μM metformin, 1 μM GW501516, or DMSO. For the treated cells, chromatin immunoprecipitation (Conventional ChIP) was performed using an antibody that recognizes the FLAG tag.

[0100] The measurement results of the relative values (%) of the DNA amounts of the Angptl4 gene, Pdk4 gene, Plin2 gene, and Ucp3 gene in the immunoprecipitates obtained by chromatin immunoprecipitation (the ratio of the chromatin amount obtained in each IP to the input chromatin amount: %) (with the DNA amount of the IgG control set to 1) are shown in Fig. 13. As a result, similar to the results observed in gene expression, it was shown that PPARδ was recruited to the vicinity of PPRE (PPAR response element) on the promoters of the Angptl4 gene, Pdk4 gene, Plin2 gene, and Ucp3 gene by metformin treatment or GW501516 treatment.

[0101] [Example 4] PPARδ is known to induce the expression of lipid metabolism-related genes and enhance fatty acid β-oxidation, especially in skeletal muscles. PPARδ agonists are expected to have the effect of increasing mitochondrial activity. Therefore, we investigated the effect of metformin on metabolism, especially mitochondrial activity. Specifically, we used C2C12 cells and measured the cellular oxygen consumption rate (OCR) using a cell metabolism measuring device (Agilemt extracellular flux analyzer).

[0102] In this metabolic measurement, four inhibitors were used to forcibly change the metabolism in the cells (Figure 14). First, Oligomycin, which inhibits Complex V of the electron transport chain, was added. Oligomycin inhibited ATP production and reduced the OCR. Next, the uncoupler FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) was added. FCCP forcibly expels hydrogen ions from the mitochondrial membrane to the outside of the membrane, eliminating the proton concentration gradient. Since a proton concentration gradient is necessary for ATP production, when the gradient is forcibly eliminated by FCCP, the TCA cycle and the electron transport chain are activated to their maximum. This allows the maximum respiration rate of the cells to be measured. It is known that the OCR at this time increases more in cells with enhanced fatty acid oxidation. Finally, Rotenone and Antimycin were added. Rotenone inhibits Complex I of the electron transport chain, and Antimycin inhibits Complex III of the electron transport chain. By adding these inhibitors, the electron transport chain was completely stopped, and the OCR of the cells was almost zero.

[0103] C2C12 cells were treated with solvent (DMSO), 100 μM metformin, or 10 μM GW501516 for 24 hours, and inhibitors were added in the order mentioned above, and OCR was measured. The results of OCR measurements are shown in Figure 14. As a result, the basal OCR (pmol / min) before inhibitor treatment did not change in either metformin or GW501516 treatment. After addition of FCCP, an increase in OCR was observed in metformin-treated and GW501516-treated cells compared to DMSO-treated cells (Figure 14, arrows). This indicates that metformin, like GW501516, increased mitochondrial metabolic activity (respiratory activity).

[0104] [Example 5] A series of synthetic PPARδ agonists (GW drugs) that have been developed so far, such as GW501516, are phenoxyacetic acid derivatives, and their basic structure is a carboxyl group (-COOH) bound to a long-chain hydrophobic group such as a nonpolar hydrocarbon. In contrast, metformin is a biguanide drug, and has no similarity in chemical structure to conventional GW drugs, and does not have either a carboxyl group or a long-chain hydrophobic group, which are essential for the specific binding of GW drugs to the ligand-binding pocket of PPARδ. In addition, while the physical properties of conventional GW drugs are characterized by acidic and nonpolar properties, metformin is basic and water-soluble. In addition, the molecular size of conventional GW drugs corresponds to the size of the ligand-binding pocket of PPARδ, but metformin is significantly smaller. For example, the molecular weights of representative synthetic agonists GW501516 and GW2331 are 453.5 and 490.3, respectively, while metformin's is 169.2, approximately one-third of that of the other two. Thus, metformin has no commonality with any of the conventional drugs that target PPARδ, and it is impossible to predict how it binds to PPARδ from the conventional agonists whose complex structures are known. Therefore, we prepared crystals of the complex between PPARδ LBD and metformin, and determined the three-dimensional structure of the complex by X-ray crystallography.

[0105] <Generation of the LBD polypeptide of PPARδ> Crystal structure analysis of the complex of metformin and a polypeptide (PPARδ-LBD) from the 170th glutamine to the 441st tyrosine (carboxyl terminus, C-terminus) of the amino acid sequence of PPARδ was performed. PPARδ-LBD was prepared as follows. First, it was expressed in Escherichia coli as a His×6-tagged polypeptide, then lysed and the insoluble fraction was separated. The separated insoluble fraction was solubilized with a solubilization solution (20 mM Tris-Cl (pH 7.5), 2 M urea, 2 mM DTT, 500 mM NaCl, 0.5% Tween20), and then the supernatant was separated by centrifugation (25,000 rpm, 45 minutes). The obtained supernatant was dialyzed to remove urea and Tween20, then purified using a Ni-affinity column (Ni-NTA Agarose, manufactured by QIAGEN), and the His×6 tag was cleaved with HRV3C. The polypeptide after cleavage was concentrated using Amicon (Amicon Ultra tube cut-off molecular weight 10,000, manufactured by Merk Millipore), and then purified using a Superdex 75pg gel filtration column (manufactured by GE Healthcare) equilibrated with a developing solution (20 mM TrisCl (pH 7.5), 500 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphate (TCEP)). The fraction containing PPARδ-LBD was concentrated to 10 mg / mL with Amicon to obtain a sample for crystallization. The purified sample was confirmed to be PPARδ-LBD by mass spectrometry (MALDI-TOF MS, manufactured by Bruker Daltonics), flash-frozen in liquid nitrogen, and stored at a low temperature of -80°C.

[0106] <Preparation of crystals> The complex crystals of PPARδ-LBD and metformin were prepared by the hanging drop type vapor diffusion equilibration method. Metformin hydrochloride (LKT Laboratories) was dissolved in distilled purified water to prepare a 100 mM metformin aqueous solution, which was mixed with the purified PPARδ-LBD sample to prepare a crystallization sample solution (PPARδ-LBD concentration 0.3 mM, PPARδ-LBD:metformin = 1:10 (molar ratio). For crystallization, 1 μL of the crystallization sample solution was mixed with a reservoir solution (40 mM Bis-Tris-Propandiol (pH 6.8), 10 mM DTT, 2.5% EDTA (1,2-Propanediol, 1 mM ethylenediaminetetraacetic acid), 0.5% HBDG (detergent n-Heptyl-β-D-thioglucoside), 200 μM KCl, 4% PEG8K), and the mixture was allowed to vapor equilibrate with the reservoir solution at 20 °C, and crystals were obtained in about 4 days.

[0107] A micrograph of the obtained PPARδ-LBD / metformin complex crystal is shown in FIG. 15(A). In the figure, the scale bar is 100 μm. The crystal has unit lattice constants of a, 49.34 Å; b, 57.88 Å; c, 59.16 Å; α, 98.07°; β, 90.08°; and γ, 107.02°, and belongs to the triclinic space group P1. The crystal was flash frozen in liquid nitrogen using 30% glycerol as a cryoprotectant.

[0108] <Obtaining three-dimensional structural coordinates> The X-ray intensity data were collected at a temperature of 100 K using an MX300HE detector at beamline BL41XU of the large synchrotron radiation facility SPring-8. The collected X-ray intensity data were subjected to various corrections using software for X-ray diffraction data processing (DENZO / SCALPACK, HKL2000 program) to obtain an X-ray intensity data set for structure analysis (resolution: 2.00 Å). Structure analysis was performed based on the published structure of PPARδ-LBD (PDB code 5U3Q) registered in the RCSB Protein Data Bank (Rutgers, UCSD), and the initial phase was determined by the molecular replacement method using a program (PHASER). The modification and reconstruction of the structure model were carried out using a molecular graphics program (COOT) and refined using a program (PHENIX). By repeating these model modifications and refinements, an atomic model of the complex with an R-factor of 18.4% and a Free R-factor of 21.4% was obtained.

[0109] Regarding the complex of PPARδ-LBD and phenformin, experiments similar to those with metformin were carried out, and crystals almost isomorphic to the complex of PPARδ-LBD and metformin were obtained (the unit cell constants are a = 49.21 Å; b = 57.64 Å; c = 107.82 Å; α = 98.02°; β = 90.03°; γ = 107.08°, belonging to the triclinic space group P1). A micrograph of the obtained crystals is shown in Fig. 15(B). In the figure, the scale bar is 100 μm. Furthermore, structure analysis was carried out using the PPARδ-LBD structure of the metformin complex. As a result, an atomic model of the complex with a resolution of 2.29 Å, an R-factor of 19.3%, and a Free R-factor of 22.0% was obtained.

[0110] <Three-dimensional structural data of the complex crystal of PPARδ-LBD and metformin> The structure of the PPARδ-LBD / metformin complex crystal is shown in Figure 16. The crystal contains two PPARδ-LBD molecules with almost identical structures (Molecule A and Molecule B in the figure), and metformin binds to the ligand-binding pocket in each domain. The surfactant HBDG used for crystallization was bound to the molecular surface of each PPARδ-LBD.

[0111] The structure of PPARδ-LBD in the complex crystal consisted of 15 α-helices H1-H12, H2', H2", and H3' and three β-strands S1, S2, and S3, and the three β-strands formed an antiparallel β-sheet (Figure 16). In the center of the molecule, there was a cavity (ligand-binding pocket) where a Y-shaped ligand could bind. This overall structure was almost identical to the basic structure of PPARδ LBD reported previously (Non-Patent Documents 7 and 8). It was also similar to the structures of PPARα LBD and PPARγ LBD.

[0112] FIG. 17 shows a metformin molecule in the ligand-binding pocket in the complex crystal. In the figure, the dashed lines indicate hydrogen bonds, and the attached numbers indicate distances. H3, H4, H11, etc. indicate α-helices forming the ligand-binding pocket. This ligand-binding pocket was Y-shaped, with three tunnel-like cavities called arms I, II, and III. The metformin molecule was bound to arm I formed by α-helices H3, H4, H11, and H12, and the two amino groups of the biguanide backbone of metformin were fixed by forming direct hydrogen bonds with the residues of the respective α-helices, Thr253 (α-helix H3), His287 (α-helix H4), His413 (α-helix H11), and Tyr437 (α-helix H12). The peptide region (Leu429-Med441) called the activation function-2 segment (AF-2 fragment), which is essential for PPAR activation, bound to the LBD in such a way that it formed α-helix H12 to cover the ligand-binding pocket. This means that the LBD of PPARδ bound to metformin adopts an active conformation. The formation of this α-helix H12 is induced by the direct interaction with metformin as described above, which corresponds well to metformin being an agonist of PPARδ.

[0113] In addition to the above-mentioned hydrogen bonds and other polar interactions, metformin fits into the narrow space at the tip of arm I, and all of its atoms are in contact with atoms of PPARδ-LBD, stabilizing the bond. The biguanide skeleton of metformin makes nonpolar contacts with Leu433, Phe246, and Met417. The two methyl groups of metformin make nonpolar contacts with Thr253, Phe291, Cys249, and Ile327. Metformin is a small molecule and is not large enough to fill the entire ligand-binding pocket of PPARδ-LBD. The tips of the two methyl groups of metformin are connected to the cavities of arm II and arm III, but these arms remain empty.

[0114] The above-mentioned characteristics of the interaction were also observed in the structure of the complex between phenformin and PPARδ-LBD. FIG. 18 shows the structure of the metformin-PPARδ-LBD complex crystal superimposed on the structure of the phenformin-PPARδ-LBD complex crystal. In the figure, the dashed lines indicate hydrogen bonds, and the attached numbers indicate distances. H3, H4, H11, and H12 indicate α-helices forming the ligand-binding pocket. The biguanide skeleton of phenformin overlaps well with the biguanide skeleton of metformin, and phenformin also formed hydrogen bonds similar to the four hydrogen bonds of metformin. The phenyl group of phenformin protrudes into a wide space at the back of the hydrophobic ligand-binding pocket, but does not make close nonpolar contact with PPARδ.

[0115] The shape of the ligand-binding pocket of hPPARδ can be drawn using the molecular graphics software PyMOL by incorporating the data in Tables 1 to 63.

[0116] [Table 1]

[0117] [Table 2]

[0118] [Table 3]

[0119] [Table 4]

[0120] [Table 5]

[0121] [Table 6]

[0122]

Table 7

[0123]

Table 8

[0124]

Table 9

[0125]

Table 10

[0126]

Table 11

[0127]

Table 12

[0128]

Table 13

[0129]

Table 14

[0130]

Table 15

[0131]

Table 16

[0132]

Table 17

[0133]

Table 18

[0134]

Table 19

[0135]

Table 20

[0136]

Table 21

[0137]

Table 22

[0138]

Table 23

[0139]

Table 24

[0140]

Table 25

[0141]

Table 26

[0142]

Table 27

[0143]

Table 28

[0144]

Table 29

[0145]

Table 30

[0146]

Table 31

[0147]

Table 32

[0148]

Table 33

[0149]

Table 34

[0150]

Table 35

[0151]

Table 36

[0152]

Table 37

[0153]

Table 38

[0154]

Table 39

[0155]

Table 40

[0156]

Table 41

[0157]

Table 42

[0158]

Table 43

[0159]

Table 44

[0160]

Table 45

[0161]

Table 46

[0162]

Table 47

[0163]

Table 48

[0164]

Table 49

[0165]

Table 50

[0166]

Table 51

[0167]

Table 52

[0168]

Table 53

[0169]

Table 54

[0170]

Table 55

[0171]

Table 56

[0172]

Table 57

[0173]

Table 58

[0174] [Table 59]

[0175] [Table 60]

[0176] [Table 61]

[0177] [Table 62]

[0178] [Table 63]

[0179] [Example 6] We examined whether metformin can improve exercise tolerance by conducting a terminal exercise tolerance test using mice.

[0180] Metformin was administered by intraperitoneal injection of metformin / PBS solution (metformin dissolved in PBS) at a metformin dose of 25 mg / kg of body weight per mouse. For the non-metformin administration group, an equal volume of PBS was intraperitoneally administered instead of metformin / PBS solution.

[0181] Training for the final exercise tolerance test was performed using a treadmill, starting at a speed of 15 m / min, increasing the speed by 1 m / min every 10 minutes, and running for 10 minutes after reaching 20 m / min (total of 1 hour). This was performed 5 times a week for 4 weeks. Metformin was administered at 10 pm each time, and training was started 12 hours later (10 am).

[0182] First, 20 10-week-old male C57BL / 6 mice were divided into 4 groups (5 mice per group). One of the four groups was neither trained nor administered metformin (hereinafter referred to as the "control group"); one was trained without metformin (hereinafter referred to as the "training group"); one was administered metformin but not trained (hereinafter referred to as the "metformin group"); and the remaining group was administered metformin and trained (hereinafter referred to as the "training + metformin group"). In order to average the motivation and preference for exercise of the mice in each group, the first exercise tolerance test was performed after training acclimation, and the mice were ranked and then grouped into the above 4 groups so that the results were equal. In addition, for the test after the training period, the running time was defined as the time it took for the electrode contacts to reach 50 times during the test (Number of Shocks: NOS50).The running time started at 15 m / min and the speed was increased by 1 m / min every 10 minutes.After reaching a maximum speed of 30 m / min, the subjects were allowed to run until they reached NOS50, to evaluate exercise tolerance.

[0183] FIG. 19 shows the measurement results of the number of electrode contacts (NOS) at each elapsed time from the start of training for each group. FIG. 19(A) shows the results for the control group (in the figure, "cont") and the metformin administration group (in the figure, "met"), and FIG. 19(B) shows the results for the training group (in the figure, "cont+train") and the training + metformin administration group (in the figure, "met+train"). As shown in FIG. 19(A), the metformin administration group did not show an improvement in exercise tolerance compared to the control group, and showed a slight tendency to decrease. In contrast, as shown in FIG. 19(B), the training + metformin administration group showed a higher exercise tolerance than the training group. From these results, it was found that while metformin alone cannot improve exercise tolerance, it can be improved by combining metformin administration and training.

[0184] [Example 7] Using the metformin / PPARδ cocrystal structure data, we searched for new PPARδ activators.

[0185] Specifically, based on the data of the cocrystal structure of metformin / PPARδ, a docking mode analysis between metformin and PPARδ was performed, and a docking mode prediction calculation was performed for known guanidine and biguanidine derivatives, taking into account the solvent effect, to search for guanidine or biguanidine derivatives that can fit into the ligand binding pocket like metformin. The prediction was performed by calculating the binding free energy based on the thermodynamic cycle, and the accuracy was improved using the trajectory of molecular dynamics calculation (MM-PBSA: Molecular Mechanic / Poisson Boltzmann Surface Area, MM-GBSA: Molecular Mechanic / Generalized Born Surface Area).

[0186] As shown in FIG. 20, the guanidino group of metformin interacts with and binds to amino acid residues in the ligand binding pocket of hPPARδ (Tyr437, Leu433, Met417, His287, Thr253, Gln250, and Phe246 are shown in the figure). His413 is not shown because it is located in front of the plane of the figure. In addition, as a result of molecular dynamics calculations, it was found that metformin can interact with more amino acid residues than those shown in FIG. 2, depending on the solvent conditions, etc. The interaction energy with each amino acid was calculated and compared with GW501516 and GW0742 (CAS No.: 317318-84-6), which are known PPARδ synthetic agonists of GW-based drugs, and the results are shown in FIG. 21. As shown in FIG. 21, metformin, like GW-based drugs, can bind to the seven amino acid residues in the ligand binding pocket of hPPARδ with the binding energy shown in the figure. In particular, the binding energy between Met417 and Phe246 was clearly larger in metformin than in the GW drugs, indicating that metformin is more stably fixed at the entrance of the ligand-binding pocket and has a higher helix-12 folding activity and thus a higher agonistic activity than the GW drugs.

[0187] By docking mode analysis, compounds were selected that have guanidino or biguanidino groups at the molecular end and that enter the ligand-binding pocket of hPPARδ as a whole molecule with the guanidino groups etc. forming hydrogen bonds with His413, His287, Thr253, and Tyr437 of hPPARδ. These selected compounds are candidate compounds for PPARδ activators.

[0188] [Example 8] Among the compounds (1-1-4), the effects of the compounds (B-1), (B-2), and (B-3) on the transcriptional regulation of PPARδ were examined. The effects on the transcriptional activity of PPARδ were measured by performing a luciferase assay on cells treated with 10 μM of each compound dissolved in DMSO.

[0189] Specifically, CV1 cells, which are cultured cells derived from African green monkey kidneys, were seeded onto a 24-well plate (1 × 10 5 cells / well) and cultured until 70% confluent. DMEM containing 10% fetal bovine serum and 1% antibiotics was used as the culture medium. After reaching the target cell density, the medium was completely removed, and then 2 μL of MH2004 (plasmid encoding firefly luciferase gene with Gal4 activation sequence upstream: 100 ng / μL), 1 μL of pRL-CMV (plasmid encoding Renilla luciferase gene directly under CMV promoter: 100 ng / μL), 2 μL of GAL4-Ppard (plasmid encoding Gal4-DNA binding region and PPARδ gene: 100 ng / μL), and 1.5 μL of PEI (polyethyleneimine: manufactured by Invitrogen) were mixed with 45 μL of culture medium "Opti-MEM (registered trademark)" (manufactured by Thermo Fisher Scientific) and cultured for about 36 hours. Thereafter, the entire medium was replaced with a medium containing a test substance, such as metformin, for which the transcriptional activation ability of PPARδ was to be examined, and the cells were then cultured for 12 hours.

[0190] The collected cells were then lysed in a cell lysis buffer, suspended in a luminescent substrate (firefly luciferin), and the luminescence of the suspension was measured using a luminometer. The remaining suspension was mixed with Renilla luciferin, and the luminescence was measured using a luminometer. The result of the luminescence amount by Renilla was used as an endogenous control for gene introduction. Finally, the luminescence amount of firefly luciferin was divided by the luminescence amount of Renilla luciferin to calculate the luminescence intensity (RLU).

[0191] The results of the luciferase assay (n=3) are shown in Table 64. The luminescence intensity of the reaction solution treated with each compound was determined as the transcription activation ability of PPARδ in each compound treatment, with the luminescence intensity of the reaction solution (control) to which an equal amount of DMSO was added being taken as 1. The relative luminescence intensity of the reaction solution to which all compounds were added was 1.1 or more, indicating that the transcription activity of PPARδ was increased. This result indicated that these compounds could be PPARδ activators.

[0192] [Table 64]

[0193] [Example 9] Of the compounds (1-1-2), compound (A-4) (1-{4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]4-methylthiazol-5-yl}methyl)thio]-2-methylbenzyl}guanidine) was synthesized and its effect on the transcriptional regulation of PPARδ was investigated.

[0194] <Synthesis of compound (A-4)> (1) Synthesis of S-(4-cyano-3-methylphenyl)ethanethioate

[0195] [ka]

[0196] A solution of 4-fluoro-2-methylbenzonitrile (CAS No.: 147754-12-9, compound 1) (8 g, 59.2 mmol) in DMF (9 mL) was added to the solution at room temperature under a nitrogen atmosphere with Na 2S (5.08 g, 65.1 mmol) was added in one portion. The reaction mixture was stirred overnight. The resulting mixture was cooled to 0° C., and then acetic anhydride (9 mL) was added dropwise, followed by stirring at room temperature for 1 hour. Ethyl acetate (100 mL) and water (30 mL) were then added to the mixture. The entire mixture was layer separated, and the organic layer was washed with saturated brine (30 mL×2), and then dried over anhydrous sodium sulfate. The solvent was then removed under reduced pressure, and the residue was purified by column chromatography (silica gel, eluted with 3%-40% ethyl acetate in hexane) to give the title compound (1.4 g, 12.4% yield) as a brown oil.

[0197] 1 H NMR (400 MHz, CDCl 3 ) δ 7.62(d,J=8.0Hz,1H),7.39(s,1H),7.33(dd,J=8.0,1.1Hz,1H),2.56(s,3H),2.45(s,3H).

[0198] (2) Synthesis of 4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl)thio]-2-methylbenzonitrile

[0199] [ka]

[0200] A mixture of 5-(chloromethyl)-2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazole (CAS No. 317319-33-8, U.S. Patent Publication 2003 / 0203947A1, compound 11) (2.23 g, 7.22 mmol), S-(4-cyano-3-methylphenyl)ethanethioate (compound 2) (1.38 g, 7.22 mmol), and potassium carbonate (1.22 g, 8.66 mmol) in methanol (20 mL) was stirred at room temperature for 1 hour. The resulting mixture was filtered under vacuum and the filter cake was collected and dissolved in dichloromethane (200 mL). The resulting organics were washed with water (30 mL), brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (1.8 g, 59.1% yield) as a white solid.

[0201] 1 H NMR (400 MHz, CDCl 3 ) δ 7.72(dd,J=13.8,9.8Hz,2H),7.67-7.61(m,1H),7.51(d,J=8.1Hz,1H),7 .22(s,1H),7.17(d,J=8.2Hz,1H),4.32(s,2H),2.51(s,3H),2.43(s,3H). MS-ESI(m / z) 423.1[M+H] + .

[0202] (3) Synthesis of 4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl)thio]-2-methylbenzaldehyde

[0203] [ka]

[0204] DIBAL-H (1.5 M in toluene, 4.69 mL, 7.04 mmol) was added dropwise to a solution of 4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl)thio]-2-methylbenzonitrile (compound 12) (1.98 g, 4.69 mmol) in dichloromethane (20 mL) at 0 °C. The resulting reaction mixture was stirred at 0 °C for 1 h and then quenched with 10% hydrochloric acid (34 mL). The resulting mixture was vigorously stirred for 30 min. The resulting mixture was then treated with 20% potassium sodium tartrate (34 mL) and the resulting mixture was vigorously stirred for another 30 min. The reaction mixture was basified to pH 9 with 15% sodium hydroxide and then extracted with dichloromethane (60 mL × 3). All the resulting organic layers were combined, washed with brine (40 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, eluted with 2%-10% ethyl acetate in hexane) to give the title compound (1.28 g, 64.0% yield) as a pale yellow solid.

[0205] 1 H NMR (400 MHz, CDCl 3 ) δ 10.19(s,1H),7.72(dd,J=15.8,7.6Hz,3H),7.63(t,J=7.6Hz,1H),7.24(s,1H),7.16(s,1H),4.35(s,2H),2.64(s,3H),2.45(s,3H). MS-ESI(m / z) 426.1[M+H] + .

[0206] (4) Synthesis of {4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl}thio)-2-methylphenyl}methanol

[0207] [ka]

[0208] A solution of 4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl)thio}-2-methylbenzaldehyde (compound 13) (1.5 g, 3.53 mmol) in methanol (15 mL) was added to sodium borohydride (NaBH 4) (295 mg, 7.76 mmol) was added portionwise at 0° C. The resulting mixture was stirred at 0° C. for 30 minutes. To the mixture was added aqueous ammonium chloride solution (20 mL), followed by extraction with ethyl acetate (60 mL×2). The organic layers were combined, washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (1.45 g, yield 96.0%) as a pale yellow solid.

[0209] 1 H NMR (400 MHz, CDCl 3 ) δ 7.75-7.67(m,2H),7.65-7.59(m,1H),7.30(d,J=7.7Hz,1H),7.19(d,J=8.5Hz,2H),4.68(d,J=5.4Hz,2H),4.21(s,2H),2.30(s,6H). MS-ESI(m / z) 428.1[M+H] + .

[0210] (5) Synthesis of 5-({[4-(chloromethyl)-3-methylphenyl]thio]methyl}-2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazole

[0211] [ka]

[0212] {4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl)thio]-2-methylphenyl}methanol (compound 14) (1.45 g, 3.39 mmol) was added dropwise to dichloromethane (14 mL) at 0° C., DMF (1 drop) and thionyl chloride (0.37 mL, 5.09 mmol) were added dropwise. The resulting mixture was stirred at 0° C. for 1 hour. Water (10 mL) was added to the mixture, and then the organic layer was separated. The collected organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the title compound (1.47 g, 97.0% yield) as a yellow solid.

[0213] 1 H NMR (400 MHz, CDCl 3 ) δ 7.76-7.67(m,2H),7.62(t,J=7.6Hz,1H),7.24(d,J=7.9Hz,1H), 7.19-7.12(m,2H),4.57(s,2H),4.23(s,2H),2.38(s,3H),2.32(s,3H). MS-ESI(m / z)446.0[M+H] + .

[0214] (6) Synthesis of 1,3-di-Boc-2-{4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl]methyl}thio)-2-methylbenzyl]guanidine

[0215] [ka]

[0216] A mixture containing 5-({[4-(chloromethyl)-3-methylphenyl)thio]methyl}-2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazole (compound 15) (1.47 g, 3.3 mmol), 1,3-bis(tert-butoxycarbonyl)guanidine (CAS No. 154476-57-0, compound 4) (1.28 g, 4.95 mmol) and potassium carbonate (685 mg, 4.95 mmol) dissolved in DMF (15 mL) was heated at 75° C. for 1 hour under nitrogen atmosphere. The mixture was then diluted with water (60 mL). The entire volume was extracted with ethyl acetate (60 mL×3). The entire volume of the collected organic layers was combined, washed with brine (30 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, eluted with 1%-10% ethyl acetate in hexanes) to give the title compound (1.6 g, 73% yield) as a pale green solid.

[0217] 1 H NMR (400 MHz, CDCl 3 ) δ 9.50(s,1H),9.35(s,1H),7.70(t,J=10.1Hz,2H),7.62(t,J=7.5Hz,1H),7.14-7.16(m,2H),6.90(d, J = 7.8 Hz, 1H),5.12(s,2H),4.19(s,2H),2.30(s,3H),2.25(s, 3H),1.46(s,9H),1.22(s,9H). MS-ESI(m / z)669.3[M+H] + .

[0218] (7) Synthesis of 1-{4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]4-methylthiazol-5-yl}methyl)thio]-2-methylbenzyl}guanidine

[0219] [ka]

[0220] A mixture of 1,3-di-Boc-2-{4-[({2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methylthiazol-5-yl}methyl)thio]-2-methylbenzyl}guanidine (compound 16) (800 mg, 1.2 mmol) dissolved in 1,4-dioxane (8 mL) and HCl / 1,4-dioxane (4.0 M, 3.0 mL) was stirred at 50 °C for 2 h. The solvent was then removed under reduced pressure, and the residue was dissolved in methanol (6 mL) and the pH was adjusted to 8-9 with aqueous sodium carbonate. Water (15 mL) was added to the resulting mixture, which was then vacuum filtered. The resulting filter cake was washed with water (10 mL) and dried under high vacuum to give the title compound (A-4) (350 mg, 62% yield) as a white solid.

[0221] 1 H NMR(300MHz,DMSO-d6): δ 7.92-7.84(m,3H),7.55-7.00(m,5H),4.48(s,2H),4.20(s,2H),2.31(s,3H),2.22(s,3H). MS-ESI(m / z) 469.2[M+H] + .

[0222] The effect of the obtained compound (A-4) on PPARδ activity was examined. Specifically, a luciferase assay was performed in the same manner as in Example 8, except that the compound (A-4) dissolved in DMSO (0.1%) was added to the reaction system as a test substance (n=3). The measurement results of the relative transcription activation ability obtained by the luciferase assay are shown in FIG. 22. As a result, it was observed that the transcription activity of PPARδ tends to increase depending on the amount of compound (A-4) added. However, when the amount added was 50 μM or more, the cells died and the PPARδ transcription activity could not be measured. These results showed that compound (A-4) can be a PPARδ activator.

[0223] [Example 10] The effect of compound (A-4) on the expression of genes induced by PPARδ was examined. Specifically, compound (A-4) was added to C2C12 cells, a myoblast cell line derived from mouse skeletal muscle, and the expression levels of angptl4 gene, pdk4 gene, and cpt1a gene (Non-Patent Document 12), among the genes induced by PPARδ, were measured. In addition, Hprt gene was used as an endogenous control, and GW0742 was used as a positive control for PPARδ activation.

[0224] First, C2C12 cells were seeded in a 24-well plate (1 × 10 5 The cells were cultured at 100 x 100 cells / well until they reached 80-90% confluence. DMEM containing 10% bovine serum and 1% antibiotics was used as the culture medium. After 36 hours of culture when the target cell density was reached, the entire medium was replaced with serum-free DMEM containing the respective concentrations of compound (A-4), and cultured for 16 hours (n=3). After culture, the medium was discarded, and trizol reagent (Invitrogen) for RNA extraction was directly added to each well to dissolve the cells, and total RNA was extracted by ethanol precipitation. Using the obtained total RNA as a template, cDNA was synthesized using the reverse transcriptase "superscript" (Biorad).

[0225] Quantitative PCR was performed using the obtained cDNA as a template. The template cDNA, primers for amplifying each gene to be measured, and quantitative PCR polymerase mix "ssoFast EvaGreen supermix" (manufactured by Biorad) were mixed, and analysis was performed using a real-time PCR detection system "CFX connect (registered trademark)" (manufactured by Biorad). From the obtained gene expression data, the expression level of each gene was divided by the expression level of an endogenous control gene to calculate a relative expression level.

[0226] The results are shown in Figures 23A-C. As a result, in the cells to which 10 μM of compound (A-4) was added, the expression levels of angptl4 gene, pdk4 gene, and cpt1a gene were increased, similarly to the cells to which GW0742 was added. This result also confirmed that compound (A-4) has the effect of activating the transcription activity of PPARδ.

Claims

1. This medicine contains a guanidine derivative or a biguanidine derivative as an active ingredient, and activates the transcription activity of PPARδ (Peroxisome proliferator-activated receptor δ), The guanidine derivative is represented by the following general formula (1): 【Chemistry 1】 [In formula (1), R １ is -(Z １ )-R ４ , a carboxy group, a nitro group, a cyano group, a hydroxy group, or an amino group; １ represents a single bond, an oxygen atom, a sulfur atom, -NH-, -N=CH-, -CO-, -CO-O-, -O-CO-, -CO-NH-, or -NH-CO-; ４ represents an aliphatic hydrocarbon group, an aromatic hydrocarbon group, or a heterocyclic group. A compound represented by the formula: The biguanidine derivative is represented by the following general formula (2): 【Chemistry 2】 [In formula (2), R ２ and R ３ are each independently a hydrogen atom, -(Z １ )-R ４ , a carboxy group, a nitro group, a cyano group, a hydroxy group, or an amino group; １ represents a single bond, an oxygen atom, a sulfur atom, -NH-, -N=CH-, -CO-, -CO-O-, -O-CO-, -CO-NH-, or -NH-CO-; ４ represents an aliphatic hydrocarbon group, an aromatic hydrocarbon group, or a heterocyclic group; R ２ and R ３ Both are -(Z １ )-R ４ In this case, they may be linked to form a ring structure. A PPARδ agonist agent, which is a compound represented by the formula:

2. It contains a guanidine derivative as an active ingredient, activates the transcription activity of PPARδ (Peroxisome proliferator-activated receptor δ), The guanidine derivative is represented by the following general formula (1-1): 【Chemistry 3】 [In formula (1-1), Z １１ represents an oxygen atom or a sulfur atom; n1 represents 0 or 1; R １１ represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, or an aromatic hydrocarbon group which may have a substituent.] (excluding L-arginine)

3. This drug contains a biguanidine derivative as an active ingredient, and activates the transcription activity of PPARδ (Peroxisome proliferator-activated receptor δ). The biguanidine derivative is represented by the following general formula (2-1): 【Chemistry 4】 [In formula (2-1), R ２１ and R ２２ each independently represents a hydrogen atom or an optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms.] A PPARδ agonist agent, which is a compound represented by the formula:

4. The PPARδ agonist agent according to claim 1, which contains one or more active ingredients selected from the group consisting of metformin, phenformin, and buformin.

5. The guanidine derivative and the biguanidine derivative can be fitted into the inside of the ligand binding pocket of PPARδ in a state where the guanidino group or the biguanidino group is hydrogen-bonded with amino acid residues corresponding to the 413th histidine, the 287th histidine, the 253rd threonine, and the 437th tyrosine of human PPARδ, among the amino acid residues constituting the inner surface of the ligand binding pocket of PPARδ, respectively. The PPARδ agonist agent according to any one of claims 1 to 3.

6. A PPARδ agonist agent described in any one of claims 1 to 5, for use in treating a disease for which activating the transcriptional activity of PPARδ is expected to have a therapeutic effect.

7. A PPARδ agonist agent described in any one of claims 1 to 5, for use in treating a disease selected from obesity, cardiomyopathy, and hepatic fibrosis.

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