S-nitrosylation inhibitor of ire1α and screening method thereof

Abstract

To provide an S-nitrosylation inhibitor of IRE1α and screening method thereof.SOLUTION: Provided is an S-nitrosylation inhibitor of IRE1α represented by the following formula (I): (A is a monovalent α,β-unsaturated carbonyl group; B and C are the same or different and either (i) or (ii): (i) B and C together form a substituted / unsubstituted monocyclic or polycyclic ring, the monocyclic or polycyclic ring containing at least a 6- or 7-membered heterocyclic ring; and (ii) B and C are the same or different and are a substituted / unsubstituted monovalent lower alkyl group, a substituted / unsubstituted monovalent aryl group, or a substituted / unsubstituted monovalent cyclic group which may contain a heteroatom, provided that B and C are not both lower alkyl groups; and L1 and L2 are the same or different and are divalent linkers having 1 to 4 carbon atoms, provided that when B and C together form a monocyclic or polycyclic ring, L1 and L2 are absent).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of S-nitrosylation of IRE1α and a screening method for the same. [Background technology]

[0002] Neurodegenerative diseases, including Parkinson's disease, are characterized by neuronal cell death. Aging is considered to be the greatest risk factor for the onset of neurodegenerative diseases. In recent years, nitric oxide (NO), whose production is promoted by aging and inflammation, has attracted attention as one of the factors that cause neuronal cell death.

[0003] Endogenous NO plays a role in the body in memory formation, vasodilation, blood pressure control, and defense against bacterial and viral attacks. However, excessive NO production due to aging and inflammation is known to cause neurological disorders such as cerebral infarction, Alzheimer's disease, and Parkinson's disease. On the other hand, a lack of NO production is also known to cause high blood pressure. Thus, it is important that NO is properly controlled in the body.

[0004] NO inhibits protein activity by oxidatively modifying proteins (S-nitrosylation), contributing to the onset of disease (Non-Patent Document 1). Drug discovery strategies targeting excessive NO production have included the development of NO synthase inhibitors (Non-Patent Documents 2 and 3) and radical scavengers (Non-Patent Document 4).

[0005] However, as mentioned above, NO has various physiological functions in the body, and NO synthase inhibitors that uniformly block the physiological functions of NO have significant effects on the periphery, particularly the circulatory system, and due to their side effects, they have not yet been put to practical use as drugs. While efforts have been made to develop NO synthase inhibitors and radical scavengers that are more selective for specific NO synthase isoforms, these are insufficiently effective against proteins that undergo S-nitrosylation. Currently, NO synthase inhibitors are only used topically for hair loss and other conditions.

[0006] The endoplasmic reticulum is an organelle responsible for protein synthesis. When various stresses or external factors disrupt the endoplasmic reticulum's protein maturation and quality control mechanisms, denatured proteins accumulate in the endoplasmic reticulum lumen, leading to a state known as endoplasmic reticulum stress. In an endoplasmic reticulum stress state where endoplasmic reticulum function is impaired, new proteins are not produced normally. For this reason, cells have a mechanism to avoid endoplasmic reticulum stress (unfolded protein response; UPR), which maintains the endoplasmic reticulum in a normal state.

[0007] Inositol-requiring enzyme 1 α (IRE1α) is one of the sensor proteins that detects ER stress and activates the UPR as follows. IRE1α is an enzyme with ribonuclease (RNase) activity that splices full-length Xbp1 mRNA (Xbp1u) in response to ER stress. Xbp1s, generated after splicing, are translated and function as the transcription factor XBP1, which induces the expression of ER stress-relieving molecules such as chaperones.

[0008] The present inventors previously discovered that NO induces endoplasmic reticulum stress (Non-Patent Document 1). Furthermore, they found that the mechanism is through the binding of NO to IRE1α (Non-Patent Document 5). Specifically, S-nitrosylation of IRE1α by NO significantly attenuates the RNase activity of IRE1α. Thus, NO not only induces endoplasmic reticulum stress but also promotes cell death by rendering cells unable to cope with stress, which is thought to contribute to neurodegenerative diseases.

[0009] Thus, APY29, sunitinib (Non-Patent Document 6, Patent Document 1), compound 3 (Non-Patent Document 7), and IXA4 (Non-Patent Document 8) have been identified as IRE1α RNase activators. However, because overactivation of IRE1α can drive cell death pathways and lead to cell carcinogenesis, there are concerns that simple activating drugs may cause serious side effects. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Uehara et al., Nature. 2006 May 25;441(7092):513-7. [Non-patent document 2] Vallance et al., Nat Rev Drug Discov. 2002 Dec;1(12):939-50. [Non-patent document 3] Minhas et al., Med Res Rev. 2020 May;40(3):823-855. [Non-patent document 4] Kuehl et al., Inflammation. 1977 Dec;2(4):285-94. [Non-patent document 5] Nakato et al., Sci Rep. 2015 Oct 8;5:14812. [Non-patent document 6] Wang et al., Nat Chem Biol. 2012 Dec;8(12):982-9. [Non-Patent Document 7] Joshi et al., Oncotarget. 2015 May 30;6(15):13019-35. [Non-patent document 8] Grandjean et al., Nat Chem Biol. 2020 Oct;16(10):1052-1061. [Patent documents]

[0011] [Patent Document 1] Special Publication 2015-532287 Summary of the Invention [Problem to be solved by the invention]

[0012] Based on this background, the present inventors have considered that controlling IRE1α activity is important for avoiding NO-induced neuronal cell death, and have conceived the present invention, which specifically inhibits S-nitrosylation of IRE1α. An objective of the present invention is to provide a therapeutic drug for neurodegenerative diseases with a novel mechanism of action and a screening method for the same. [Means for solving the problem]

[0013] As a result of extensive research, the present inventors have discovered that NO-induced S-nitrosylation of IRE1α inhibits splicing from Xbp1u to Xbp1s, blocking the pathway responsible for alleviating endoplasmic reticulum stress and inducing cell death. The inventors have found that inhibiting NO-induced S-nitrosylation of IRE1α is important for avoiding NO-induced cell death, leading to the completion of the present invention. Specifically, the present invention includes the following aspects.

[0014] Section 1. The following formula (1) [ka] A is a monovalent α,β-unsaturated carbonyl group; B and C are the same or different and are either (i) or (ii): (i) B and C together form a monocyclic or polycyclic ring, and the monocyclic or polycyclic ring contains at least a 6- or 7-membered heterocyclic ring and may be substituted with one or more substituents; (ii) B and C are the same or different and are each an optionally substituted monovalent lower alkyl group, an optionally substituted monovalent aryl group, or an optionally substituted monovalent cyclic group which may contain a heteroatom, and B and C are not both lower alkyl groups; L1 and L2 are the same or different and are divalent linkers having 1 to 4 carbon atoms, provided that when B and C are joined together to form a monocycle or polycycle, L1 and L2 do not exist. or a salt thereof as an active ingredient, an S-nitrosylation inhibitor of inositol-requiring enzyme 1α (IRE1α). Section 2. The S-nitrosylation inhibitor of IRE1α described in claim 1, wherein in the formula (1), A is a monovalent α,β-unsaturated carbonyl group including an alkenyl group which may be substituted with a substituent, or an alkynyl group substituted with a lower alkyl group. Section 3. The S-nitrosylation inhibitor of IRE1α described in claim 1, wherein in the formula (1), B and C together form the monocycle consisting of a heterocycle containing two or more N atoms or O atoms. Section 4. The S-nitrosylation inhibitor of IRE1α described in claim 1, wherein in the formula (1), B and C are the same or different and are a monovalent lower alkyl group substituted with a carboxylic acid amide group, a monovalent aryl group substituted with a halogen group, or a monovalent heterocyclic group in which a hydrogen atom bonded to a heteroatom is substituted with an acyl group. Section 5. The compound is represented by the following formula (2): [ka] (X is a divalent lower alkyl group optionally substituted with a carboxyl group, or a divalent monocyclic or polycyclic group containing at least a 6-membered heterocyclic or aromatic ring; Y 1 and Y 2 are the same or different and represent a carboxyl group, a carboxylic acid amide group, a monovalent lower alkyl group, a halogen atom, or a carbamoyl group, n1 represents an integer of 0 to 3, and n2 represents an integer of 0 to 4. The S-nitrosylation inhibitor of IRE1α according to claim 1, which is a compound represented by: Section 6. The compound is represented by the following formula (3): [ka] (X is a monocyclic or polycyclic group containing at least a divalent lower alkyl group, an aryl group, or a heteroaryl group, which may be substituted with a carboxyl group, or which may be substituted with a halogen atom; Y 1 and Y 2 are the same or different and each represents a carboxyl group, a carboxylic acid amide group, a monovalent lower alkyl group, a halogen atom, or a carbamoyl group; Z is a lower alkyl group; n1 is an integer of 0 to 3; and n2 is an integer of 0 to 3. The S-nitrosylation inhibitor of IRE1α according to claim 5, which is a compound represented by the formula: Section 7. The compound is represented by the following formula (4): [ka] (X is a divalent lower alkyl group optionally substituted with a carboxyl group, or a divalent monocyclic or polycyclic group containing at least a 6-membered heterocycle or aromatic ring; L3 is a divalent linker having 1 to 4 carbon atoms, the carbon optionally being substituted with a heteroatom; and T is an aryl group optionally substituted with a halogen.) The S-nitrosylation inhibitor of IRE1α according to claim 1, which is a compound represented by: Section 8. The compound is represented by the following formula (1): [ka] (A is a monovalent α,β-unsaturated carbonyl group containing an alkynyl group substituted with a lower alkyl group, B and C together form a monocyclic or polycyclic ring, and the monocyclic or polycyclic ring contains at least a 6- or 7-membered heterocyclic ring and may be substituted with one or more substituents. The S-nitrosylation inhibitor of IRE1α according to claim 1, which is a compound represented by: Section 9. The compound is represented by the following formula (5): [ka] An S-nitrosylation inhibitor of IRE1α, comprising a compound represented by the formula: or a salt thereof as an active ingredient. Section 10. The S-nitrosylation inhibitor of IRE1α according to claims 1 to 9, which is a therapeutic agent for a neurodegenerative disease. Section 11. The therapeutic agent for neurodegenerative diseases according to claim 10, wherein the neurodegenerative disease is Parkinson's disease. Section 12. An S-nitrosylation inhibitor of IRE1α according to claims 1 to 9, used for the treatment of diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, hepatic steatosis, liver fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, cardiac fibrosis or rheumatoid arthritis. Section 13. A method for screening for an S-nitrosylation inhibitor of IRE1α, comprising the steps of culturing cells containing IRE1α in the presence of a nitric oxide generator and a candidate substance, and detecting Xbp1u and Xbp1s. [Effects of the Invention]

[0015] The present invention provides an inhibitor of S-nitrosylation of IRE1α. This inhibitor can prevent NO-induced cell death by inhibiting NO-induced S-nitrosylation of IRE1α. The present invention provides a therapeutic agent for neurodegenerative diseases that avoids the problems of the prior art by utilizing a novel mechanism of action that inhibits only NO-induced S-nitrosylation of IRE1α.

[0016] The IRE1α S-nitrosylation inhibitors of the present invention inhibit only the NO-induced S-nitrosylation of IRE1α without interfering with the RNase activity of IRE1α or the physiological role of NO in vivo, and are therefore advantageous because they maintain the function of IRE1α even in the presence of NO and can reduce side effects (toxicity). Thus, the IRE1α S-nitrosylation inhibitors of the present invention have advantageous effects that set them apart from existing drugs that regulate NO or IRE1α themselves. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the endoplasmic reticulum stress response mechanism of IRE1α. [Figure 2] FIG. 1 shows the mechanism by which NO regulates IRE1α activity. [Figure 3] FIG. 1 shows a method for detecting S-nitrosylated IRE1α. [Figure 4] FIG. 1 shows the effect of Compound 2-26 on SNO-IRE1α formation. [Figure 5] FIG. 1 shows a method for measuring NO in a culture medium. [Figure 6] FIG. 1 shows the effect of Compound 2-26 on NO in the medium. [Figure 7] FIG. 10 shows the effect of Compound 2-26 on NO-induced decrease in IRE1α RNase activity. [Figure 8] FIG. 1 shows the effect of Compound 2-26 on IRE1α RNase activity. [Figure 9] FIG. 1 shows the effect of Compound 2-26 on NO-induced cell death. [Figure 10] FIG. 1 shows the effect of Compound 2-26 on MPP+-induced cell death. [Figure 11] FIG. 1 shows the modification site of IRE1α Cys931 by Compound 2-26. [Figure 12] FIG. 1 shows the modification site of IRE1α Cys951 by Compound 2-26. DETAILED DESCRIPTION OF THE INVENTION

[0018] Figure 1 shows the ER stress response mechanism of IRE1α according to the present invention. Accumulation of denatured proteins in the ER lumen induces ER stress, activating the sensor protein IRE1α, which splices Xbp1u to Xbp1s. Xbp1s is translated and functions as the transcription factor XBP1, which induces the expression of ER stress-relieving molecules such as chaperones, thereby enabling cells to survive.

[0019] The mechanism by which NO regulates IRE1α activity is shown in Figure 2. When IRE1α is S-nitrosylated, IRE1α is not activated even when endoplasmic reticulum stress is induced, resulting in the persistence of endoplasmic reticulum stress and cell death.

[0020] The present invention presents a drug with a new mechanism of action that targets S-nitrosylation of IRE1α. The drug of the present invention is characterized by inhibiting only S-nitrosylation of IRE1α (Figure 4) without affecting NO in vivo (Figure 6) or activating IRE1α RNase activity (Figure 8). The drug of the present invention, identified starting from in silico virtual screening, is expected to act on the S-nitrosylation site of IRE1α. Therefore, this result, in which the drug does not exhibit the same behavior as NO despite having the same site of action, was completely unexpected. The drug of the present invention restores IRE1α RNase activity attenuated by NO (Figure 7), and exhibits an inhibitory effect on NO-induced neuronal cell death (Figure 9) and an inhibitory effect on MPP. + The present invention shows an inhibitory effect on neuronal cell death induced by α-glucan (FIG. 10), which has advantageous effects over the prior art.

[0021] The S-nitrosylation inhibitor of IRE1α in the present invention has an inhibitory effect on S-nitrosylation by NO by covalently binding to the C931 or C951 residue of IRE1α, more preferably the C931 residue.

[0022] The meaning of each term used in this specification is explained below. Unless otherwise specified, each term has the same meaning whether used alone or in combination with other terms. The term "consisting of" means having only the constituent elements. The term "comprising" is meant to be open-ended and not to exclude unlisted elements.

[0023] The IRE1α S-nitrosylation inhibitor of the present invention comprises a compound represented by general formula (1) or a salt thereof as an active ingredient.

[0024] Formula (1) [ka] A is a monovalent α,β-unsaturated carbonyl group, B and C together form a monocyclic or polycyclic ring, said monocyclic or polycyclic ring containing at least a 6- or 7-membered heterocyclic ring, and optionally substituted with one or more substituents; When B and C are taken together to form a monocyclic or polycyclic ring, L1 and L2 are absent.

[0025] In a preferred embodiment of the present invention, A is a monovalent α,β-unsaturated carbonyl group including an alkenyl group which may be substituted with a substituent, or an alkynyl group substituted with a lower alkyl group, and the lower alkyl group is preferably a methyl group; B and C together form a monocyclic or polycyclic ring, said monocyclic or polycyclic ring containing at least a 6- or 7-membered heterocyclic ring, and optionally substituted with one or more substituents; When B and C are taken together to form a monocyclic or polycyclic ring, L1 and L2 are absent.

[0026] In another preferred embodiment of the invention, A is a monovalent α,β-unsaturated carbonyl group, B and C together form a monocyclic ring consisting of a heterocyclic ring containing two or more N atoms or O atoms, the monocyclic ring containing at least a 6- or 7-membered heterocyclic ring and optionally substituted with one or more substituents; When B and C are taken together to form a monocyclic or polycyclic ring, L1 and L2 are absent.

[0027] In another preferred embodiment of the present invention, the compound is represented by the following formula (5): [ka] or a salt thereof as an active ingredient.

[0028] In another preferred embodiment of the present invention, the compound is represented by the following formula (6): [ka] or a salt thereof as an active ingredient.

[0029] In another preferred embodiment of the present invention, A is a monovalent α,β-unsaturated carbonyl group; L1 and L2 are the same or different and are divalent linkers having 1 to 4 carbon atoms; B and C are the same or different and are an optionally substituted monovalent lower alkyl group, an optionally substituted monovalent aryl group, or an optionally substituted monovalent cyclic group which may contain a heteroatom; and B and C are not simultaneously lower alkyl groups.

[0030] In another preferred embodiment of the present invention, A is a monovalent α,β-unsaturated carbonyl group; L1 and L2 are the same or different and are divalent linkers having 1 to 4 carbon atoms; B and C are the same or different and are a monovalent lower alkyl group substituted with a carboxylic acid amide group, a monovalent aryl group substituted with a halogen group, or a monovalent heterocyclic group in which a hydrogen atom bonded to a heteroatom is substituted with an acyl group; and B and C are not both lower alkyl groups. The lower alkyl group is preferably a methyl group. The halogen is preferably chlorine or fluorine.

[0031] In another preferred embodiment of the present invention, the IRE1α S-nitrosylation inhibitor of the present invention comprises a compound represented by general formula (7) or a salt thereof as an active ingredient. [ka] A is a monovalent α,β-unsaturated carbonyl group, and M 1is a monovalent aryl group which may be substituted with a halogen group, and the halogen is preferably chlorine or fluorine.

[0032] In another preferred embodiment of the present invention, the IRE1α S-nitrosylation inhibitor of the present invention comprises a compound represented by general formula (8) or a salt thereof as an active ingredient. [ka] M 2 is a monovalent aryl group which may be substituted with a halogen group, and the halogen is preferably chlorine or fluorine.

[0033] An αβ-unsaturated carbonyl refers to a carbon-carbon double bond and a carbon-carbon triple bond adjacent to an aldehyde or ketone.

[0034] The alkyl group includes a straight-chain or branched hydrocarbon group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, and n-decyl. Of these, methyl is preferred.

[0035] Examples of the lower alkyl group include straight-chain or branched C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, etc. Among these, methyl is preferred.

[0036] The term "alkenyl" refers to a linear or branched hydrocarbon group having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms, and having one or more double bonds at any position. Examples of alkenyl include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, and pentadecenyl. Of these, vinyl and allyl are preferred.

[0037] The alkenyl may be unsubstituted or substituted with one type of substituent. The substituent is one type of group selected from lower alkyl, COOH, and monocyclic or polycyclic substituents containing at least a 6-membered heterocycle or aromatic ring. The lower alkyl is preferably methyl. The heterocycle or aromatic ring may be substituted with a halogen.

[0038] Alkynyl includes linear or branched hydrocarbon groups having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms, and having one or more triple bonds at any position. Examples include ethynyl and propynyl. Of these, propynyl is preferred.

[0039] The alkynyl may be unsubstituted or substituted with a lower alkyl group, preferably methyl.

[0040] The aromatic ring means a monocyclic or polycyclic aromatic hydrocarbon ring.

[0041] The heterocycle means a monocyclic or polycyclic hydrocarbon ring having one or more identical or different heteroatoms selected from O, N and S in the ring.

[0042] The heteroatom is selected from N, O and S, and is preferably N or O. The number of heteroatoms is, for example, 1 to 3, and preferably 2.

[0043] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, with a fluorine atom and a chlorine atom being preferred.

[0044] Examples of the substituent for A include OH, NH, COOH, NO, NO, carbamoyl, sulfamoyl, halogen atoms, lower alkyl groups, aryl groups, lower alkoxy groups, aryloxy groups, lower alkylthio groups, mono-lower alkylamino groups, di-lower alkylamino groups, acyl groups, acyloxy groups, acylamino groups, lower alkoxycarbonyl groups, cycloalkyl groups, lower alkenyl groups, lower alkynyl groups, mono-lower alkyl-substituted carbamoyl groups, di-lower alkyl-substituted carbamoyl groups, and lower alkoxycarbonylamino groups. Among these, COOH, methyl groups, aryl groups, and heteroaryl groups are preferred. The methyl group is preferably substituted at the α-position of the αβ-carbonyl. The COOH, aryl groups, and heteroaryl groups are preferably substituted at the β-position. The aryl and heteroaryl groups may be substituted with halogen. The number of substituents is, for example, 0 to 10, preferably 1 to 3.

[0045] When B and C together form a monocyclic or polycyclic ring, examples of the substituent include OH, NH, COOH, NO, NO, carbamoyl, sulfamoyl, halogen atoms, lower alkyl groups, aryl groups, lower alkoxy groups, aryloxy groups, lower alkylthio groups, mono-lower alkylamino groups, di-lower alkylamino groups, acyl groups, acyloxy groups, acylamino groups, lower alkoxycarbonyl groups, cycloalkyl groups, lower alkenyl groups, lower alkynyl groups, mono-lower alkyl-substituted carbamoyl groups, di-lower alkyl-substituted carbamoyl groups, and lower alkoxycarbonylamino groups. Among these, COOH, methyl groups, aryl groups, and heteroaryl groups are preferred. The number of substituents is, for example, 0 to 10, preferably 1 to 3.

[0046] When B and C are taken together to form a monocycle, it preferably contains two or more heteroatoms selected from N and O, and more preferably has a piperazine structure containing N at the 1st and 4th positions of the heterocycle.

[0047] When B and C are different, examples of the substituent for B include OH, NH, COOH, NO, NO, carbamoyl, sulfamoyl, halogen atoms, lower alkyl, aryl, lower alkoxy, aryloxy, lower alkylthio, mono-lower alkylamino, di-lower alkylamino, acyl, acyloxy, acylamino, lower alkoxycarbonyl, cycloalkyl, lower alkenyl, lower alkynyl, mono-lower alkyl-substituted carbamoyl, di-lower alkyl-substituted carbamoyl, lower alkoxycarbonylamino, etc. Among these, lower alkyl and aryl groups are preferred, and methyl and halogen-substituted aryl groups are more preferred.

[0048] When B and C are different, examples of the substituent for C include OH, NH2, COOH, NO, NO2, a carboxylic acid amide group, a carbamoyl group, a sulfamoyl group, a halogen atom, a lower alkyl group, an aryl group, a lower alkoxy group, an aryloxy group, a lower alkylthio group, a mono-lower alkylamino group, a di-lower alkylamino group, an acyl group, an acyloxy group, an acylamino group, a lower alkoxycarbonyl group, a cycloalkyl group, a lower alkenyl group, a lower alkynyl group, a mono-lower alkyl-substituted carbamoyl group, a di-lower alkyl-substituted carbamoyl group, and a lower alkoxycarbonylamino group. Among these, substitution with a carboxylic acid amide group is preferred. The carboxylic acid amide may have a piperidine structure.

[0049] Carboxylic acid amides include RC(=O)-NR 1 R 2 This refers to a compound having the structure shown below, and includes primary amides, secondary amides, and tertiary amides.

[0050] The aryl group refers to a monocyclic or polycyclic group consisting of a 5- or 6-membered aromatic hydrocarbon ring, and specific examples include phenyl, naphthyl, fluorenyl, anthryl, biphenylyl, tetrahydronaphthyl, chromanyl, 2,3-dihydro-1,4-dioxanaphthalenyl, indanyl, and phenanthryl. Phenyl is preferred, and the phenyl is more preferably substituted with a halogen atom, and most preferably substituted with a chlorine atom.

[0051] The heteroaryl group refers to a monocyclic or polycyclic group consisting of a 5- or 6-membered aromatic ring containing 1 to 2 heteroatoms selected from N and O. In the case of a polycyclic group, at least one ring must be aromatic. Specific examples include pyrrolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, quinolyl, benzo[d][1,3]dioxole, benzo[b]thienyl, and benzimidazolyl. Among these, pyrazinyl and benzo[d][1,3]dioxole are particularly preferred.

[0052] Examples of lower alkoxy groups include straight-chain or branched C1-6 alkoxy groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isopentyloxy, and hexyloxy.

[0053] Aryloxy groups include phenyloxy, naphthyloxy, fluorenyloxy, anthryloxy, biphenylyloxy, tetrahydronaphthyloxy, chromanyloxy, 2,3-dihydro-1,4-dioxanaphthalenyloxy, indanyloxy, and phenanthryloxy.

[0054] Examples of lower alkylthio groups include straight-chain or branched C1-6 alkylthio groups such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio, isopentylthio, and hexylthio.

[0055] Mono-lower alkylamino groups include amino groups mono-substituted with C1-6 alkyl, such as methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, tert-butylamino, n-pentylamino, isopentylamino, and hexylamino.

[0056] Di-lower alkylamino groups include amino groups di-substituted with C1-6 alkyl, such as dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-tert-butylamino, di-n-pentylamino, diisopentylamino, and dihexylamino.

[0057] The acyl group includes C1-6 alkylcarbonyl, arylcarbonyl, and aryl-substituted C1-4 alkylcarbonyl.

[0058] Examples of C1-6 alkylcarbonyl include methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl, isopentylcarbonyl, and hexylcarbonyl. Arylcarbonyl includes phenylcarbonyl, naphthylcarbonyl, fluorenylcarbonyl, anthrylcarbonyl, biphenylylcarbonyl, tetrahydronaphthylcarbonyl, chromanylcarbonyl, 2,3-dihydro-1,4-dioxanaphthalenylcarbonyl, indanylcarbonyl and phenanthrylcarbonyl.

[0059] Examples of the aryl-substituted C1-4 alkylcarbonyl include benzylcarbonyl, naphthylmethylcarbonyl, fluorenylmethylcarbonyl, anthrylmethylcarbonyl, biphenylylmethylcarbonyl, tetrahydronaphthylmethylcarbonyl, chromanylmethylcarbonyl, 2,3-dihydro-1,4-dioxanaphthalenylmethylcarbonyl, indanylmethylcarbonyl, and phenanthrylmethylcarbonyl, phenethylcarbonyl, naphthylethylcarbonyl, fluorenylethylcarbonyl, anthrylethylcarbonyl, biphenylylethylcarbonyl, tetrahydronaphthylethylcarbonyl, chromanylethylcarbonyl, 2,3-dihydro-1,4-dioxanaphthalenylethylcarbonyl, indanylethylcarbonyl, and phenanthrylethylcarbonyl.

[0060] The acyloxy group includes C1-6 alkylcarbonyloxy, arylcarbonyloxy, and aryl-substituted C1-4 alkylcarbonyloxy.

[0061] Specific examples of C1-6 alkylcarbonyloxy include methylcarbonyloxy, ethylcarbonyloxy, n-propylcarbonyloxy, isopropylcarbonyloxy, n-butylcarbonyloxy, isobutylcarbonyloxy, tert-butylcarbonyloxy, n-pentylcarbonyloxy, isopentylcarbonyloxy and hexylcarbonyloxy.

[0062] Specific examples of arylcarbonyloxy include phenylcarbonyloxy, naphthylcarbonyloxy, fluorenylcarbonyloxy, anthrylcarbonyloxy, biphenylylcarbonyloxy, tetrahydronaphthylcarbonyloxy, chromanylcarbonyloxy, 2,3-dihydro-1,4-dioxanaphthalenylcarbonyloxy, indanylcarbonyloxy, and phenanthrylcarbonyloxy.

[0063] Specific examples of the aryl-substituted C1-4 alkylcarbonyloxy include benzylcarbonyloxy, naphthylmethylcarbonyloxy, fluorenylmethylcarbonyloxy, anthrylmethylcarbonyloxy, biphenylylmethylcarbonyloxy, tetrahydronaphthylmethylcarbonyloxy, chromanylmethylcarbonyloxy, 2,3-dihydro-1,4-dioxanaphthalenylmethylcarbonyloxy, indanylmethylcarbonyloxy, and phenanthrylmethylcarbonyloxy, phenethylcarbonyloxy, naphthylethylcarbonyloxy, fluorenylethylcarbonyloxy, anthrylethylcarbonyloxy, biphenylylethylcarbonyloxy, tetrahydronaphthylethylcarbonyloxy, chromanylethylcarbonyloxy, 2,3-dihydro-1,4-dioxanaphthalenylethylcarbonyloxy, indanylethylcarbonyloxy, and phenanthrylethylcarbonyloxy.

[0064] The acylamino group includes C1-6 alkylcarbonylamino, arylcarbonylamino, and aryl-substituted C1-4 alkylcarbonylamino.

[0065] Specific examples of C1-6 alkylcarbonylamino include methylcarbonylamino, ethylcarbonylamino, n-propylcarbonylamino, isopropylcarbonylamino, n-butylcarbonylamino, isobutylcarbonylamino, tert-butylcarbonylamino, n-pentylcarbonylamino, isopentylcarbonylamino and hexylcarbonylamino.

[0066] Specific examples of arylcarbonylamino include phenylcarbonylamino, naphthylcarbonylamino, fluorenylcarbonylamino, anthrylcarbonylamino, biphenylylcarbonylamino, tetrahydronaphthylcarbonylamino, chromanylcarbonylamino, 2,3-dihydro-1,4-dioxanaphthalenylcarbonylamino, indanylcarbonylamino and phenanthrylcarbonylamino.

[0067] Specific examples of the aryl-substituted C1-4 alkylcarbonylamino include benzylcarbonylamino, naphthylmethylcarbonylamino, fluorenylmethylcarbonylamino, anthrylmethylcarbonylamino, biphenylylmethylcarbonylamino, tetrahydronaphthylmethylcarbonylamino, chromanylmethylcarbonylamino, 2,3-dihydro-1,4-dioxanaphthalenylmethylcarbonylamino, indanylmethylcarbonylamino, phenanthrylmethylcarbonylamino, phenethylcarbonylamino, naphthylethylcarbonylamino, fluorenylethylcarbonylamino, anthrylethylcarbonylamino, biphenylylethylcarbonylamino, tetrahydronaphthylethylcarbonylamino, chromanylethylcarbonylamino, 2,3-dihydro-1,4-dioxanaphthalenylethylcarbonylamino, indanylethylcarbonylamino, and phenanthrylethylcarbonylamino.

[0068] Examples of lower alkoxycarbonyl groups include straight-chain or branched C alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, n-pentyloxycarbonyl, isopentyloxycarbonyl, and hexyloxycarbonyl.

[0069] Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0070] Examples of lower alkenyl groups include C2-6 alkenyl groups such as vinyl, allyl, 1-propenyl, 2-methyl-2-propenyl, isopropenyl, 1-, 2- or 3-butenyl, 2-, 3- or 4-pentenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 5-hexenyl, 1-cyclopentenyl, 1-cyclohexenyl, and 3-methyl-3-butenyl.

[0071] Examples of lower alkynyl groups include C2-6 alkynyl groups such as ethynyl, 1- or 2-propynyl, 1-, 2- or 3-butynyl, and 1-methyl-2-propynyl.

[0072] Examples of mono-lower alkyl-substituted carbamoyl groups include carbamoyl groups mono-substituted with C alkyl, such as methylcarbamoyl, ethylcarbamoyl, n-propylcarbamoyl, isopropylcarbamoyl, n-butylcarbamoyl, isobutylcarbamoyl, tert-butylcarbamoyl, n-pentylcarbamoyl, isopentylcarbamoyl, and hexylcarbamoyl.

[0073] Di-lower alkyl-substituted carbamoyl groups include carbamoyl groups di-substituted with C alkyl, such as dimethylcarbamoyl, diethylcarbamoyl, di-n-propylcarbamoyl, diisopropylcarbamoyl, di-n-butylcarbamoyl, diisobutylcarbamoyl, di-tert-butylcarbamoyl, di-n-pentylcarbamoyl, diisopentylcarbamoyl, and dihexylcarbamoyl.

[0074] Lower alkoxycarbonylamino groups include methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, isopropoxycarbonylamino, butoxycarbonylamino, isobutoxycarbonylamino, tert-butoxycarbonylamino, pentyloxycarbonylamino, isopentyloxycarbonylamino, hexyloxycarbonylamino and the like.

[0075] The compounds of the general formula (1) of the present invention include known compounds and novel compounds, and Compound 2-26 can be synthesized, for example, as shown in the following formula (9). Step 1: N-5-Quinolinylacetamide (CAS RN 42464-80-2) was prepared according to literature reports (Manas Chakrabarty et al.). Details are as follows: To a solution of 5-aminoquinoline (purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan, 1.00 g, 6.9 mmol) in pyridine (7.0 ml), acetic anhydride (0.978 ml, 10.4 mmol) was added dropwise. The mixture was stirred at room temperature for 1.5 hours until the starting material completely disappeared, as indicated by TLC. The crude compound was filtered, washed with toluene, and purified by recrystallization from diethyl ether to give N-5-quinolinylacetamide (0.68 g, 3.7 mmol) as a white solid in 53% yield. Step 2: N-(1,2,3,4-tetrahydro-5-quinolinyl)acetamide (CAS RN 156694-10-9) was prepared according to a literature report (Dipanjan Bhattacharyya et al.). The details are as follows: A mixture of N-5-quinolinylacetamide (0.41 g, 2.2 mmol), boronic acid (0.02 g, 0.3 mmol), and diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate (1.38 g, 5.4 mmol) in 1,2-dichloroethane (7.0 ml) was placed in a preheated oil bath (60 °C) and stirred for 13 h until the starting material had completely disappeared, as indicated by TLC. The crude compound was filtered, washed with hexane, and purified by column chromatography on silica gel (hexane: EtOAc = 1:1 to 1:3) to give pure N-(1,2,3,4-tetrahydro-5-quinolinyl)acetamide (0.25 g, 1.3 mmol) as a yellow solid in 61% yield. Step 3: N-[1,2,3,4-tetrahydro-1-(1-oxo-2-propen-1-yl)-5-quinolinyl]acetamide (CAS RN 2196076-49-8) was prepared according to a literature report (Jonas Eriksson et al.). Details are as follows: A solution of N-(1,2,3,4-tetrahydro-5-quinolinyl)acetamide (0.14 g, 0.8 mmol) and sodium carbonate (0.10 g, 0.9 mmol) in acetone (1 mL) and water (1 mL) was cooled to below 5 °C in an ice bath. Acryloyl chloride (0.07 mL, 0.9 mmol) was added dropwise to the solution with vigorous stirring for 10 min. The reaction mixture was warmed to room temperature and stirred for 14 h until the starting material had completely disappeared, as indicated by TLC. The precipitate was dissolved in ethyl acetate, dried over sodium sulfate, and the solvent was purified by column chromatography on silica gel to give pure N-[1,2,3,4-tetrahydro-1-(1-oxo-2-propen-1-yl)-5-quinolinyl]acetamide (0.20 g, 0.8 mmol) as a white oil in nearly 100% yield, which crystallized upon cooling at 4 °C for 72 h to give a white solid (mp 104-110 °C).1H NMR (400 MHz, (CD3)2SO) δ : 1. 80-1.87 (m, 2H), 2.05 (s, 3H), 2.58 (t, J=6.7 Hz, 2H), 3.71 (t, J=6.5 Hz, 2H), 5.70 (dd, J=10.3, 2.2 Hz, 1H), 6.21 (dd, J=16. 8, 2.2 Hz, 1H), 6.51 (dd, J = 16.7, 10.3 Hz, 1H), 6.93 (s, 1H), 7.15 (t, J=8.0 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 9.40 (s, 1H); 13C NMR (400 MHz, CDCl3) δ: 22.45, 23.38, 23.73, 42.76, 121.86, 122.57, 125.79, 126.57, 127.77, 129.72, 135.24, 138.76, 165.56, 169.20; HRMS (ESI) m / z: C14H16N2NaO2 [M+Na]+: 267.1109, 267.1109. [ka]

[0076] When synthesizing derivatives of Compound 2 and Compound 2-26, the feasibility of synthesis is examined based on the simplicity of the reaction and process, yield, etc. It is also preferable to synthesize derivatives while taking into consideration the cost, stability (photodegradability, hygroscopicity, etc.) and danger of the raw materials and reaction reagents.

[0077] The IRE1α S-nitrosylation inhibitors of the present invention are expected to have the effect of inhibiting NO-induced S-nitrosylation by Michael addition of the α,β-unsaturated carbonyl structure in the compound structure to the C931 or C951 residue of IRE1α. More preferably, the IRE1α S-nitrosylation inhibitors of the present invention have the effect of inhibiting NO-induced S-nitrosylation by specifically Michael addition of the α,β-unsaturated carbonyl structure in the compound structure to the C931 residue of IRE1α.

[0078] The composition of the present invention containing an S-nitrosylation inhibitor is a composition for treating neurodegenerative diseases, diabetes, hepatic steatosis, hepatic fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, cardiac fibrosis, or rheumatoid arthritis, containing a compound that specifically inhibits IRE1α. Administration of this pharmaceutical composition to humans specifically inhibits NO-induced S-nitrosylation of IRE1α, resulting in normal splicing of Xbp1u to Xbp1s, a cascade responsible for endoplasmic reticulum stress, and resulting in the alleviation of endoplasmic reticulum stress and cell survival.

[0079] The screening method for IRE1α S-nitrosylation inhibitors of the present invention comprises the steps of culturing cells in the presence of a nitric oxide (NO) generator and a candidate substance, and detecting S-nitrosylated IRE1α. Examples of NO generators include S-nitrosocysteine ​​(SNOC) and S-nitrosoglutathione (GSNO). [Example]

[0080] This invention is further illustrated by the following examples, which should not be construed as further limiting.

[0081] 1. Identification of compounds that inhibit IRE1α S-nitrosylation by virtual screening The screening flow for IRE1α S-nitrosylation inhibitors of the present invention is shown below. First, we performed in silico docking simulations using CovDock to predict compounds that could covalently bind to the S-nitrosylation sites C931 or C951 of IRE1α from Enamine's stock compound library (approximately 860 compounds). Based on docking scores and visual analysis, we identified 35 hit compounds that could bind to C931. Next, we performed virtual screening using in silico FRED docking simulation (2017 version) to predict compounds that could non-covalently bind to the S-nitrosylation sites C931 or C951 of IRE1α from Namiki-Shoji's entire in-stock compound library (approximately 4,000,000 compounds). We then performed further docking simulations using GLIDE for the top 10,000 compounds with FRED docking scores. We then visually analyzed the top 100 docking scores, and identified 25 compounds with the potential to bind to C931 and 15 compounds with the potential to bind to C951 as hit compounds. 1-3. Through the virtual screening of 1-1 and 1-2 above, 75 hit compounds with docking scores of -4.306 kcal / mol or less were obtained. Of these hit compounds, the 11 compounds with the highest docking scores were evaluated for their effect on Xbp1 splicing (rescuing Xbp1 splicing activity) using the cell-based method described in 2 below, leading to the identification of Compound 2. In addition, Compounds 4, 8, 9, 10, and 11 were obtained. 1-4. Furthermore, we performed in silico docking simulations using CovDock to search for compounds similar to Compound 2 that could interact with the S-nitrosylation site (C931) of IRE1α from the stock compound libraries (approximately 4,000,000 compounds) of Enamine (860 compounds), Namiki (approximately 4,000,000 compounds), and Chemdiv (approximately 2,000 compounds). We obtained 130 hit compounds with docking scores below -0.044 kcal / mol. The top 81 compounds were further evaluated for their effects on Xbp1 splicing using the cell-based approach described in Section 2 below, leading to the identification of Compound 2-26. Compounds 2-1, 2-66, and 2-71 were also identified. 1-5. Finally, in the bioassay described in 3., we investigated the effects of MPP on S-nitrosylation of IRE1α and its role in inducing Parkinson's disease-like phenotypes via NO or NO production. + The effect on induced cell death was assessed.

[0082] Next, we attempted to synthesize derivatives of the hit compounds obtained above. When synthesizing derivatives, we considered not only the inhibitory activity of the compound but also the stability (photodegradability and hygroscopicity) and cost (inexpensive availability or synthesis), hazards, and ease of synthesis (e.g., simplicity and yield of the reaction and process, and the lack of special reactions (e.g., anhydrous reactions or reactions under pressure)) of the raw materials and reaction reagents, thereby selecting a more suitable structure. Specifically, favorable structures can be obtained by considering factors such as: X in formula (10) having a halogen or A1 to A3 having an alkyl; the raw materials being inexpensive; the synthesized product being a novel compound; avoiding the PAINS skeleton; including a fluorine atom (F) because of its lipophilicity and increased electronegativity; and considering the amino acid residues around the S-nitrosylation site. [ka]

[0083] 2.Rescue evaluation of Xbp1 splicing activity of compounds obtained by virtual screening To evaluate the IRE1α S-nitrosylation inhibitors of the present invention, cells are cultured in the presence of a nitric oxide (NO) generator and a candidate substance, and S-nitrosylated IRE1α is detected. Examples of NO generators include S-nitrosocysteine ​​(SNOC) and S-nitrosoglutathione (GSNO). The concentration of the NO generator in the culture medium is 200 μM. In the examples, "old SNOC" refers to SNOC that has ceased to release NO several days after preparation.

[0084] IRE1α activity includes IRE1α ribonuclease (RNase) activity. Full-length Xbp1 mRNA (Xbp1u) is spliced ​​in an IRE1α RNase-dependent manner to produce Xbp1s, encoding the active form of XBP1. IRE1α RNase activity can be assessed by RT-PCR followed by electrophoresis to quantify Xbp1s expression. After normalization with ACTB (a housekeeping gene), the levels of Xbp1s expression under various conditions can be compared. Therefore, to verify whether the reduction in Xbp1 splicing resulting from NO-induced inhibition of IRE1α RNase activity was rescued by the addition of compounds identified through virtual screening, we calculated the percentage increase in Xbp1s by adding the compounds, relative to the NO-induced decrease in Xbp1s under conditions in which Xbp1s were induced by the endoplasmic reticulum stress-inducing agent (tunicamycin). Compounds that induced splicing when added alone were excluded from further analysis. The results are shown in Table 1. In the table, the docking score (kcal / mol) indicates the results of the docking simulation, and the smaller the value, the stronger the binding to the target is predicted to be.

[0085] [Table 1] TIFF2025147648000015.tif179170TIFF2025147648000016.tif177170TIFF20251476480 00017.tif179170TIFF2025147648000018.tif179170TIFF2025147648000019.tif204170

[0086] 3. Bioassay using hit compounds 2-26 Next, the candidate substance "Compound 2-26 (Cpd 2-26)," which had the highest activity in the above splice rescue experiment, was subjected to a bioassay.

[0087] S-nitrosylated IRE1α can be detected by Western blot analysis using an antibody after the biotin switch method shown in Figure 3. The development and principles of the biotin switch method can be described in a previous paper (Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH. Nat Cell Biol. 2001 Feb;3(2):193-7).

[0088] NO in the culture medium can be analyzed using the Griess method shown in Figure 5. NO is oxidized, and most of it is converted to stable nitrite ions (NO 2- This method is used to measure NO 2- This method indirectly quantifies the NO concentration in the culture medium by measuring the NO concentration.

[0089] Example 1 SH-SY5Y cells were cultured at 37°C for 1 hour in the presence of Cpd 2-26 (0 μM (DMSO only, control), 1 μM, 5 μM, or 10 μM), and then Old SNOC or SNOC (200 μM) was added and cultured at 37°C for 30 minutes. The degree of S-nitrosylation of IRE1α was measured using the biotin-switch method shown in Figure 3, which uses biotin and avidin. The results are shown in Figure 4. Cpd 2-26 was found to inhibit S-nitrosylation of IRE1α in a concentration-dependent manner.

[0090] Example 2 SH-SY5Y cells were cultured at 37°C for 1 hour in the presence of Cpd 2-26 (0 μM (DMSO only, control), 1 μM, 5 μM, 10 μM), and then Old SNOC or SNOC (200 μM) was added and cultured at 37°C for 30 minutes. NO in the medium was then measured. 2- The concentrations were quantified according to the Griess method shown in Figure 5. The results are shown in Figure 6. It was revealed that Cpd 2-26 did not affect NO in the medium at concentrations that inhibited S-nitrosylation of IRE1α.

[0091] Example 3 SH-SY5Y cells were cultured at 37°C for 1 hour in the presence of Cpd 2-26 (0 μM (DMSO only, control), 1 μM, 5 μM, or 10 μM). After that, cells were cultured at 37°C for 30 minutes with old SNOC (-) or SNOC (200 μM, +). Cells were then cultured at 37°C for 2 hours with the endoplasmic reticulum stress inducer tunicamycin (Tm) at 0 μg / mL (DMSO only, control, -) or 10 μg / mL (+). Expression of Xbp1u, Xbp1s, and ACTB was measured by RT-PCR and electrophoresis. The results are shown in Figure 7. Cpd 2-26 concentration-dependently rescued NO-induced suppression of Xbp1s expression.

[0092] Example 4 SH-SY5Y cells were cultured at 37°C for 1 hour in the presence of Cpd 2-26 (0 μM (DMSO only, control, -), 10 μM (+)). After adding Tm (0 μg / mL (DMSO only, control, -), 10 μg / mL (+)), the cells were cultured at 37°C for 2 hours. The expression levels of Xbp1u, Xbp1s, and ACTB were measured by RT-PCR and electrophoresis. The results are shown in Figure 8. Cpd 2-26 did not affect Xbp1s expression at concentrations that restored NO-induced suppression of Xbp1s expression.

[0093] Example 5 SH-SY5Y cells were cultured at 37°C for 1 hour in the presence of Cpd 2-26 (0 μM (DMSO only, control), 1 μM, 5 μM, or 10 μM). After that, glutathione (GSH, control) or GSNO (200 μM) was added and cultured at 37°C for 24 hours. Cell morphology was observed by phase contrast observation. The results are shown in Figure 9 (left). The percentage of PI-positive cells (= dead cells) relative to the total cells (detected by Hoechst staining) was calculated. The results are shown in Figure 9 (right). Cpd 2-26 was found to inhibit NO-induced neuronal cell death in a concentration-dependent manner.

[0094] Example 6 Previous studies (Nakato et al., Sci Rep. 2015 Oct 8;5:14812.) have shown that NO induces neuronal death through a decrease in IRE1α RNase activity. Furthermore, MPP induces Parkinson's disease-like phenotypes through NO production. + (Przedborski et al., Proc Natl Acad Sci U S A. 1996 May 14;93(10):4565-71.) has also been shown to reduce IRE1α RNase activity. + Whether the product of the present invention can prevent cell death induced by β-glucan was evaluated by observing cell morphology using phase contrast observation and detecting dead cells using propidium iodide (PI) staining.

[0095] SH-SY5Y cells were cultured at 37°C for 1 hour in the presence of Cpd 2-26 (0 μM (DMSO only, control), 0.1 μM, 1 μM, 10 μM), and then MPP + The cells were cultured at 37°C for 24 hours after the addition of DMSO (0 mM (DMSO only, control), 2 mM), and the cell morphology was observed by phase contrast observation. The results are shown in the left side of Figure 10. The ratio of PI-positive cells (= dead cells) to the total cells (detected by Hoechst staining) was calculated. The results are shown in the right side of Figure 10. Cpd 2-26 inhibited MPP in a concentration-dependent manner. + It was found that it suppresses induced neuronal cell death.

[0096] Example 7 A recombinant protein (1 μg) containing the cytoplasmic domain of IRE1α was reacted with Cpd 2-26 (0 μM (DMSO only, control), 1000 μM) at 37°C for 1.5 hours, and the modification site of Cpd 2-26 on IRE1α was identified by LC-MS / MS. The results are shown in Figures 11 and 12. Cpd 2-26 was found to modify the cysteine ​​(Cys) residue, which is the S-nitrosylation site of IRE1α.

[0097] Problems with the prior art include "1. NO synthase inhibitors uniformly block the physiological effects of NO, raising concerns about side effects, particularly on the circulatory system." However, as shown in Figure 6, the present invention demonstrates that it does not affect NO, thereby avoiding problem 1. Regarding problem 2, "NO synthase inhibitors and radical scavengers may be insufficiently effective against proteins that undergo S-nitrosylation," as shown in Figures 4 and 7, the present invention fully inhibits S-nitrosylation and restores IRE1α RNase activity against the target protein IRE1α, thereby avoiding problem 2. Furthermore, regarding problem 3, "Because overactivation of IRE1α can drive cell death pathways and lead to cell carcinogenesis, simple activating drugs are likely to cause serious side effects," as shown in Figure 8, the present invention has been confirmed to not induce IRE1α RNase activity at concentrations that exhibit S-nitrosylation inhibitory effects and IRE1α RNase activity restoration effects, thereby avoiding problem 3. In summary, the present invention inhibits only NO modification of IRE1α without affecting its RNase activity or NO. Therefore, its mechanism of action is distinct from existing drugs that regulate NO or IRE1α, which can contribute to both physiological and pathological conditions. It is expected to be a drug that efficiently inhibits neuronal death while minimizing side effects (toxicity). Furthermore, previous studies have confirmed S-nitrosylation of IRE1α and reduced IRE1α RNase activity in diabetes (Yang et al., Science. 2015 Jul 31;349(6247):500-6.) and hepatic steatosis (Wang et al., Sci Signal. 2018 May 15;11(530):eaao4617.). Therefore, the present invention has potential applications for these diseases. Additionally, as described in a prior patent (JP 2023-036864), increased IRE1α RNase activity may contribute to the treatment or prevention of liver fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, or cardiac fibrosis through the promotion of cellular senescence. The present invention may also be applicable when reduced RNase activity associated with S-nitrosylation of IRE1α is involved in the pathogenesis of these conditions.

Claims

1. The following formula (I) 【Chemical 1】 A is a monovalent α,β-unsaturated carbonyl group; B and C are the same or different and are either (i) or (ii): (i) B and C together form a monocyclic or polycyclic ring, and the monocyclic or polycyclic ring contains at least a 6- or 7-membered heterocyclic ring and may be substituted with one or more substituents; (ii) B and C are the same or different and are each an optionally substituted monovalent lower alkyl group, an optionally substituted monovalent aryl group, or an optionally substituted monovalent cyclic group which may contain a heteroatom, and B and C are not both lower alkyl groups; L1 and L2 are the same or different and are divalent linkers having 1 to 4 carbon atoms, provided that when B and C are joined together to form a monocycle or polycycle, L1 and L2 do not exist. or a salt thereof as an active ingredient, is an S-nitrosylation inhibitor of inositol-requiring enzyme 1α (IRE1α).

2. The S-nitrosylation inhibitor of IRE1α according to claim 1, wherein in formula (I), A is a monovalent α,β-unsaturated carbonyl group containing an alkenyl group which may be substituted with a substituent, or an alkynyl group substituted with a lower alkyl group.

3. The S-nitrosylation inhibitor of IRE1α described in claim 1, wherein in formula (I), B and C together form the monocycle consisting of a heterocycle containing two or more N atoms or O atoms.

4. The S-nitrosylation inhibitor of IRE1α described in claim 1, wherein in formula (I), B and C are the same or different and are a monovalent lower alkyl group substituted with a carboxylic acid amide group, a monovalent aryl group substituted with a halogen group, or a monovalent heterocyclic group in which a hydrogen atom bonded to a heteroatom is substituted with an acyl group.

5. The compound is represented by the following formula (II): 【Chemistry 2】 (X is a divalent lower alkyl group which may be substituted with a carboxyl group, or a divalent monocyclic or polycyclic group which contains at least a 6-membered heterocyclic or aromatic ring; Y 1 and Y 2 are the same or different and represent a carboxyl group, a carboxylic acid amide group, a monovalent lower alkyl group, a halogen atom, or a carbamoyl group, n1 represents an integer of 0 to 3, and n2 represents an integer of 0 to 4. The S-nitrosylation inhibitor of IRE1α according to claim 1, which is a compound represented by the formula:

6. The compound is represented by the following formula (III): 【Chemistry 3】 (X is a monocyclic or polycyclic group containing at least a divalent lower alkyl group, an aryl group, or a heteroaryl group, which may be substituted with a carboxyl group, or which may be substituted with a halogen atom; Y 1 and Y 2 are the same or different and are a carboxyl group, a carboxylic acid amide group, a monovalent lower alkyl group, a halogen atom, or a carbamoyl group; Z is a lower alkyl group; n1 is an integer of 0 to 3; and n2 is an integer of 0 to 3. The S-nitrosylation inhibitor of IRE1α according to claim 5, which is a compound represented by the formula:

7. The compound is represented by the following formula (IV): 【Chemistry 4】 (X is a divalent lower alkyl group optionally substituted with a carboxyl group, or a divalent monocyclic or polycyclic group containing at least a 6-membered heterocycle or aromatic ring; L3 is a divalent linker having 1 to 4 carbon atoms, the carbon optionally being substituted with a heteroatom; and T is an aryl group optionally substituted with a halogen.) The S-nitrosylation inhibitor of IRE1α according to claim 1, which is a compound represented by the formula:

8. The compound is represented by the following formula (I): 【Chemistry 5】 (A is a monovalent α,β-unsaturated carbonyl group containing an alkynyl group substituted with a lower alkyl group, B and C together form a monocyclic or polycyclic ring, and the monocyclic or polycyclic ring contains at least a 6- or 7-membered heterocyclic ring and may be substituted with one or more substituents. The S-nitrosylation inhibitor of IRE1α according to claim 1, which is a compound represented by the formula:

9. The following formula (V) 【Chemistry 6】 An S-nitrosylation inhibitor of IRE1α, comprising a compound represented by the formula: or a salt thereof as an active ingredient.

10. An IRE1α S-nitrosylation inhibitor described in any one of claims 1 to 9, which is a therapeutic agent for neurodegenerative diseases.

11. The therapeutic agent for neurodegenerative diseases according to claim 10, wherein the neurodegenerative disease is Parkinson's disease.

12. An S-nitrosylation inhibitor of IRE1α described in any one of claims 1 to 9, used for the treatment of diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, hepatic steatosis, liver fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, cardiac fibrosis or rheumatoid arthritis.

13. A method for screening for an S-nitrosylation inhibitor of IRE1α, comprising the steps of culturing cells containing IRE1α in the presence of a nitric oxide generator and a candidate substance, and detecting Xbp1u and Xbp1s.

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