Agents for preventing or treating hypoxic injury, ischemia-reperfusion injury, or inflammation, agents for protecting cells for transplantation, and agents for preserving living tissue

Abstract

To provide an agent for the prevention or treatment of hypoxic disorder, ischemia-reperfusion injury or inflammation, a cell protectant for transplantation, and a biopreservative.SOLUTION: The present invention provides an agent for the prevention or treatment of hypoxic disorder, ischemia-reperfusion injury or inflammation, a cell protectant for transplantation, and a biopreservative, each of which comprises at least one selected from a heterocyclic compound represented by the formula (I) (where each symbol is as set forth in the specifications) or a salt thereof, an isothiocyanate compound represented by the formula S=C=N-R5 (II) (where each symbol is as set forth in the specifications), and a TRPA1 agonist as an active ingredient.SELECTED DRAWING: Figure 10A

Description

[Technical field]

[0001] The present invention relates to an agent for preventing or treating hypoxic injury, ischemia-reperfusion injury or inflammation, an agent for protecting cells for transplantation, and an agent for biopreservation. [Background technology]

[0002] It is believed that humans and animals have acquired the ability to increase the chances of survival when faced with a crisis during the course of evolution. There are many known cases where humans who were lost in snowy mountains or other places were found in a state of hypothermia and recovered even though several hours had passed since their cardiac arrest. In such cases, it is possible that a latent endogenous individual protective effect was induced by some kind of stimulus. Some mammals, such as bears and squirrels, have the ability to hibernate. During hibernation, these animals can maintain life with a reduced body temperature and oxygen consumption. When oxygen supply stops due to cardiac arrest, cells in tissues with high oxygen demand, such as the brain, suffer irreversible damage. On the other hand, resumption of blood flow after cardiac arrest causes the generation of reactive oxygen species, which causes significant damage to tissues. Such hypoxic damage and ischemia-reperfusion damage can be alleviated by hypothermia therapy, which artificially induces hypothermia in individuals. It is also known that resistance to inflammation and ischemia-reperfusion damage increases in animals in a hibernating state (Non-Patent Documents 1-3).

[0003] Fear is an emotion induced when the brain judges that danger is approaching, and it induces escape behavior and freezing behavior that reduces the probability of being detected by a natural enemy, as well as various types of physiological responses. When a phobic patient faints after being presented with a fearful stimulus, their heart rate drops by nearly 50% (Non-Patent Document 4). In this way, fear emotion is also linked to the induction of strong physiological responses in humans. If humans or animals in danger can enhance the protective effects of tissues and individuals by inducing anti-inflammatory and immune control responses in addition to hypothermia and hypometabolism, it would be possible to increase the chances of survival. However, no technology has been developed to induce fear emotion or latent intrinsic individual protective effects. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent No. 5350496 [Non-patent literature]

[0005] [Non-Patent Document 1] N Engl J Med 346:612-613, 2002 [Non-Patent Document 2] Lancet Neurol 2: 410-416, 2003 [Non-Patent Document 3] Nat Rev Neurosci 13: 267-278, 2012 [Non-Patent Document 4] Psychosom Med 23: 493-507, 1961 [Non-Patent Document 5] Nature 450: 503-508, 2007 [Non-Patent Document 6] Cell 163: 1153-1164, 2015 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a technology for inducing fear emotion and latent endogenous individual protective effects, and to provide a preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury or inflammation, a cell protective agent for transplantation, and a biopreservative. [Means for solving the problem]

[0007] Fear emotion is controlled by innate and acquired mechanisms. The inventors have elucidated that innate and acquired odor information in the nasal cavity is transmitted to the brain by separate neural circuits to control behavior (Non-Patent Document 5). This discovery has proven the existence of neural circuits responsible for innate behavior control in response to odors, and as a result, the conventional wisdom that behavior in response to odors is determined by acquired learning and experience has been overturned. If the response to odors is controlled innately, it is possible to develop a technology to control behavior in a desired way by developing odor molecules that act on this genetic mechanism. In fact, the inventors have succeeded in developing "thiazoline-related fear odors (tFOs)", artificial odor molecules that induce extremely strong innate fear emotions, for the first time in the world (Patent Document 1, Non-Patent Document 6). The development of tFOs has made it possible to clarify in detail the control mechanism of innate fear emotion and the physiological response induced by innate fear emotion, which have been difficult to elucidate until now.

[0008] Both innate and acquired fear stimuli induced freezing behavior at the same level. However, when physiological responses were used as indicators, it was revealed that only innate fear stimuli induced a decrease in body temperature and heart rate. Furthermore, it was revealed that hypoxic injury, ischemia-reperfusion injury, and inflammation could be suppressed by sniffing or intraperitoneally administering a group of compounds (heterocyclic compounds and isothiocyanate compounds) involved in the induction of innate fear and latent endogenous individual protective action represented by the following general formula of the present invention. On the other hand, the present inventors have elucidated that compounds that induce fear emotionality and latent endogenous individual protective action have the property of activating TRPA1 (transient receptor potential ankyrin 1), and that fear emotionality and latent endogenous individual protective action are partially suppressed in TRPA1 knockout mice. These results indicate that fear emotionality and latent endogenous individual protective action are partly controlled via the TRPA1 gene. The TRPA1 gene is present in a wide range of organisms, from mammals such as humans and livestock to birds, fish, and insects, which are targets for the use of the technology of the present invention for the prevention or treatment of hypoxia injury, ischemia-reperfusion injury, or inflammation, and the cell protective agent for transplantation.

[0009] That is, the present invention relates to the following. [1] Formula (I)

[0010] [ka]

[0011] (In the formula, Ring A is a 5- to 7-membered heterocycle containing 1 or 2 heteroatoms selected from a nitrogen atom, an optionally oxidized sulfur atom, and an oxygen atom; R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, C 1-6 Alkyl group, halogen atom, amino group, -SH, C 1-6 Alkylthio group, C 2-6 Alkenylthio group, C 1-6Alkyl-carbonyl group, formyl group, C 6-10 Aryl group, C 1-6 an alkoxycarbonyl group, a 5- or 6-membered heteroaryl group, or an oxo group; R 1 and R 2 may be bonded to each other to form an optionally substituted 5- or 6-membered ring; n is 0, 1, or 2. or a salt thereof, and Formula (II) S=C=NR 5 (II) (In the formula, R 5 is C 1-6 Alkyl group, C 1-6 Haloalkyl group, C 2-6 Alkenyl group, C 6-10 an aryl group, or a 5- or 6-membered heteroaryl group; 1. A preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury or inflammation, a cell protective agent for transplantation, or a biopreservative agent, comprising as an active ingredient at least one selected from the isothiocyanate compounds represented by the following formula: [2] The agent according to [1], wherein ring A is thiazoline, thiazole, thiazolidine, thiomorpholine, thiophene, pyrrole, morpholine, azepane, pyridine, pyrazine, furan, 2,3-dihydro-4H-1,4-thiazine, or imidazole. [3] The agent according to [1] or [2], wherein the active ingredient is a heterocyclic compound represented by formula (I) or a salt thereof. [4] The agent according to [1], wherein the active ingredient is an isothiocyanate compound represented by formula (II). [5] The agent according to any one of [1] to [4], which is an agent for preventing or treating hypoxic injury, ischemia-reperfusion injury, or inflammation. [6] The agent described in any one of [1] to [4], which is a cell protective agent for transplantation. [7] The agent described in any one of [1] to [4], which is a biopreservative. [8] The agent according to any one of [1] to [7] for nasal administration. [9] A preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury, or inflammation, which contains a TRPA1 agonist as an active ingredient.

[10] A cell protective agent for transplants containing a TRPA1 agonist as an active ingredient.

[11] A biopreservative containing a TRPA1 agonist as an active ingredient.

[12] The agent according to any one of [9] to

[11] for nasal administration.

[13] At least one compound selected from the heterocyclic compound represented by formula (I) or a salt thereof, and the isothiocyanate compound represented by formula (II) for use in the prevention or treatment of hypoxic injury, ischemia-reperfusion injury, or inflammation.

[14] Use of at least one compound selected from the heterocyclic compound represented by formula (I) or a salt thereof, and the isothiocyanate compound represented by formula (II) as a cell protective agent or biopreservative for transplantation.

[15] A TRPA1 agonist for use in the prevention or treatment of hypoxia, ischemia-reperfusion injury, or inflammation.

[16] Use of TRPA1 agonists as cell protectants or biopreservatives for transplantation.

[17] Use of at least one compound selected from the heterocyclic compound represented by formula (I) or a salt thereof, and the isothiocyanate compound represented by formula (II) for producing an agent for preventing or treating hypoxic injury, ischemia-reperfusion injury, or inflammation, an agent for protecting cells for transplantation, or an agent for preserving tissue.

[18] Use of a TRPA1 agonist for producing an agent for preventing or treating hypoxic injury, ischemia-reperfusion injury, or inflammation, a cell protective agent for transplantation, or a biopreservation agent.

[19] A method for preventing or treating hypoxic injury, ischemia-reperfusion injury, or inflammation in a mammal, comprising administering to the mammal an effective amount of at least one compound selected from a heterocyclic compound represented by formula (I) or a salt thereof, and an isothiocyanate compound represented by formula (II).

[20] A method for protecting cells for transplantation, comprising contacting cells for transplantation with at least one compound selected from the heterocyclic compound represented by formula (I) or a salt thereof, and the isothiocyanate compound represented by formula (II).

[21] A method for preserving a living organism, comprising contacting the living organism with at least one compound selected from the heterocyclic compound represented by formula (I) or a salt thereof, and the isothiocyanate compound represented by formula (II).

[22] A method for preventing or treating hypoxic injury, ischemia-reperfusion injury, or inflammation in a mammal, comprising administering an effective amount of a TRPA1 agonist to the mammal.

[23] A method for protecting cells for transplantation, comprising contacting the cells for transplantation with a TRPA1 agonist.

[24] A method for preserving a living organism, comprising contacting the living organism with a TRPA1 agonist. Effect of the Invention

[0012] Fear is an emotion induced by the brain when it senses a dangerous situation, and induces behaviors and physiological responses that increase the survival rate of humans and animals in danger. Hypothermia therapy, which artificially lowers body temperature and metabolism, has the effect of improving the prognosis of emergency patients. Therefore, it is purposeful for the brain and nervous system, which sense fear or a crisis signal, to induce a decrease in body temperature and metabolism throughout the body, and to control inflammation and immune responses, thereby acquiring tissue and individual protection. However, the mechanism that controls such fear emotion and latent endogenous individual protection has not been elucidated. The technology of the present invention induces behaviors that characterize innate fear and latent endogenous individual protection, physiological responses such as hypothermia and hypometabolism, suppresses inflammation and immune responses, and at the same time provides strong resistance to hypoxia injury, ischemia-reperfusion injury, and inflammation, by administering a compound that satisfies specific chemical structure rules. The compound used in the technology of the present invention is effective when it is used as a volatile odor molecule to activate receptors or is taken into the body, or when it is administered directly into the body by injection or other methods. The effects are hypothermia, low metabolism, suppression of oxygen consumption, low heart rate, induction of anti-inflammatory response, protective effect on tissues and individuals under hypoxic conditions, protective effect against ischemia-reperfusion injury, protective effect against inflammation, etc. This technology can provide therapeutic agents for hypoxic injury, ischemia-reperfusion injury, and inflammation in individuals, protective agents for organs for transplantation, and biopreservatives. [Brief description of the drawings]

[0013] [Figure 1] A: Changes in body surface temperature over time (mean ± standard error) when exposed to 2MT (2-methyl-2-thiazoline) or odor molecules that induce acquired fear (anis-FS+). B: Average changes in body surface temperature over 20 min during the presentation of 2MT or odor molecules that induce acquired fear (anis-FS+). [Diagram 2] FIG. 1 shows the average change in body surface temperature upon presentation of various types of thiazoline analogues or odor molecules that induce acquired fear (Anis-FS+). [Diagram 3] A: Mean ± standard error of immobility behavior (freezing behavior) when various types of thiazoline analogues were intraperitoneally injected. B: Mean ± standard error of change in body surface temperature when various types of thiazoline analogues were intraperitoneally injected. [Figure 4] A: Changes in core body temperature over time (mean ± standard error) when exposed to 2MT or odor molecules that induce acquired fear (anis-FS+). B: Average changes in core body temperature over 20 min while exposed to 2MT or odor molecules that induce acquired fear (anis-FS+). [Diagram 5] A: Time course of heart rate changes (mean ± standard error) when exposed to 2MT or odor molecules that induce acquired fear (anis-FS+). B: Average change in heart rate over 20 min during the presentation of 2MT or odor molecules that induce acquired fear (anis-FS+). [Figure 6] A: A graph showing the change in blood flow over time (mean ± standard error) when subjects were exposed to 2MT or an odor molecule that induces acquired fear (Anis-FS+). B: A graph showing the average skin blood flow when subjects were presented with no odor, 2MT, or an odor molecule that induces acquired fear (Anis-FS+). [Figure 7]A: Changes in respiratory rate over time (mean ± standard error) when subjects were exposed to 2MT or spice odor (Eug). B: Average respiratory rate when subjects were presented with no odor, 2MT, or spice odor (Eug). [Figure 8] A: Changes in oxygen consumption per unit weight of mice over time (mean ± standard error) when presented with 2MT and when given no odor (saline). B: Average oxygen consumption per unit weight of mice when presented with 2MT and when given no odor. [Figure 9] A: A Kaplan-Meier curve showing the survival time of mice in 4% oxygen when 2MT was presented and when there was no odor. B: A graph showing the change in core body temperature over time (mean ± standard error) when odor was presented under the same conditions as in Example A. [Figure 10A] FIG. 1 shows the survival time (mean±standard error) of mice in 4% oxygen after intraperitoneal injection of each heterocyclic compound. [Figure 10B] FIG. 10 shows the structural formulas of the compounds used in Example 10 in the order of the graph in FIG. 10A. [Figure 11A] FIG. 1 shows the survival time (mean±standard error) of mice in 4% oxygen when each isothiocyanate compound was intraperitoneally injected. [Figure 11B] FIG. 11B shows the structural formulas of the compounds used in Example 11 in the order of the graph in FIG. 11A. [Figure 12] FIG. 1 shows Kaplan-Meier curves of survival time in 4% oxygen for mice administered thiomorpholine and saline. [Figure 13] The survival time of each individual mouse after intraperitoneal injection of each of the solutions shown in the figure is plotted. [Figure 14]A: Photographs showing the results of daily observation of changes in the wound area for each individual under odorless conditions. B: Photographs showing the results of daily observation of changes in the wound area for each individual under conditions in which 2MT was presented. C: A figure quantifying the area of ​​the wound observed in A and B. D: A figure showing the change in deep body temperature over time (mean ± standard error) when odor was presented under the same conditions as in the example AC. [Figure 15] This figure shows the average change in body surface temperature when control mice (trpa1+ / -; n=5) and Trpa1 knockout mice (trpa1- / -; n=8) were presented with 2MT. [Figure 16] A: Changes in core body temperature over time when 2MT was presented to control mice (trpa1+ / -; n=7) and Trpa1 knockout mice (trpa1- / -; n=7). B: Changes in core body temperature over time when 2MT was presented to control mice (trpa1+ / -) and Trpa1 knockout mice (trpa1- / -). [Figure 17] FIG. 1 shows the average survival time in 4% oxygen when control mice (Hetero) and Trpa1 knockout mice (KO) were presented with saline or 2MT. [Figure 18] FIG. 1 shows the amount of TNF-α (mean±standard error) in the blood when each heterocyclic compound or isothiocyanate compound was intraperitoneally injected into sepsis model mice. [Figure 19] A: IL-1β levels (mean ± standard error) in the blood of sepsis model mice after intraperitoneal injection of 2MT. B: IL-10 levels (mean ± standard error) in the blood of sepsis model mice after intraperitoneal injection of 2MT. [Figure 20] FIG. 1 shows the amount of HMGB1 (mean±standard error) in the blood when 2MT was intraperitoneally injected into sepsis model mice. [Figure 21] FIG. 1 shows the survival time in sepsis model mice after intraperitoneal injection of 2MT, as a Kaplan-Meier curve and a bar graph (mean±standard error). [Figure 22]FIG. 1 shows tissue staining of the brain in a mouse model of cerebral ischemia-reperfusion injury, and quantification of the area of ​​damaged regions. [Figure 23] FIG. 1 shows the survival time (mean ± standard error) in 4% oxygen when each heterocyclic compound was intraperitoneally administered to control mice (Trpa1+ / +) and Trpa1 knockout mice (Trpa1- / -). [Figure 24] FIG. 1 shows the survival time (mean ± standard error) in 4% oxygen when thiomorpholine was administered intraperitoneally to control mice (Trpa1+ / +) and Trpa1 knockout mice (Trpa1- / -). [Diagram 25] FIG. 1 shows the change in oxygen consumption over time (mean ± standard error) when 2MT was presented to control mice (Trpa1+ / -) and Trpa1 knockout mice (Trpa1- / -). [Figure 26] A: Survival time (mean ± SEM) in 4% oxygen after administration of each Trpa1 agonist. B: Survival time (mean ± SEM) in 4% oxygen after administration of acetaminophen to control mice (Trpa1+ / +, Trpa1+ / -) and Trpa1 knockout mice (Trpa1- / -). [Figure 27] A: Changes in body surface temperature over time (mean ± standard error) when 4-Ethyl-2-methyl-2-thiazoline (4E2MT) was intraperitoneally injected into control mice (Trpa1+ / -) and Trpa1 knockout mice (Trpa1- / -). B: Survival time (mean ± standard error) in 4% oxygen when 4E2MT was intraperitoneally injected into control mice (Trpa1+ / -) and Trpa1 knockout mice (Trpa1- / -). C: Blood TNFα levels (mean ± standard error) when LPS (Lipopolysaccharide) and 4E2MT were administered to control mice (Trpa1+ / +, Trpa1+ / -) and Trpa1 knockout mice (Trpa1- / -). [Figure 28]FIG. 1 shows the time course of oxygen consumption (mean±standard error) when thiomorpholine (TMO) and 2MT were intraperitoneally administered. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Ring A in formula (I) represents a 5- to 7-membered heterocycle containing 1 or 2 heteroatoms selected from a nitrogen atom, an optionally oxidized sulfur atom, and an oxygen atom. Ring A is preferably a 5- to 7-membered heterocycle containing 1 or 2 heteroatoms selected from a nitrogen atom and an optionally oxidized sulfur atom. Ring A is more preferably a 5- to 7-membered heterocycle containing a nitrogen atom and an optionally oxidized sulfur atom. The number of members of ring A is more preferably 5 or 6.

[0015] Examples of the heterocycle include, but are not limited to, pyrrole, pyridine, pyridazine, pyrimidine, pyrazine, piperazine, pyrrolidine, hexahydropyridazine, imidazole, imidazolidine, piperidine, thiophene, thiolane, tetrahydro-2H-thiopyran, thiazoline (e.g., 2-thiazoline, 3-thiazoline, 4-thiazoline), thiazole, thiazolidine, isothiazole, isothiazoline, thiomorpholine, thiadiazoline, thiadiazole, thiadiazolidine, 1,3-thiazine, 5,6-dihydro-4H-1,3-thiazine, 2,3-dihydro-4H-1,4-thiazine, furan, 2H-pyran, 4H-pyran, oxazole, isoxazole, morpholine, oxazoline, and azepane. Preferred are thiazoline (e.g., 2-thiazoline, 3-thiazoline, 4-thiazoline), thiazole, thiazolidine, thiomorpholine, thiophene, pyrrole, morpholine, azepane, pyridine, pyrazine, furan, 2,3-dihydro-4H-1,4-thiazine, or imidazole, and more preferred are thiazoline (e.g., 2-thiazoline), thiazole, thiazolidine, thiomorpholine, thiophene, or 2,3-dihydro-4H-1,4-thiazine.

[0016] The "halogen atom" used herein is preferably selected from a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

[0017] The "C" used here 1-6 "Alkyl group" (when used as a group or part of a group) means a straight or branched chain alkyl group having 1 to 6 carbon atoms. 1-6 Examples of the alkyl group include, but are not limited to, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a 1-methylpropyl group (sec-butyl group), a 2-methylpropyl group (isobutyl group), a tert-butyl group, a pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 2,2-dimethylpropyl group, a 1,2-dimethylpropyl group, a 1-ethylpropyl group, a hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 4-methylpentyl group, a 1,1-dimethylbutyl group, a 2,2-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 1-ethylbutyl group, a 2-ethylbutyl group, and a 1-ethyl-2-methylpropyl group. 1-6 Examples of the alkyl group include C 1-4 Examples of the alkyl group include an alkyl group (a straight or branched alkyl group having 1 to 4 carbon atoms), and more preferred are a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and a sec-butyl group, and particularly preferred is a methyl group.

[0018] The "C" used here 1-6 "Haloalkyl group" means a C substituted with 1 to 5 halogen groups. 1-6 It means an alkyl group, and when there are two or more halogeno groups, the types of the halogeno groups may be the same or different. Examples of the halogeno group include a fluoro group, a chloro group, and a bromo group. 1-6Examples of haloalkyl groups include, but are not limited to, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a chlorodifluoromethyl group, a 1-fluoroethyl group, a 2-fluoroethyl group, a 2-chloroethyl group, a 2-bromoethyl group, a 1,1-difluoroethyl group, a 1,2-difluoroethyl group, a 2,2,2-trifluoroethyl group, a 1,1,2,2-tetrafluoroethyl group, a 1,1,2,2,2-pentafluoroethyl group, a 1-fluoropropyl group, a 1,1-difluoropropyl group, a 2,2-difluoropropyl group, a 3-fluoropropyl group, a 3,3,3-trifluoropropyl group, a 4-fluorobutyl group, a 4,4,4-trifluorobutyl group, a 5-fluoropentyl group, a 5,5,5-trifluoropentyl group, a 6-fluorohexyl group, and a 6,6,6-trifluorohexyl group.

[0019] The "C" used here 2-6 "Alkenyl group" (when used as a group or part of a group) means a straight or branched chain alkenyl group having 2 to 6 carbon atoms. 2-6 Alkenyl groups include, but are not limited to, vinyl, allyl, prop-1-enyl, but-1-en-1-yl, but-2-en-1-yl, pent-4-en-1-yl, 2-methylallyl and the like.

[0020] The "C" used here 1-6 "Alkoxy group" (when used as a group or part of a group) means a straight or branched chain alkoxy group having 1 to 6 carbon atoms. 1-6 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 2,2-dimethylpropoxy, 1,2-dimethylpropoxy, 1-ethylpropoxy, and hexyloxy groups.

[0021] The "C" used here 1-6 "Alkylthio group" is C 1-6 It means an -SH group substituted with an alkyl group. 1-6 Examples of alkylthio groups include, but are not limited to, methylthio groups, ethylthio groups, propylthio groups, and butylthio groups.

[0022] The "C" used here 2-6 Alkenylthio group" is C 2-6 It means an -SH group substituted with alkenyl. 2-6 Examples of alkenylthio groups include, but are not limited to, vinylthio, allylthio, prop-1-enylthio, but-1-en-1-ylthio, but-2-en-1-ylthio, pent-4-en-1-ylthio, and 2-methylallylthio groups.

[0023] The "C" used here 1-6 "Alkyl-carbonyl group" is C 1-6 It means a carbonyl group to which an alkyl group is bonded. 1-6 Examples of alkyl-carbonyl groups include, but are not limited to, acetyl, propionyl, butyryl, isobutyryl, valeryl, and hexanoyl groups.

[0024] The "C" used here 1-6 "Alkoxycarbonyl group" is C 1-6 It means a carbonyl group to which an alkoxy group is bonded. 1-6 Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl groups, ethoxycarbonyl groups, propoxycarbonyl groups, isopropoxycarbonyl groups, and butoxycarbonyl groups.

[0025] The "C" used here 6-10 "Aryl group" means an aromatic hydrocarbon group having 6 to 10 carbon atoms. 6-10 Examples of aryl groups include, but are not limited to, phenyl groups, naphthyl groups (1-naphthyl groups, 2-naphthyl groups), and the like.

[0026] The term "5- or 6-membered heteroaryl group" as used herein means a 5- or 6-membered heteroaryl group containing at least one (preferably 1 to 3, more preferably 1 or 2) heteroatom selected from a nitrogen atom, an optionally oxidized sulfur atom, and an oxygen atom. The 5- or 6-membered heteroaryl group is preferably a 5- or 6-membered heteroaryl group containing 1 or 2 heteroatoms selected from a nitrogen atom and an optionally oxidized sulfur atom.

[0027] Examples of 5- or 6-membered heteroaryl groups include, but are not limited to, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, imidazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, furyl, oxazolyl, and isoxazolyl groups. Preferred are pyridyl and thienyl groups.

[0028] As used herein, the term "oxo" (when used as a group or part of a group) refers to the group ═O.

[0029] As used herein, "optionally oxidized sulfur atom" means S, SO, or SO2.

[0030] R 1 and R 2 The "5- or 6-membered ring" in the "optionally substituted 5- or 6-membered ring" formed by bonding together means a 5- or 6-membered ring containing at least one heteroatom (preferably 1 to 3, more preferably 1 or 2) selected from a nitrogen atom, an optionally oxidized sulfur atom, and an oxygen atom. Examples of the 5- or 6-membered ring include a benzene ring and a tetrahydropyrimidine ring. The 5- or 6-membered ring may be substituted, and examples of the substituent include, for example, C 1-6 Alkyl group, halogen atom, amino group, -SH, C 1-6 Alkylthio group, C 2-6 Alkenylthio group, C 1-6 Alkyl-carbonyl group, formyl group, C 1-6The substituents are preferably selected from the group consisting of alkoxycarbonyl, oxo, and the like. 1-6 and 1 to 4 substituents selected from alkyl groups (eg, methyl) and oxo groups.

[0031] In formula (I), preferably, R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, C 1-6 Alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl), halogen atoms (e.g., chlorine atoms), amino groups, -SH, C 1-6 Alkylthio group (e.g., methylthio), C 2-6 Alkenylthio group (e.g., allylthio), C 1-6 Alkyl-carbonyl group (e.g., acetyl), formyl group, C 6-10 R is an aryl group (e.g., phenyl), a 5- or 6-membered heteroaryl group (e.g., thienyl), or an oxo group; 1 and R 2 may be bonded to each other to form an optionally substituted 5- or 6-membered ring (eg, a benzene ring, a tetrahydropyrimidine ring).

[0032] In formula (I), when n=1 or 2, R 1 , R 2 , R 3 and R 4 In formula (I), when n=0, R 1 , R 2 and R 3 It is preferable that at least one of the groups is not a hydrogen atom.

[0033] In the present invention, examples of the heterocyclic compound of formula (I) suitable for use as an active ingredient include 2-Methyl-2-thiazoline (2MT), 2,4,5-trimethylthiazole, 2-aminothiazole, Thiomorpholine, 2-ethylpyrrole, 2-acetylthiophene, 3-chlorothiophene, 2,3-dimethylthiomorpholine, 2,6-dimethylthiomorpholine, 2-methylthiomorpholine, 2-(methylthio)-2-thiazoline, 2-methylthiophene, 2-sec-butyl-2-thiazoline (SBT), 4-ethyl-2-methyl-2-thiazoline (4E2MT), 2-amino-2-thiazoline, 2-ethylthiophene, 2,4,5-trimethyl-3-thiazoline (TMT), 2-methylthiazole, 3-methylpyrrole, Thiomorpholine 1,1-dioxide, 2-isopropyl-4-methylthiazole, Azepan, 2,6-dimethylpyridine (2,6-lutidine), 2-ethyl-4-methylthiazole, 2-mercaptothiazole, Thiazole, 2,4-dimethylpyridine, 2-propylthiazole, 4-phenylthiomorpholine 1,1-dioxide, 2-(arylthio)-2-thiazoline, 5-methylthiazole, Thiazolidinedione, 2-methylbenzo[d]thiazole, 2-chlorothiazole, 2,4-dimethyl-1H-pyrrole, Caffeine, 2-acetylthiazole, 2,3-diethylpyrazine, 2-isobutylthiazole, 2-(2-thienyl)benzothiazole, 3,4-dimethylpyridine, 2-ethylfuran, 3-methylthiophene, 2H-1,4-benzothiazin-3(4H)-one, 5-Thiazolecarboxaldehyde (5-formylthiazole), 2,6-dimethylpyrazine, 2,2-dimethylthiazolidine, 2,3-dimethylpyridine, 3-methylpyridine, Morpholine, 2-thiophenecarboxaldehyde, 2,5-dimethyl-2-thiazoline, 2-Ethyl-2-thiazoline 2-Ethyl-3,5-dimethylpyrazine These include, but are not limited to:

[0034] In the present invention, examples of the isothiocyanate compound of formula (II) suitable for use as an active ingredient include Methallyl isothiocyanate, Allyl isothiocyanate, ethyl isothiocyanate, 2-chloroethyl isothiocyanate, 3-pyridyl isothiocyanate, phenyl isothiocyanate, 4-penten-1-yl isothiocyanate, butyl isothiocyanate, Propyl isothiocyanate These include, but are not limited to:

[0035] In the present invention, a TRPA1 agonist refers to a substance that activates TRPA1. In the present invention, examples of suitable TRPA1 agonists that can be used as an active ingredient include: 5-methylthiazole, 2-ethylfuran, 4-ethyl-2-methyl-2-thiazoline, 2-methylthiophene, 2,3-diethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-acetylthiophene, 2,6-lutidine (2,6-dimethylpyridine), Thiomorpholine, Acetaminophen, Allyl isothiocyanate, Delta 9-Tetrahydrocannabinol These include, but are not limited to:

[0036] In the present invention, the heterocyclic compound of formula (I) and the isothiocyanate compound of formula (II) used as active ingredients include substances generally known as reagents, and commercially available products can be used, or they can be obtained by a method known per se. The use of the heterocyclic compound of formula (I) and the isothiocyanate compound of formula (II) as a preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury, or inflammation, a cell protective agent for transplantation, or a biopreservative has not been disclosed or suggested so far.

[0037] Preferred examples of the heterocyclic compound represented by formula (I) include compounds represented by the following formulas (A) to (D) or salts thereof.

[0038] [ka]

[0039] (In the formula, X 1 is S, O, or N(R 16 ) and; X 2 is N or CR 12 and; X 3 is S, SO2, O, or -(CH2)2-; X 4 is N or CR 15 and;

[0040] [ka]

[0041] represents a single or double bond; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each independently a hydrogen atom, C 1-6 Alkyl group, halogen atom, amino group, -SH, C 1-6 Alkylthio group, C 2-6 Alkenylthio group, C 1-6 Alkyl-carbonyl group, formyl group, C 6-10 Aryl group, C 1-6 R is an alkoxycarbonyl group, a 5- or 6-membered heteroaryl group, or an oxo group; 13 and R 14 are bonded to each other to form a benzene ring or 1-6 may form a tetrahydropyrimidine ring optionally substituted with 1 to 4 substituents selected from an alkyl group and an oxo group; provided that in formula (A), R 11 and R 12 is not an oxo group; in formula (A),

[0042] [ka]

[0043] When represents a double bond, R 13 and R 14 is not an oxo group; In formula (D), R 11 , R 12 , R 13 , R 14 and R 15 is not an oxo group, and in formula (B), R 11 and R 12 may combine to form an oxo group.

[0044] In formulae (A) to (D), preferably, R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each independently a hydrogen atom, C 1-6 Alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl), halogen atoms (e.g., chlorine atoms), amino groups, -SH, C 1-6 Alkylthio group (e.g., methylthio), C 2-6 Alkenylthio group (e.g., allylthio), C 1-6 Alkyl-carbonyl group (e.g., acetyl), formyl group, C 6-10 an aryl group (e.g., phenyl), a 5- or 6-membered heteroaryl group (e.g., thienyl), or an oxo group; R 13 and R 14 are bonded to each other to form a benzene ring or 1-6 A tetrahydropyrimidine ring may be formed which may be substituted with 1 to 4 substituents selected from an alkyl group and an oxo group.

[0045] The salt of the compound according to the present invention may be any pharma- ceutically acceptable salt, and examples thereof include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts and calcium salts; ammonium salts such as dimethylammonium salts and triethylammonium salts; inorganic acid salts such as hydrochlorides, perchlorates, sulfates and nitrates; and organic acid salts such as acetates and methanesulfonates.

[0046] In the present invention, hypoxic injury refers to a disease caused by hypoxia, such as hypoxia, hypoxic encephalopathy, neonatal hypoxia, hypoxic-ischemic encephalopathy, hypoxic hypoxia, ischemic hypoxia, congestive hypoxia, altitude sickness, cerebral infarction, myocardial infarction, renal failure, heart failure, diabetic vascular disease, arteriosclerosis obliterans, ulcer, spinal cord injury, optic nerve injury, photoreceptor injury, and neuropathy.

[0047] In the present invention, ischemia-reperfusion injury refers to injury caused to cells or tissues of an ischemic organ by reperfusion accompanying the resumption of blood flow to the organ in an ischemic state, such as ischemia-reperfusion injury after reperfusion therapy for myocardial infarction, cerebral infarction, mesenteric vascular occlusion, etc., or after organ transplantation, bedsores, etc.

[0048] In the present invention, inflammation refers to a pathological inflammatory state in the whole body or tissues induced by trauma, invasion by pathogens, chemical irritation, radiation damage, etc. Examples of inflammation include sepsis, encephalitis, meningitis, arteritis, sinusitis, rhinitis, pneumonia, bronchitis, stomatitis, esophagitis, gastritis, enteritis, hepatitis, myositis, dermatitis, arthritis, nephritis, adrenal inflammation, lymphangitis, rheumatoid arthritis, psoriasis, osteoporosis, Crohn's disease, etc.

[0049] In the present invention, the term "cell protective agent for transplantation" refers to a drug used to protect cells for transplantation (including organs and tissues). Examples of cells include organs and tissues such as the heart, lungs, kidneys, liver, bone marrow, pancreas, skin, bones, veins, arteries, corneas, blood vessels, small intestine, large intestine, brain, spinal cord, smooth muscles, skeletal muscles, ovaries, testes, uterus, and umbilical cord, as well as cells derived therefrom.

[0050] In the present invention, a biopreservative can be used to preserve the whole or part of a living organism or maintain its freshness when storing or transporting the organism as food. Such organisms include organisms such as animals, plants, and seafood that are used as food. In the present invention, a living organism refers to the whole or part of an organism such as animals, plants, and seafood that are used as food (e.g., fish, shellfish, livestock, vegetables, fruits, etc.).

[0051] The heterocyclic compound represented by the formula (I) or a salt thereof, or the isothiocyanate compound represented by the formula (II) (hereinafter also referred to as the compound of the present invention) can be used as a preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury, or inflammation by vaporizing and inhaling it, or by administering it to the body. Alternatively, the compound of the present invention can be administered to a donor from whom an organ for transplant is extracted, or can be added to a preservation solution for the extracted organ for transplant (including tissues and cells) to be used as a cell protective agent for transplantation. In addition, the compound can be used as a biopreservative by adding it to a preservation solution for animals, plants, seafood, etc., which are provided as food.

[0052] Method of administration The compound of the present invention can be administered to animals, including humans, that have developed or may develop hypoxic injury, ischemia-reperfusion injury, or inflammation, for the purpose of preventing the onset of injury or alleviating symptoms. The compound of the present invention can also be administered to the body of an organ transplant donor for the purpose of tissue protection. A gas generated at a concentration of 0.1 to 100,000 ppm (or 10 to 100,000 ppm) derived from the compound of the present invention can be inhaled through the nasal cavity or lungs using a gas mask or a device with a similar function. Alternatively, the compound of the present invention can be orally administered at a dose of 1 μg / kg to 5,000 mg / kg. Alternatively, the compound of the present invention can be injected into the body at a dose of 1 μg / kg to 5,000 mg / kg by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, intraarterial injection, intrathecal injection, intraperitoneal injection, or the like. The frequency of administration can be a single administration, or continuous administration at regular intervals or continuous administration at different time intervals. When the cell protective agent for transplantation of the present invention is added to a preservation solution for organs for transplantation (including tissues and cells), the concentration of the compound of the present invention in the preservation solution is preferably 1 μg / l to 5,000 mg / l. Examples of animals that are subjects of administration or organ transplant donors include mammals (humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, horses, monkeys, etc.). When the bioprotective agent of the present invention is added to a preservation solution for storing or transporting food, the concentration of the compound of the present invention in the preservation solution is preferably 1 μg / l to 5,000 mg / l. Examples of organisms that are subjects of administration include mammals, fish, birds, and insects.

[0053] When the compound of the present invention is used as a preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury, or inflammation, a cell protective agent for transplantation, or a biopreservative (hereinafter also referred to as the agent of the present invention), a pharma- ceutical acceptable additive can be added as necessary. That is, the agent of the present invention can be used as a composition (pharmaceutical composition) containing the compound of the present invention and a pharma- ceutical acceptable additive.

[0054] Specific examples of pharma- ceutically acceptable excipients include, but are not limited to, antioxidants, preservatives, colorants, flavoring agents, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, carriers, excipients, and / or pharmaceutical adjuvants.

[0055] The formulation of the agent of the present invention is not particularly limited, and examples thereof include liquids, injections, sustained-release agents, sprays, etc. The solvent used to formulate the agent of the present invention into the above-mentioned preparations may be either aqueous or non-aqueous.

[0056] Injections can be prepared by methods well known in the art. For example, after dissolving in a suitable solvent (such as physiological saline, a buffer solution such as PBS, sterilized water, etc.), filtration sterilization using a filter or the like is performed, and then filling into a sterile container (such as an ampoule, etc.) to prepare an injection. This injection may contain a conventional pharmaceutical carrier as necessary. A non-invasive catheter-based administration method may also be used. Carriers that can be used in the present invention include neutral buffered saline, saline containing serum albumin, etc.

[0057] The present invention will be described in more detail and specifically below with reference to examples and experimental examples, but the present invention is not limited to these examples and experimental examples. EXAMPLES

[0058] Example 1: Body surface temperature reduction induced by thiazoline analogues Experimental Method The effects of thiazoline analogues and odor molecules inducing acquired fear on body surface temperature were analyzed. Three-month-old male C57 / BL6N mice were anesthetized with pentobarbital (50 mg / kg, ip) 2-3 days before the test, and the hair on their backs was removed with depilatory cream. On the day of the test, each mouse was placed in a test cage and allowed to acclimate for 10 minutes. After that, the mice were presented with filter paper soaked with 271 μmol of thiazoline analogues (2-methyl-2-thiazoline; 2MT) or odor molecules inducing acquired fear (anisole). The change in body surface temperature on the back was analyzed using a thermography camera (NEC Avio). The change in body surface temperature was calculated as the difference between the average body surface temperature for 10 minutes when water was presented and the average body surface temperature for 20 minutes when each odor molecule was presented. The change in body surface temperature without odor was set to zero. For the mice that had been exposed to the odor that induced acquired fear, they were given an association between electric shock and the odor of anisole one day before the measurements, so that the mice were taught to feel acquired fear toward the odor of anisole.

[0059] result The results are shown in Figure 1. Figure 1A: Thiazoline analogues (2MT) or odor molecules that induce acquired fear (anis-FS) were administered to mice. + The change in body surface temperature over time (mean ± standard error) when subjects were exposed to the scent of Figure 1B: Thiazoline analogues (2MT) or odor molecules that induce acquired fear (anis-FS) + The graph shows the average change in body surface temperature over a 20-minute period when the odorant was presented. The graph shows the mean ± standard error, and a Student's t-test was performed on the change in body surface temperature when no odor was presented and when the odor was presented. *** indicates a significant difference at p<0.001. A thiazoline analogue (2MT), which induces innate fear, caused a clear decrease in body surface temperature, whereas an odor molecule (anis-FS+), which induces acquired fear, had no significant effect on body surface temperature.

[0060] Example 2: Body surface temperature reduction induced by various thiazoline analogues Experimental Method The effects of various thiazoline analogues on body surface temperature were analyzed in the same manner as in Example 1. As a control, the effects of odor molecules that induce acquired fear were also analyzed. Experimental compounds (the names in parentheses indicate those in Figure 2) 2-methylthiazoline 2,5-dimethylthiazoline 2,2-dimethylthiazolidine 2,4,5-trimethyl-3-thiazoline (TMT) Thiazole 2-ethylthiazoline Thiomorpholine

[0061] result The results are shown in Figure 2. Figure 2: Various types of thiazoline analogues or odor molecules that induce acquired fear (Anis-FS) + The graph shows the average change in body surface temperature when odor molecules that induce acquired fear and thiazoline analogues were presented. Student's t-test was performed on the change in body surface temperature when odor molecules that induce acquired fear and thiazoline analogues were presented. *** indicates a significant difference at p<0.001 (n≧8 for each). Various types of thiazoline analogues have been shown to have the effect of significantly lowering body surface temperature.

[0062] Example 3: Freezing behavior and decrease in body surface temperature induced by intraperitoneal administration of various thiazoline analogues Experimental Method The effects of intraperitoneal administration of various types of thiazoline analogues on the freezing behavior (immobility time) and body surface temperature of mice were analyzed. Odor molecules were administered by intraperitoneal injection of 100 μl (approximately 40 mg / kg, ip) of a solution diluted 100-fold with saline, and body surface temperature was measured in the same manner as in Example 1. Freezing behavior was measured using freezing behavior analysis software (Freeze Frame2) and calculated as the percentage (%) of immobility time for 20 minutes after odor molecule administration. As controls, saline (saline) and an odor molecule (thiophene) that does not induce innate fear were also administered intraperitoneally, and the changes in freezing behavior and body surface temperature were also analyzed. Experimental compounds (the names in parentheses are those in Figure 3) Thiophene 5-Methylthiazole 2-sec-Butyl-2-thiazoline (SBT) 2,4,5-trimethyl-3-thiazoline (TMT) 2-methylthiazoline Thiomorpholine 2,2-dimethylthiazolidine 2-methyl-4-ethylthiazoline 2-ethylthiophene

[0063] result The results are shown in Figure 3. Figure 3A: Mean ± standard error of immobility behavior (freezing behavior) when various types of thiazoline analogues were intraperitoneally injected. Immobility behavior in the absence of odor (saline) was analyzed as a control, and a Student's t-test was performed on immobility behavior in the absence of odor and the presence of odor. ** indicates a significant difference at p<0.01, *** indicates a significant difference at p<0.001, and ns indicates no significant difference at p>0.05. Figure 3B: The mean ± standard error of the change in body surface temperature when various types of thiazoline analogues were intraperitoneally injected is shown. The change in body surface temperature without odor (saline) was analyzed as a control, and a Student's t-test was performed on the change in body surface temperature without odor and with odor. ** indicates a significant difference at p<0.01, *** indicates a significant difference at p<0.001, and ns indicates no significant difference at p>0.05. Thiazoline analogues induced freezing behavior when administered intraperitoneally, and many of them decreased the body surface temperature when administered intraperitoneally.

[0064] Example 4: Reduction in core body temperature induced by thiazoline analogues Experimental Method The effects of thiazoline analogues and odor molecules that induce acquired fear on core body temperature were analyzed. A wireless bio-parameter measuring device (Physiotel) was implanted in the abdomen of male C57 / BL6N mice approximately 3 months old. The mice were allowed to recover for approximately 10 days after surgery before undergoing the following experiment. Each mouse was placed in a test cage and, after 10 minutes of acclimation, presented with a water-soaked filter paper for 10 minutes, followed by a filter paper soaked with 271 μmol of a thiazoline analogue (2-methyl-2-thiazoline; 2MT) or an odor molecule that induces acquired fear (anisole) for 20 minutes. Body temperature information was recorded every 10 seconds using Dataquest ART software (DataScience international) during this period. The change in deep body temperature was calculated as the difference between the average deep body temperature during the 10 minutes when water was presented and the average deep body temperature during the 20 minutes when each odor molecule was presented, and the change in deep body temperature without odor was set to 0. For the mice that were exposed to the odor that induces acquired fear, they were given an association lesson between electric shock and the odor of anisole one day before the measurements, so that the mice were taught to feel acquired fear toward the odor of anisole.

[0065] result The results are shown in Figure 4. Figure 4A: Thiazoline analogues (2MT) or odor molecules that induce acquired fear (anis-FS) were administered to mice. + The change in body core temperature over time (mean ± standard error) when mice were exposed to the scent of Figure 4B: Thiazoline analogues (2MT) or odor molecules that induce acquired fear (anis-FS) + The graph shows the average change in deep body temperature over a 20-minute period when the odor was presented. The graph shows the mean ± standard error, and a Student's t-test was performed on the change in body surface temperature when no odor was presented (control) and when the odor was presented. *** indicates a significant difference at p<0.001. A thiazoline analogue (2MT) caused a clear decrease in core body temperature, whereas an odor molecule that induces acquired fear (anis-FS+) significantly increased surface body temperature.

[0066] Example 5: Heart rate reduction induced by thiazoline analogues Experimental Method The effects of thiazoline analogues and odor molecules that induce acquired fear on heart rate were analyzed. A wireless bioparameter measuring device (Physiotel) was implanted into the abdomen of male C57 / BL6N mice approximately 3 months old. The mice were allowed to recover for approximately 10 days after surgery before undergoing the following experiment. Each mouse was placed in a test cage and, after 10 minutes of acclimation, presented with water-soaked filter paper for 10 minutes. Then, presented with filter paper soaked with 271 μmol of a thiazoline analogue (2-methyl-2-thiazoline; 2MT) or an odor molecule that induces acquired fear (anisole) for 20 minutes. Heart rate information during this period was recorded every 10 seconds using Dataquest ART software (DataScience international). The change in heart rate was calculated as the difference between the heart rate during the 10 minutes when water was presented and the average heart rate during the 20 minutes when each odor molecule was presented, and the change in heart rate without odor was set to zero. For the mice that had been exposed to the odor that induced acquired fear, they were given an association between electric shock and the odor of anisole one day before the measurements, so that the mice were taught to feel acquired fear toward the odor of anisole.

[0067] result The results are shown in Figure 5. Figure 5A: Thiazoline analogues (2MT) or odor molecules that induce acquired fear (anis-FS) + The change in heart rate over time (mean ± standard error) when subjects were exposed to the scent of Figure 5B: Thiazoline analogues (2MT) or odor molecules that induce acquired fear (anis-FS) + The graph shows the average change in heart rate over a 20-minute period when the odor was presented. The graph shows the mean ± standard error, and a Student's t-test was performed on the change in body surface temperature when no odor was presented (control) and when the odor was presented. *** indicates a significant difference at p<0.001. The thiazoline analogue (2MT) caused a clear decrease in heart rate, halving it within 3 minutes, whereas the odor molecule that induces acquired fear (anis-FS+) had no significant effect on heart rate.

[0068] Example 6: Reduction in skin blood flow induced by thiazoline analogues Experimental Method We analyzed the effects of thiazoline analogues and odor molecules inducing acquired fear on skin blood flow. Three-month-old male C57 / BL6N mice were anesthetized with pentobarbital (50 mg / kg, ip) about 5 days before the test, and the hair on their backs was removed with a depilatory cream. A mouse restraining device with an observation window in a 50 ml tube was created, and mice were trained to enter the restraining device from the day after hair removal, and this procedure was continued until the day before the test to acclimate them. On the day of the test, mice were placed in the restraining device one by one, and a laser Doppler probe was attached to the mouse's shaved back with adhesive tape. After confirming that the blood flow signal was stable, (1) no odor for 10 minutes, (2) Eugenol odor was presented for 10 minutes, and (3) a thiazoline analogue (2MT) or an odor inducing acquired fear (Anis-FS+) was presented for 20 minutes, and the changes in blood flow were measured. Odors were presented by bringing filter paper soaked in 271 μmol of odor molecules close to the nose of the mouse. Blood flow was measured every 10 seconds using a laser Doppler blood flow meter (ADVANCE ALF21D). The average skin blood flow was calculated by averaging the blood flow during the presentation of no odor (10 min) and the presentation of thiazoline analogues or acquired fear-inducing odors (20 min), and was expressed as a relative value to the average skin blood flow during the presentation of no odor (100%).

[0069] result The results are shown in Figure 6. FIG. 6A: Changes in blood flow over time (mean ± standard error) when mice were exposed to a thiazoline analogue (2MT) or an odor molecule that induces acquired fear (Anis-FS+) are shown (n≧8). FIG. 6B: No odor, thiazoline analogue (2MT), or odor molecule that induces acquired fear (Anis-FS) +The bar graph shows the average skin blood flow when odor was presented. The graph shows the mean ± standard error, and a Student's t-test was performed on the blood flow when no odor was presented and when odor was presented. ** indicates a significant difference at p<0.01, and *** indicates a significant difference at p<0.001. Both thiazoline analogues and odor molecules that induce acquired fear reduced skin blood flow. The two possible mechanisms by which thiazoline analogues induce a decrease in body temperature are promotion of heat exchange by increasing skin blood flow and a decrease in heat production. However, the results of this study suggest that thiazoline analogues may induce a decrease in heat production rather than promotion of heat exchange.

[0070] Example 7: Reduction in respiratory rate induced by thiazoline analogues Experimental Method The effects of thiazoline analogues and odor molecules that induce acquired fear on respiratory rate were analyzed. Respiratory rate was analyzed using a pulse oximeter (Mouse Oxplus; STARR Life Science). Approximately 3-month-old C57 / BL6N male mice were habituated by repeatedly attaching an oximeter probe to their necks about 1 week before the test. Two days before the test, the mice were anesthetized with pentobarbital (50 mg / kg, ip) and the hair on their necks was removed with depilatory cream. On the day of the test, the oximeter probe was attached to the hairless neck of the mice, and after confirming that the signal was stable, a baseline without odor was measured for 10 minutes. Then, the change in respiratory rate was measured for 20 minutes when a thiazoline analogue (2MT) or a neutral odor (Eugenol; Eug) was presented (n=6 for each). Each odor was presented by placing filter paper soaked in 271 μmol of the odor into the test cage. The oximeter signal was acquired at 1 Hz. The average respiratory rate was calculated by averaging the respiratory rates during 10 minutes without odor and 20 minutes after odor presentation.

[0071] result The results are shown in Figure 7. FIG. 7A shows the change in respiratory rate over time (mean ± standard error) when mice were exposed to a thiazoline analogue (2MT) or the odor of a spice (Eug) (n=6). Figure 7B: Bar graphs show the average baseline respiratory rate (ctrl) without odor and the average respiratory rate when odor (Eug or 2MT) was presented for the group presented with the spice odor (Eug) and the group presented with a thiazoline analogue (2MT). The graphs show the mean ± standard error values, and a Student's t-test was performed on the respiratory rates of the group presented with 2MT and the group presented with Eug. ** indicates a significant difference at p<0.01, and ns indicates no significant difference at p>0.05. It was revealed that the respiratory rate was reduced by about half when treated with thiazoline analogues. A drop in body temperature and metabolic rate is observed in animals during hibernation, and it is well known that the respiratory rate of animals during hibernation also decreases. It was revealed that thiazoline analogues also cause a decrease in the respiratory rate, similar to that during hibernation.

[0072] Example 8: Reduction in oxygen consumption induced by thiazoline analogues Experimental Method The effect of thiazoline analogues on oxygen consumption was analyzed. Oxygen consumption was analyzed using a small animal energy metabolism measuring device (ARCO-2000; Arco Systems). Approximately 3-month-old C57 / BL6N male mice were placed individually in the measurement chamber, and after habituation for approximately 2 hours, a thiazoline analogue (2MT) was presented. Odor was presented by placing filter paper soaked in 100 μl (104 mmol) of odor molecules into the measurement chamber. As a control, a saline-soaked filter paper was also presented to the mice. Oxygen consumption was measured every minute. The average oxygen consumption was calculated as the average of the 10 min before and 20 min after odor presentation.

[0073] result The results are shown in Figure 8. FIG. 8A: The change over time in oxygen consumption per mouse body weight (mean ± standard error) when a thiazoline analogue (2MT) was presented and when no odor was present (saline) was presented (n=8). Figure 8B: Bar graph showing the average oxygen consumption per body weight of mice when presented with a thiazoline analogue (2MT) and when no odor was administered. The graph shows the mean ± standard error, and a Student's t-test was performed on the oxygen consumption when presented with 2MT and when presented with saline. *** indicates a significant difference at p<0.001, and ns indicates no significant difference at p>0.05. In the control (saline) condition, oxygen consumption increased when a novel substance (filter paper) was presented. In contrast, oxygen consumption significantly decreased when a thiazoline analogue (2MT) was presented. It is known that oxygen consumption decreases in animals during hibernation due to a decrease in metabolic rate, and it was shown that a similar change was also caused by a thiazoline analogue.

[0074] Example 9: Hypoxia resistance induced by thiazoline analogues Experimental Method Approximately 3-month-old C57 / BL6N male mice were placed in a tightly sealed breeding cage, and a filter paper soaked with a total of 100 μl (104 mmol) of a thiazoline analogue compound (2MT) was placed in the breeding cage, and the odor was presented for 50 minutes. After the odor presentation, the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured up to 30 minutes. As a control, the survival time of mice presented with a filter paper soaked in water was also measured (no odor). In addition, the change in body core temperature when the odor was presented under the same conditions as in this example was measured using the same experimental method as in Example 4.

[0075] result The results are shown in Figure 9. FIG. 9A: Kaplan-Meier curves show the survival time of mice in 4% oxygen when presented with a thiazoline analogue (2MT) and when there was no odor (n=6 for each). Figure 9B: The change in core body temperature over time (mean ± standard error) when odor was presented under the same conditions as in the example of Figure 9A (2MT, n=8; no odor, n=6). In the control condition without odor, all individuals died within 20 minutes in 4% oxygen, whereas in the condition where a thiazoline analogue (2MT) was presented, many individuals survived even after 30 minutes. A significant drop in body core temperature was also observed under the odor presentation condition used in this example. It was shown that thiazoline analogues induce hypoxia resistance.

[0076] Example 10: Hypoxia resistance induced by various heterocyclic compounds Experimental Method We analyzed the effect of intraperitoneal administration of various heterocyclic compounds on the survival time of mice in a hypoxic environment. The odor molecules were administered intraperitoneally at 200 μl (approximately 80 mg / kg, ip) of a solution diluted 100-fold with saline. After 30 minutes, the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured up to 30 minutes. As a control, the survival time of mice that received intraperitoneal injections of saline (control) was measured.

[0077] result The results are shown in Figure 10A. Figure 10A: Bar graphs show the survival time (mean ± standard error) of mice in 4% oxygen after intraperitoneal injection of each heterocyclic compound. The survival time is shown as a relative value (%), with the mean survival time of the control group injected intraperitoneally with saline set as 100% (n=4). Student's t-test was performed on the survival time between the control group and each compound-administered group. * indicates a significant difference at p<0.05, ** indicates p<0.01, and *** indicates p<0.001. FIG. 10B: The structural formulas of the compounds used in Example 10 are shown in the order of the graph. A wide variety of heterocyclic compounds have been shown to confer hypoxia resistance.

[0078] Example 11: Hypoxic resistance induced by isothiocyanate compounds Experimental Method Using the same method as in Example 10, the effect of intraperitoneal administration (about 80 mg / kg, ip) of various isothiocyanate compounds on the survival time of mice in a hypoxic environment was analyzed.

[0079] result The results are shown in Figure 11A. Figure 11A: Bar graphs show the survival time (mean ± standard error) of mice in 4% oxygen after intraperitoneal injection of each compound. The survival time is shown as a relative value (%), with the mean survival time of the control group injected intraperitoneally with saline set as 100% (n = 4). Student's t-test was performed on the survival time between the control group and each compound-administered group. * indicates a significant difference with p < 0.05, ** with p < 0.01, and *** with p < 0.001. FIG. 11B: The structural formulas of the compounds used in Example 11 are shown in the order of the graph. Various isothiocyanate compounds have been shown to induce hypoxia resistance.

[0080] Example 12: Hypoxia resistance induced by intraperitoneal administration of thiomorpholine Experimental Method Three-month-old male C57 / BL6N mice were intraperitoneally injected with 200 μl (approximately 80 mg / kg, ip) of thiomorpholine solution diluted 100-fold with saline, and 30 minutes later, the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured. As a control, the survival time of mice that received an intraperitoneal injection of saline was also measured.

[0081] result The results are shown in Figure 12. FIG. 12: Kaplan-Meier curves showing survival times in 4% oxygen for mice treated with thiomorpholine and saline (n=6). Thiomorpholine has been shown to confer hypoxia resistance.

[0082] Example 13: Effect of intraperitoneal administration of NaHS on survival time under 4% oxygen conditions Experimental Method Three-month-old male C57 / BL6N mice were intraperitoneally injected with 200 μl of NaHS solution, and 30 minutes later, the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured for up to 30 minutes. NaHS solutions were used at four different concentrations: 0.1%, 0.125%, 0.05%, and 0.0125%. Of these, mice died within a few minutes when the 0.1% solution was intraperitoneally injected, so survival times were measured at the remaining three concentrations. As controls, saline and 2-methylthiophene (1% solution) were intraperitoneally injected, and their survival times in 4% oxygen were analyzed.

[0083] result The results are shown in Figure 13. Figure 13: The survival time of each individual mouse intraperitoneally injected with each solution shown in Figure 13 was plotted. Comparison of survival time between the two groups indicated by dotted lines was performed using Student's t-test. *** indicates a significant difference at p<0.001, and ns indicates no significant difference at p>0.05. It has been reported that NaHS, a donor of H2S, has a preventive effect against ischemia-reperfusion injury (Yu et al., Cell Physiol Biochem 36: 1539-1551, 2015). However, mice died immediately after injection of high concentrations of NaHS, and low concentrations did not show hypoxia resistance. In contrast, all mice administered 2-methylthiophene, a thiazoline analogue, survived in 4% oxygen for 30 minutes of observation.

[0084] Example 14: Effect of thiazoline analogues on ischemia-reperfusion injury Experimental Method A mouse model of skin ischemia-reperfusion injury was created according to the method of Uchiyama et al. (Uchiyama et al., Sci Rep 5: 9072, 2015). Male C57 / BL6N mice aged about 3 months were depilated from their backs 2-3 days before the test using the same method as in Example 1. On the day of the test, the mice were placed in tightly sealed cages one by one, and filter paper soaked with a total of 100 μl (104 mmol) of a thiazoline analogue compound (2MT) was placed in the cage, and the odor was presented for 30 minutes. As a control, filter paper soaked with water was presented for 30 minutes (no odor). After 30 minutes, the mouse was removed from the cage, the skin on the back was sandwiched between two circular magnets, and the mouse was returned to the sealed cage. Filter paper soaked with 100 μl (104 mmol) of 2MT or water was placed in the sealed cage, and the mouse was left for 12 hours. After 12 hours, the mouse was taken out of the sealed cage, the magnet was removed, and the mouse was transferred to a normal breeding cage without odor. The condition of the skin on the back that had been sandwiched between the magnets was recorded by taking photographs every day. The area of ​​the wound was quantified based on the acquired images using image analysis software (Photoshop, Adobe). In addition, the change in deep body temperature when the odor was presented under the same conditions as in this example was measured using the same experimental method as in Example 2.

[0085] result The results are shown in Figure 14. FIG. 14A shows the results of daily observations of changes in the wound area for each individual mouse under odorless conditions (n=5). FIG. 14B shows the results of daily observation of changes in the wound area for each individual mouse under conditions in which 2MT was presented (n=5). Figure 14C: The wound area observed in A and B was quantified. The graph shows the change in wound area over time (mean ± standard error) as a relative value, with the mean wound area after 3 days in the absence of odor set to 100%. A Student's t-test was performed on the wound areas observed on the same day when 2MT was presented and when there was no odor. ** indicates a significant difference at p<0.01, and *** indicates a significant difference at p<0.001. Figure 14D: Changes in core body temperature over time (mean ± standard error) when odors were presented under the same conditions as in the AC examples (n = 6 each). In the odorless control, pressure ulcers were formed when the skin was clamped between magnets for 12 hours, whereas the formation of pressure ulcers was significantly reduced when 2MT was exposed to the smell. A significant drop in core body temperature was also observed under the odor presentation conditions used in this example. Pressure ulcers are caused by an ischemic state caused by continuous pressure. It was shown that thiazoline-related compounds have the effect of reducing and preventing ischemia-reperfusion injury.

[0086] Example 15: Changes in body surface temperature in Trpa1 knockout mice in response to thiazoline analogues Experimental Method Using male Trpa1 knockout mice aged about 3 months and their heterozygous littermate mice, changes in body surface temperature induced by a thiazoline analogue (2MT) were analyzed in the same manner as in Example 1. The changes in body surface temperature were calculated as the difference between the average body surface temperature during 20 min of 2MT presentation minus the average body surface temperature during habituation without odor.

[0087] result The results are shown in Figure 15. Figure 15: Control mice (trpa1 + / - ; n = 5) and Trpa1 knockout mice (trpa1 - / - The average change in body surface temperature when a thiazoline analogue (2MT) was presented to 8 mice (n=8) was shown. Student's t-test was performed on the change in body surface temperature between control mice and knockout mice. ** indicates a significant difference at p<0.01. The decrease in body surface temperature induced by thiazoline analogue (2MT) was not observed in Trpa1 knockout mice, suggesting that the decrease in body surface temperature induced by 2MT is mediated by Trpa1.

[0088] Example 16: Changes in core body temperature in Trpa1 knockout mice in response to thiazoline analogues Experimental Method Using male Trpa1 knockout mice and their heterozygous littermate mice aged approximately 3 months, changes in body core temperature induced by a thiazoline analogue (2MT) were analyzed in the same manner as in Example 2. The changes in body core temperature were calculated as the difference between the average body core temperature during 20 min of 2MT presentation minus the average body core temperature during acclimation without odor.

[0089] result The results are shown in Figure 16. Figure 16A shows the time course of changes in core body temperature when control mice (trpa1+ / -; n=7) and Trpa1 knockout mice (trpa1- / -; n=7) were presented with 2MT. FIG16B: Control mouse (trpa1 + / - ) and Trpa1 knockout mice (trpa1 - / - The average change in body core temperature when 2MT was presented to control mice was shown. Student's t-test was performed on the change in body core temperature between control mice and knockout mice. *** indicates a significant difference at p<0.001. In Trpa1 knockout mice, the decrease in core body temperature induced by a thiazoline analogue (2MT) was not observed, suggesting that the decrease in core body temperature by 2MT is mediated by Trpa1.

[0090] Example 17: Hypoxia resistance induced by thiazoline analogues in Trpa1 knockout mice Experimental Method Approximately 3-month-old male Trpa1 knockout mice and their littermate heterozygotes were presented with a thiazoline analogue (2MT) for 10 minutes, after which the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured for up to 30 minutes. The odor was presented by placing filter paper soaked in 271 μmol of 2MT in the cage. As a control, an experiment was also conducted in which filter paper soaked in saline was presented (no odor).

[0091] result The results are shown in Figure 17. Figure 17: The average survival time in 4% oxygen when saline or 2MT was presented to control mice (Hetero) and Trpa1 knockout mice (KO) (n=6 for no odor hetero, n=7 for others). Student's t-test was performed on the survival time when saline and 2MT were presented to each mouse. * indicates significant difference at p<0.05, ns indicates no significant difference at p>0.05. Unlike control mice, Trpa1 knockout mice did not show a significant increase in survival time in 4% oxygen after treatment with a thiazoline analogue (2MT), suggesting that the hypoxic resistance induced by the thiazoline analogue is mediated by Trpa1.

[0092] Example 18: Suppressive effects of various heterocyclic and isothiocyanate compounds on TNF-α levels in blood in a sepsis model Experimental Method A sepsis model was created by intraperitoneally administering lipopolysaccharide (LPS) (0.6 mg / kg) to male Balb / c mice aged approximately 2-3 months. Immediately after LPS administration, 200 μl (approximately 80 mg / kg) of a solution of various heterocyclic compounds and isothiocyanate compounds diluted 100-fold with physiological saline was administered intraperitoneally. Blood was collected 60 minutes after administration of LPS and compounds, EDTA plasma was prepared, and the blood levels of TNF-α, an inflammatory cytokine, were measured by ELISA.

[0093] result The results are shown in Figure 18. In Figure 18, "4-Ethyl-2-methyl-thiazoline" means 4-ethyl-2-methyl-2-thiazoline. The blood TNF-α levels (mean ± standard error) after administration of each compound are shown in a bar graph. The TNF-α levels are shown as relative values ​​(%), with the mean TNF-α level in the case of administration of only LPS and no compound (saline) set at 100% (n ≥ 4). Student's t-test was performed for the TNF-α levels between the control group (saline) and each compound-administered group. * indicates significant difference at p < 0.5, ** at p < 0.01, and *** at p < 0.001. The administration of various types of heterocyclic and isothiocyanate compounds reduced the blood concentration of TNF-α, an inflammatory cytokine, indicating that these compounds have anti-inflammatory effects in sepsis model animals.

[0094] Example 19: Effects of thiazoline analogues on the production of pro-inflammatory and anti-inflammatory cytokines in a sepsis model Experimental Method Sepsis model mice were prepared in the same manner as in Example 18. Immediately after LPS administration, 200 μl (approximately 80 mg / kg) of a thiazoline analogue (2MT) solution diluted 100-fold with physiological saline was administered intraperitoneally. Blood was collected 1 hour and 4 hours after administration of LPS and the thiazoline analogue (2MT), EDTA plasma was prepared, and the blood levels of the inflammatory cytokine Interleukin-1β (IL-1β) and the anti-inflammatory cytokine Interleukin-10 (IL-10) were measured by ELISA.

[0095] result The results are shown in Figure 19. The amount of IL-1β (Fig. 19A) and IL-10 (Fig. 19B) (all mean ± standard error) when thiazoline analogues (2MT) were administered and when saline was administered as a control are shown in bar graphs (n=6). Student's t-test was performed for each condition between the control group (saline) and the thiazoline analogues (2MT) administration group. * indicates a significant difference at p<0.05, ** indicates a significant difference at p<0.01. The administration of thiazoline analogues reduced the blood concentration of IL-1β, an inflammatory cytokine, and conversely increased the blood concentration of IL-10, an anti-inflammatory cytokine, demonstrating that thiazoline analogues have an anti-inflammatory effect in sepsis model animals.

[0096] Example 20: Inhibitory effect of thiazoline analogues on inflammatory mediator HMGB1 in a sepsis model Experimental Method Sepsis model mice were prepared in the same manner as in Example 18. Immediately after LPS administration, 200 μl (approximately 80 mg / kg) of a thiazoline analogue (2MT) solution diluted 100-fold with physiological saline was administered intraperitoneally. 16 hours after administration of LPS and thiazoline analogue (2MT), blood was collected, EDTA plasma was prepared, and the blood level of the inflammatory mediator High Mobility Group Box 1 (HMGB1) was measured by ELISA.

[0097] result The results are shown in Figure 20. The blood HMGB1 levels (mean ± standard error) when a thiazoline analogue (2MT) was administered and when saline was administered as a control are shown in a bar graph (n=6). A Student's t-test was performed between the control group (saline) and the thiazoline analogue (2MT) administration group. *** indicates a significant difference at p<0.001. Since administration of a thiazoline analogue reduced the blood concentration of HMGB1, an inflammatory mediator, it was demonstrated that thiazoline analogues have an anti-inflammatory effect in sepsis model animals.

[0098] Example 21: Effect of intraperitoneal administration of thiazoline analogues on survival time of septic model animals Experimental Method Sepsis model mice were prepared in the same manner as in Example 18. Immediately after LPS administration, 200 μl (approximately 80 mg / kg) of a thiazoline analogue (2MT) solution diluted 100-fold with physiological saline was administered intraperitoneally. The survival time of mice was measured when the thiazoline analogue (2MT) was administered and when physiological saline (saline) was administered as a control.

[0099] result The results are shown in Figure 21. Survival curves are shown for the control group (saline) and the group administered a thiazoline analogue (2MT) (left). The average survival time (mean ± standard error) for each group is shown in a bar graph (n=10; right). Student's t-test was performed between the control group and the group administered a thiazoline analogue (2MT). ** indicates a significant difference at p<0.01. It was shown that administration of a thiazoline analogue has the effect of extending the survival time of sepsis model animals.

[0100] Example 22: Effect of thiazoline analogues on cerebral ischemia-reperfusion injury Experimental Method The common carotid arteries of both sides of C57 / BL6 mice (approximately 3 months old) were clipped to induce cerebral ischemic injury, and the clips were removed 30 minutes later to allow blood reperfusion. During reperfusion, 200 μl (approximately 80 mg / kg) of a thiazoline analogue (2MT) solution or saline (control) was administered intraperitoneally. Two days after reperfusion, the mouse brain tissue was removed, sliced, and the injured area was analyzed by antibody staining for microtubule-associated protein 2 (MAP2).

[0101] result The results are shown in Figure 22. The experimental protocol is shown at the top of the figure. Representative examples of brain sections stained with MAP2 antibodies from control and thiazoline analogue (2MT)-treated animals are shown on the left of the figure. In the figure, damaged areas of the brain were observed as areas not stained with MAP2 (black). In addition, the area of ​​areas not stained with MAP2 in the control group and the thiazoline analogue (2MT)-treated group, i.e., the area of ​​damaged areas (mean ± standard error) are shown in a bar graph on the right of the figure (saline: n = 9, 2MT: n = 8). Student's t-test was performed between the control group and the thiazoline analogue (2MT)-treated group. * indicates a significant difference at p < 0.05. It was shown that administration of a thiazoline analogue at the time of reperfusion has the effect of reducing cerebral ischemia-reperfusion injury.

[0102] Example 23: Induction of hypoxia resistance by Trpa1 against administration of heterocyclic compounds Experimental Method Male Trpa1 knockout and wild-type mice aged approximately 3 to 6 months were intraperitoneally injected with 200 μl (approximately 80 mg / kg) of various heterocyclic compounds diluted 100-fold with saline. Thirty minutes later, the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured up to 30 minutes.

[0103] result The results are shown in Figure 23. In Figure 23, "4-Ethyl-2-methyl-thiazoline" means 4-ethyl-2-methyl-2-thiazoline. Figure 23: Wild-type mice (Trpa1 + / + ) and Trpa1 knockout mice (Trpa1 - / -The bar graph shows the survival time (mean ± standard error) in 4% oxygen when saline or each heterocyclic compound was administered to 4 mice (n = 4). Student's t-test was performed between the control group (saline) and the compound-administered group for each condition. ** indicates significant difference at p < 0.01, *** indicates significant difference at p < 0.001, and ns indicates no significant difference at p > 0.05. Various types of heterocyclic compounds have the effect of significantly increasing survival time under hypoxic conditions when administered to wild-type mice, but such an effect was not observed in Trpa1 knockout mice. Therefore, it was suggested that hypoxia resistance induced by these heterocyclic compounds is induced by Trpa1.

[0104] Example 24: Induction effect of hypoxia resistance against thiomorpholine administration by Trpa1 Experimental Method Male wild-type and Trpa1 knockout mice aged approximately 3 to 6 months were intraperitoneally injected with 200 μl (approximately 80 mg / kg) of a heterocyclic compound (thiomorpholine) diluted 100-fold with saline. Thirty minutes later, the mice were placed in a tight box with an oxygen concentration adjusted to 4%, and their survival time was measured.

[0105] result The results are shown in Figure 24. Figure 24: Wild-type mice (Trpa1 + / + ) and Trpa1 knockout mice (Trpa1 - / - The bar graph shows the survival time (mean ± standard error) in 4% oxygen when thiomorpholine was administered to 6 mice (n=6). Student's t-test was performed between the control group (saline) and the compound-administered group. *** indicates a significant difference at p<0.001. When the heterocyclic compound (thiomorpholine) was administered to wild-type mice, it had the effect of greatly extending survival time under hypoxic conditions, but survival time was significantly reduced in Trpa1 knockout mice compared to wild-type mice. Therefore, it was demonstrated that hypoxia resistance induced by thiomorpholine is induced by Trpa1.

[0106] Example 25: Induction effect of Trpa1 on suppression of oxygen consumption by administration of thiazoline analogues Experimental Method The oxygen consumption was measured using a small animal energy metabolism measuring device in the same manner as in Example 8. Approximately 3- to 6-month-old Trpa1 knockout mice and their littermate heterozygotes were placed in the measurement chamber of the small animal energy metabolism measuring device, and after habituation for approximately 2 hours, a thiazoline analogue (2MT) was presented. The odor was presented by placing two pieces of filter paper soaked with 271 μmol of the thiazoline analogue (2MT) into the measurement chamber.

[0107] result The results are shown in Figure 25. FIG. 25: Control mice (Trpa1) reacted with a thiazoline analogue (2MT) + / - ; n = 9) and Trpa1 knockout mice (Trpa1 - / - The change in oxygen consumption per body weight over time (mean ± standard error) is shown for mice (n=7). The odor was presented at 10 minutes in the figure. In control mice, oxygen consumption was reduced by the presentation of a thiazoline analogue (2MT), whereas no reduction in oxygen consumption was observed in Trpa1 knockout mice. This indicates that the reduction in oxygen consumption by thiazoline analogues is mediated by Trpa1.

[0108] Example 26: Induction of hypoxia resistance by administration of Trpa1 agonist Experimental Method Male C57 / BL6N mice, approximately 3 months of age, were treated with saline or a known Trpa1 agonist, delta9-tetrahydrocannabinol (Δ9-TTC), to 9Mice were intraperitoneally administered 100 mg / kg of 1-THC, 40 mg / kg of AITC, or 300 mg / kg of acetaminophen (APAP). Thirty minutes later, the mice were placed in a tight box with an oxygen concentration of 4% and the survival time of the mice was measured for up to 30 minutes. For APAP, the survival time of male Trpa1 knockout mice and their littermate heterozygotes aged 3 to 6 months in a 4% oxygen environment was also measured.

[0109] result The results are shown in Figure 26. FIG. 26A: Bar graphs showing survival times (mean ± standard error) in 4% oxygen after administration of saline or various Trpa1 agonists to wild-type mice (n = 22 for saline, Δ 9 -THC, AITC: n=8, APAP: n=7). Student's t-test was performed between the control group and the Trpa1 agonist-administered group. ** indicates a significant difference at p<0.01, *** indicates a significant difference at p<0.001. Administration of the Trpa1 agonist was found to have the effect of extending survival time in a hypoxic environment. FIG. 26B: Control mice (Trpa1) after intraperitoneal administration of Trpa1 agonist (APAP) + / - ) and Trpa1 knockout mice (Trpa1 - / - ) survival time (mean ± standard error) in 4% oxygen is shown in a bar graph (n=8). Student's t-test was performed between the control group (saline) and the Trpa1 agonist-treated group (APAP). ** indicates significant difference at p<0.01, and ns indicates no significant difference at p>0.05. The Trpa1 agonist (APAP) had the effect of extending survival time in a hypoxic environment in control mice, whereas no such effect was observed in Trpa1 knockout mice. It was demonstrated that the induction of hypoxia resistance by the Trpa1 agonist is mediated by Trpa1.

[0110] Example 27: Effects of Trpa1 knockout mice on body surface area changes, hypoxia resistance and anti-inflammatory effects induced by thiazoline analogues Experimental Method A thiazoline analogue (4-ethyl-2-methyl-2-thiazoline; 4E2MT) was administered intraperitoneally to male Trpa1 knockout mice and their heterozygous littermate mice aged approximately 3 to 6 months. The changes in body surface temperature, survival time in a hypoxic environment, and TNF-α production in the blood upon LPS administration were measured using methods similar to those described in Examples 3, 10, and 18, respectively.

[0111] result The results are shown in Figure 27. FIG. 27A: Control mice (Trpa1) upon intraperitoneal injection of a thiazoline analogue (4E2MT). + / ‐ ) and Trpa1 knockout mice (Trpa1 ‐ / ‐ ) shows the time-dependent change in body surface temperature (mean ± standard error). The thiazoline analogue compound (4E2MT) was administered at 10 minutes. The result of a Student's t-test on the average surface temperature 30 minutes after administration of the thiazoline analogue compound (4E2MT) between control mice and Trpa1 knockout mice is shown in the figure. *** indicates a significant difference at p<0.001. It was revealed that the decrease in body surface temperature when the thiazoline analogue compound was administered intraperitoneally was not observed in Trpa1 knockout mice. FIG. 27B: Control mice (Trpa1) upon intraperitoneal injection of a thiazoline analogue (4E2MT). + / ‐ ) and Trpa1 knockout mice (Trpa1 ‐ / ‐ The survival time (mean ± standard error) of Trpa1 knockout mice in 4% oxygen is shown in a bar graph. A Student's t-test was performed between control mice and Trpa1 knockout mice. ** indicates a significant difference at p<0.01. It was revealed that the effect of intraperitoneal injection of thiazoline analogues in extending survival time in a hypoxic environment was suppressed in Trpa1 knockout mice. FIG. 27C: Control mice (Trpa1) were intraperitoneally injected with a thiazoline analogue (4E2MT) at the same time as intraperitoneal administration of LPS. + / + , Trpa1 + / ‐ ) and Trpa1 knockout mice (Trpa1‐ / ‐ The bar graph shows the amount of TNF-α in the blood (mean ± standard error) one hour after administration of Trpa1 + / + We also measured the amount of TNF-α in the blood when LPS and saline were administered to mice. ** indicates a significant difference at p<0.01, and *** indicates a significant difference at p<0.001. It was shown that administration of a thiazoline analogue (4E2MT) to sepsis model mice reduces the amount of TNF-α, an inflammatory cytokine, in the blood, but this effect is suppressed in Trpa1 knockout mice. The results in Figures 27A-C indicate that the reduction in body surface temperature, hypoxia resistance, and anti-inflammatory effects induced by thiazoline analogues are mediated by Trpa1.

[0112] Example 28: Inhibitory effect of oxygen consumption induced by heterocyclic compounds Experimental Method The change in oxygen consumption when heterocyclic compounds (2MT and thiomorpholine (TMO)) were intraperitoneally injected into approximately 3-month-old C57 / BL6N mice was measured in the same manner as described in Example 8.

[0113] result The results are shown in Figure 28. Figure 28: Changes in oxygen consumption over time (mean ± standard error) when heterocyclic compounds (2MT, TMO) and saline (saline) were administered (n=4). Heterocyclic compounds (2MT, TMO) and saline (saline) were administered at the time points indicated by the arrows in the figure. It was shown that administration of heterocyclic compounds has the effect of reducing oxygen consumption. [Industrial Applicability]

[0114] The compound of the present invention induces hypothermia, low metabolism, suppression of oxygen consumption, low heart rate and anti-inflammatory response, and exhibits protective effects on tissues and individuals under hypoxic conditions, protective effects against ischemia-reperfusion injury and protective effects against inflammation, and is therefore useful as a preventive or therapeutic agent for hypoxic injury, ischemia-reperfusion injury or inflammation, a cell protective agent for transplantation, and a biopreservative.

[0115] This application is based on patent application No. 2018-049761 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. Formula (I) 【Chemistry 1】 (In the formula, Ring A is a 2-thiazoline; R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom, C １－６ Alkyl group, C １－６ Alkylthio group, or C ２－６ an alkenylthio group; n is 0) 2. A preventive or therapeutic agent for ischemia-reperfusion injury, comprising as an active ingredient a heterocyclic compound represented by the following formula:

2. A heterocyclic compound represented by formula (I): 2-methyl-2-thiazoline, 2-(methylthio)-2-thiazoline, 2-sec-butyl-2-thiazoline, 4-ethyl-2-methyl-2-thiazoline, 2-(allylthio)-2-thiazoline, 2,5-dimethyl-2-thiazoline, and 2-Ethyl-2-thiazoline The agent according to claim 1 , which is selected from the group consisting of

3. The agent according to claim 1 or 2, wherein the heterocyclic compound represented by formula (I) is 2-methyl-2-thiazoline.

4. The agent according to any one of claims 1 to 3, for nasal administration.

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