glutaminase 1 inhibitor

Abstract

To provide: a glutaminase 1 inhibitor; a pharmaceutical composition, a food and beverage composition or a cosmetic composition containing the inhibitor as an active ingredient; and a new compound useful as an active ingredient of a glutaminase 1 inhibitor.SOLUTION: The glutaminase 1 inhibitor contains as an active ingredient a compound represented by the general formula (I) in the figure, a pharmaceutically acceptable salt thereof, or a solvate of them.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a glutaminase 1 inhibitor and a pharmaceutical composition, food or drink composition, or cosmetic composition containing the same as an active ingredient. The present invention also relates to a novel compound useful as an active ingredient of a glutaminase 1 inhibitor. [Background technology]

[0002] Glutaminase is an enzyme that produces glutamic acid from glutamine. There are two types of glutaminase: kidney-type glutaminase (referred to as "glutaminase 1"), which is expressed in the kidney, and liver-type glutaminase (referred to as "glutaminase 2"), which is expressed in the liver. Hereinafter, glutaminase 1 will be referred to as "GLS1," glutaminase 2 as "GLS2," and both will be collectively referred to as "GLS."

[0003] GLS1 is widely distributed in the kidney, small intestine, and brain, while GLS2 is found only in the liver. Glutamine is the most abundant free amino acid in the body and is known to play a role in regulating many processes, including metabolism, protein synthesis and degradation, and insulin resistance (Non-Patent Document 1). GLS has also been found to be overexpressed in various cancer cells, and GLS inhibitors targeting this protein, such as CB-839, have been developed as pharmaceuticals (Non-Patent Document 2). GLS inhibitors such as CB-839 are known to have anti-inflammatory effects and are useful as anti-inflammatory agents for the prevention, amelioration, or treatment of inflammatory diseases (Patent Document 1). Furthermore, these GLS inhibitors are known to have anti-obesity effects and are useful as anti-obesity agents for the prevention, amelioration, or treatment of metabolic syndrome (Patent Document 2). Furthermore, recent research suggests that inhibiting GLS1 may have a protective effect against aging (Non-Patent Document 3).

[0004] Thus, the importance of GLS as a drug target is increasing today, and new GLS inhibitors are needed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-29412 [Patent Document 2] Japanese Patent Application Publication No. 2020-28239 [Non-patent literature]

[0006] [Non-Patent Document 1] Molecular mechanisms of glutamine action, J. Cell. Physiol., 2005, 204(2): 392-401. [Non-patent document 2] Antitumor activity of the Glutaminase inhibitor CB-839 in triple-negative breast cancer. Mol Cancer Ther. 2014 Apr; 13 (4):890-901. [Non-patent document 3] Senolysis by Glutaminolysis inhibition ameliorates various age-associated disorders. Science. 2021 Jan 15; 371 (6526):265-270. Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a GLS1 inhibitor. Another objective of the present invention is to provide a pharmaceutical composition, food or drink composition, or cosmetic composition that is useful for preventing or ameliorating diseases or pathological conditions that are effectively treated by inhibiting GLS1. Another object of the present invention is to provide novel compounds that are useful as GLS1 inhibitors or as active ingredients of the above-mentioned pharmaceutical compositions, food and drink compositions, or cosmetic compositions. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, the present inventors searched for candidate GLS1 inhibitor compounds from a commercially available compound database by multi-stage in silico screening (in silico screening consisting of a primary screening using a 3D pharmacophore obtained from the complex crystal structure of glutaminase and a known GLS1 inhibitor, filtering by Lipinski's "Rule of Five", a secondary screening using molecular docking calculations, filtering by binding free energy calculations, filtering based on docking poses, and molecular similarity analysis based on chemical structure). As a result, they found that the compound represented by the following formula has GLS1 inhibitory activity. [ka]

[0009] Based on the above findings, the present invention was completed as a result of further investigations to find compounds that have GLS1 inhibitory activity similar to the above compounds, and has the following embodiments.

[0010] (I) GLS1 inhibitor (I-1) A GLS1 inhibitor comprising a compound represented by general formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient: [ka] [In formula (I), X is CH2 or C=O; n represents an integer of 0 to 2, provided that when X is C═O, n is 1. Y is a divalent 5-membered heterocyclic group having at least two nitrogen atoms; R 1 is a hydrogen atom, a halogen atom, a haloalkyl group, an alkoxy group, an alkyl group, or a hydroxyl group; R 2 represents a group represented by any one of the following formulas (a) to (c): [ka] (In formula (a), A represents a hydroxyl group or an oxygen atom; A double line consisting of a dotted line and a solid line represents a single bond when A is a hydroxyl group, and a double bond when A is an oxygen atom. The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I). [ka] (In formula (b), B and D are the same or different and each represents a hydrogen atom, an alkyl group, or an alkoxy group; The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I). [ka] (In formula (c), E represents an oxygen atom or an alkylene group in which one hydrogen atom may be substituted with a hydroxyl group; The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I). When n is 0, R1 is not an alkoxy group; When R1 is a hydroxyl group, R 2 is a group represented by formula (b) or (c).

[0011] (I-2) A GLS1 inhibitor according to (I-1), wherein in the compound represented by formula (I), Y is a pyrazole group or an imidazole group, preferably a pyrazole group.

[0012] (I-3) The compound represented by formula (I) is R 2 is a group represented by formula (a), In formula (a), A represents a hydroxyl group, and the double line consisting of a dotted line and a solid line represents a single bond. A GLS1 inhibitor according to (I-1) or (I-2), wherein X is CH2 and n is an integer of 0 to 2. (I-4) The compound represented by formula (I) is R 2 is a group represented by formula (b), A GLS1 inhibitor according to (I-1) or (I-2), wherein X is CH2 and n is an integer of 0 to 2. (I-5) The compound represented by formula (I) is R 2 is a group represented by formula (c), A GLS1 inhibitor according to (I-1) or (I-2), wherein X is CH2 and n is 0 to 2.

[0013] (II) Pharmaceutical compositions, food and beverage compositions, or cosmetic compositions (II-1) A pharmaceutical composition, a food or drink composition, or a cosmetic composition, which contains as an active ingredient a GLS1 inhibitor according to any one of (I-1) to (I-5). (II-2) A pharmaceutical composition, food or beverage composition, or cosmetic composition according to (II-1), which is used for the prevention, amelioration, or treatment of inflammation, diseases resulting from inflammation, obesity, metabolic syndrome, aging, or cancer.

[0014] (III) Compounds having GLS1 inhibitory activity (III-1) A compound having GLS1 inhibitory activity represented by general formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof: [ka] [In formula (I), X is CH2 or C=O; n represents an integer of 0 to 2, provided that when X is C═O, n is 1. Y is a divalent 5-membered heterocyclic group having at least two nitrogen atoms; R 1 is a hydrogen atom, a halogen atom, a haloalkyl group, an alkoxy group, an alkyl group, or a hydroxyl group; R 2 represents a group represented by any one of the following formulas (a) to (c): [ka] (In formula (a), A represents a hydroxyl group or an oxygen atom; A double line consisting of a dotted line and a solid line represents a single bond when A is a hydroxyl group, and a double bond when A is an oxygen atom. The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I). [ka] (In formula (b), B and D are the same or different and each represents a hydrogen atom, an alkyl group, or an alkoxy group; The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I). [ka] (In formula (c), E represents an oxygen atom or an alkylene group in which one hydrogen atom may be substituted with a hydroxyl group; The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I). When n is 0, R1 is not an alkoxy group; When R1 is a hydroxyl group, R 2 is a group represented by formula (b) or (c). However, N-(4-hydroxycyclohexyl)-3-phenyl-1H-pyrazole-5-carboxamide is excluded.

[0015] (III-2) The compound according to (III-1), wherein Y is a pyrazole group or an imidazole group, preferably a pyrazole group.

[0016] (III-3) The above R 2 is a group represented by formula (a), In formula (a), A represents a hydroxyl group, and the double line consisting of a dotted line and a solid line represents a single bond. The compound according to (III-1) or (III-2), wherein X is CH2 and n is an integer of 0 to 2. (III-4) The compound represented by formula (I) is R 2 is a group represented by formula (b), The compound according to (III-1) or (III-2), wherein X is CH2 and n is an integer of 0 to 2. (III-5) The compound represented by formula (I) is R 2 is a group represented by formula (c), The compound according to (III-1) or (III-2), wherein X is CH2 and n is 0 to 2. [Effects of the Invention]

[0017] The present invention provides a novel GLS1 inhibitor, which is useful as an active ingredient in pharmaceutical compositions, food and beverage compositions, or cosmetic compositions for preventing or ameliorating diseases or pathological conditions that are effectively treated by inhibiting GLS1 (e.g., inflammation, diseases or pathological conditions caused by inflammation, obesity, metabolic syndrome, aging, cancer, etc.). Therefore, the pharmaceutical composition, food and beverage composition, or cosmetic composition of the present invention, which contains a GLS1 inhibitor as an active ingredient, is useful as it has the effect of preventing or ameliorating, for example, inflammation, diseases and pathologies caused by inflammation, obesity, metabolic syndrome, aging, or cancer, by inhibiting GLS1. Furthermore, the compounds provided by the present invention are expected to inhibit GLS1 and thereby exhibit effects of preventing or ameliorating, for example, inflammation, diseases and conditions resulting from inflammation, obesity, metabolic syndrome, aging, or cancer, and are useful as the GLS1 inhibitors and as active ingredients of pharmaceutical compositions, food and beverage compositions, or cosmetic compositions. [Brief explanation of the drawings]

[0018] [Figure 1-1] The structural formulas of candidate GLS1 inhibitor compounds whose GLS1 inhibitory activity was evaluated in the experimental examples are shown below. [Figure 1-2] The structural formulas of candidate GLS1 inhibitor compounds whose GLS1 inhibitory activity was evaluated in the experimental examples are shown below. [Figure 1-3] The structural formulas of candidate GLS1 inhibitor compounds whose GLS1 inhibitory activity was evaluated in the experimental examples are shown below. [Figure 2](A) Structural formulas of candidate GLS1 inhibitor compounds 1 to 8, whose GLS1 inhibitory activity was evaluated in Experimental Example 1, are shown. (B) Results of measuring glutamic acid (Glu) production rate (%) using mouse kidney extract for candidate GLS1 inhibitor compounds 1 to 8, known GLS1 inhibitors (DON, CB-839), and a control (DMSO). [Figure 3] In Experimental Example 2, the Glu production rate (%) was measured using mouse kidney extract for candidate GLS1 inhibitor compounds, known GLS1 inhibitors (DON, CB-839), and a control (DMSO). [Figure 4] The results of Experimental Example 3 show the results of measuring the glutamate production rate (%) using mouse recombinant GLS1 for candidate GLS1 inhibitors (pyrazole derivatives 5i and 8a), known GLS1 inhibitors (DON, CB-839), and a control (DMSO). P values ​​were calculated using the Tukey-Kramer HSD test. n = 3. *P < 0.05 (vs Ct). [Figure 5] The results of Experimental Example 3 show the results of measuring the glutamate production rate (%) using human recombinant GLS1 for candidate GLS1 inhibitors (pyrazole derivatives 5i and 8a), known GLS1 inhibitors (DON, CB-839), and a control (DMSO). P values ​​were calculated using the Tukey-Kramer HSD test. n = 3. *P < 0.05 (vs Ct). [Figure 6] In Experimental Example 4, the pyrazole derivatives 5i and 8a were evaluated for their growth inhibitory activity against a human breast cancer cell line (MCF7 cells). The results are shown below. n=3 [Figure 7] In Experimental Example 4, the pyrazole derivatives 5i and 8a were evaluated for their growth inhibitory activity against acute myeloid leukemia-derived cell line (MOLM13 cells). The results are shown below. n=3 DETAILED DESCRIPTION OF THE INVENTION

[0019] (I) GLS1 inhibitor The GLS1 inhibitor of the present invention is characterized in that it contains, as an active ingredient, a compound represented by the following formula (I) (hereinafter also referred to as "compound (I)"), a pharmaceutically acceptable salt thereof, or a solvate thereof: [ka]

[0020] In formula (I), X represents CH2 or C=O, preferably CH2. When X is CH2, n is an integer of 0 to 2. Preferably, n is 0 or 1. When X is C=O, n is 1.

[0021] In formula (I), Y is a divalent 5-membered heterocyclic group having at least two nitrogen atoms (hereinafter, also referred to simply as "N-containing 5-membered heterocyclic group"). The N-containing 5-membered heterocyclic group may be a divalent 5-membered heterocyclic group having two or three nitrogen atoms in the ring and a carbon atom bonding to an adjacent group, but is preferably an N-containing 5-membered unsaturated heterocyclic group. The N-containing 5-membered heterocyclic group may have a carbon atom or a heteroatom such as an oxygen atom or a sulfur atom as a bond other than the nitrogen atom. For example, examples of N-containing 5-membered unsaturated heterocyclic groups in which the atoms other than the two nitrogen atoms are carbon atoms include a pyrazole group and an imidazole group; examples of N-containing 5-membered unsaturated heterocyclic groups in which the atoms other than the two nitrogen atoms are oxygen atoms include a furazan group; and examples of N-containing 5-membered unsaturated heterocyclic groups having three nitrogen atoms include a triazole group. The N-containing 5-membered heterocyclic group is preferably a pyrazole group or an imidazole group in which the atoms other than the two nitrogen atoms are carbon atoms, and more preferably a pyrazole group.

[0022] The two bonds of the N-containing 5-membered heterocyclic group are preferably located as far apart as possible. For example, when the bond of the N-containing 5-membered heterocyclic group bonded to the X group, which is part of the main skeleton of the compound represented by formula (I), is taken as the 1st position, the other bond is preferably located at the 3rd or 4th position.

[0023] In formula (I), R 1 is a hydrogen atom, a halogen atom, a haloalkyl group, an alkoxy group, an alkyl group, or a hydroxyl group. Although not limited thereto, it is preferably a hydrogen atom, a halogen atom, a haloalkyl group, or an alkoxy group, and more preferably a hydrogen atom or a halogen atom. In addition, in formula (I), when n is 0, R1 is preferably a group other than an alkoxy group, more preferably a hydrogen atom, a halogen atom, or a hydroxyl group. When R1 is a hydroxyl group, R 2 is preferably a group represented by the formula (b) or (c) described below, and more preferably a group represented by the formula (b).

[0024] R 1 Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferred are a fluorine atom and a chlorine atom.

[0025] R 1 The haloalkyl group represented by the formula (I) includes a straight-chain or branched alkyl group having 1 to 6 carbon atoms (C1-6) and 1 to 3 halogen atoms. A trihaloC1-6 alkyl group having 3 halogen atoms is preferred, and a trihaloC1-3 alkyl group is more preferred. Examples of the trihaloC1-6 alkyl group include a trifluoromethyl group, a trichloromethyl group, a 2,2,2-trifluoroethyl group, a 2,2,2-trichloroethyl group, and a 3,3,3-trifluoropropyl group. A trifluoromethyl group is preferred.

[0026] R 1Although not limited thereto, preferred examples of the alkyl group represented by the formula (I) include linear or branched alkyl groups having 1 to 6 carbon atoms (C1 to C6). More preferred are C1 to C3 alkyl groups. These alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl groups. Although not limited thereto, preferred are methyl, ethyl, and tert-butyl groups, and more preferred is methyl.

[0027] R 1 The alkoxy group represented by the formula (I) is a group having a structure in which an alkyl group is bonded to an oxygen atom. The alkyl group contained in this structure is also as described above, and is preferably a C1-3 alkyl group. Specific examples of the alkoxy group include a methoxy group and an ethoxy group. A methoxy group is preferred.

[0028] In formula (I), R on the phenyl group 1 The position of may be any of the ortho, meta, or para positions relative to the X group bonded to the benzene ring. Although not limited thereto, when n is 0, the para position is preferred. When n is 1, the para position, meta position, and ortho position are preferred in that order, and the para position is more preferred.

[0029] In formula (I), R 2 is a group represented by any one of the following formulas (a) to (c). A group represented by formula (a) : [ka] In formula (a), A represents a hydroxyl group or an oxygen atom. When A is a hydroxyl group, the double line consisting of a dotted line and a solid line in formula (a) represents a single bond. When A is an oxygen atom, the double line consisting of a dotted line and a solid line in formula (a) means a double bond. In formula (a), the asterisk represents the bond to the carbon atom of the carbonyl group in formula (I).

[0030] A group represented by formula (b) : [ka] In formula (b), B and D are the same or different and each represent a hydrogen atom, an alkyl group, or an alkoxy group. Here, the alkyl group is the above-mentioned R 1 As with the alkyl group represented by the formula (I), preferred is a C1 to C6 linear or branched alkyl group. More preferred is a C1 to C3 linear alkyl group, and specific examples thereof include a methyl group and an ethyl group. The alkoxy group may also be the above-mentioned R 1 As with the alkoxy group represented by the formula: Figure imgf000012_0001 and Figure imgf000012_0002, suitable examples include alkoxy groups having a C1-3 alkyl group, preferably a methoxy group and an ethoxy group, and more preferably a methoxy group. Combinations of B and D include, but are not limited to, a combination of a hydrogen atom and an alkyl group, a combination of alkyl groups, a combination of an alkyl group and an alkoxy group, and a combination of a hydrogen atom and an alkoxy group. In formula (b), the asterisk represents the bond to the carbon atom of the carbonyl group in formula (I).

[0031] A group represented by formula (c) : [ka] In formula (c), E represents an oxygen atom or an alkylene group. The alkylene group includes, but is not limited to, an alkylene group having 1 to 3 carbon atoms, preferably 1 to 2 carbon atoms. One of the hydrogen atoms of the alkylene group may be substituted with a hydroxyl group. In formula (c), the asterisk represents the bond to the carbon atom of the carbonyl group in formula (I).

[0032] Preferred compounds of Compound (I) include the following compounds: (A)R 2 is a group represented by formula (a) (collectively referred to as "compound a"): Compound a includes the following compounds: (a1) In formula (a), A represents a hydroxyl group; the double line consisting of a dotted line and a solid line represents a single bond; X represents CH; n represents an integer of 0 to 2; Y represents a pyrazole group having bonds at the 3- and 5-positions; R 1 is a hydrogen atom, a halogen atom, a haloalkyl group, an alkoxy group, or an alkyl group. Among these, in formula (a), A is a hydroxyl group; the double line consisting of a dotted line and a solid line is a single bond; X is CH; n is 0 or 1; Y is a pyrazole group having bonds at the 3- and 5-positions; R 1 is a hydrogen atom or a halogen atom.

[0033] These compounds specifically include the following compounds: All isomers of the compounds are included, regardless of stereoisomerism. Compound 1: N-(4-hydroxycyclohexyl)-3-phenyl-1H-pyrazole-5-carboxamide Compound 2: 3-(4-fluorophenyl)-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide Compound 3: 3-benzyl-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide Compound 4: 3-(4-fluorobenzyl)-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide Compound 5: 3-(4-chlorobenzyl)-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide Compound 6: 3-(2-chlorobenzyl)-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide Compound 7: 3-(3-chlorobenzyl)-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide Compound 8: N-(4-hydroxycyclohexyl)-3-(4-(trifluoromethyl)benzyl)-1H-pyrazole-5-carboxamide Compound 9: N-(4-hydroxycyclohexyl)-3-(3-methoxybenzyl)-1H-pyrazole-5-carboxamide Compound 10: N-(4-hydroxycyclohexyl)-3-(4-methoxybenzyl)-1H-pyrazole-5-carboxamide Compound 11: N-(4-hydroxycyclohexyl)-3-(2-methoxybenzyl)-1H-pyrazole-5-carboxamide Compound 12: N-(4-hydroxycyclohexyl)-3-(4-methylbenzyl)-1H-pyrazole-5-carboxamide Compound 13: N-(4-hydroxycyclohexyl)-3-phenethyl-1H-pyrazole-5-carboxamide

[0034] (a2) In formula (a), A represents an oxygen atom; the double line consisting of a dotted line and a solid line represents a double bond; X represents CH; n represents 0 to 2, preferably 1; Y represents a pyrazole group having bonds at the 3- and 5-positions; R 1 is a hydrogen atom, compound. The compounds specifically include the following compounds: Regardless of stereoisomers, the compounds also include all isomers. Compound 14: 3-benzyl-N-(4-oxocyclohexyl)-1H-pyrazole-5-carboxamide

[0035] (a3) In formula (a), A represents a hydroxyl group; the double line consisting of a dotted line and a solid line represents a single bond; X represents C=O; n represents 1; Y represents a pyrazole group having bonds at the 3- and 5-positions; R 1 is a hydrogen atom, compound. The compounds specifically include the following compounds: Regardless of stereoisomers, the compounds also include all isomers. Compound 15: 3-benzoyl-N-(4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide

[0036] (a4) In formula (a), A represents a hydroxyl group; the double line consisting of a dotted line and a solid line represents a single bond; X represents CH; n represents 0 to 2, preferably 1; Y represents an imidazole group having bonds at the 2- and 5-positions; R 1 is a hydrogen atom, compound. The compounds specifically include the following compounds: Regardless of stereoisomers, the compounds also include all isomers. Compound 16: N-(4-hydroxycyclohexyl)-5-phenyl-1H-imidazole-2-carboxamide

[0037] (B)R 2 is a group represented by formula (b) (collectively referred to as "compound b"): Compound b includes the following compounds: (b) In formula (b), B and D are the same or different and each represent a hydrogen atom, an alkyl group, or an alkoxy group; X represents CH; n represents an integer of 0 to 2; Y represents a pyrazole group having bonds at the 3- and 5-positions; R 1 is a hydrogen atom or a hydroxyl group. The compounds specifically include the following compounds: Regardless of stereoisomers, the compounds also include all isomers. Compound 17: 3-(4-hydroxyphenyl)-N-methyl-1H-pyrazole-5-carboxamide Compound 18: 3-benzyl-N-methyl-1H-pyrazole-5-carboxamide Compound 19: 3-benzyl-N,N-dimethyl-1H-pyrazole-5-carboxamide Compound 20: 3-benzyl-N-methoxy-N-methyl-1H-pyrazole-5-carboxamide

[0038] (C)R 2 is a group represented by formula (c) (collectively referred to as "compound c"): Compound c includes the following compounds: (c) In formula (c), E represents an oxygen atom or an alkylene group in which one hydrogen atom may be substituted with a hydroxyl group; X represents CH; n represents 0 to 2; Y represents a pyrazole group having bonds at the 3- and 5-positions; R 1 is a hydrogen atom, compound. The compounds specifically include the following compounds: Regardless of stereoisomers, the compounds also include all isomers. Compound 21: (3-benzyl-1H-pyrazol-5-yl)(piperidin-1-yl)methanone Compound 22: azepan-1-yl(3-benzyl-1H-pyrazol-5-yl)methanone Compound 23: (3-benzyl-1H-pyrazol-5-yl)(morpholino)methanone Compound 24: (3-benzyl-1H-pyrazol-5-yl)(4-hydroxypiperidin-1-yl)methanone

[0039] The aforementioned compound (I) can take the form of a pharmaceutically acceptable salt, and such salt can be used in the same manner as the free form of compound (I). Pharmaceutically acceptable salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, or boric acid (inorganic acid salts); and salts with organic acids such as formic acid, acetic acid, lactic acid, fumaric acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, or tosylic acid (organic acid salts). However, they are not limited to these. These salts can be prepared by conventional means. Furthermore, the aforementioned compound (I) and its pharmaceutically acceptable salts may be solvates. Solvates also include hydrates.

[0040] Compound (I), a pharmaceutically acceptable salt thereof, or a solvate thereof (hereinafter, these are also collectively referred to as "compound (I), etc.") can be produced based on the description of the production examples described below.

[0041] Compound (I) and the like obtained by these production methods are isolated and purified from the reaction mixture by applying known isolation and / or purification means, such as distillation, recrystallization, solvent extraction, column chromatography, ion exchange chromatography, gel chromatography, affinity chromatography, preparative thin layer chromatography, etc.

[0042] Compound (I) and the like have GLS1 inhibitory activity, as shown in Experimental Examples 2 and 3 described below.

[0043] The GLS1 inhibitor of the present invention may consist solely of the aforementioned compound (I), etc., or may contain other components, such as carriers and additives, that are commonly used in the relevant fields (biochemistry, pharmaceuticals, cosmetics, food). The specific types of carriers and additives and their blending ratios are not particularly limited, as long as the GLS1 inhibitor exerts GLS1 inhibitory activity based on compound (I), etc. For example, the types of carriers and additives and their blending ratios can be appropriately selected with reference to the types and blending ratios of the carriers and additives described in the pharmaceutical composition section below.

[0044] (II) Pharmaceutical Composition The pharmaceutical composition of the present invention (hereinafter referred to as "the pharmaceutical composition") is characterized by containing the aforementioned GLS1 inhibitor, specifically, the aforementioned compound (I), a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient.

[0045] Based on the GLS1 inhibitory activity of the aforementioned compound (I), the present pharmaceutical composition can be applied to diseases or symptoms (including conditions) that can be effectively treated by inhibiting GLS1. Diseases or symptoms that can be treated with the present pharmaceutical composition include inflammation, diseases caused by inflammation, obesity, metabolic syndrome, aging, and cancer.

[0046] The pharmaceutical composition can be suitably used for the prevention or treatment of such diseases or symptoms. In the present invention, "treatment" not only means curing the disease or symptoms, but also includes alleviating or partially improving them. In the present invention, "prevention" also includes inhibiting the progression of the disease or symptoms and inhibiting recurrence. In particular, the prevention or treatment of cancer includes reducing cancer tissue, preventing or inhibiting cancer progression, and suppressing cancer recurrence by inhibiting the proliferation of cancer cells.

[0047] In the present invention, the term "cancer" is broadly interpreted and is used interchangeably with the term "malignant tumor." Furthermore, before a pathological diagnosis is confirmed, i.e., before a tumor is determined to be benign or malignant, the term may collectively include benign tumors, borderline benign / malignant lesions, and malignant tumors. Generally, cancers are named after the organ or tissue from which they originated, and the main types include tongue cancer, gum cancer, pharyngeal cancer, maxillary cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, liver cancer, biliary tract cancer, gallbladder cancer, pancreatic cancer, lung cancer, breast cancer, thyroid cancer, adrenal cancer, pituitary tumor, pineal gland tumor, uterine cancer, ovarian cancer, vaginal cancer, bladder cancer, kidney cancer, prostate cancer, urethral cancer, retinoblastoma, conjunctival cancer, neuroblastoma, glioma, glioblastoma, skin cancer, medulloblastoma, leukemia, malignant lymphoma, testicular tumor, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, liposarcoma, chondrosarcoma, Ewing's sarcoma, multiple myeloma, and acute myeloid leukemia. Depending on the location of the organ in which they originate, cancers are further classified into upper, mid, and lower pharyngeal cancer, upper, middle, and lower esophageal cancer, gastric cardia cancer, gastric pyloric cancer, cervical cancer, and uterine cancer, but these are not limitative and are included in the description of "cancer" in the present invention. The pharmaceutical composition of the present invention used in anticancer therapy (also referred to as an "anticancer pharmaceutical composition") is effective against "cancer" in general, but may be particularly preferably used against breast cancer and acute myeloid leukemia. In particular, among compound (I) and the like, compounds 1 and 4, and their pharmaceutically acceptable salts and solvates, have excellent anticancer effects, particularly in inhibiting the proliferation of cancer cells in breast cancer and / or acute myeloid leukemia.

[0048] The term "anticancer pharmaceutical composition" refers to a pharmaceutical composition that exhibits a therapeutic or preventive effect against cancer, which is a target disease or pathology. Preferably, it refers to a pharmaceutical composition that exhibits a therapeutic effect. Therapeutic effects include alleviating (alleviating) symptoms characteristic of cancer or associated symptoms, and preventing or delaying the worsening of symptoms. The latter can be considered a type of preventive effect in that it prevents the condition from becoming severe. As such, therapeutic and preventive effects are concepts that partially overlap, making it difficult to clearly distinguish between them, and there is little practical benefit in doing so. A typical preventive effect is preventing or delaying the recurrence (onset) of symptoms characteristic of cancer. Note that any composition that exhibits a therapeutic effect, a preventive effect, or both against cancer falls under the category of an anticancer pharmaceutical composition. Furthermore, the therapeutic or preventive effect against cancer provided by Compound (I) and the like may also include the improvement of cancer complications (e.g., cachexia). In other words, "anticancer" and "cancer treatment" as used herein may include not only the effects of inhibiting proliferation or shrinking the cancer tissue itself, but also the improvement of complications (preferably cachexia). The "anti-cancer pharmaceutical composition" of the present invention can also be used in combination with known anti-cancer agents or anti-cancer agents that will be developed in the future.

[0049] The present pharmaceutical composition may consist solely of the aforementioned compound (I), etc., or may be prepared by combining it with other pharmaceutically acceptable ingredients and preparing it in a form suitable for the desired use, such as the route of administration or the method of administration, by a known method. The present pharmaceutical composition can be formulated according to conventional methods, except for the addition of compound (I), etc., as the active ingredient. When formulated, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, surfactants, lubricants, diluents, coating agents, sugar-coating agents, flavoring agents, emulsifying / solubilizing / dispersing agents, pH adjusters, isotonic agents, solubilizing agents, flavorings, coloring agents, solubilizing aids, physiological saline, etc.) may be contained. The dosage form when formulated is not particularly limited, and includes tablets, powders, fine granules, granules, capsules, syrups, liquids, suspensions, emulsions, jellies, injections, topical preparations, inhalants, nasal drops, eye drops, and suppositories.

[0050] The present pharmaceutical composition contains, as an active ingredient, Compound (I) etc. in an amount necessary to obtain the expected preventive or therapeutic effect (i.e., a therapeutically effective amount). The amount of Compound (I) etc. in the present pharmaceutical composition varies depending on the dosage form, but can be set, for example, within the range of about 0.01% by mass to about 99.9% by mass so as to achieve the desired dosage.

[0051] The pharmaceutical composition may be administered to a subject by oral or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on its dosage form. These administration routes are not mutually exclusive, and any two or more of them may be used in combination (e.g., intravenous injection simultaneously with oral administration or after a predetermined time has elapsed). Local administration may be used instead of systemic administration. The active ingredient may be administered to target tissues specifically using a drug delivery system (DDS). The "subject" here is not particularly limited and includes humans and non-human mammals (including pet animals, livestock, and laboratory animals, such as mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, chickens, and quails) in need of treatment or prevention. Humans are preferred.

[0052] The dosage of the pharmaceutical composition generally varies depending on the patient's symptoms, age, sex, and weight, but those skilled in the art can determine an appropriate dosage. For example, oral administration can be, but is not limited to, administered to an adult at a dose of, for example, about 0.01 mg to 1000 mg per day, either once or in several divided doses. Furthermore, parenteral administration can be, for example, administered at a dose of about 0.01 mg to 1000 mg per day by subcutaneous injection, intramuscular injection, or intravenous injection. Administration schedules can be, for example, once to several times per day, once every two days, or once every three days. When determining the administration schedule, consideration can be given to the patient's symptoms and the duration of effect of the active ingredient.

[0053] (III) Food and drink composition The food and beverage composition of the present invention (hereinafter referred to as "the food and beverage composition") is characterized by containing the aforementioned GLS1 inhibitor, specifically, the compound (I), a pharmaceutically acceptable salt thereof, or a solvate thereof. The present food and beverage composition can be prepared by, but is not limited to, blending the compound (I) etc. in the manufacturing process of a conventionally known food and beverage, or by adding the compound (I) etc. to a conventionally known food and beverage. The present food and drink composition can be effectively used for, but not limited to, anti-obesity and prevention or amelioration of metabolic syndrome.

[0054] (IV) Cosmetic compositions The cosmetic composition according to the present invention (hereinafter referred to as "the present cosmetic composition") is characterized by containing the above-mentioned GLS1 inhibitor, specifically, the above-mentioned compound (I), a pharmaceutically acceptable salt thereof, or a solvate thereof. The present cosmetic composition also includes medicated cosmetics that fall under the category of quasi-drugs. The present cosmetic composition can be prepared by, but is not limited to, blending the compound (I) etc. in the manufacturing process of conventionally known cosmetics (including medicated cosmetics). Alternatively, the present cosmetic composition can be prepared by adding the compound (I) etc. to conventionally known cosmetic ingredients. The present cosmetic composition is useful for, but not limited to, anti-aging, specifically for improving wrinkles and preventing aging.

[0055] As described above, in this specification, the terms "comprise" and "contain" include the meanings of "consist of" and "substantially consist of." Furthermore, the present invention is not limited to the above-described embodiments, and can be freely modified within the scope of the present invention. [Example]

[0056] The present invention will be explained below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0057] Production Example 1: Synthesis of GLS1 inhibitor candidate compound GLS1 inhibitor candidate compounds (compounds 1 to 14, 17 to 24, and 3a) were produced according to the procedure shown in the following scheme. [ka]

[0058] (1) Synthesis of compounds 2a to 2o Under an Ar atmosphere, sodium bis(trimethylsilyl)amide (0.79 mL, 1.9 M in THF, 1.50 mmol) was added to a solution of methyl ketone (1.00 mmol) (compounds 1a–1o) in tetrahydrofuran (5 mL) at −78°C, followed by stirring at −78°C for 30 min. Diethyl oxalate (0.27 mL, 2.00 mmol) was then added at −78°C, followed by warming to room temperature and stirring at room temperature for 2 h. After completion of the reaction, 10% aqueous HCl (3 mL) was added. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (1.5 mL × 3) and combined with the organic layer. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The resulting ketoester was used in the next reaction without further purification. Under an Ar atmosphere, hydrazine monohydrate (0.07 mL, 1.50 mmol) was added to a solution of the resulting ketoester in ethanol (5 mL) at room temperature, followed by stirring under reflux for 4 hours. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (5 mL) and water (3 mL) was added. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (1.5 mL × 3) and combined with the organic layer. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 7:1 to 1:1) to obtain the desired pyrazole esters (compounds 2a-2o).

[0059] (2) Synthesis of Compounds 1 to 14, 18 to 24, and Compound 3a Under an Ar atmosphere, a solution of pyrazole ester (compounds 2a-2o) (0.50 mmol) in ethanol (3 mL) was added with 10% aqueous NaOH (1.5 mL) at room temperature and stirred under reflux for 2 hours. After completion of the reaction, the ethanol was evaporated, and 10% aqueous HCl (3 mL) was added. The aqueous layer was extracted with ethyl acetate (1.5 mL × 3). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting carboxylic acid was used in the next reaction without further purification. To a solution of the resulting carboxylic acid in dichloromethane (3 mL) under an Ar atmosphere, the corresponding amine (0.75 mmol), triethylamine (0.11 mL, 0.75 mmol), 1-hydroxybenzotriazole (101 mg, 0.75 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (143 mg, 0.75 mmol) were added sequentially at room temperature, followed by stirring at room temperature for 20 hours. After completion of the reaction, the solvent was removed using a rotary evaporator, and the resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 7:1 to 1:5) to obtain the desired compounds (Compounds 1-14, 18-24, and Compound 3a).

[0060] (3) Synthesis of Compound 17 Under an Ar atmosphere, a solution of amide (compound 3a) (0.50 mmol) in methanol (3 mL) was added with 10% aqueous HCl (1.5 mL) at room temperature and stirred under reflux for 30 minutes. After completion of the reaction, the solvent was removed using a rotary evaporator, and the resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 1:1 to 1:5) to obtain the desired compound (compound 17).

[0061] The properties of each compound synthesized by the above method are shown below.

[0062] Compound 1: N-(trans-4-hydroxycyclohexyl)-3-phenyl-1H- Pyrazole-5-carboxamide 1 H-NMR (400 MHz, CD3OD) δ: 1.37-1.51 (4H, m), 2.00-2.06 (4H, m), 3.58-3.61 (1H, m), 3.83-3.90 (1H, m), 7.02 (1H, s), 7.38-7.47 (3H, m), 7.69-7.79 (2H, m). [1]

[0063] Compound 2: 3-(4-fluorophenyl)-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 237-239 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.37-1.51 (4H, m), 1.99-2.02 (4H, m), 3.56-3.58 (1H, m), 3.85-3.87 (1H, m), 7.00 (1H, s), 7.19 (2H, dr), 7.74 (2H, dr); 13 C-NMR (100 MHz, DMSO-d6) δ: 30.29, 34.21, 47.23, 54.95, 68.18, 68.30, 102.42, 115.77, 115.87 (d, J = 21.9 Hz), 127.28, 140.35, 160.60, 161.92 (d, J = 243.2 Hz); IR (KBr): 3300, 3113, 2930, 1638, 1547, 1508, 1454, 1234, 839 cm -1 ; MS (EI): m / z 303 (M + ); HRMS: Calcd for C 16 H 18 FN3O2303.1383, Found 303.1388.

[0064] Compound 3: 3-benzyl-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 94-96 ℃; 1H-NMR (400 MHz, CD3OD) δ: 1.29-1.46 (4H, m), 1.96-1.98 (4H, m), 3.49-3.61 (1H, m), 3.74-3.87 (1H, m), 4.02 (2H, s), 6.47 (1H, s), 7.20-7.32 (5H, m); 13 C-NMR (100 MHz, CD3OD) δ: 31.44, 32.57, 34.84, 70.35, 105.47, 127.64, 129.56, 129.66, 139.67, 145.39, 148.11, 164.23; IR (KBr): 3315, 2855, 1647, 1943, 1456, 1238 cm -1 ; MS (EI): m / z 299 (M + ); HRMS: Calcd for C 17 H 21 N3O2299.1634, Found 299.1629.

[0065] Compound 4: 3-(4-fluorobenzyl)-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 95-96 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.34-1.46 (4H, m), 1.96-1.98 (4H, m), 3.47-3.64 (1H, m), 3.73-3.87 (1H, m), 4.01 (2H, s), 6.47 (1H, s), 7.00-7.04 (2H, m), 7.22-7.25 (2H, m); 13 C-NMR (100 MHz, CD3OD) δ: 31.48, 31.64, 34.90, 70.42, 105.40, 116.29 (d, J = 21.9 Hz), 131.34 (d, J = 8.9 Hz), 135.60, 145.49, 148.15, 163.15 (d, J = 242.1 Hz), 164,36; IR (KBr): 3300, 3215, 2932, 1636, 1541, 1508, 1456, 1223 cm -1 ; MS (EI): m / z 317 (M +); HRMS: Calcd for C 17 H 20 FN3O2317.1540, Found 317.1541.

[0066] Compound 5: 3-(4-cyclobenzyl)-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 88-89 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.34-1.45 (4H, m), 1.96-1.98 (4H, m), 3.49-3.61 (1H, m), 3.73-3.87 (1H, m), 4.01 (2H, s), 6.48 (1H, s), 7.21 (2H, d, J = 8.4 Hz), 7.30 (2H, d, J = 8.4 Hz); 13 C-NMR (100 MHz, CD3OD) δ: 29.56, 31.49, 31.74, 32.14, 34.92, 70.43, 105.50, 129.76, 131.23, 133.57, 138.47, 145.04, 148.26, 164.24; IR (KBr): 3317, 3300, 3263, 2930, 2864, 1638, 1545, 1491, 1456, 1088 cm -1 ; MS (EI): m / z 333 (M + ); HRMS: Calcd for C 17 H 20 ClN3O2333.1244, Found 333.1235.

[0067] Compound 6: 3-(2-cyclobenzyl)-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 96-97 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.32-1.43 (4H, m), 1.96-1.98 (4H, m), 3.50-3.61 (1H, m), 3.74-3.84 (1H, m), 4.15 (2H, s), 6.42 (1H, s), 7.21-7.31 (3H, m), 7.39-7.42 (1H, m); 13C-NMR (100 MHz, DMSO-d6) δ: 28.94, 29.65, 30.33, 34.27, 47.04, 68.32, 104.32, 127.57, 128.71, 129.41, 130.86, 132.96, 136.14, 141.78, 147.22, 161.00; IR (KBr): 3375, 3315, 3138, 2934, 2860, 1638, 1541, 1454 cm -1 ; MS (EI): m / z 333 (M + ); HRMS: Calcd for C 17 H 20 ClN3O2333.1244, Found 333.1241.

[0068] Compound 7: 3-(3-cyclobenzyl)-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 83-85 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.29-1.46 (4H, m), 1.96-1.98 (4H, m), 3.55-3.56 (1H, m), 3.71-3.88 (1H, m), 4.02 (2H, s), 6.51 (1H, s), 7.15-7.30 (4H, m); 13 C-NMR (100 MHz, CD3OD) δ: 29.56, 31.47, 32.58, 34.91, 49.13, 70.42, 105.52, 127.80, 128.05, 129.64, 131.20, 135.44, 142.26, 146.59, 163.43; IR (KBr): 3377, 3254, 3078, 2932, 2855, 1638, 1545, 1456, 1431, 1078 cm -1 ; MS (EI): m / z 333 (M + ); HRMS: Calcd for C 17 H 20 ClN3O2333.1244, Found 333.1249.

[0069] Compound 8: N-(trans-4-hydroxycyclohexyl)-3-(4-(trifluoromethyl)benzyl)-1H-pyrazole-5-carboxamide mp: 94-96 ℃;1 H-NMR (400 MHz, CD3OD) δ: 1.34-1.46 (4H, m), 1.96-1.98 (4H, m), 3.47-3.64 (1H, m), 3.73-3.87 (1H, m), 4.12 (2H, s), 6.51 (1H, s), 7.42 (2H, d, J = 8.0 Hz), 7.61 (2H, d, J = 8.0 Hz); 13 C-NMR (100 MHz, CD3OD) δ: 31.48, 32.18, 34.91, 70.42, 105.67, 125.76 (q, J = 270.0 Hz), 126.57, 130.28, 144.25, 144.51, 148.35, 164.16; IR (KBr): 3308, 3263, 2937, 1638, 1541, 1456, 1327, 1124, 1067 cm -1 ; MS (EI): m / z 367 (M + ); HRMS: Calcd for C 18 H 20 F3N3O2367.1508, Found 367.1507.

[0070] Compound 9: N-(trans-4-hydroxycyclohexyl)-3-(3-methoxybenzyl)-1H-pyrazole-5-carboxamide mp: 65-67 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.29-1.46 (4H, m), 1.96-1.98 (4H, m), 3.55-3.58 (1H, m), 3.76-3.80 (4H, m), 3.99 (2H, s), 6.48 (1H, s), 6.79-6.81 (3H, m), 7.20 (2H, t, J = 8.0 Hz); 13C-NMR (100 MHz, CD3OD) δ: 31.47, 32.47, 34.89, 55.61, 70.40, 105.46, 113.07, 115.31, 121.86, 130.69, 141.69, 145.51, 148.16, 161.41, 164.28; IR (KBr): 3373, 3360, 3283, 2999, 2901, 2858, 1612, 1508, 1491, 1456, 1508 cm -1 ; MS (EI): m / z 329 (M + ); HRMS: Calcd for C 18 H 23 N3O3329.1739, Found 329.1743.

[0071] Compound 10: N-(trans-4-hydroxycyclohexyl)-3-(4-methoxybenzyl)-1H-pyrazole-5-carboxamide mp: 75-77 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.33-1.41 (4H, m), 1.96-1.98 (4H, m), 3.50-3.61 (1H, m), 3.76-3.82 (4H, m), 3.95 (2H, s), 6.45 (1H, s), 6.86 (2H, d, J = 8.6 Hz), 7.14 (2H, d, J= 8.6 Hz); 13 C-NMR (100 MHz, CD3OD) δ: 31.49, 31.63, 34.90, 55.70, 70.42, 105.30, 115.09, 130.59, 131.55, 146.10, 148.10, 159.98, 164.32; IR (KBr): 3261, 2930, 1647, 1541, 1508, 1456, 1246 cm -1 ; MS (EI): m / z 329 (M + ); HRMS: Calcd for C 18 H 23 N3O3329.1739, Found 329.1746.

[0072] Compound 11: N-(trans-4-hydroxycyclohexyl)-3-(2-methoxybenzyl)-1H-pyrazole-5-carboxamide mp: 71-72 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 1.24-1.49 (4H, m), 1.98-2.08 (4H, m), 3.59-3.66 (1H, m), 3.86-3.94 (4H, m), 3.97 (2H, s), 6.61 (1H, s), 6.90-6.93 (2H, m), 7.15 (1H, dd, J = 8.0, 2.4 Hz), 7.23-7.28 (1H, m); 13 C-NMR (100 MHz, CDCl3) δ: 26.90, 29.24, 30.74, 31.74, 33.87, 47.26, 55.47, 69.65, 104.67, 110.66, 120.91, 126.21, 128.39, 130.13, 144.74, 146.62, 156.87, 161.58; IR (KBr): 3412, 3337, 3227, 1641, 1551, 1495, 1456, 1246 cm -1 ; MS (EI): m / z 329 (M + ); HRMS: Calcd for C 18 H 23 N3O3329.1739, Found 329.1738.

[0073] Compound 12: N-(trans-4-hydroxycyclohexyl)-3-(4-methylbenzyl)-1H-pyrazole-5-carboxamide mp: 86-87 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.34-1.46 (4H, m), 1.96-1.98 (4H, m), 2.29 (3H, s), 3.47-3.63 (1H, m), 3.72-3.86 (1H, m), 3.96 (2H, s), 6.45 (1H, s), 7.08-7.14 (4H, m); 13C-NMR (100 MHz, CD3OD) δ: 21.08, 29.57, 31.48, 32.13, 34.89, 70.37, 105.37, 129.48, 130.28, 136.53, 137.35, 145.94, 148.15, 164.30; IR (KBr): 3327, 3234, 3177, 2925, 2864, 1645, 1543, 1516, 1456, 1082 cm -1 ; MS (EI): m / z 313 (M + ); HRMS: Calcd for C 18 H 23 N3O2313.1790, Found 313.1790.

[0074] Compound 13: N-(trans-4-hydroxycyclohexyl)-3-phenethyl-1H-pyrazole-5-carboxamide mp: 91-93 ℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.34-1.47 (4H, m), 1.97-1.99 (4H, m), 2.89-3.02 (4H, m), 3.55-3.57 (1H, m), 3.75-3.88 (1H, m), 6.48 (1H, m), 7.14-7.18 (3H, m), 7.25 (2H, t, J = 3.6 Hz); 13 C-NMR (100 MHz, CD3OD) δ: 28.36, 31.51, 34.88, 36.49, 49.16, 70.39, 104.85, 127.23, 129.42, 129.45, 142.02, 146.06, 148.01, 164.45; IR (KBr): 3368, 3179, 2930, 2864, 1649, 1543, 1456, 1086, 700 cm -1 ; MS (EI): m / z 313 (M + ); HRMS: Calcd for C 18 H 23 N3O2313.1790, Found 313.1781.

[0075] Compound 14: 3-benzyl-N-(4-oxocyclohexyl)-1H-pyrazole-5-carboxamide mp: 131-132 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 1.73-1.84 (2H, m), 2.27-2.31 (2H, m), 2.40-2.53 (4H, m), 4.04 (2H, s), 4.35-4.44 (1H, m), 6.62 (1H, s), 6.80 (1H, d, J = 6.8 Hz), 7.22 (2H, d, J = 6.8 Hz), 7.26 (1H, tt, J = 6.8, 1.6 Hz), 7.32 (2H, tt, J = 6.8, 1.6 Hz); 13 C-NMR (100 MHz, CDCl3) δ: 29.25, 31.76, 32.20, 39.09, 46.03, 105.04, 126.96, 128.58, 128.81, 137.44, 144.92, 146.26, 161.70, 210.11; IR (KBr): 3300, 3192, 1715, 1638, 1545, 1456 cm -1 ; MS (EI): m / z 297 (M + ); HRMS: Calcd for C 17 H 19 N3O2297.1477, Found 297.1486.

[0076] Compound 17: 3-(4-hydroxyphenyl)-N-methyl-1H-pyrazole-5-carboxamide mp: 177-179 ℃; 1 H-NMR (400 MHz, DMSO-d6) δ: 2.74 (3H, d, J = 4.0 Hz), 6.82 (2H, d, J = 8.4 Hz), 6.89 (1H, s), 7.58 (2H, d, J = 8.4 Hz), 8.18 (1H, d, J = 3.6 Hz); 13 C-NMR (100 MHz, DMSO-d6) δ: 25.71, 101.23, 115.82, 120.97, 126.87, 145.29, 145.73, 157.85, 161.54; IR (KBr): 3219, 3107, 3042, 1655, 1614, 1508 cm -1; MS (EI): m / z 217 (M + ); HRMS: Calcd for C 11 H 11 N3O2217.0851, Found 217.0841.

[0077] Compound 18: 3-benzyl-N-methyl-1N-pyrazole-5-carboxamide mp: 168-170 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 2.95 (3H, d, J = 4.8 Hz), 4.03 (2H, s), 6.58 (1H, s), 6.80 (1H, s), 7.19-7.34 (5H, m); 13 C-NMR (100 MHz, DMSO-d6) δ: 25.56, 31.00, 103.95, 126.48, 128.49, 128.56, 138.80, 143.42, 147.18, 162.46; IR (KBr): 3084, 1638, 1570 cm -1 ; MS (EI): m / z 215 (M + ); HRMS: Calcd for C 12 H 13 N3O 215.1059, Found 215.1055.

[0078] Compound 19: 3-benzyl-N,N-dimethyl-1N,N-dimethyl-1H-pyrazole-5-carboxamide mp: 149-151 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 3.10 (3H, s), 3.27 (3H, s), 4.03 (2H, s), 6.35 (1H, s), 7.21-7.24 (3H, m), 7.31 (2H, t, J = 7.4 Hz); 13 C-NMR (100 MHz, CDCl3) δ: 33.43, 36.28, 38.79, 106.46, 126.51, 128.59, 128.71, 138.69, 141.21, 148.27, 162.54; IR (KBr): 3128, 2968, 2860, 1599, 1495, 1398, 719 cm -1; MS (EI): m / z 229 (M + ); HRMS: Calcd for C 13 H 15 N3O 229.1215, Found 229.1219.

[0079] Compound 20: 3-benzyl-N-methoxy-N-methyl-1H-pyrazole-5-carboxamide 1 H-NMR (400 MHz, CDCl3) δ: 3.36 (3H, s), 3.73 (3H, s), 4.07 (2H, s), 6,57 (1H, s), 7.20-7.32 (5H, m); 13 C-NMR (100 MHz, CDCl3) δ: 32.93, 34.27, 61.46, 107.88, 126.30, 128.47, 128.70, 135.97, 139.33, 152.05, 159.72; IR (neat): 3213, 3155, 1626, 1558, 1495, 1456, 1435 cm -1 ; MS (EI): m / z 245 (M + ); HRMS: Calcd for C 13 H 15 N3O2245.1164, Found 245.1161.

[0080] Compound 21: (3-benzyl-1H-pyrazol-5-yl)(piperidin-1-yl)methanone mp: 151-153 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 1.61-1.70 (6H, m), 3.59-3.83 (4H, m), 4.03 (2H, s), 6.31 (1H, s), 7.22-7.26 (4H, m), 7.29-7.33 (2H, m); 13C-NMR (100 MHz, CDCl3) δ: 24.60, 25,63, 26.59, 33.32, 43.65, 48.02, 105.93, 126.63, 128.67, 128.76, 138.47, 142.18, 147.64, 161.49; IR (KBr): 3146, 2924, 1593, 1506, 1421, 1250, 725 cm -1 ; MS (EI): m / z 269 (M + ); HRMS: Calcd for C 16 H 19 N3O 269.1528, Found 269.1537.

[0081] Compound 22: Azepan-1-yl(3-benzyl-1H-pyrazol-5-yl)methanone mp: 145-147 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 1.58-1.59 (4H, m), 1.77-1.80 (4H, m), 3.65 (2H, t, J= 6.0 Hz), 3.72 (2H, t, J = 6.0 Hz), 4.05 (2H, s), 6.31 (1H, s), 7.22-7.26 (3H, m), 7.31 (2H, t, J = 7.2 Hz); 13 C-NMR (100 MHz, CDCl3) δ: 26.88, 27.27, 27.52, 29.42, 33.57, 46.72, 48.68, 106.00, 126.48, 128.58, 128.71, 138.77, 141.09, 148.80, 162.13; IR (KBr): 3171, 3128, 3086, 2978, 2924, 2853, 1576, 1510, 1454, 1421, 719 cm -1 ; MS (EI): m / z 283 (M + ); HRMS: Calcd for C 17 H 21 N3O 283.1685, Found 283.1675.

[0082] Compound 23: (3-benzyl-1H-pyrazol-5-yl)(morpholino)methanone mp: 122-124 ℃; 1 H-NMR (400 MHz, CDCl3) δ: 3.72-3.91 (8H, m), 4.05 (2H, s), 6.41 (1H, s), 7.22-7.35 (5H, m); 13 C-NMR (100 MHz, CDCl3) δ: 32.61, 42.81, 47.47, 66.90, 106.70, 126.78, 128.766, 128.73, 137.89, 143.80, 145.76, 162.22; IR (KBr): 3119, 2966, 2853, 1585, 1499, 1238, 1109 cm -1 ; MS (EI): m / z 271 (M + ); HRMS: Calcd for C 15 H 17 N3O2271.1321, Found 271.1322.

[0083] Compound 24: (3-benzyl-1H-pyrazol-5-yl)(4-hydroxypiperidin-1-yl)methanone 1 H-NMR (400 MHz, CD3OD) δ: 1.44-1.59 (2H, m), 1.81-1.97 (2H, m), 3.30-3.48 (2H, m), 3.85-3.91 (1H, m), 4.03 (2H, s), 4.09-4.31 (2H, m), 6.32 (1H, s), 7.20-7.32 (5H, m); 13 C-NMR (100 MHz, CD3OD) δ: 32.40, 34.83, 35.67, 41.15, 45.79, 67.81, 106.66, 127.71, 129.60, 129.71, 139.64, 145.00, 148.08, 165.72; IR (neat): 3676, 3387, 3327, 3294, 3179, 2955, 1611, 1597, 1506, 1495 cm -1 ; MS (EI): m / z 285 (M + ); HRMS: Calcd for C 16 H 19N3O2285.1477, Found 285.1468.

[0084] (4) Synthesis of Compound 15 [ka]

[0085] Selenium dioxide (420 mg, 3.78 mmol) was added to a solution of acetophenone 4 (300 mg, 2.50 mmol) in a 1,4-dioxane and water (15 mL, 3:1) mixture under an Ar atmosphere at room temperature, followed by stirring under reflux for 18 hours. After cooling, the reaction mixture was filtered and the solvent was removed using a rotary evaporator. Water (5 mL) was added to the resulting residue, and the mixture was further stirred under reflux for 5 hours. After completion of the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 1:1) to give gem-diol 5 (85 mg, 22%, 0.55 mmol). To a solution of gem-diol 5 (78 mg, 0.51 mmol) in dimethyl sulfoxide (2.5 mL) under an Ar atmosphere, p-toluenesulfonyl hydrazide (114 mg, 0.61 mmol), ethyl acrylate (0.08 mL, 0.77 mmol), and cesium carbonate (500 mg, 1.54 mmol) were added sequentially and the mixture was stirred at 100 °C for 5 h. After completion of the reaction, water (3 mL) was added and the mixture was diluted with ethyl acetate (5 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (1.5 mL × 3) and combined with the organic layer. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 5:1) to give pyrazole ester 6 (27 mg, 22%, 0.11 mmol).

[0086] Under an Ar atmosphere, a solution of pyrazole ester 6 (27 mg, 0.12 mmol) in ethanol (3 mL) was added with 10% aqueous NaOH (1.5 mL) at room temperature and stirred under reflux for 2 hours. After completion of the reaction, the ethanol was evaporated, and 10% aqueous HCl (3 mL) was added. The aqueous layer was extracted with ethyl acetate (1.5 mL × 3). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting carboxylic acid was used in the next reaction without further purification. To a solution of the resulting carboxylic acid in dichloromethane (3 mL) under Ar atmosphere, the corresponding trans-4-aminocyclohexanol (22 mg, 0.19 mmol), triethylamine (0.03 mL, 0.75 mmol), 1-hydroxybenzotriazole (25 mg, 0.75 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (36 mg, 0.75 mmol) were added sequentially at room temperature, followed by stirring at room temperature for 20 h. After completion of the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:acetone = 1:3) to give the desired compound 15 (17 mg, 44% in 2 steps, 0.06 mmol).

[0087] Compound 15: 3-Benzoyl-N-(trans-4-hydroxycyclohexyl)-1H-pyrazole-5-carboxamide mp: 83-84℃; 1 H-NMR (400 MHz, CDCl3) δ: 1.31-1.53 ​​(4H, m), 2.03-2.14 (4H, m), 3.64-3.71 (1H, m), 3.93-4.02 (1H, m), 6.76 (1H, d, J = 7.6 Hz), 7.36 (1H, s), 7.54 (2H, t, J = 7.6 Hz), 7.66 (1H, t, J = 7.6 Hz), 8.01 (2H, d, J = 7.6 Hz); 1313C-NMR (100 MHz, acetone-d6) δ: 31.34, 48.75, 49.75, 55.47, 69.78, 109.57, 128.98, 129.32, 130.57, 131.25, 133.96, 137.96, 159.74, 186.18; IR (KBr): 3433, 3344, 2856, 1647, 1545, 1319 cm -1 ; MS (EI): m / z 313 (M + ); HRMS: Calcd for C 17 H 19 N3O3 313.1426, Found 313.1423.

[0088] (5) Synthesis of Compound 16

Chem.

[0089] To a solution of 2-bromo-1-phenylethanone 7 (1 g, 5.02 mmol) in chloroform (15 mL) under an Ar atmosphere, hexamethylenetetramine (845 mg, 6.03 mmol) was added at room temperature, followed by stirring at 60 °C for 6 h. After cooling, the precipitated solid was collected by filtration. The resulting solid was dissolved in a mixture of ethanol and concentrated hydrochloric acid (20 mL, 3:1) and heated to reflux and stirred for 16 h. After completion of the reaction, the solvent was removed using a rotary evaporator, and the resulting hydrochloride salt was used in the next reaction without further purification. To a solution of the resulting hydrochloride salt in acetic acid (15 mL) under an Ar atmosphere, ethyl thiooxamate (669 mg, 5.02 mmol) and sodium acetate (989 mg, 12.06 mmol) were added sequentially at room temperature, followed by stirring at reflux for 19 h. After the reaction was complete, the reaction mixture was diluted with ethyl acetate (20 mL) and saturated aqueous sodium bicarbonate (20 mL) was added. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (5 mL × 3) and combined with the organic layer. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 3:1) to give the desired imidazole ester 8 (608 mg, 56% in 3 steps, 2.81 mmol).

[0090] Under an Ar atmosphere, a solution of imidazole ester 8 (220 mg, 1.02 mmol) in ethanol (5 mL) was added with 10% aqueous NaOH (2.5 mL) at room temperature and stirred under reflux for 2 hours. After completion of the reaction, the ethanol was evaporated, and 10% aqueous HCl (5 mL) was added. The aqueous layer was extracted with ethyl acetate (1.5 mL × 3). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting carboxylic acid was used in the next reaction without further purification. To a solution of the resulting carboxylic acid in dichloromethane (5 mL) under Ar atmosphere, the corresponding trans-4-aminocyclohexanol (175 mg, 1.52 mmol), triethylamine (0.21 mL, 1.52 mmol), 1-hydroxybenzotriazole (206 mg, 1.52 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (292 mg, 1.52 mmol) were added sequentially at room temperature, followed by stirring at room temperature for 20 h. After completion of the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:acetone = 1:3) to give the desired compound 16 (171 mg, 59% in 2 steps, 0.60 mmol).

[0091] Compound 17: N-(trans-4-hydroxycyclohexyl)-5-phenyl-1H-imidazole-2-carboxamide mp: 178-180℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.37-1.53 ​​(4H, m), 1.99-2.05 (4H, m), 3.57-3.63 (1H, m), 3.80-3.86 (1H, m), 7.27 (1H, t, J = 7.2 Hz), 7.39 (2H, t, J = 7.2 Hz), 7.57 (1H, s), 7.76 (1H, d, J = 7.2 Hz); 13C-NMR (100 MHz, CD3OD) δ: 31.43, 34.75, 49.30, 70.28, 111.49, 118.65, 126.20, 127.16, 128.24, 128.50, 129.82, 133.98, 142.52, 159.58; IR (KBr): 3311, 3281, 3204, 1653, 1634, 1549, 1456 cm -1 ; MS (EI): m / z 285 (M + ); HRMS: Calcd for C 17 H 20 N2O2285.1477, Found 285.1484.

[0092] (6) Synthesis of Comparative Compounds 1 and 2 [ka]

[0093] Synthesis of comparative compound 1 Under an Ar atmosphere, a solution of ethyl 4-bromo-1H-pyrrole-2-carboxylate 9 (93 mg, 0.43 mmol) in acetonitrile (2 mL) was added with di-tert-butyl dicarbonate (121 mg, 0.56 mmol) and N,N-dimethylaminopyridine (10 mg, 0.09 mmol) sequentially at room temperature, followed by stirring at room temperature for 23 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (3 mL) was added. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (1.5 mL × 3) and combined with the organic layer. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 10:1) to yield the desired Boc-protected product (111 mg, 82%, 0.35 mmol). Phenylboronic acid (60 mg, 0.50 mmol) and tetrakis(triphenylphosphine)palladium (12 mg, 0.01 mmol) were added sequentially to a solution of the Boc-protected compound (63 mg, 0.20 mmol) in N,N-dimethylformamide and saturated aqueous sodium carbonate (2 mL, 3:1) under an Ar atmosphere at room temperature, followed by stirring at 110 °C for 40 h. After completion of the reaction, the solvent was removed using a rotary evaporator, and the resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 2:1) to give the desired pyrrole ester 10 (26 mg, 62%, 0.12 mmol).

[0094] Under an Ar atmosphere, a solution of pyrrole ester 10 (34 mg, 0.16 mmol) in ethanol (1 mL) was added with 10% aqueous NaOH (0.5 mL) at room temperature and stirred under reflux for 2 hours. After completion of the reaction, the ethanol was evaporated, and 10% aqueous HCl (1 mL) was added. The aqueous layer was extracted with ethyl acetate (1 mL × 5). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting carboxylic acid was used in the next reaction without further purification. To a solution of the resulting carboxylic acid in dichloromethane (3 mL) under Ar atmosphere, the corresponding trans-4-aminocyclohexanol (28 mg, 0.24 mmol), triethylamine (0.03 mL, 0.24 mmol), 1-hydroxybenzotriazole (32 mg, 0.24 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (46 mg, 0.24 mmol) were added sequentially at room temperature, followed by stirring at room temperature for 20 h. After completion of the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:acetone = 1:3) to give the desired comparative compound 1 (27 mg, 58% in 2 steps, 0.10 mmol).

[0095] Comparative compound 1: N-(trans-4-hydroxycyclohexyl)-4-phenyl-1H-pyrrole-2-carboxamide mp: 241-243℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.36-1.50 (4H, m), 1.97-2.01 (4H, m), 3.54-3.59 (1H, m), 3.81-3.82 (1H, m), 7.12-7.16 (2H, m), 7.25 (1H, d, J = 1.2 Hz), 7.30 (2H, t, J = 8.0 Hz), 7.53 (2H, dd, J = 8.0, 1.2 Hz); 13 C-NMR (100 MHz, CD3OD) δ: 31.78, 35.11, 49.17, 70.60, 108.94, 119.59, 125.95, 126.71, 127.05, 127.95, 129.68, 136.79, 162.95; IR (KBr): 3439, 3302, 2936, 1622, 1570, 1541, 756 cm -1 ; MS (EI): m / z 284 (M + ); HRMS: Calcd for C 17 H 20 N2O2284.1525.

[0096] Synthesis of comparative compound 2 Phenylboronic acid (80 mg, 0.65 mmol) and tetrakis(triphenylphosphine)palladium (25 mg, 0.02 mmol) were added sequentially to a solution of ethyl 5-bromo-1H-pyrrole-2-carboxylate 11 (95 mg, 0.44 mmol) in toluene and saturated aqueous sodium carbonate (2 mL, 3:1) under an Ar atmosphere at room temperature, followed by stirring at 100 °C for 20 h. After completion of the reaction, the solvent was removed using a rotary evaporator, and the resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 2:1) to give the desired pyrrole ester 12 (40 mg, 43%, 0.19 mmol). Under an Ar atmosphere, a solution of pyrrole ester 12 (37 mg, 0.17 mmol) in ethanol (1 mL) was added with 10% aqueous NaOH (0.5 mL) at room temperature and stirred under reflux for 2 hours. After completion of the reaction, the ethanol was evaporated, and 10% aqueous HCl (1 mL) was added. The aqueous layer was extracted with ethyl acetate (1 mL × 5). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated using a rotary evaporator. The resulting carboxylic acid was used in the next reaction without further purification. To a solution of the resulting carboxylic acid in dichloromethane (3 mL) under Ar atmosphere, the corresponding trans-4-aminocyclohexanol (31 mg, 0.27 mmol), triethylamine (0.04 mL, 0.27 mmol), 1-hydroxybenzotriazole (37 mg, 0.27 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (52 mg, 0.27 mmol) were added sequentially at room temperature, followed by stirring at room temperature for 20 h. After completion of the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (n-hexane:acetone = 1:3) to give the desired comparative compound 2 (32 mg, 62% in 2 steps, 0.11 mmol).

[0097] Comparative compound 2: N-(trans-4-hydroxycyclohexyl)-5-phenyl-1H-pyrrole-2-carboxamide mp: 232-234℃; 1 H-NMR (400 MHz, CD3OD) δ: 1.35-1.49 (4H, m), 1.99-2.01 (4H, m), 3.55-2.57 (1H, m), 3.75-3.90 (1H, m), 6.52 (1H, d, J = 4.0 Hz), 6.87 (1H, d, J = 4.0 Hz), 7.24 (1H, t, J= 7.6 Hz), 7.38 (1H, t, J = 7.6. Hz), 7.66 (2H, dd, J = 7.6, 1.2 Hz); 13IR (KBr): 3414, 3319, 3227, 1624, 1541, 1458, 1339, 1273, 756 cm -1 ; MS (EI): m / z 284 (M + ); HRMS: Calcd for C 17 H 20 N2O2284.1525, Found 284.1521.

[0098] The structural formulas of the compounds synthesized by the above method are shown in Figures 1-1, 1-2 and 1-3.

[0099] Experimental example 1. Experimental Materials (1) Reagents 6-Diazo-5-oxo-L-norleucine (DON) (Wako Pure Chemical Industries, Ltd.), CB-839 (Selleck), 3-Phenyl-1H-1,2,4-triazol-5-amine (1) (Vitas M Chemical, Hong Kong), compound (2) (7127371, Otava Chemicals, Vilniaus, Lithuania), compound (3) (P2001S-209195, Pharmeks, Moscow, Russia), N-(Pyridin-2-ylmethyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxamide (4) (F5791-2259, Life Chemicals, Ontario, Canada), 5-(Furan-2-yl)-N-[(pyridin-3-yl)methyl]-1H-pyrazole-3-carboxamide (5) Z317489074, Enamine, Kiev, Ukraine), 5-(Furan-2-yl)-N-(pyridin-2-ylmethyl)-1H-pyrazole-3-carboxamide (6) (Z317488928, Enamine, Kiev, Ukraine), N-((3-(2-Oxo-1,2-dihydropyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)-1H-pyrazole-5-carboxamide (7) (Life Chemicals), and 2-hydroxy-4-oxo-N-(pyridin-2-ylmethyl)-4H-pyrido[1,2-a]pyrimidine-3-carboxamide (8) (ChemDiv) were all dissolved in DMSO.

[0100] (2) Experimental animals Mice (C57BL / 6JJcl, male, 8 weeks old, Sankyo Laboservice Co., Ltd.) were fed standard solid food (CLEA Rodent Diet CE-2 for mice, rats, and hamsters, CLEA Japan, Inc.) and tap water ad libitum. They were housed in a laboratory animal room under a 12-hour light-dark cycle (8:00 AM to 8:00 PM) and controlled temperature and humidity (24°C, 30%). This experiment was conducted in accordance with the Toyama University Animal Experiment Handling Regulations, with due consideration given to animal ethics, after approval by the Animal Care and Use Committee.

[0101] 2. Preparation of mouse recombinant GLS1 A 2.0-kb DNA fragment of mouse GLS1 was amplified from a mouse brain cDNA library using primers G1U1 (5'-cccatatgatgcggctgcgaggctcggcg-3' (SEQ ID NO: 1), NdeI site) and G1-204L1 (5'-ggggatccttatagcaacccgtcgagattcttg-3' (SEQ ID NO: 2), BamHI site) and KOD-Neo DNA polymerase (TOYOBO). The amplified DNA fragment was subcloned into the EcoRV site of pLITMUS28 (NEB) to obtain plasmid pLITG1. After confirming the DNA sequence using a Genetic Analyzer (ABI), the NdeI-BamHI DNA fragment of pLITG1 was cloned into the NdeI and BamHI sites of the pOPHLT expression vector to obtain pOPG1. pOPG1 was transformed into Origami B (DE3) competent cells (Novagen). 5 μl of glycerol stock (pO-Gls1-204 in Origami), 5 ml of LB, and 5 μl of ampicillin were mixed and shaken at 37° C. overnight.

[0102] 10 g of Bacto Peptone (Thermo), 5 g of dried yeast extract (Wako Pure Chemical Industries), and 5 g of sodium chloride were added to 1 L of deionized water and sterilized to prepare 500 ml of LB medium. 500 μl of ampicillin and 2 ml of culture medium were then added and the mixture was shaken at 37°C until an OD 600 nm of 0.6 was reached. 2.5 ml of 100 mM IPTG (isopropyl-β-D(-)-thiogalactopyranoside) (TaKaRa) was added and the mixture was shaken at 25°C for at least 16 hours to induce expression of the His-tagged recombinant protein. The mixture was centrifuged at 5,000 rpm for 15 min at 4°C, the supernatant was discarded, and the mixture was suspended in 30 ml of 50 mM Tris-HCl (pH 8.0). The mixture was then centrifuged at 10,000 rpm for 5 min at 25°C, the supernatant was discarded, and the pellet was stored at -80°C. The pellet was purified using a HiTag FF column (Pharmacia) and subjected to Ni column purification. It was then subjected to 10% SDS-PAGE and stained with Quick CBB Plus (Wako Pure Chemical Industries) to identify the fraction containing the target protein. The fraction containing the target protein was placed in a dialysis tube (Spectra / Por, REPLIGEN) and dialyzed overnight at 4°C in dialysis buffer (50 mM Tris-phosphate pH 8.0, 1 mM DTT, 10% glycerol). The solution containing GLS1 was concentrated to approximately 1 mg / mL using a centrifugal tube. After confirming the enzyme activity, it was used in experiments.

[0103] 3. Preparation of human recombinant GLS1 DNA encoding the amino acid sequence of hGLS1 (residues 73-669) was inserted into the NdeI and BamHI sites of the pOPTH plasmid (Obita et al., 2007), and the resulting plasmid was transformed into Escherichia coli (strain SoruBL21). When the culture reached an OD600nm of 0.5, IPTG was added to a final concentration of 0.5 mM to induce hGLS1 expression. After overnight incubation at 37°C, cells were harvested by centrifugation at 4°C and resuspended in a buffer containing 20 mM Tris-HCl (pH 8.0 at 4°C), 500 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol. Cells were lysed by sonication on ice, followed by centrifugation at 10,200 × g for 60 minutes at 4°C to obtain the supernatant. The His-tagged hGLS1 protein was purified on a Ni-NTA column and then by gel filtration chromatography on a Superdex HiLoad 16 / 60 column equilibrated with a buffer containing 20 mM Tris (pH 8.0, 4°C) and 100 mM NaCl. The purified protein consists of hGLS1 (residues 73-669) and an N-terminal His-tag.

[0104] 4. Evaluation of glutaminase 1 inhibitory activity (1) Evaluation of glutamate production inhibitory effect using mouse kidney lysate Approximately 50 mg of mouse kidney was homogenized with homogenization buffer (0.25 M sucrose, 5 mM Tris, 1 mM EDTA·2Na, pH 7.4) using a BioMasher III (Nippi Corporation). The supernatant was centrifuged at 6000 x g for 30 seconds at 4°C. Absorbance was then measured at 595 nm using the Bradford method with a Quick Start Protein Assay (5000201JA, BIO-RAD) on an iMark microplate reader (BIO-RAD). The homogenate sample (5 mg / mL) was mixed with a GLS1 inhibitor candidate compound (test compound) dissolved in DMSO or solvent alone (control), and reaction buffer (27.5 mM Tris, 0.11 mM EDTA·2Na, 110 mM KH2PO4, pH 8.6) and incubated at 37°C for 20 minutes. L-glutamine solution (40 mM) was added and the mixture was incubated at 37°C for 10 minutes, after which 10 μL of 3N HCl was added to stop the reaction. Glu concentration was measured by measuring absorbance at 560 nm using the L-glutamic acid measurement kit "Yamasa" NEO (Yamasa Shoyu Co., Ltd.) with a multifunction plate reader (GENios, Tecan). Glu concentration was calculated according to the kit instructions using the following formula:

[0105] L-glutamic acid concentration (mg / L) = (Sample measurement value - Blank)) / (STD measurement value - Blank) x 250

[0106] (2) Evaluation of the inhibitory effect on Glu production using mouse and human recombinant GLS1 A 10.5 μL reaction mixture (Tris-acetate (50 mM, pH 8.6), KPO4 (150 mM, pH 8.0), EDTA (0.2 mM, pH 8.0)) was incubated at 37°C for 20 minutes with 2.5 μL of a GLS1 inhibitor candidate compound (test compound) dissolved in DMSO or solvent alone (control), and 32 μL of recombinant Gls1 stock. L-glutamine solution (20 mM) was added to each reaction mixture and incubated at 37°C for 1 hour. Glu concentrations were measured by measuring the absorbance at 555 nm using the Yamasa NEO L-glutamate assay kit on a UV-visible spectrophotometer (GeneQuant 1300, Biochrom).

[0107] 5.Statistical analysis Measurement data are expressed as mean ± standard error. Statistical analysis of the experiments was performed using JMP Pro version 15.2.0 (SAS Institute Inc, NC, USA) using the Tukey-Kramer honest significant difference test. p < 0.05 was considered statistically significant.

[0108] Experimental Example 1: GLS1 inhibitory activity of compounds selected by in silico screening Eight compounds (Figure 2(A)) were selected as candidate GLS1 inhibitors from a commercially available compound database through a primary screening using a 3D pharmacophore obtained from the complex crystal structure of glutaminase and a known GLS1 inhibitor, filtering by Lipinski's "Rule of Five", secondary screening using molecular docking calculations, filtering by binding free energy calculations, filtering based on docking poses, and molecular similarity analysis based on chemical structure.

[0109] These candidate compounds were evaluated for GLS1 inhibitory activity by measuring glutamate (Glu) production rates using mouse kidney lysate. As positive control compounds, DON (6-Diazo-5-oxo-L-norleucine), a known GLS1 inhibitor shown in the formula below, and CB-839 were used.

[0110] [ka]

[0111] [ka]

[0112] DON and CB-839 were dissolved in DMSO to a final concentration of 1 mM. The eight candidate compounds were dissolved in DMSO to a final concentration of 10 mM. As a control, the Glu production rate was measured in the same manner using a system in which only the solvent was added instead of the compounds.

[0113] The results are shown in Figure 2(B). As shown in the figure, candidate compound 3 had the strongest GLS1 inhibitory activity, reducing Glu production by 53% compared to the control. From the correlation between the structures of candidate compounds 1 to 8 and their GLS1 inhibitory activities, it was inferred that the pyrazole ring and the carbonyl group in the amide moiety in the structure of candidate compound 3 are important for GLS1 inhibitory activity.

[0114] Experimental Example 2: Structure-activity relationship In Experimental Example 1, candidate compound 3 exhibited approximately 50% GLS1 inhibitory activity, and therefore, multiple compounds were synthesized using candidate compound 3 as a lead compound. Then, for these compounds, the glutamic acid (Glu) production rate was determined using an evaluation method using mouse kidney lysate, and the GLS1 inhibitory activity was evaluated.

[0115] Using GLS1 inhibitory activity as an index, the structure-activity relationship with the lead compound (candidate compound 3) was examined from the following four points. 1) Modification of the nitrogen substituent at the amide moiety 2) Conversion of the substituents on the benzene ring 3) Increasing or decreasing the number of carbon atoms in the linker between the benzene ring and the pyrazole ring 4) Conversion of the pyrazole ring into other heterocycles.

[0116] Table 1 shows the correspondence between the symbols of the compounds used in the experiment and the numbers of the compounds described above. [Table 1]

[0117] (1) Modification of the nitrogen substituent at the amide moiety Following the procedure described in the above-mentioned Preparation Example, amide derivatives 2a to 2l having a secondary or tertiary amino moiety or a cyclic amine were synthesized by condensation reaction of the carboxylic acid of known compound (1) with an amine (Scheme 1), and their GLS1 inhibitory activity was evaluated (Figures 3(A) and (B)).

[0118] [ka] As a result, all amide derivatives except for 2c, in which R is an NHPh group, exhibited GLS1 inhibitory activity. However, amide derivative 2f, in which R is a pyrrolidinyl group, exhibited low GLS1 inhibitory activity. Among these amide derivatives, amide derivative 2j, in which R is a trans-4-hydroxycyclohexylamino group, exhibited the strongest GLS1 inhibitory activity, and the Glu production rate at 10 mM was 99% lower than that of the control (Figure 3(B)).

[0119] (2) Transformation of the benzene ring substituent Next, the structure of the amide moiety was fixed, and the hydrogen atom of the benzene ring was substituted with another atom or a functional group (R), and the GLS1 inhibitory activity was evaluated (Figures 3(A) and (B)). Specifically, as described in the above Preparation Example, commercially available phenylacetones 3a to 3j were converted to ethyl esters 4a to 4j in two steps (Scheme 2). Next, the ester moiety of the ethyl esters 4a to 4j was hydrolyzed, and the resulting carboxylic acids were subjected to a condensation reaction to obtain derivatives 5a to 5j. Furthermore, by deprotecting the methoxymethyl group of derivative 5j, derivative 5k, which has an OH group at the 4-position of the benzene ring, was obtained. The GLS1 inhibitory activity of these derivatives 5a to 5i and 5k was evaluated (Figure 3(B)).

[0120] [ka]

[0121] As shown in Figure 3(B), all derivatives exhibited GLS1 inhibitory activity. Among these, derivative 5i, in which the hydrogen atom at position 4 of the benzene ring was replaced with a fluorine atom, exhibited the strongest GLS1 inhibitory activity, with its Glu production rate at a 10 mM concentration being 90% lower than that of the control. However, the GLS1 inhibitory activity of derivative 5i was lower than that of the amide derivative 2j, suggesting that the introduction of a substituent into the benzene ring tends to decrease GLS1 inhibitory activity.

[0122] (3) Increasing or decreasing the number of carbon atoms in the linker between the benzene ring and the pyrazole ring To investigate the effect of increasing or decreasing the number of carbon atoms in the linker between the benzene ring and the pyrazole ring, two derivatives, 8a and 8b, were synthesized. Specifically, as shown in Scheme 3, phenyl methyl ketone 6a and benzyl acetone 6b were first converted to amide derivative 8a, in which the benzene ring and the pyrazole ring are directly bonded, and amide 8b, which has two linker carbon atoms (ethylene groups) between the two rings, using a method similar to that described in Scheme 2.

[0123] [ka]

[0124] The GLS1 inhibitory activity of these amide derivatives (8a and 8b) was evaluated (FIGS. 3(A) and (B)). As shown in FIG. 3(B), both amide derivatives 8a and 8b were found to have GLS1 inhibitory activity. However, compared to the GLS1 inhibitory activity of amide derivative 8a, the GLS1 inhibitory activity of amide derivative 8b was lower, indicating that the GLS1 inhibitory activity depends on the number of carbon atoms (length) of the linker, and that the GLS1 inhibitory activity tends to be higher when the number of carbon atoms (number n) of the linker is smaller, preferably when n = 0.

[0125] (4) Substitution of the pyrazole ring with other heterocycles As shown in Scheme 4 below, imidazole derivative 10 was synthesized from the known ester compound 8 prepared from 2-bromoacetophenone.

[0126] [ka]

[0127] Furthermore, as shown in Scheme 5 below, pyrrole derivatives 13 and 14 were similarly synthesized from known ester compounds 10 and 12, respectively.

[0128] [ka]

[0129] The GLS1 inhibitory activity of these derivatives 13 and 14 was evaluated (FIG. 3(B)). As shown in Figure 3(B), imidazole derivative 10 had relatively high GLS1 inhibitory activity, although it was slightly lower than the corresponding pyrazole derivative 8a. In contrast, the corresponding pyrrole derivatives 13 and 14 exhibited significantly reduced GLS1 inhibitory activity. This suggests that the heterocycle (pyrazole ring or imidazole ring) containing two nitrogen atoms as the heterocycle forming the basic skeleton is important for GLS1 inhibitory activity.

[0130] From the above results, as shown in FIG. 3(B), among the compounds evaluated above, pyrazole derivatives 2j, 5i, and 8a in particular were found to have exceptionally high GLS1 inhibitory activity.

[0131] Experimental Example 3: Evaluation of GLS1 inhibitory activity using recombinant GLS1 The GLS1 inhibitory activity of pyrazole derivatives 5i and 8a, which were confirmed to have high GLS1 inhibitory activity in Experimental Example 2, was reevaluated using mouse and human recombinant GLS1 (FIGS. 4 and 5). In the evaluation using mouse recombinant GLS1, the positive controls DON and CB-839 showed IC50 The IC values ​​for DON were approximately 1 mM and for CB-839 were between 10 and 100 nM, consistent with previous reports. As shown in Figure 4, both pyrazole derivatives 5i and 8a significantly suppressed Glu production at 10 mM, which was inhibitory activity in mouse kidney extracts. 50 It was confirmed that the concentration of these compounds was between 1 and 10 mM. Furthermore, as shown in Figure 5, all of these compounds significantly suppressed Glu production in human recombinant GLS1 at 10 mM, demonstrating high GLS1 inhibitory activity.

[0132] Experimental Example 3: Evaluation of anti-cancer activity GLS1 has been implicated in the progression of various cancers, and inhibiting glutamine metabolism through GLS1 inhibition is known to suppress the growth and proliferation of cancer cells. Currently, clinical trials of various compounds with GLS1 inhibitory activity are being conducted in patients with renal cell carcinoma, colorectal cancer, non-small cell lung cancer, melanoma, and advanced myelodysplastic syndrome, and therapeutic effects on various types of cancer are expected.

[0133] Therefore, the pyrazole derivatives 5i and 8a, which have particularly high GLS1 inhibitory activity, were used to evaluate their cancer cell proliferation inhibitory effects as representative compounds with GLS1 inhibitory activity confirmed in Experimental Example 2. The cancer cells used were a human breast adenocarcinoma-derived cell line (MCF7) and an acute myeloid leukemia-derived cell line (MOLM13).

[0134] The experiment was carried out using 3 × 10 3 The pyrazole derivatives 5i and 8a were added to MCF7 cells (cells / ml) at various concentrations and cultured. The cell number was counted on the 7th day from the start of culture, and the cell growth inhibitory effect was evaluated as the ratio to the cell number (100%) of the control (DMSO added instead of the pyrazole derivative) (n=3). For MCF7 cells, the medium was changed on the 1st, 3rd, and 6th days of culture.

[0135] The results for the human breast adenocarcinoma-derived cell line (MCF7) and acute myeloid leukemia-derived cell line (MOLM13) are shown in Figures 6 and 7, respectively. As shown in Figure 6, pyrazole derivatives 5i and 8a inhibited cell proliferation in MCF7 cells by 40% and 67%, respectively, at 100 μM (IC 50 [μM]: 5i = 77.5, 8a = 93.1). As shown in Figure 7, pyrazole derivatives 5i and 8a inhibited cell proliferation in MOLM13 cells by 60% and 92.5%, respectively, at 100 μM (IC 50 [μM]: 5i = 58.6, 8a = 62.1).

[0136] As shown by these results, the compounds having GLS1 inhibitory activity confirmed in Experimental Example 2, as represented by pyrazole derivatives 5i and 8a, exhibit the effect of suppressing the proliferation of cancer cells based on their GLS1 inhibitory activity, and have been confirmed to be effective in anticancer therapy, such as for the prevention and treatment of cancer, the inhibition of cancer progression, and the prevention of cancer recurrence. [Sequence List Free Text]

[0137] SEQ ID NO: 1 is the nucleotide sequence of primer G1U1 (NdeI site) used to prepare mouse recombinant GLS1, and SEQ ID NO: 2 is the nucleotide sequence of primer G1-204L1 (BamHI site) used to prepare the same.

Claims

1. A glutaminase 1 inhibitor comprising a compound represented by general formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient: 【Chemistry 1】 [In formula (I), X is CH ２ or C=O; n represents an integer of 0 to 2, provided that when X is C═O, n is 1. Y represents an unsubstituted pyrazole group having bonds at the 3- and 5-positions, or an unsubstituted imidazole group having bonds at the 2- and 5-positions; R １ represents a hydrogen atom, a halogen atom, a haloalkyl group, an alkoxy group, an alkyl group, or a hydroxyl group; R ２ represents a group represented by any one of the following formulas (a) to (c): 【Chemistry 2】 (In formula (a), A represents a hydroxyl group or an oxygen atom; A double line consisting of a dotted line and a solid line represents a single bond when A is a hydroxyl group, and a double bond when A is an oxygen atom. The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I); 【Transformation 3】 (In formula (b), B and D are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group, and the asterisk represents the bond to the carbon atom of the carbonyl group in formula (I); 【Chemistry 4】 (In formula (c), E represents an oxygen atom or an alkylene group in which one hydrogen atom may be substituted with a hydroxyl group; The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I); When n is 0, R1 is not an alkoxy group; When R1 is a hydroxyl group, R ２ is a group represented by formula (b) or (c).

2. In the compound represented by formula (I), R ２ is a group represented by formula (a), in which A is a hydroxyl group, and the double line consisting of a dotted line and a solid line is a single bond; X is CH ２ , n is an integer from 0 to 2; The glutaminase 1 inhibitor according to claim 1.

3. In the compound represented by formula (I), R ２ is a group represented by formula (b), and X is CH ２ , n is an integer from 0 to 2; The glutaminase 1 inhibitor according to claim 1.

4. In the compound represented by formula (I), R ２ is a group represented by formula (c), and X is CH ２ , n is 0 to 2; The glutaminase 1 inhibitor according to claim 1.

5. A pharmaceutical composition, food or drink composition, or cosmetic composition, comprising the glutaminase 1 inhibitor according to any one of claims 1 to 4 as an active ingredient.

6. 6. The pharmaceutical composition, food or beverage composition, or cosmetic composition according to claim 5, which is used for the prevention, amelioration, or treatment of inflammation, diseases caused by inflammation, obesity, metabolic syndrome, aging, or cancer.

7. A compound having glutaminase 1 inhibitory activity represented by general formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof: 【Transformation 5】 [In formula (I), X is CH ２ or C=O; n represents an integer of 0 to 2, provided that when X is C═O, n is 1. Y is an unsubstituted pyrazole group having bonds at the 3- and 5-positions, or an unsubstituted imidazole group having bonds at the 2- and 5-positions; R １ represents a hydrogen atom, a halogen atom, a haloalkyl group, an alkoxy group, an alkyl group, or a hydroxyl group; R ２ represents a group represented by any one of the following formulas (a) to (c): 【Transformation 6】 (In formula (a), A represents a hydroxyl group or an oxygen atom; A double line consisting of a dotted line and a solid line represents a single bond when A is a hydroxyl group, and a double bond when A is an oxygen atom. The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I); 【Transformation 7】 (In formula (b), B and D are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group, and the asterisk represents the bond to the carbon atom of the carbonyl group in formula (I); 【Transformation 8】 (In formula (c), E represents an oxygen atom or an alkylene group in which one hydrogen atom may be substituted with a hydroxyl group; The asterisk represents the bond to the carbon atom of the carbonyl group in formula (I); When n is 0, R1 is not an alkoxy group; When R1 is a hydroxyl group, R ２ is a group represented by formula (b) or (c). However, N-(4-hydroxycyclohexyl)-3-phenyl-1H-pyrazole-5-carboxamide is excluded.

8. The R ２ is a group represented by formula (a), In formula (a), A represents a hydroxyl group, and the double line consisting of a dotted line and a solid line represents a single bond. X is CH ２ , n is an integer from 0 to 2; The compound according to claim 7.

9. In the compound represented by formula (I), R ２ is a group represented by formula (b), and X is CH ２ , n is an integer from 0 to 2; The compound according to claim 7.

10. In the compound represented by formula (I), R ２ is a group represented by formula (c), and X is CH ２ , n is 0 to 2; The compound according to claim 7.

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