Prophylactic or therapeutic agent for hypoxia disorder, ischemia-reperfusion disorder or inflammation, cell protective agent for transplantation, and biological preservative

Abstract

The present invention provides a prophylactic or therapeutic agent for hypoxia disorders, ischemia-reperfusion disorders or inflammation, a cell protective agent for transplantation, and a biological preservative. The present invention pertains to: a prophylactic or therapeutic agent for hypoxia disorders, ischemia-reperfusion disorders or inflammation; a cell protective agent for transplantation; and a biological preservative, each of which contains, as an active ingredient, at least one selected from the group consisting of: a heterocyclic compound represented by formula (I) (wherein each symbol in the formula is as defined in the description) or a salt thereof; an isothiocyanate compound represented by formula S = C = N-R5 (II) (wherein the symbols are as described in the specification); and a TRPA1 agonist. # imgabs0 #

Description

This application is a divisional application of the Chinese patent application No. 201980019788.1 (application date: March 15, 2019, invention name: preventive or therapeutic agent for hypoxia disorder, ischemia-reperfusion disorder or inflammation, cell protectant for transplantation, and biological preservative). Technical Field

[0001] The present invention relates to a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, and a biological preservative. Background Art

[0002] Humans and animals may have acquired the ability to increase their chances of survival in times of crisis during the course of evolution. There are many known cases where people who were found in a low-temperature state in snowy mountains and other places were able to recover even after several hours of cardiac arrest. In such cases, there may be some kind of stimulus that triggers the potential intrinsic individual protection. Some mammals such as bears and squirrels have the ability to hibernate. During hibernation, these animals can maintain life in a state of reduced body temperature and oxygen consumption. When oxygen supply is stopped due to cardiac arrest, irreversible damage is caused to cells in high oxygen-consuming tissues such as the brain. On the other hand, the restoration of blood flow after cardiac arrest causes the production of reactive oxygen species, etc., which causes great damage to tissues. Hypothermia therapy, which artificially induces low body temperature in individuals, can alleviate such hypoxic disorders and ischemia-reperfusion disorders. In addition, it is known that animals in a hibernating state have enhanced resistance to inflammation, ischemia-reperfusion disorders, etc. (Non-patent literature 1-3).

[0003] Fear is an emotion induced by the brain when it judges that danger is approaching; fear can induce escape behavior and freezing behavior (Freezing behavior), the latter of which can reduce the possibility of being discovered by natural enemies, and also induce a variety of physiological reactions. When a phobic patient faints due to the application of fear stimulation, the heart rate will drop by nearly 50% (Non-patent Document 4). Such fear emotions are also associated with inducing strong physiological reactions in humans. If people or animals in crisis can induce anti-inflammatory and immune control reactions in addition to low body temperature and low metabolism to enhance the protection of tissues and individuals, the survival rate can be improved. However, no technology that can induce fear emotions or potential intrinsic individual protection has yet to be developed. Prior art literature Patent Literature

[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 literature 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 Problems to be solved by the invention

[0006] The purpose of the present invention is to provide a technology for inducing fear-induced or potential intrinsic individual protective effects, and to provide a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, and a biological preservative. Means of solving the problem

[0007] Fear emotion is controlled by innate and acquired mechanisms. The inventor has found that in the nasal cavity, innate and acquired smell information is transmitted to the brain to control behavior by separated neural circuits (non-patent document 5). This discovery proves the existence of the neural circuit responsible for the innate behavior control of smell, thus overturning the common sense all along, that is, the behavior for smell is determined by acquired learning and experience. If the reaction to smell is innately controlled, then the technology that can control behavior by will can be developed by developing the odor molecules that act on this genetic mechanism. In fact, the inventor has successfully developed "thiazoline-related fear odors (thiazoline-related fear odors, tFOs)" for the first time in the world, which is a kind of artificial odor molecule that can cause extremely strong innate fear emotion (patent document 1, non-patent document 6). The development of tFOs makes it possible to explain in detail the control mechanism of the innate fear emotion that is difficult to explain so far and the physiological reaction caused by the innate fear emotion.

[0008] Both innate and acquired fear stimuli caused by olfaction cause similar degrees of freezing behavior. However, it is known that only innate fear stimulation can cause a decrease in body temperature and heart rate if physiological reactions are used as indicators. In addition, it is known that for the compound groups (heterocyclic compounds and isothiocyanate compounds) related to innate fear or potential intrinsic individual protection represented by the following general formula of the present invention, by smelling or by intraperitoneal administration of these compounds, hypoxia, ischemia-reperfusion disorders and inflammation can be inhibited. On the other hand, the inventors found that the compounds that induce fear effects or potential intrinsic individual protection have the characteristics of activating TRPA1 (transient receptor potential ankyrin 1), and explained that in TRPA1 knockout mice, fear emotions and potential intrinsic individual protection are partially inhibited. These results show that a part of fear emotions and potential intrinsic individual protection is controlled by the TRPA1 gene. The TRPA1 gene is widely present in organisms, including humans, livestock and other mammals, birds, fish and even insects, in which the technology of the present invention can prevent or treat hypoxia, ischemia-reperfusion disorders or inflammation or use cell protectants for transplantation.

[0009] That is, the present invention relates to the following. 1. A preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, or a biological preservative, comprising at least one selected from the following as an active ingredient:

[0010] [Chemical formula 1]

[0011] In formula (I), ring A is a 5- to 7-membered heterocyclic ring 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 Each is independently: hydrogen atom, C 1-6 Alkyl, halogen atom, amino, -SH, C 1-6 Alkylthio, C 2-6 Alkenylthio, C 1-6 Alkyl-carbonyl, formyl, C 6-10 Aryl, C 1-6 Alkoxycarbonyl, 5- or 6-membered heteroaryl, or oxo; R 1 and R 2 may combine with each other to form an optionally substituted 5- or 6-membered ring; n is 0, 1, or 2; and, Isothiocyanate compound represented by formula (II) S=C=NR 5(II) In formula (II), R 5 C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 6-10 2. The agent according to item 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 item 1 or 2, wherein the active ingredient is a heterocyclic compound represented by formula (I) or a salt thereof. 4. The agent according to item 1, wherein the active ingredient is an isothiocyanate compound represented by formula (II). 5. The agent according to any one of Items 1 to 4, which is a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder, or inflammation. 6. The agent according to any one of items 1 to 4, which is a cell-protective agent for transplantation. 7. The agent according to any one of items 1 to 4, which is a biological preservative. 8. The agent according to any one of Items 1 to 7, which is for nasal administration. 9. A preventive or therapeutic agent for hypoxia disorder, ischemia-reperfusion disorder or inflammation, comprising a TRPA1 agonist as an active ingredient. 10. A cell protectant for transplantation, comprising a TRPA1 agonist as an active ingredient. 11. A biological preservative comprising a TRPA1 agonist as an active ingredient. 12. The agent according to any one of Items 9 to 11, which is for nasal administration. 13. At least one compound selected from the group consisting of the heterocyclic compound represented by the aforementioned formula (I) or a salt thereof and the isothiocyanate compound represented by the aforementioned formula (II), for use in the prevention or treatment of hypoxic disorders, ischemic reperfusion disorders or inflammation. 14. Use of at least one compound selected from the group consisting of the heterocyclic compound represented by the aforementioned formula (I) or a salt thereof and the isothiocyanate compound represented by the aforementioned formula (II) as a cell protectant for transplantation or a biological preservative. 15. A TRPA1 agonist for use in preventing or treating hypoxia, ischemia-reperfusion disorder or inflammation. 16. Use of TRPA1 agonists as cell protectants for transplantation or biological preservatives. 17. Use of at least one compound selected from the heterocyclic compound represented by the aforementioned formula (I) or its salt and the isothiocyanate compound represented by the aforementioned formula (II) in the preparation of a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, or a biological preservative. 18. Use of a TRPA1 agonist in the preparation of a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, or a biological preservative. 19. A method for preventing or treating hypoxia, ischemia-reperfusion disorder or inflammation in mammals, comprising administering to the mammal an effective amount of at least one compound selected from the heterocyclic compound represented by the aforementioned formula (I) or a salt thereof and the isothiocyanate compound represented by the aforementioned formula (II). 20. A method for protecting cells for transplantation, comprising contacting the cells for transplantation with at least one compound selected from the group consisting of the heterocyclic compound represented by the aforementioned formula (I) or a salt thereof and the isothiocyanate compound represented by the aforementioned formula (II). 21. A method for preserving an organism, comprising contacting the organism with at least one compound selected from the group consisting of a heterocyclic compound represented by the aforementioned formula (I) or a salt thereof and an isothiocyanate compound represented by the aforementioned formula (II). 22. A method for preventing or treating hypoxia, ischemia-reperfusion disorder 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 an organism, comprising contacting the organism with a TRPA1 agonist. [Effects of the Invention]

[0012] Fear is an emotion induced by the brain after it perceives a dangerous situation. Fear can induce behavioral and physiological reactions, increasing the chances of survival 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, the brain and nervous system that perceive fear and crisis signals induce a decrease in body temperature and metabolism, or control inflammation and immune response, thereby obtaining a protective effect on tissues and individuals. Such a function is in line with the purpose. However, the regulatory mechanism of this fear emotion and potential intrinsic individual protective effect has not yet been elucidated. By administering compounds that meet specific chemical structure rules, the technology of the present invention can induce behaviors and physiological reactions such as hypothermia and low metabolism characterized by innate fear and potential intrinsic individual protective effects, while inhibiting inflammation and immune response, and producing strong resistance to hypoxia, ischemia-reperfusion disorder and inflammation. The compounds used in the technology of the present invention enter the body by activating receptors as volatile odor molecules, or by directly applying them to the body by methods such as injection to exert their effects. Its effects include low body temperature, low metabolism, suppression of oxygen consumption, low heart rate, induction of anti-inflammatory response, protection of tissues and individuals under hypoxic conditions, protection against ischemia-reperfusion disorders, protection against inflammation, etc. Through this technology, therapeutic agents for hypoxia disorders, ischemia-reperfusion disorders and inflammation, protective agents for organ transplantation, and biological preservatives can be provided to individuals. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [ Figure 1 A: Shows the effect of sniffing 2MT (2-methyl-2-thiazoline) or an acquired fear-inducing odor molecule (anis-FS + ) over time (mean ± standard error). B: Exposure to 2MT or an odor molecule that induces acquired fear (anis-FS + )The average change in body surface temperature over 20 minutes. [ Figure 2 ] showed that exposure to various thiazoline-related compounds or odor molecules that induce acquired fear (Anis-FS + ) is a graph showing the average change in body surface temperature when [ Figure 3 ]A: A graph showing the mean ± standard error of "immobility behavior" ("freezing behavior") when various thiazoline-related compounds were intraperitoneally injected. B: A graph showing the mean ± standard error of changes in body surface temperature when various thiazoline-related compounds were intraperitoneally injected. [ Figure 4 A: Shows that smelling 2MT may induce acquired fear (anis-FS +A: The time-dependent changes in deep body temperature during exposure to 2MT or an odor molecule that induces acquired fear (anis-FS + ) Graph showing the average change in deep body temperature during a 20-minute period. [ Figure 5 A: Shows that smelling 2MT may induce acquired fear (anis-FS + ) (mean ± standard error). B: Shows the changes in heart rate over time after exposure to 2MT or odor molecules that induce acquired fear (anis-FS + )A graph of the average change in heart rate over a 20-minute period. [ Figure 6 A: Shows that sniffing 2MT may induce acquired fear odor molecules (Anis-FS + ) over time (mean ± standard error). B: Shows the blood flow changes over time (mean ± standard error) when exposed to no odor and 2MT or odor molecules that induce acquired fear (Anis-FS + ) plot of average skin blood flow at . [ Figure 7 A: A graph showing the temporal changes (mean ± standard error) in respiratory rate when sniffing 2MT or the odor of a fragrance (Eug). B: A graph showing the average respiratory rate when exposed to no odor and 2MT or the odor of a fragrance (Eug). [ Figure 8 A: A graph showing the time-dependent changes (mean ± standard error) in oxygen consumption per unit body weight of mice exposed to 2MT and odorless (saline saline). B: A graph showing the average oxygen consumption per unit body weight of mice exposed to 2MT and odorless. [ Fig. 9 A: A graph showing the survival time of mice under 4% oxygen conditions when exposed to 2MT and when there is no odor (expressed as a Kaplan-Meier curve). B: A graph showing the temporal changes in deep body temperature when odor exposure is performed under the same conditions as in Example A (mean ± standard error). [ Fig. 10A ] A graph showing the survival time (mean ± standard error) of mice injected intraperitoneally with each heterocyclic compound under 4% oxygen conditions. [ Fig. 10B ]according to Fig. 10A The diagrams showing the structural formulas of the compounds used in Example 10 are shown in the diagrammatic order. [ Fig.11A ] A graph showing the survival time (mean ± standard error) of mice injected intraperitoneally with each isothiocyanate compound under 4% oxygen conditions. [ Fig. 11B ]according to Fig.11AThe diagrams showing the structural formulas of the compounds used in Example 11 are shown in the order of diagrams. [ Fig.12 ] A graph showing the survival time (expressed as a Kaplan-Meier curve) of mice administered with thiomorpholine and normal saline under 4% oxygen conditions. [ Fig.13 ]The survival time of mice injected intraperitoneally with each solution shown in the figure is plotted separately for each individual. [ Fig.14 A: Photograph of the results of daily observation of changes in the wound site for each individual under odor-free conditions. B: Photograph of the results of daily observation of changes in the wound site for each individual under 2MT conditions. C: Graph of quantitative analysis of the areas of the wound sites observed in A and B. D: Graph of changes in deep body temperature over time (mean ± standard error) when odor exposure was performed under the same conditions as in Examples AC. [ Fig.15 ] showed that control mice (trpa1 + / - ; n = 5) and Trpa1 knockout mice (trpa1 - / - ; n=8) Graph of the mean changes in body surface temperature during exposure to 2MT. [ Fig.16 ]A: shows control mice (trpa1 + / - ; n = 7) and Trpa1 knockout mice (trpa1 - / - ; n = 7) Graph showing the temporal changes in deep body temperature when exposed to 2MT. B: Shows the change in deep body temperature of control mice (trpa1 + / - ) and Trpa1 knockout mice (trpa1 - / - ) Plot of the mean deep body temperature changes during exposure to 2 MT. [ Fig.17 ] Graph showing the mean survival time of control mice (Hetero) and Trpa1 knockout mice (KO) exposed to saline or 2MT under 4% oxygen conditions. [ Fig.18 ] A graph showing the amount of TNF-α in the blood (mean ± standard error) when each heterocyclic compound or isothiocyanate compound was intraperitoneally injected into sepsis model mice. [ Fig.19 ]A: A graph showing the amount of IL-1β in the blood (mean ± standard error) when 2MT was intraperitoneally injected into sepsis model mice. B: A graph showing the amount of IL-10 in the blood (mean ± standard error) when 2MT was intraperitoneally injected into sepsis model mice. [ Fig. 20 ] A graph showing the amount of HMGB1 in the blood (mean ± standard error) when 2MT was intraperitoneally injected into sepsis model mice. [ Fig.21 ] A graph showing the survival time (expressed as Kaplan-Meier curve and bar graph (mean ± standard error)) when 2MT was intraperitoneally injected into sepsis model mice. [ Fig. 22 ] A diagram showing brain tissue staining in cerebral ischemia-reperfusion disorder model mice and quantification of the area of ​​the damaged region. [ Fig.23 ] showed that the control mice (Trpa1 + / + ) and Trpa1 knockout mice (Trpa1 - / - ) A graph showing the survival time (mean ± standard error) under 4% oxygen conditions when each heterocyclic compound was intraperitoneally administered. [ Fig.24 ] showed that the control mice (Trpa1 + / + ) and Trpa1 knockout mice (Trpa1 - / - ) Survival time (mean ± standard error) under 4% oxygen condition when thiomorpholine was intraperitoneally administered [ Fig.25 ] showed that control mice (Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / - ) is a graph showing the time course of oxygen consumption when exposed to 2MT (mean ± standard error). [ Fig.26 A: A graph showing the survival time (mean ± standard error) under 4% oxygen conditions when each Trpa1 agonist was administered. B: A graph showing the survival time (mean ± standard error) when each Trpa1 agonist was administered to control mice (Trpa1 + / + , Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / - ) Graph showing the survival time (mean ± SE) under 4% oxygen conditions when acetaminophen was administered. [ Fig. 27 ]A: shows the expression of Trpa1 in control mice + / - ) and Trpa1 knockout mice (Trpa1 - / - ) shows the time-dependent changes in body surface temperature (mean ± standard error) when 4E2MT (4-ethyl-2-methyl-2-thiazoline) was intraperitoneally injected. B: shows the time-dependent changes in body surface temperature (mean ± standard error) when 4E2MT (4-ethyl-2-methyl-2-thiazoline) was intraperitoneally injected into control mice (Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / - ) Survival time (mean ± standard error) under 4% oxygen conditions when 4E2MT was intraperitoneally injected. C: shows the survival time (mean ± standard error) of control mice (Trpa1 + / + , Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / -) A graph showing the amount of TNFα in the blood (mean ± standard error) when LPS (lipopolysaccharide) and 4E2MT were administered. [ Fig.28 ] A graph showing the time course of oxygen consumption (mean ± standard error) when thiomorpholine (TMO) and 2MT were intraperitoneally administered. DETAILED DESCRIPTION

[0014] Ring A of formula (I) represents a 5- to 7-membered heterocyclic ring 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 heterocyclic ring 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 heterocyclic ring 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 aforementioned heterocycles are, for example, but 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, isothiazolin, thiomorpholine, thiadiazoline, thiadiazole, thiadiazolidine, 1,3-thiazinane, 5,6-dihydro-4H-1,3-thiazine, 2,3-dihydro-4H-1,4-thiazine, furan, 2H-pyran, 4H-pyran, oxazole, isoxazole, morpholine, oxazoline, azepane, and the like. 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, more preferably 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 1-6 "Alkyl" (when used as a group or a part of a group) refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. 1-6The alkyl group may be exemplified by methyl, ethyl, propyl, isopropyl, butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), tert-butyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1-ethyl-2-methylpropyl, etc., but is not limited thereto. Preferred C 1-6 Alkyl is, for example, C 1-4 The alkyl group (a linear or branched alkyl group having 1 to 4 carbon atoms) is more preferably 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 preferably a methyl group.

[0018] The “C 1-6 "Haloalkyl" refers to a C 1-6 When there are two or more halogen groups in the alkyl group, the types of the halogen groups may be the same or different. Examples of the halogen group include fluoro, chloro, bromo, and the like. 1-6 The haloalkyl group is exemplified by fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 2,2,2-trifluoroethyl, 1,1,2,2-tetrafluoroethyl, 1,1,2,2,2-pentafluoroethyl, 1-fluoropropyl, 1,1-difluoropropyl, 2,2-difluoropropyl, 3-fluoropropyl, 3,3,3-trifluoropropyl, 4-fluorobutyl, 4,4,4-trifluorobutyl, 5-fluoropentyl, 5,5,5-trifluoropentyl, 6-fluorohexyl, 6,6,6-trifluorohexyl, and the like, but is not limited thereto.

[0019] The “C 2-6 "Alkenyl" (when used as a group or a part of a group) refers to a straight chain or branched alkenyl group having 2 to 6 carbon atoms. 2-6 Examples of the alkenyl group include 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, but are not limited thereto.

[0020] The “C 1-6 "Alkoxy" (when used as a group or a part of a group) means a straight chain or branched chain alkoxy group having 1 to 6 carbon atoms. 1-6Examples of the alkoxy group include 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, hexyloxy, and the like, but are not limited thereto.

[0021] The “C 1-6 "Alkylthio" refers to a C 1-6 Alkyl-substituted -SH group. 1-6 The alkylthio group may be exemplified by methylthio, ethylthio, propylthio, butylthio and the like, but is not limited thereto.

[0022] The “C 2-6 "Alkenylthio" refers to a C 2-6 Alkenyl-substituted -SH group. 2-6 The alkenylthio group can be exemplified by vinylthio, allylthio, prop-1-enylthio, but-1-en-1-ylthio, but-2-en-1-ylthio, pent-4-en-1-ylthio, 2-methylallylthio and the like, but is not limited thereto.

[0023] The “C 1-6 "Alkyl-carbonyl" refers to a group with a C 1-6 The carbonyl group of an alkyl group. 1-6 The alkyl-carbonyl group may be exemplified by acetyl, propionyl, butyryl, isobutyryl, valeryl, hexanoyl and the like, but is not limited thereto.

[0024] The “C 1-6 "Alkoxycarbonyl" refers to a group having a C 1-6 The carbonyl group of an alkoxy group. 1-6 The alkoxycarbonyl group can be exemplified by methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl and the like, but is not limited thereto.

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

[0026] The "5- or 6-membered heteroaryl group" used herein refers to 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 the 5- or 6-membered heteroaryl group include, but are not limited to, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, imidazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, furyl, oxazolyl, isoxazolyl, etc., and preferably pyridyl, thienyl, etc.

[0028] As used herein, the term "oxo" (when used as a group or part of a group) refers to the =0 group.

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

[0030] R 1 and R 2 The "5- or 6-membered ring" of the "optionally substituted 5- or 6-membered ring" formed by combining with each other refers to a 5- or 6-membered ring containing at least one (preferably 1 to 3, more preferably 1 or 2) heteroatom selected from nitrogen atoms, optionally oxidized sulfur atoms and oxygen atoms. The aforementioned 5- or 6-membered ring may be exemplified by a benzene ring, a tetrahydropyrimidine ring, etc. The aforementioned 5- or 6-membered ring may be optionally substituted, and the substituent may be exemplified by a substituent selected from C 1-6 Alkyl, halogen atom, amino, -SH, C 1-6 Alkylthio, C 2-6 Alkenylthio, C 1-6 Alkyl-carbonyl, formyl, C 1-6 1 to 4 (preferably 1 or 2) substituents of alkoxycarbonyl, oxo, etc. As the substituent, preferably selected from C 1-6 1 to 4 substituents of the alkyl group (eg, methyl group) and the oxo group.

[0031] In formula (I), preferably, R 1 , R 2 , R 3 and R 4 Each is independently a hydrogen atom, C 1-6 Alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl), halogen atom (e.g., chlorine atom), amino group, -SH, C 1-6 Alkylthio (eg methylthio), C 2-6 Alkenylthio (eg allylthio), C 1-6 Alkyl-carbonyl (e.g. acetyl), formyl, C 6-10 Aryl (eg, phenyl), 5- or 6-membered heteroaryl (eg, thienyl), or oxo; R 1 and R 2 They may combine with 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 R4 It is preferred that at least one of them is not a hydrogen atom. In formula (I), when n=0, R 1 , R 2 and R 3 It is preferred that at least one of them is not a hydrogen atom.

[0033] In the present invention, suitable heterocyclic compounds of formula (I) used as active ingredients include 2-methyl-2-thiazoline (2MT), 2,4,5-Trimethylthiazole, 2-aminothiazole, Thiomorph, 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, azepane, 2,6-Dimethylpyridine (2,6-lutidine), 2-ethyl-4-methylthiazole, 2-mercaptothiazole, Thiazole, 2,4-Dimethylpyridine, 2-propylthiazole, 4-phenylthiomorpholine 1,1-dioxide, 2-(allylthio)-2-thiazoline, 5-methylthiazole, Thiazolidine, 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, But it is not limited to this.

[0034] In the present invention, suitable isothiocyanate compounds of formula (II) used as an active ingredient include Methylallyl isothiocyanate, Allyl isothiocyanate, Ethyl isothiocyanate, 2-Chloroethyl isothiocyanate, 3-Pyridyl isothiocyanate, Phenyl isothiocyanate, 4-Penten-1-yl isothiocyanate, Butyl isothiocyanate, Propyl isothiocyanate, But it is not limited to this.

[0035] In the present invention, a TRPA1 agonist refers to a substance that activates TRPA1. In the present invention, suitable TRPA1 agonists used as active ingredients are, for example, 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), Thiomorph, Acetaminophen, Allyl isothiocyanate But it is not limited to this.

[0036] In the present invention, the heterocyclic compound of formula (I) and the isothiocyanate compound of formula (II) used as the active ingredient contain substances known as reagents, commercially available substances can be used, or the substances can be obtained by methods known per se. So far, it has never been disclosed or taught to use the heterocyclic compound of formula (I) and the isothiocyanate compound of formula (II) as a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, or a biological preservative.

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

[0038] [Chemical formula 2]

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

[0040] [Chemical formula 3]

[0041] represents a single or double bond; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 Each is independently a hydrogen atom, C 1-6 Alkyl, halogen atom, amino, -SH, C 1-6 Alkylthio, C 2-6 Alkenylthio, C 1-6 Alkyl-carbonyl, formyl, C 6-10 Aryl, C 1-6 Alkoxycarbonyl, 5- or 6-membered heteroaryl, or oxo; R 13 and R 14 may be combined with each other to form a benzene ring, or to form a 1-6 A tetrahydropyrimidine ring substituted with 1 to 4 substituents of an alkyl group and an oxo group; however, R 11 and R 12 Not an oxo group; When in formula (A),

[0042] [Chemical formula 4]

[0043] When it represents a double bond, R 13 and R 14 It is not an oxo group; In formula (D), R 11 , R 12, R 13 , R 14 and R 15 is not an oxo group, R in formula (B) 11 and R 12 can form an oxo group together)

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

[0045] The salt of the compound involved in the present invention may be a pharmaceutically acceptable salt, for example, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salts such as dimethylammonium salt and triethylammonium salt; inorganic acid salts such as hydrochloride, perchlorate, sulfate and nitrate; organic acid salts such as acetate and methanesulfonate, etc.

[0046] In the present invention, hypoxic disorder refers to a disease caused by hypoxia. Examples thereof include: hypoxia, cerebral hypoxia, neonatal hypoxia, hypoxic-ischemic encephalopathy, hypoxic hypoxia, ischemic hypoxia, congestive hypoxia, mountain sickness, cerebral infarction, myocardial infarction, renal failure, heart failure, diabetic vascular injury, occlusive arteriosclerosis, ulcer, spinal cord injury, optic nerve injury, optic cell injury, nerve injury, etc.

[0047] In the present invention, the so-called ischemia-reperfusion disorder refers to damage in cells or tissues of ischemic organs due to reperfusion accompanying the recovery of blood flow to the organs under ischemic conditions. Examples thereof include ischemia-reperfusion disorder after reperfusion treatment of 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 of the whole body or tissue caused by trauma, invasion of pathogens, stimulation of chemical substances, radiation damage, etc. For example, sepsis, encephalitis, meningitis, arteritis, sinusitis, rhinitis, pneumonia, bronchitis, oral ulcer, esophagitis, gastritis, enteritis, hepatitis, myositis, dermatitis, arthritis, nephritis, adrenalitis, lymphangitis, rheumatoid arthritis, psoriasis, osteoporosis, Crohn's disease, etc.

[0049] In the present invention, the so-called transplant cell protectant refers to a drug used to protect cells (including organs and tissues) for transplantation. 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 intestines, large intestines, brain, spinal cord, smooth muscles, skeletal muscles, ovaries, testicles, uterus, umbilical cord, and cells derived therefrom.

[0050] In the present invention, the so-called biological preservative refers to a substance used to preserve the whole body or part of the organism when the organism is stored or transported as food, so as to maintain freshness. The organism can be exemplified by animals, plants, or aquatic organisms used as food. In the present invention, the organism refers to the whole or part of animals, plants, or aquatic organisms used as food (for example, fish, shellfish, livestock, vegetables, fruits, etc.).

[0051] By gasifying the heterocyclic compound or its salt shown in the above formula (I), or the isothiocyanate compound (hereinafter referred to as the compound of the present invention) shown in the above formula (II) so as to be inhaled, or by the mode of in vivo administration, it is used as a preventive or therapeutic agent for hypoxia, ischemia-reperfusion disorder or inflammation. Alternatively, the compound of the present invention can be administered to the donor of the organ for transplantation, or it can be used as a transplant cell protectant in the form of adding to the preservation solution of the transplanted organ (including tissues and cells). In addition, it can be used as a biological preservative by adding to the preservation solution of animals, plants, aquatic products, etc. supplied as food.

[0052] Application method For animals including humans that have or may have hypoxia, ischemia-reperfusion disorders, and inflammation, the compounds of the present invention may be administered for the purpose of preventing the occurrence of disorders or alleviating symptoms. In addition, for the purpose of tissue protection, the compounds of the present invention may be administered to the body of organ transplant donors. A gas mask or a device with similar functions may be used to inhale the gas derived from the compounds of the present invention at a concentration of 0.1 to 100,000ppm (or 10 to 100,000ppm) via the nasal cavity or lungs. Alternatively, the compounds of the present invention may be orally administered in an amount of 1μg / kg to 5,000mg / kg. Alternatively, the compounds of the present invention may be injected into the body in an amount of 1μg / kg to 5,000mg / kg by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, intraarterial injection, spinal cavity injection, intraperitoneal injection, etc. The frequency of administration may 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 the preservation solution of the organ 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. Animals that are the subject of administration or organ transplant donors include mammals (humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, horses, monkeys, etc.). When the organism protective agent of the present invention is added to the 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. Organisms that are the subject 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 hypoxia, ischemia-reperfusion disorder or inflammation, a cell protectant for transplantation, or a biological preservative (hereinafter also referred to as the agent of the present invention), a pharmaceutically acceptable additive may be used as needed. That is, the medicament of the present invention can be used as a composition (pharmaceutical composition) containing the compound of the present invention and a pharmaceutically acceptable additive.

[0054] Specific examples of pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, expanders, buffers, delivery vehicles, diluents, carriers, excipients and / or pharmaceutical adjuvants, but are not limited thereto.

[0055] The preparation form of the agent of the present invention is not particularly limited, and examples thereof include liquid preparations, injections, sustained-release preparations, sprays, etc. In order to formulate the agent of the present invention in the above preparation form, the solvent used may be aqueous or non-aqueous.

[0056] The injection can be prepared by methods known in the art. For example, after being dissolved in an appropriate solvent (physiological saline, buffer such as PBS, sterile water, etc.), it can be sterilized by filtration through a filter, etc., and then filled in a sterile container (such as an ampoule, etc.) to prepare the injection. Such an injection may contain a conventional pharmaceutical carrier as required. An administration method using a non-invasive catheter can also be used. As carriers available in the present invention, neutral buffered physiological saline, or physiological saline containing serum albumin, etc. can be exemplified.

[0057] The following examples and experimental examples are given to further explain the present invention in detail and specifically, but the examples and experimental examples do not limit the present invention. [Example]

[0058] Example 1: Decrease in body surface temperature induced by thiazoline-related compounds Experimental methods The effects of thiazoline-related compounds and acquired fear-inducing odor molecules on body surface temperature were analyzed. C57 / BL6N male mice aged approximately 3 months were anesthetized with pentobarbital (50 mg / kg, ip) 2-3 days before the experiment, and the hair on the back was removed with a depilatory cream. On the day of the experiment, each mouse was placed in a test cage. After 10 minutes of acclimatization, filter paper soaked with 271 μmol of thiazoline-related compounds (2-methyl-2-thiazoline; 2MT) or acquired fear-inducing odor molecules (anisole) was placed in the test cage for exposure, and the changes in body surface temperature on the back were analyzed using a thermal imager (NEC Avio). The value of the change in body surface temperature was calculated as follows: the difference between the average body surface temperature after 10 minutes of exposure to water and the average body surface temperature after 20 minutes of exposure to each odor molecule, with the change in body surface temperature in the absence of odor as 0. The mice that were to smell the acquired fear-inducing odor were trained with electric shock and the odor of anisole one day before the measurement, so that the mice could feel the acquired fear of the odor of anisole through the learning.

[0059] result The results are Figure 1 Displayed in. Figure 1 A: Shows the effect of sniffing a thiazoline-related compound (2MT) or an acquired fear-inducing odor molecule (anis-FS + ) over time (mean ± standard error) (n≧8). Figure 1 B: Shows the changes in the expression of odor molecules after exposure to a thiazoline-related compound (2MT) or an acquired fear-inducing odor molecule (anis-FS +) Average change in body surface temperature for 20 minutes. The graph shows the mean ± standard error, and the change in body surface temperature when there is no odor and when exposed to the odor was tested by Student's t-test. *** indicates a significant difference with p < 0.001. Thiazoline-related compounds (2MT) that induce innate fear clearly caused a decrease in body surface temperature. In contrast, odor molecules (anis-FS) that induce acquired fear + ) did not significantly affect body surface temperature.

[0060] Example 2: Decrease in body surface temperature induced by various thiazoline-related compounds Experimental methods The effects of various thiazoline-related compounds 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. The experimental compounds (in brackets Figure 2 Mark in 2-Methylthiazoline 2,5-dimethylthiazoline 2,2-dimethylthiazolidine 2,4,5-Trimethyl-3-thiazoline(TMT) Thiazole 2-ethylthiazoline Thiomorpholine

[0061] result The results are Figure 2 Displayed in. Figure 2 : Exposure to various thiazoline-related compounds or odor molecules that induce acquired fear (Anis-FS + ). The graph shows the mean ± standard error. Student's t-test was performed on the changes in body surface temperature when exposed to odor molecules that induce acquired fear and thiazoline-related compounds. *** indicates p<0.001, which is significantly different (each n≧8). This indicates that various thiazoline-related compounds have a significant effect of lowering body surface temperature.

[0062] Example 3: Freezing behavior and decrease in body surface temperature induced by intraperitoneal administration of various thiazoline-related compounds Experimental methods The effects of various thiazoline-related compounds on freezing behavior (immobility time) and body surface temperature of mice when administered intraperitoneally were analyzed. The administration of the odor molecules was performed by intraperitoneal injection of 100 μl of a solution diluted 100 times with saline (about 40 mg / kg, ip), and the measurement of body surface temperature was performed in the same manner as in Example 1. Freezing behavior was measured using freezing behavior analysis software (Freeze Frame2) and calculated as the ratio (%) of the immobility time 20 minutes after the administration of the odor molecules. As a control, the changes in freezing behavior and body surface temperature when saline (saline) and odor molecules that do not induce innate fear (thiophene) were administered intraperitoneally were also analyzed. Experimental compounds (shown in brackets Figure 3 Mark in 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 Figure 3 Displayed in. Figure 3 A: Shows the mean ± standard error of immobility (freezing behavior) when various thiazoline-related compounds were intraperitoneally injected. As a control, immobility without odor (saline) was analyzed, and Student's t-test was performed on immobility without odor and with odor. ** indicates p<0.01, *** indicates p<0.001 and significant difference, ns indicates p>0.05 and no significant difference. Figure 3B: Mean ± standard error of changes in body surface temperature when various thiazoline-related compounds were intraperitoneally injected is shown. Changes in body surface temperature without odor (saline) were analyzed as a control, and Student's t-test was performed on changes in body surface temperature without odor and with odor. ** indicates p<0.01, *** indicates p<0.001 and significant difference, and ns indicates p>0.05 and no significant difference. Intraperitoneal administration of thiazoline-related compounds induced freezing behavior. In addition, a decrease in body surface temperature was observed by intraperitoneal administration of several compounds of thiazoline-related compounds.

[0064] Example 4: Decreased Deep Body Temperature Induced by Thiazoline-Related Compounds Experimental methods The effects of thiazoline-related compounds and odor molecules that induce acquired fear on deep body temperature were analyzed. A wireless bioparameter measuring device (manufactured by Physiotel) was embedded in the abdomen of C57 / BL6N male mice of about 3 months of age. The following experiment was performed on the mice after a recovery period of about 10 days after surgery. Each mouse was placed in a test cage, and after acclimation for 10 minutes, it was exposed to filter paper soaked in water for 10 minutes. Then, the filter paper soaked with 271 μmol of thiazoline-related compounds (2-methyl-2-thiazoline; 2MT) or odor molecules that induce acquired fear (anisole) was exposed for 20 minutes, during which the body temperature data was recorded every 10 seconds using Dataquest ART software (DataScience international). The value of the change in deep body temperature was calculated as follows: the difference between the average deep body temperature after 10 minutes of exposure to water and the average deep body temperature after 20 minutes of exposure to each odor molecule, with the change in deep body temperature in the absence of odor as 0. The mice that were to smell the acquired fear-inducing odor were trained with electric shock and the odor of anisole one day before the measurement, so that the mice could feel the acquired fear of the odor of anisole through the learning.

[0065] result The results are Figure 4 Displayed in. Figure 4 A: Shows the effect of sniffing a thiazoline-related compound (2MT) or an acquired fear-inducing odor molecule (anis-FS + ) over time (mean ± standard error) (n≧8). Figure 4 B: Shows the changes in the expression of odor molecules after exposure to a thiazoline-related compound (2MT) or an acquired fear-inducing odor molecule (anis-FS +) Average change in deep body temperature over 20 minutes. The graph shows the mean ± standard error, and Student's t-test was performed on the change in body surface temperature when there was no odor (control) and when exposed to odor. *** indicates a significant difference at p < 0.001. Thiazoline-related compounds (2MT) clearly caused a decrease in deep body temperature. In contrast, the odor molecule that induces acquired fear (anis-FS + ) significantly increases body surface temperature.

[0066] Example 5: Heart rate reduction induced by thiazoline-related compounds Experimental methods The effects of thiazoline-related compounds and odor molecules that induce acquired fear on heart rate were analyzed. A wireless bioparameter measuring device (manufactured by Physiotel) was embedded in the abdomen of C57 / BL6N male mice of about 3 months old. The following experiment was performed on the mice after a recovery period of about 10 days after surgery. Each mouse was placed in a test cage, and after acclimating to the environment for 10 minutes, it was exposed to filter paper soaked in water for 10 minutes. Then, the filter paper soaked with 271 μmol of thiazoline-related compounds (2-methyl-2-thiazoline; 2MT) or odor molecules that induce acquired fear (anisole) was exposed for 20 minutes, during which the heart rate data was recorded every 10 seconds using Dataquest ART software (DataScience international). The value of the heart rate change was calculated as follows: the difference between the heart rate exposed to water for 10 minutes and the average heart rate when exposed to each odor molecule for 20 minutes, and the change in heart rate when there was no odor was regarded as 0. For mice that smelled an odor that can induce acquired fear, an association learning between electric shock and anisole odor was performed one day before measurement to make the mice learn to develop acquired fear of anisole odor.

[0067] result The results are Figure 5 Displayed in. Figure 5 A: Shows the effect of sniffing a thiazoline-related compound (2MT) or an acquired fear-inducing odor molecule (anis-FS + ) over time (mean ± standard error) (n≧8). Figure 5 B: Shows the changes in the expression of odor molecules after exposure to a thiazoline-related compound (2MT) or an acquired fear-inducing odor molecule (anis-FS + ) Average change in heart rate for 20 minutes. The graph shows the mean ± standard error, and Student's t-test was performed on the changes in body surface temperature when there was no odor (control) and when exposed to odor. *** indicates a significant difference at p < 0.001. Thiazoline-related compounds (2MT) clearly caused a decrease in heart rate, for example, the heart rate was halved within 3 minutes. In contrast, the odor molecule that induces acquired fear (anis-FS + ) had no significant effect on heart rate.

[0068] Example 6: Reduction of skin blood flow induced by thiazoline-related compounds Experimental methods The effects of thiazoline-related compounds and odor molecules that induce acquired fear on skin blood flow were analyzed. About 5 days before the experiment, C57 / BL6N male mice aged about 3 months were anesthetized with pentobarbital (50 mg / kg, ip) and the hair on their backs was removed with a depilatory cream. A mouse fixture with an observation window in a 50 ml tube was made, and the mice were placed in the fixture for training from the day after depilation. This operation was performed until the day before the experiment to adapt to the environment. On the day of the experiment, each mouse was placed in the fixture, and a laser Doppler probe was attached to the mouse's depilated back with tape. After confirming that the blood flow signal was stable, (1) no odor exposure for 10 minutes, (2) exposure to the odor of eugenol for 10 minutes, (3) exposure to thiazoline-related compounds (2MT) or an odor that induces acquired fear (Anis-FS + ) for 20 minutes, and the changes in blood flow were measured. The odor exposure was performed by bringing filter paper soaked with 271 μmol of odor molecules close to the nose tip of the mouse. The blood flow was measured every 10 seconds using a laser Doppler blood flowmeter (ADVANCE ALF21D). The average skin blood flow was calculated by averaging the blood flow in the absence of odor (10 minutes) and exposure to thiazoline-related compounds or odors that induce acquired fear (20 minutes). The average skin blood flow in the absence of odor was taken as 100%, and the blood flow was expressed as a relative value.

[0069] result The results are Figure 6 Displayed in. Figure 6 A: Shows the effect of sniffing thiazoline-related compounds (2MT) or odor molecules that induce acquired fear (Anis-FS + ) over time (mean ± standard error) (n≧8). Figure 6 B: Bar graph showing the presence of odorless and odor-exposed thiazoline-related compounds (2MT) or learned fear-inducing odor molecules (Anis-FS +). The graph shows the mean ± standard error, and the blood flow in the absence of odor and exposure to odor was tested by Student's t test. ** indicates a significant difference of p<0.01, and *** indicates a significant difference of p<0.001. Thiazoline-related compounds and odor molecules that induce acquired fear jointly reduce skin blood flow. The mechanism by which thiazoline-related compounds induce hypothermia is believed to be through increasing skin blood flow, thereby promoting heat exchange, and reducing heat generation. However, the results of this experiment suggest that thiazoline-related compounds may not induce the promotion of heat exchange, but only induce the reduction of heat generation.

[0070] Example 7: Reduction in respiratory rate induced by thiazoline-related compounds Experimental methods The effects of thiazoline-related compounds 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). For C57 / BL6N male mice aged about 3 months, the operation of attaching the oximeter probe to the neck was repeated from about 1 week before the experiment to acclimate the mice to the environment. Two days before the experiment, the mice were anesthetized with pentobarbital (50 mg / kg, ip) and the hair on the neck was removed with a depilatory cream. On the day of the experiment, the oximeter probe was attached to the depilated neck of the mouse, and after confirming that the signal was stable, the baseline without odor was measured for 10 minutes, and the change in respiratory rate when exposed to thiazoline-related compounds (2MT) or neutral odor (eugenol; Eug) was measured for 20 minutes (n=6 each). Exposure to each odor was implemented by placing filter paper soaked with 271 μmol of odor molecules in the test cage. The signal of the oximeter was obtained at 1 Hz. Mean respiratory rate is shown as the average of the respiratory rate for 10 min without odor and 20 min after odor exposure.

[0071] result The results are Figure 7 Displayed in. Figure 7 A: Shown are the changes over time in respiratory rate when sniffing the odor of a thiazoline-related compound (2MT) or a fragrance (Eug) (mean ± standard error) (n=6). Figure 7B: For the group exposed to the fragrance odor (Eug) and the group exposed to the thiazoline-related compound (2MT), the average respiratory frequency of the baseline without odor (control ctrl) and the average respiratory frequency when exposed to the odor (Eug or 2MT) are shown in the form of a bar graph. The graph shows the value of the mean ± standard error, and the respiratory frequency of the group exposed to 2MT and the group exposed to Eug was subjected to a student's t test. ** indicates that p<0.01 is significantly different, and ns indicates that p>0.05 is not significantly different. The results showed that thiazoline-related compounds reduced respiratory rate by about half. Although lower body temperature and reduced metabolic capacity have been observed in some hibernating animals, it is well known that animals in hibernation have reduced respiratory rate. This suggests that thiazoline-related compounds also cause a reduction in respiratory rate, just as in hibernation.

[0072] Example 8: Reduction of oxygen consumption induced by thiazoline-related compounds Experimental methods The effect of thiazoline-related compounds on oxygen consumption was analyzed. Oxygen consumption was analyzed using an energy metabolism measuring device for small animals (ARCO-2000; Arco System). Each C57 / BL6N male mouse, approximately 3 months old, was placed in a measuring compartment and exposed to a thiazoline-related compound (2MT) after acclimating to the environment for approximately 2 hours. Odor exposure was performed by placing 100 μl (104 mmol) of odor molecules in the measuring compartment. As a control, mice were exposed using filter paper soaked in saline (Saline). Oxygen consumption was measured every minute. The average oxygen consumption was calculated using the average of the 10 minutes before and 20 minutes after odor exposure.

[0073] result The results are Figure 8 Displayed in. Figure 8 A: Shows the temporal changes in oxygen consumption per unit body weight of mice exposed to a thiazoline-related compound (2MT) and no odor (Saline) (mean ± standard error) (n=8). Figure 8 B: The average oxygen consumption per unit body weight of mice exposed to thiazoline-related compounds (2MT) and without odor is shown in the form of a bar graph. The graph shows the value of the mean ± standard error, and the oxygen consumption when exposed to 2MT and when exposed to Saline was tested by Student's t test. *** indicates a significant difference with p < 0.001, and ns indicates no significant difference with p > 0.05. Under the control (saline) condition, exposure to the novel substance (filter paper) resulted in an increase in oxygen consumption. In contrast, exposure to a thiazoline-related compound (2MT) significantly reduced oxygen consumption. It is well known that animals in hibernation experience a decrease in metabolic capacity and a concomitant decrease in oxygen consumption, and this experimental result suggests that thiazoline-related compounds can cause the same changes.

[0074] Example 9: Hypoxia resistance induced by thiazoline-related compounds Experimental methods Each C57 / BL6N male mouse of about 3 months old was placed in a highly sealed breeding cage, and a filter paper soaked with a total of 100 μl (104 mmol) of thiazoline-related compounds (2MT) was placed in the breeding cage, and the mice were exposed to the odor for 50 minutes. After the odor exposure, 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. As a control, the mice were exposed with filter paper soaked in water and the survival time of the mice was measured (odorless noodor). In addition, the changes in deep body temperature when the odor was exposed under the same conditions as in Example 4 were measured.

[0075] result The results are Fig. 9 Displayed in. Fig. 9 A: Kaplan-Meier curves showing the survival time of mice exposed to thiazoline-related compounds (2MT) and no odor under 4% oxygen conditions (n=6 for each). Fig. 9 B: Shows the Fig. 9 Changes in deep body temperature over time when odor exposure was performed under the same conditions as in Example A (mean ± standard error) (2MT, n = 8; no odor, n = 6). Under the condition of the odorless control, all individuals died within 20 minutes in 4% oxygen; in contrast, under the condition of exposure to the thiazoline-related compound (2MT), many individuals still survived even after 30 minutes. A significant decrease in deep body temperature was also observed under the odor exposure condition used in this example. This indicates that the thiazoline-related compound produces hypoxia resistance.

[0076] Example 10: Hypoxia resistance induced by various heterocyclic compounds Experimental methods The effects of intraperitoneal administration of various heterocyclic compounds on the survival time of mice in an oxygen-deficient environment were analyzed. The odor molecules were administered by intraperitoneal injection of 200 μl of a solution diluted 100 times with saline (approximately 80 mg / kg, ip), and 30 minutes later, the mice were placed in a sealed box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured for up to 30 minutes. As a control, the survival time of mice injected intraperitoneally with saline (control) was measured.

[0077] result The results are Fig. 10A Displayed in. Fig. 10A : The survival time of mice under 4% oxygen condition when each heterocyclic compound was intraperitoneally injected is shown in the form of a bar graph (mean ± standard error). The average survival time of intraperitoneally injected saline (control) is taken as 100%, and the survival time is expressed as its relative value (%) (n = 4). The survival time between the control group and the group administered with each compound was subjected to a Student's t-test. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 and there is a significant difference. Fig. 10B : The structural formulas of the compounds used in Example 10 are shown in order. It was shown that various heterocyclic compounds caused hypoxia resistance.

[0078] Example 11: Hypoxia resistance induced by isothiocyanate compounds Experimental methods In the same manner as in Example 10, the effects of intraperitoneal administration of various isothiocyanate compounds (about 80 mg / kg, ip) on the survival time of mice in an oxygen-deficient environment were analyzed.

[0079] result The results are Fig.11A Displayed in. Fig.11A : The survival time of mice under 4% oxygen condition when each compound was intraperitoneally injected is shown in the form of a bar graph (mean ± standard error). The average survival time of intraperitoneally injected saline (control) is taken as 100%, and the survival time is expressed as its relative value (%) (n = 4). The survival time between the control group and the group administered with each compound was subjected to a Student's t-test. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 and there is a significant difference. Fig. 11B : The structural formulas of the compounds used in Example 11 are shown in order. It was shown that various isothiocyanate compounds caused hypoxia resistance.

[0080] Example 12: Experimental Method for Hypoxia Resistance Induced by Intraperitoneal Administration of Thiomorpholine 200 μl of thiomorpholine solution diluted 100 times with saline (about 80 mg / kg, ip) was intraperitoneally injected into C57 / BL6N male mice of about 3 months old. After 30 minutes, the mice were placed in a sealed 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 injected with saline intraperitoneally was measured.

[0081] result The results are Fig.12 Displayed in. Fig.12 : The survival time of mice administered with thiomorpholine and saline under 4% oxygen condition is represented by Kaplan-Meier curve (n=6). It was shown that the use of thiomorph resulted in hypoxia resistance.

[0082] Example 13: Effect of intraperitoneal administration of NaHS on survival time under 4% oxygen conditions Experimental methods 200 μl of NaHS solution was injected intraperitoneally into C57 / BL6N male mice of about 3 months old. After 30 minutes, the mice were placed in a sealed box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured for up to 30 minutes. Four concentrations of NaHS solution were used: 0.1%, 0.125%, 0.05%, and 0.0125%. Among them, since the intraperitoneal injection of 0.1% solution caused the death of mice within a few minutes, only the survival time at the other three concentrations was measured. As a control, the survival time under 4% oxygen conditions when saline and 2-methylthiophene (1% solution) were injected intraperitoneally was analyzed.

[0083] result The results are Fig.13 Displayed in. Fig.13 : Intraperitoneal injection Fig.13 After each solution was added, the survival time of each individual mouse was plotted. The survival time between the two groups indicated by the dotted line was compared using the Student's t-test. *** indicates a significant difference at p<0.001, and ns indicates a non-significant difference at p>0.05. It has been reported that NaHS, as a H2S donor, has the effect of preventing ischemia-reperfusion disorders (Yu et al., Cell Physiol Biochem 36: 1539-1551, 2015). However, mice died quickly after high-concentration injection of NaHS, while no hypoxia resistance was observed at low concentrations. In contrast, by administering 2-methylthiophene, one of the thiazoline-related compounds, to mice, all individuals survived under 4% oxygen conditions after 30 minutes of observation.

[0084] Example 14: Experimental method for the effect of thiazoline-related compounds on alleviating ischemia-reperfusion disorder The skin ischemia-reperfusion disorder model mice were prepared as follows using the method of Uchiyama et al. (Uchiyama et al., Sci Rep 5: 9072, 2015). 2-3 days before the test, the hair on the back of C57 / BL6N male mice of about 3 months old was removed in the same manner as in Example 1. On the day of the test, each mouse was placed in a highly sealed cage, and a filter paper soaked with a total of 100 μl (104 mmol) of thiazoline-related compounds (2MT) was placed in the breeding cage, and the mice were exposed to the odor for 30 minutes. As a control, the filter paper soaked in water was exposed for 30 minutes (no odor). After 30 minutes, the mice were taken out of the cage, the skin on the back was clamped with two circular magnets, and the sealed cage was returned. 100 μl (104 mmol) of 2MT or filter paper soaked in water was placed in the sealed cage, and the mice were placed in the cage for 12 hours. After 12 hours, the mice were taken out of the cage, the magnets were removed, and the mice were transferred to a normal breeding cage without odor. The state of the skin on the back clamped by the magnet was recorded by taking pictures every day. The area of ​​the wound was quantified based on the obtained image using image analysis software (Photoshop, Adobe). In addition, according to the same experimental method as in Example 2, the change in deep body temperature when exposed to odor under the same conditions in this example was measured.

[0085] result The results are Fig.14 Displayed in. Fig.14 A: Shows the daily observation results of changes in wound sites under odor-free conditions for each individual (n=5). Fig.14 B: Shows the daily observation results of changes in wound sites for each individual exposed to 2MT conditions (n=5). Fig.14C: Quantification of the area of ​​the wound site observed in A and B. The graph shows the change in the area of ​​the wound site over time (mean ± standard error), and the average value of the area of ​​the wound site under odorless conditions after 3 days is taken as 100%, and the wound site area is expressed as a relative value. The Student's t-test was performed on the area of ​​the wound site when exposed to 2MT and when there was no odor observed on the same day. ** indicates p<0.01, *** indicates p<0.001 and there is a significant difference. Fig.14 D: shows the temporal changes in deep body temperature when odor exposure was performed under the same conditions as in Examples AC (mean±standard error) (n=6 for each). In the odorless control, clamping the skin with a magnet for 12 hours produced bedsores; in contrast, the formation of bedsores was significantly reduced under the condition of sniffing 2MT. A significant decrease in deep body temperature was also observed under the odor exposure conditions used in this example. Continuous compression of bedsores can cause ischemic states. This shows that thiazoline-related compounds have the effect of alleviating and preventing ischemic reperfusion disorders.

[0086] Example 15: Changes in body surface temperature caused by thiazoline-related compounds in Trpa1 knockout mice Experimental methods Male Trpa1 knockout mice and their littermates were used at about 3 months of age to analyze changes in body surface temperature induced by thiazoline-related compounds (2MT) using the same method as in Example 1. The average body surface temperature after 20 minutes of exposure to 2MT was subtracted from the average body surface temperature after acclimatization to an odorless environment, and the difference was taken as the change in body surface temperature.

[0087] result The results are Fig.15 Displayed in. Fig.15 : Shown are control mice (trpa1 + / - ; n = 5) and Trpa1 knockout mice (trpa1 - / - ; n=8) Average value of changes in body surface temperature when exposed to thiazoline-related compounds (2MT). Student's t-test was performed on changes in body surface temperature of control mice and knockout mice. ** indicates p<0.01, which is significantly different. The decrease in body surface temperature induced by thiazoline-related compounds (2MT) was not observed in Trpa1 knockout mice, suggesting that the decrease in body surface temperature caused by 2MT is induced through Trpa1.

[0088] Example 16: Changes in deep body temperature caused by thiazoline-related compounds in Trpa1 knockout mice Experimental methods Male Trpa1 knockout mice and their littermates of heterozygous mice at about 3 months of age were used to analyze the changes in deep body temperature induced by thiazoline-related compounds (2MT) in the same manner as in Example 2. The change in deep body temperature was calculated as follows: the average value of the deep body temperature after 20 minutes of exposure to 2MT was subtracted from the average value of the deep body temperature after acclimatization to an odorless environment, and the difference was used as the change in deep body temperature.

[0089] result The results are Fig.16 Displayed in. Fig.16 A: Shows the temporal changes in deep body temperature of control mice (trpa1+ / -; n=7) and Trpa1 knockout mice (trpa1- / -; n=7) when exposed to 2MT. Fig.16 B: shows control mice (trpa1 + / - ) and Trpa1 knockout mice (trpa1 - / - ) Mean value of the change in deep body temperature when exposed to 2MT. Student's t-test was performed on the change in deep body temperature of control mice and knockout mice. *** indicates p<0.001, which is a significant difference. The decrease in deep body temperature induced by thiazoline-related compounds (2MT) was not observed in Trpa1 knockout mice, suggesting that the decrease in deep body temperature caused by 2MT is induced through Trpa1.

[0090] Example 17: Experimental Method for the Resistance of Trpa1 Knockout Mice to Hypoxia Induced by Thiazoline-Related Compounds Male Trpa1 knockout mice and their littermates were exposed to a thiazoline-related compound (2MT) for 10 minutes. The mice were then placed in a sealed box with an oxygen concentration adjusted to 4% and their survival time was measured for up to 30 minutes. Mice were exposed to the odor by placing filter paper soaked with 271 μmol of 2MT in a cage. For the control experiment, filter paper soaked with saline (no odor) was used for exposure.

[0091] result The results are Fig.17 Displayed in. Fig.17 : Shows the mean survival time of control mice (Hetero) and Trpa1 knockout mice (KO) exposed to saline or 2MT under 4% oxygen conditions (n=6 for hetero and n=7 for other mice). For each mouse, the survival time when exposed to saline and 2MT was tested by Student's t test. * indicates p<0.05 and there is a significant difference, ns indicates p>0.05 and there is no significant difference. Unlike control mice, Trpa1 knockout mice did not have a significantly increased survival time under 4% oxygen conditions even when exposed to 2MT, suggesting that 2MT-induced hypoxia resistance is mediated by Trpa1.

[0092] Example 18: Inhibitory effects of various heterocyclic compounds and isothiocyanate compounds on TNF-α in blood in a sepsis model Experimental methods Lipopolysaccharide (LPS) (0.6 mg / kg) was intraperitoneally administered to male Balb / c mice of about 2-3 months old to prepare a sepsis model. Immediately after LPS administration, 200 μl (about 80 mg / kg) of a solution of various heterocyclic compounds and isothiocyanate compounds diluted 100 times with saline was intraperitoneally administered. Blood was collected 60 minutes after LPS and compound administration, and EDTA plasma was prepared. The amount of inflammatory cytokine TNF-α in the blood was measured by ELISA.

[0093] result The results are Fig.18 Displayed in. Fig.18 The "4-Ethyl-2-methyl-thiazoline" in the formula refers to 4-ethyl-2-methyl-2-thiazoline. The amount of TNF-α in the blood when each compound was administered is shown in the form of a bar graph (mean ± standard error). The average amount of TNF-α when only LPS was administered without administering the compound (saline) was taken as 100%, and the amount of TNF-α was expressed as its relative value (%) (n≧4). A student's t-test was performed for the amount of TNF-α between the control group (saline) and the groups administered with each compound. * indicates p<0.5, ** indicates p<0.01, and *** indicates p<0.001 and has a significant difference. Since the blood concentration of the inflammatory cytokine TNF-α was reduced by administering various heterocyclic compounds and isothiocyanate compounds, it was shown that the compound had an anti-inflammatory effect in sepsis model animals.

[0094] Example 19: Effects of Thiazoline-related Compounds on the Production of Inflammatory Cytokines and Anti-inflammatory Cytokines in Sepsis Models Experimental methods A sepsis model mouse was prepared in the same manner as in Example 18. Immediately after LPS administration, 200 μl (about 80 mg / kg) of a solution of a thiazoline-related compound (2MT) diluted 100 times with physiological saline was intraperitoneally administered. Blood was collected 1 hour and 4 hours after LPS and thiazoline-related compound (2MT) administration, EDTA plasma was prepared, and the amount of inflammatory cytokine interleukin-1β (IL-1β) and anti-inflammatory cytokine interleukin-10 (IL-10) in the blood was measured by ELISA.

[0095] result The results are Fig.19 Displayed in. The IL-1β levels when the thiazoline-related compound (2MT) was administered and when saline was administered as a control were shown in the form of a bar graph ( Fig.19 A), IL-10 amount ( Fig.19 B) (all means ± standard error) (n = 6). Student's t-test was performed under each condition between the control group (saline) and the thiazoline-related compound (2MT) administration group. * indicates p < 0.05, ** indicates p < 0.01 and there is a significant difference. Since the administration of thiazoline-related compounds reduces the blood concentration of the inflammatory cytokine IL-1β and increases the blood concentration of the anti-inflammatory cytokine IL-10, it shows that thiazoline-related compounds have an anti-inflammatory effect in sepsis model animals.

[0096] Example 20: Inhibitory Effects of Thiazoline-Related Compounds on Inflammatory Mediator HMGB1 in Sepsis Model Experimental methods The sepsis model mice were prepared in the same manner as in Example 18. Immediately after LPS administration, 200 μl (about 80 mg / kg) of a thiazoline-related compound (2MT) solution diluted 100 times with physiological saline was intraperitoneally administered. Blood was collected 16 hours after the administration of LPS and thiazoline-related compound (2MT), EDTA plasma was prepared, and the amount of inflammatory mediator High Mobility Group Box 1 (HMGB1) in the blood was measured by ELISA.

[0097] result The results are Fig. 20 Displayed in. The amount of HMGB1 in the blood when the thiazoline-related compound (2MT) was administered and when saline (saline) was administered as a control was shown in the form of a bar graph (n=6). A student's t-test was performed between the control group (saline) and the thiazoline-related compound (2MT) administration group. *** indicates a significant difference at p<0.001. The administration of the thiazoline-related compound reduced the blood concentration of the inflammatory mediator HMGB1, indicating that the thiazoline-related compound has an anti-inflammatory effect in sepsis model animals.

[0098] Example 21: Effect of intraperitoneal administration of thiazoline-related compounds on the prolongation of survival time in sepsis model animals Experimental methods The sepsis model mice were prepared in the same manner as in Example 18. Immediately after LPS administration, 200 μl (about 80 mg / kg) of a thiazoline-related compound (2MT) solution diluted 100 times with physiological saline was intraperitoneally administered. The survival time of mice administered with the thiazoline-related compound (2MT) and with physiological saline as a control was measured.

[0099] result The results are Fig.21 Displayed in. Survival curves of the control group (saline) and the group administered with a thiazoline-related compound (2MT) are shown (left figure). In addition, the average survival time (mean ± standard error) of each group is shown in the form of a bar graph (n = 10; right figure). A student's t test was performed between the control group and the group administered with a thiazoline-related compound (2MT). ** indicates that p < 0.01 and there is a significant difference. This indicates that the administration of a thiazoline-related compound has the effect of prolonging the survival time of sepsis model animals.

[0100] Example 22: Effect of Thiazoline-related Compounds on Alleviating Cerebral Ischemia-Reperfusion Disorder Experimental methods The common carotid arteries on both sides of C57 / BL6 mice of about 3 months old were clamped to induce cerebral ischemia, and the clamps were removed after 30 minutes to allow blood to be reperfused. During reperfusion, 200 μl (about 80 mg / kg) of a thiazoline-related compound (2MT) solution or saline as a control was intraperitoneally administered. Two days after reperfusion, the brain tissue of the mice was removed, brain sections were prepared, and the damaged area was analyzed by staining with an antibody to microtubule-associated protein 2 (MAP2).

[0101] result The results are Fig. 22 Displayed in. The experimental scheme is shown at the top of the figure. Representative examples of MAP2 antibody staining of brain sections of animals administered with control and thiazoline-related compounds (2MT) are shown on the left side of the figure. In the figure, the damaged area of ​​the brain is observed to be the area not stained with MAP2 (black). In addition, the right side of the figure shows the area of ​​the area not stained with MAP2 in the control group and the thiazoline-related compound (2MT) administration group in the form of a bar graph, that is, the area of ​​the damaged area (mean ± standard error) (n=9 for saline and n=8 for 2MT). A Student's t-test was performed between the control group and the thiazoline-related compound (2MT) administration group. *Indicates a significant difference of p<0.05. This shows that the administration of thiazoline-related compounds during reperfusion has the effect of alleviating cerebral ischemia-reperfusion disorders.

[0102] Example 23: Experimental method for the effect of Trpa1 on the induction of hypoxia resistance by administration of heterocyclic compounds Male Trpa1 knockout mice and wild-type mice aged approximately 3 to 6 months were intraperitoneally injected with 200 μl (approximately 80 mg / kg) of various heterocyclic compound solutions diluted 100 times with physiological saline. After 30 minutes, the mice were placed in a sealed box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured for up to 30 minutes.

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

[0104] Example 24: Effect of Trpa1 on the induction of hypoxia resistance induced by thiomorph Experimental methods Male wild-type mice and Trpa1 knockout mice aged about 3 to 6 months were intraperitoneally injected with 200 μl (about 80 mg / kg) of a heterocyclic compound (thiomorpholine) diluted 100 times with normal saline. After 30 minutes, the mice were placed in a sealed box with an oxygen concentration adjusted to 4%, and the survival time of the mice was measured.

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

[0106] Example 25: Effect of Trpa1 on the Induction of Oxygen Consumption Inhibition by Administration of Thiazoline-Related Compounds Experimental methods The oxygen consumption was measured using the same small animal energy metabolism measuring device as in Example 8. Each Trpa1 knockout mouse and its littermate heterozygous mouse, about 3 to 6 months old, were placed in the measuring compartment of the small animal energy metabolism measuring device, and after acclimating to the environment for about 2 hours, they were exposed to the thiazoline-related compound (2MT). The odor exposure was performed by placing 2 filter papers soaked with 271 μmol of the thiazoline-related compound (2MT) into the measuring compartment.

[0107] result The results are Fig.25 Displayed in. Fig.25 : Shows the changes in control mice (Trpa1 + / - ; n = 9) and Trpa1 knockout mice (Trpa1 - / - ; n = 7) of oxygen consumption per unit body weight over time (mean ± standard error). Odor exposure was performed at the 10-minute time point in the figure. In control mice, exposure to thiazoline-related compounds (2MT) reduced oxygen consumption, whereas no reduction in oxygen consumption was observed in Trpa1 knockout mice. Therefore, it is shown that the reduction in oxygen consumption caused by thiazoline-related compounds is mediated by Trpa1.

[0108] Example 26: Effect of administration of Trpa1 agonist on induction of hypoxia resistance Experimental methods Physiological saline or known Trpa1 agonist allyl isothiocyanate (AITC; 40 mg / kg) or acetaminophen (APAP; 300 mg / kg) were intraperitoneally administered to male C57 / BL6N mice at about 3 months of age, and 30 minutes later, the mice were placed in a sealed box adjusted to 4% oxygen concentration, 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 littermates at about 3 to 6 months of age was measured in the same 4% oxygen environment.

[0109] result The results are Fig.26 Displayed in. Fig.26 A: The survival time (mean ± standard error) of wild-type mice under 4% oxygen conditions when saline or various Trpa1 agonists were administered is shown in the form of a bar graph (n=22 for saline, n=8 for AITC, and n=7 for APAP). Student's t-test was performed between the control group and the Trpa1 agonist-administered group. ** indicates a significant difference of p<0.01, and *** indicates a significant difference of p<0.001. The effect of prolonging the survival time under hypoxic environment was observed by administering Trpa1 agonists. Fig.26 B: The bar graph shows the control mice (Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / - ) Survival time under 4% oxygen conditions (mean ± standard error) (n = 8). Student's t-test was performed between the control group (saline) and the Trpa1 agonist administration group (APAP). ** indicates a significant difference at p < 0.01, and ns indicates no significant difference at p > 0.05. The Trpa1 agonist (APAP) has the effect of prolonging the survival time under hypoxia in the control mice, but in contrast, such an effect was not observed in the Trpa1 knockout mice. This suggests that the induction of hypoxia resistance by the Trpa1 agonist is mediated by Trpa1.

[0110] Example 27: Effects of Trpa1 knockout mice on changes in body surface temperature, hypoxia resistance and anti-inflammatory effects induced by thiazoline-related compounds Experimental methods The changes in body surface temperature, survival time in an oxygen-deficient environment, and TNF-α produced in the blood when LPS was administered were measured by the same methods as in Examples 3, 10, and 18 for male Trpa1 knockout mice aged approximately 3 to 6 months and their littermates' heterozygous mice when a thiazoline-related compound (4-ethyl-2-methyl-2-thiazoline; 4E2MT) was intraperitoneally administered.

[0111] result The results are Fig. 27 Displayed in. Fig. 27 A: shows the control mice (Trpa1 / - ) and Trpa1 knockout mice (Trpa1 - / - ) over time (mean ± standard error). The administration of the thiazoline-related compound (4E2MT) was performed at a time point of 10 minutes. For the average surface temperature 30 minutes after the administration of the thiazoline-related compound (4E2MT), a student's t-test was performed between the control mice and the Trpa1 knockout mice, and the results are shown in the figure. *** indicates p<0.001, which is significantly different. It has been confirmed that no decrease in body surface temperature was observed when the thiazoline-related compound was administered intraperitoneally in the Trpa1 knockout mice. Fig. 27 B: The bar graph shows the control mice (Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / - ) Survival time under 4% oxygen conditions (mean ± standard error). Student's t-test was performed between control mice and Trpa1 knockout mice. ** indicates p<0.01, which indicates a significant difference. It was confirmed that intraperitoneal injection of thiazoline-related compounds in Trpa1 knockout mice inhibited the effect of prolonging survival time under hypoxia. Fig. 27 C: The bar graph shows the control mice (Trpa1 + / + , Trpa1 + / - ) and Trpa1 knockout mice (Trpa1 - / - ) TNF-α level in blood 1 hour after administration (mean ± standard error). + / +) The amount of TNF-α in the blood was measured when LPS and saline were administered. ** indicates p<0.01, *** indicates p<0.001 and there is a significant difference. Administration of a thiazoline-related compound (4E2MT) to sepsis model mice showed a decrease in the amount of inflammatory cytokine TNF-α in the blood, and this effect was suppressed in Trpa1 knockout mice. according to Fig. 27 The results of AC indicate that the decrease in body surface temperature, hypoxia resistance, and anti-inflammatory effects induced by thiazoline-related compounds are all mediated by Trpa1.

[0112] Example 28: Oxygen consumption inhibition effect induced by heterocyclic compounds Experimental methods In the same manner as described in Example 8, changes in oxygen consumption were measured when heterocyclic compounds (2MT and thiomorpholine (TMO)) were intraperitoneally injected into C57 / BL6N mice of about 3 months of age.

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

[0114] The compounds of the present invention can be used as preventive or therapeutic agents for hypoxic disorders, ischemic reperfusion disorders or inflammation, cell protectants for transplantation, and biological preservatives because they show the effects of inducing hypothermia, low metabolism, inhibition of oxygen consumption, low heart rate, anti-inflammatory response, protective effects on tissues or individuals under hypoxic conditions, protective effects on ischemic reperfusion disorders, and protective effects on inflammation.

[0115] This application is based on Japanese Patent Application No. 2018-049761 filed in Japan, and all the contents are incorporated into this specification.

Claims

1. Use of at least one compound selected from the group consisting of a heterocyclic compound represented by formula (I) or a salt thereof and an isothiocyanate compound represented by formula (II) in the preparation of an agent for preventing or treating ischemia-reperfusion disorder, wherein: - The heterocyclic compound represented by formula (I) is [Chemical Formula 1] In formula (I), Ring A is a 5- to 7-membered heterocyclic ring 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 Each is independently a hydrogen atom, C 1-6 Alkyl group, halogen atom, Amino, -SH, C 1-6 Alkylthio, C 2-6 Alkenylthio, C 1-6 Alkyl-carbonyl, formyl, C 6-10 Aryl, C 1-6 Alkoxycarbonyl, 5- or 6-membered heteroaryl, or oxo; R 1 and R 2 may combine with each other to form an optionally substituted 5- or 6-membered ring; n is 0, 1, or 2; and -The isothiocyanate compound represented by formula (II) is S=C=NR 5 (II) In formula (II), R 5 C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 6-10 Aryl, or 5- or 6-membered heteroaryl.

2. The use according to claim 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 use according to claim 1, wherein The compound is a heterocyclic compound represented by formula (I) or a salt thereof.

4. The use according to claim 1, wherein The compound is an isothiocyanate compound represented by formula (II). 5 . The use according to any one of claims 1 to 4 , wherein the agent for preventing or treating ischemia-reperfusion disorder is for nasal administration.

6. Use of a TRPA1 agonist in the preparation of a preventive or therapeutic agent for ischemia-reperfusion disorder.

7. The use according to claim 6, wherein the preventive or therapeutic agent for ischemia-reperfusion disorder is for nasal administration.

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