WO2019037689A1 - Compound for treating ischemia-reperfusion injuries - Google Patents

Compound for treating ischemia-reperfusion injuries Download PDF

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WO2019037689A1
WO2019037689A1 PCT/CN2018/101397 CN2018101397W WO2019037689A1 WO 2019037689 A1 WO2019037689 A1 WO 2019037689A1 CN 2018101397 W CN2018101397 W CN 2018101397W WO 2019037689 A1 WO2019037689 A1 WO 2019037689A1
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alkyl
alkoxy
ischemia
phenyl
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Chinese (zh)
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李红良
张晓晶
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武汉大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A—HUMAN NECESSITIES
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    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
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    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
    • C07C311/44—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
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    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
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    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings

Definitions

  • the invention belongs to the technical field of medicinal chemistry, in particular to an ALOX12 inhibitor, for example, a compound of the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, for preparing an ischemia-reperfusion injury and
  • an ALOX12 inhibitor for example, a compound of the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, for preparing an ischemia-reperfusion injury
  • drugs for related diseases, inflammatory diseases, and cell death-related diseases particularly drugs for treating body damage caused by ischemia-reperfusion of organs such as liver, heart, and kidney.
  • Ischemia-Reperfusion Injury is the first concept proposed by Jennings in 1960. It refers to blood reperfusion after tissue and organ ischemia, which not only fails to restore the function of tissues and organs, but also increases the dysfunction and structure of tissues and organs. damage. Ischemia-reperfusion injury can occur in many important organs including heart, liver, lung, kidney, gastrointestinal tract, and the like.
  • Hepatic Ischemia Reperfusion Injury is a common pathological process in liver surgery. It is more common in pathological and physiological processes such as shock, liver surgery requiring liver blood flow, and liver transplantation.
  • liver transplantation, thrombolytic therapy and hepatic occlusion surgery have been carried out more and more, although liver protection, surgical techniques and intraoperative monitoring are improving, but ischemia and reperfusion
  • the liver injury is still the main cause of postoperative organ dysfunction, graft failure and even patient death.
  • liver tissue cells undergo a series of metabolic, structural and functional damages, which are easy to induce liver failure, which is one of the main factors affecting disease prognosis, surgical success rate and patient survival rate.
  • Acute coronary artery obstructive disease is one of the main causes of death of cardiovascular and cerebrovascular diseases.
  • bypass surgery intervention, and thrombolysis
  • the mortality rate of patients with acute myocardial infarction is still high.
  • One of the most important reasons is that there is no effective way to inhibit the blood flow of ischemic myocardium. Caused by ischemia-reperfusion injury.
  • the kidney is also a high perfusion organ, sensitive to ischemia and ischemia-reperfusion. Renal ischemia-reperfusion injury is an important injury link of ischemic acute renal failure, and also a limiting factor affecting early recovery of renal function in kidney transplantation.
  • organ ischemia-reperfusion injury such as inflammatory cytokines (TNF- ⁇ and IL), oxygen free radicals, calcium overload, microcirculatory disorders, energy metabolism disorders, etc. Blood time, tissue demand for oxygen, establishment of collateral circulation, and electrolyte concentration.
  • TNF- ⁇ and IL inflammatory cytokines
  • ALOX12 is involved in the occurrence and development of organ ischemia-reperfusion injury, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, It can treat and prevent ischemia-reperfusion injury, especially for ischemia, reperfusion injury of liver, heart and kidney. It has excellent therapeutic and preventive effects especially in liver ischemia-reperfusion injury.
  • the present invention can be accomplished by significantly inhibiting the inflammatory reaction and cell death during ischemia-reperfusion.
  • the present invention provides the use of an ALOX12 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, and cell death-related diseases.
  • the ALOX12 inhibitor is a compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof.
  • the present invention provides a compound represented by the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, for the preparation of a disease for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, and cell death-related diseases. Use in medicine.
  • the present invention also provides a method for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, or cell death-related diseases, characterized in that a drug containing an ALOX12 inhibitor is administered to a patient in need thereof.
  • the ALOX12 inhibitor is a compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof.
  • R 1 and R 2 are independently selected from the group consisting of H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, An aryl group, a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl , Br, hydroxy, C 1-6 alkoxy;
  • R 3 is selected from aryl and heteroaryl, and the above group is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, hydroxy, C 1-6 alkoxy, C 1-6 alkoxycarbonyl, cycloalkyl, aryl, piperazinyl, piperidinyl, pyridyl, morpholinyl, pyrrole An alkyl group, a pyrazolidinyl group, an imidazolidinyl group, and a thiomorpholinyl group; the above group C 1-6 alkyl group, C 2-6 alkenyl group, C 2-6 alkynyl group, C 1-6 alkoxy group, Cycloalkyl, aryl, piperazinyl, piperidinyl, pyridyl, morpholinyl, pyrrolidinyl, pyrazolidinyl,
  • the alkyl group means a straight or branched alkyl group having 1 to 6 carbon atoms, and the alkyl group is, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group or a t-butyl group. , sec-butyl, pentyl, neopentyl.
  • the alkenyl group means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as a vinyl group, a propenyl group, an isopropenyl group.
  • the alkynyl group refers to a straight or branched alkynyl group having 2 to 6 carbon atoms, such as an ethynyl group, a propynyl group, a butynyl group.
  • the alkoxy group means a straight or branched alkoxy group having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, Tert-butoxy, sec-butoxy.
  • the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
  • the aryl group refers to a monocyclic or polycyclic aromatic group having 6 to 20 (preferably 6 to 14) carbon atoms, and representative aryl groups include phenyl, naphthyl, anthryl and the like.
  • the heteroaryl group means a monocyclic or polycyclic aromatic group having 1 to 20 carbon atoms and 1 to 4 hetero atoms selected from N, O, and S.
  • the heteroaryl group may be a single ring having 3 to 7 ring atoms (2 to 6 carbon atoms and 1 to 3 hetero atoms selected from N, O, S) or having 7 to 10 ring atoms (4-9) A bicyclic ring of one carbon atom and one to three hetero atoms selected from N, O, and S.
  • Heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, Furanyl, thienyl, pyrrolyl and the like.
  • R 3 is selected from the group consisting of phenyl, naphthyl, thiazolyl, benzothiazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thienyl, benzothienyl, pyridyl, quinolyl, isoquinoline a group, an oxazolyl group, a furyl group, a benzofuranyl group, a pyrrolyl group, a pyrazolyl group, a pyrazinyl group, a pyrimidinyl group, a triazinyl group, a fluorenyl group, a fluorenyl group, each of which is optionally one or more Substituted from the group: C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, hydroxy, C 1-6 alkoxy, C 1- 6 alkoxycarbonyl, phenyl, cycl
  • R 3 is selected from the group consisting of phenyl, naphthyl, thiazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, thienyl, quinolyl, isoquinolinyl, pyridyl
  • the above group is optionally substituted by one or more substituents selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxycarbonyl, F, Cl, Br, hydroxy Phenyl, piperazinyl, piperidinyl, morpholinyl, C1-6 alkoxycarbonylpiperidinyl.
  • R 3 is selected from the group consisting of thiazolyl, 2-benzothiazolyl, 2-benzoxazolyl, 2-benzimidazolyl, 4-methyl-2-benzothiazolyl, thienyl, 4-methyl-2-thiazolyl, 5-methyl-2-thiazolyl, 4,5-dimethyl-2-thiazolyl, phenyl, 1-naphthyl, 2-naphthyl, 1,4- Biphenyl, 3-piperazine-phenyl, 4-piperidine-phenyl, 4-piperazin-3-pyridyl, 4-methyl-3-pyridyl, 3-pyridyl, 2-pyridyl, 3-tert-Butyl-phenyl, 6-methoxy-2-benzothiazolyl, 6-fluoro-2-benzothiazolyl, 4-phenyl-2-thiazolyl, 3-morpholine-phenyl 4-(N-tert-Butoxycarbonyl)piperidinyl-3
  • R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
  • R 1 when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl;
  • the compound of formula (I) is a compound of formula (II) below,
  • X is selected from O, S, NH or C; and R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 Alkynyl, F, Cl, Br, amino, aryl, heteroaryl, each of which is optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl , C 2-6 alkynyl, F, Cl, Br, hydroxy, C 1-6 alkoxy; preferably, R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl; R 4 , R 5 , R 6 , R 7 are the same or different and are independently selected from H, halogen, C 1-6 alkoxy or C 1-6 alkyl.
  • R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
  • R 1 when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
  • the compound of formula (I) is a compound of formula (III) below,
  • R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, aryl a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br , hydroxy, C 1-6 alkoxy.
  • R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl;
  • R 8 , R 9 are independently selected from H, halo, C 1-6 Alkoxy, C 1-6 alkyl, phenyl, C 1-6 alkylphenyl, C 1-6 alkoxyphenyl, halogen substituted phenyl.
  • R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
  • R 1 when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
  • the compound of formula (I) is a compound of formula (IV) below,
  • R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, aryl a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br , hydroxy, C 1-6 alkoxy; preferably, R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl; R 10 , R 11 are independently Is selected from H, halogen, C 1-6 alkoxy, C 1-6 alkyl, optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted piperazinyl, said substituent It is a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 alkoxycarbonyl group
  • R 10 and R 11 are independently selected from the group consisting of H, tert-butyl, isopropyl, phenyl, piperazinyl, 4-piperidinyl, 4-tert-butyloxycarbonylpiperidinyl.
  • R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
  • R 1 when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
  • the compound of formula (I) is preferably the following specific compound:
  • the compound of the formula (I) is further preferably a specific compound as follows:
  • the compound of formula (I) is (also referred to as ML355),
  • pharmaceutically acceptable salt refers to a derivative of a pharmaceutically active compound wherein the parent compound is modified by preparing an acid or base salt thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, basic or organic salts of acidic residues such as carboxylic acids, and the like.
  • Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from, for example, non-toxic inorganic or organic acids.
  • these conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and salts prepared from organic acids, such as acetic acid, propionic acid, Succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, methanesulfonic acid, toluenesulfonic acid, salicylic acid, p-aminobenzenesulfonic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.
  • salts prepared from organic acids such as acetic acid, propionic acid, Succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, methanesulfonic acid, to
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compound of the formula (I) of the present invention can be synthesized according to the method described in US Pat. No. 1,00, 1955, A1, the entire disclosure of which is incorporated herein by reference.
  • the ischemia-reperfusion injury and related diseases are preferably liver ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia-reperfusion injury and related diseases; more preferably liver Ischemia-reperfusion injury and related diseases.
  • the ischemia-reperfusion injury is preferably liver ischemia-reperfusion injury, cardiac ischemia-reperfusion injury, renal ischemia-reperfusion injury; more preferably liver ischemia-reperfusion injury.
  • Inflammatory factors of hepatic ischemia-reperfusion injury and related diseases include, but are not limited to, liver cysts, liver transplantation, thrombolytic therapy, hepatic occlusion, and hepatic coma.
  • Cardiac ischemia-reperfusion injury and related factors include, but are not limited to, myocardial infarction, myocardial infarction, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, coronary artery Bypass.
  • causes of renal ischemia-reperfusion injury and related diseases include, but are not limited to, kidney transplantation, renal cysts, and renal vascular surgery.
  • the inflammatory diseases include, but are not limited to, hepatitis, myocarditis, endocarditis, and nephritis.
  • the medicament further comprises a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrators, etc. .
  • the diluent is, for example, lactose, starch, cellulose derivative, inorganic calcium salt, sorbitol or the like.
  • the binder is, for example, starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone or the like.
  • the antioxidant is, for example, vitamin E, sodium hydrogen sulfite, sodium sulfite, butylated hydroxyanisole or the like.
  • the pH adjusting agent is, for example, hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate or the like.
  • the preservative is, for example, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, m-cresol, benzalkonium chloride or the like.
  • the lubricant is, for example, magnesium stearate, finely divided silica gel, talc, or the like.
  • the disintegrant is, for example, starch, methyl cellulose, xanthan gum, croscarmellose sodium or the like.
  • the dosage form of the medicament of the present invention may be in the form of an oral preparation, such as a tablet, a capsule, a pill, a powder, a granule, a suspension, a syrup, etc.; or a dosage form for injection administration, such as an injection solution, a powder injection, etc., Intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage form forms used are well known to those of ordinary skill in the pharmaceutical arts.
  • the medicament of the invention can be administered to any animal that develops or has developed ischemia-reperfusion injury.
  • animals include human and non-human animals such as pets or livestock.
  • the medicament of the present invention can be administered to a subject by a route known in the art including, but not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal. , buccal, sublingual, intranasal, transdermal, etc.
  • the dosage of the ALOX12 inhibitor to be administered will depend on the age, health and weight of the recipient, the type of combination, the frequency of treatment, the route of administration, and the like.
  • the drug can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three or four times daily.
  • the drug can be administered before the ischemia-reperfusion injury occurs or after ischemia-reperfusion injury occurs, for example, before, during, and after surgery of organs such as the liver, heart, and kidney.
  • the dose can be administered one or more times, and the administration time can be from one day to several months or longer.
  • a single dose of the ALOX12 inhibitor can vary widely from about 0.0001 to about 10,000 mg per patient per day. The range may more particularly be from about 0.001 mg/kg to 100 mg/kg body weight per day for adults (about 60 kg), such as from 0.01 mg/kg to 10 mg/kg, or from 0.1 mg/kg to 1 mg/kg.
  • the medicament can also be administered in combination with other drugs for treating ischemia-reperfusion-related injury, inflammatory diseases, and cell death-related diseases.
  • the inventors of the present invention have found that the compounds of formula (I) have said therapeutic activity and are associated with their ALOX12 inhibitors.
  • the inventors of the present invention found in the study that the expression levels of ALOX12 protein and mRNA expression in the tissues of liver ischemia-reperfusion injury were significantly increased, and the magnitude and significance of the changes were much higher than other members of ALOX (for example, ALOX5, ALOX15). ), indicating that ALOX12 is associated with ischemia-reperfusion injury, especially liver ischemia-reperfusion injury.
  • ALOX12 promotes decreased activity and inflammatory response of hepatocytes, heart cells, and kidney cells after hypoxia and reoxygenation, while low expression of ALOX12 attenuates hypoxia and reoxygenation.
  • Reduced activity of liver cells, heart cells, and kidney cells, as well as inflammatory responses suggest that ALOX12 promotes the development of ischemia-reperfusion injury, as well as inflammatory and cell-dead diseases in these organs, inhibiting the activity of ALOX12 to treat ischemia. Perfusion damage, as well as inflammatory and cell death diseases in these organs.
  • Figure 1A and 1B In the liver ischemia-reperfusion injury, after ML355 1mg/kg, 2mg/kg, 3mg/kg, the serum ALT, AST test results (ns represent P ⁇ 0.05, * represents 0.01 ⁇ P ⁇ 0.05, ** represents P ⁇ 0.01).
  • Figure 2 Hepatic HE staining micrographs of mice after administration of ML355 1 mg/kg, 2 mg/kg, 3 mg/kg in hepatic ischemia-reperfusion injury.
  • Figure 3A and 3B In the liver ischemia-reperfusion injury, after ML355 3 mg/kg, the serum ALT and AST results were detected over time (ns represent P ⁇ 0.05, * represents 0.01 ⁇ P ⁇ 0.05,* * represents P ⁇ 0.01).
  • Figure 4 TUNEL staining of liver tissue over time after administration of ML355 3 mg/kg in hepatic ischemia-reperfusion injury, in which white cells represent apoptotic cells.
  • Fig. 5A and Fig. 5B Immunofluorescence staining of liver Mac1 and Ly6G positive inflammatory cells over time in liver hepatic ischemia-reperfusion injury after administration of ML355 3 mg/kg, in which light gray cells represent inflammatory cells.
  • Fig. 6A and Fig. 6B are the results of detection of CK and LDH over time after administration of ML355 3 mg/kg in cardiac ischemia-reperfusion injury (ns represent P ⁇ 0.05, * represents 0.01 ⁇ P ⁇ 0.05, ** represents P ⁇ 0.01).
  • FIG. 7 TTC staining results of reperfusion for 24 hours after administration of ML355 3 mg/kg in cardiac ischemia-reperfusion injury.
  • the white area in the figure represents the tissue infarct area.
  • FIG 8 RT-PCR results of Tnf and Il6 mRNA expression in heart tissue after reperfusion for 24 hours after administration of ML355 3 mg/kg in cardiac ischemia-reperfusion injury (** represents P ⁇ 0.01)
  • Figure 9 RT-PCR results of Bcl2 and Bax mRNA expression in heart tissue after reperfusion for 24 hours after administration of ML355 3mg/kg in cardiac ischemia-reperfusion injury (* represents 0.01 ⁇ P ⁇ 0.05)
  • Fig. 10A and Fig. 10B are the results of detection of BUN and Scr over time after administration of ML355 3 mg/kg in renal ischemia-reperfusion injury (n.s. represents P ⁇ 0.05, * represents 0.01 ⁇ P ⁇ 0.05).
  • Fig. 11A is a graph showing the results of RT-PCR detection of mRNA expression levels of ALOX12, ALOX5, and ALOX15 in hepatic ischemia-reperfusion injury (n.s. indicates P ⁇ 0.05, ** indicates P ⁇ 0.01).
  • Fig. 11B is a graph showing the results of Western-blot expression of protein expression of ALOX12, ALOX5 and ALOX15 in hepatic ischemia-reperfusion injury.
  • GAPDH is a control standard.
  • Figure 12 Identification of ALOX12 protein expression after L02 cells were transfected with GFP and ALOX12 overexpression lentivirus.
  • GAPDH is a control standard.
  • FIG. 13 LDH release detection results in L02 cell injury induced by H/R treatment by ALOX12 overexpression (n.s. indicates P ⁇ 0.05, * indicates 0.01 ⁇ P ⁇ 0.05, ** indicates P ⁇ 0.01).
  • Figure 14 Detection results of inflammatory factor Il-6, Tnf- ⁇ and chemokine Ccl2, Cxcl10 mRNA in L02 cell injury induced by H/R treatment by ALOX12 overexpression (ns indicates P ⁇ 0.05, * indicates 0.01 ⁇ P ⁇ 0.05, ** indicates P ⁇ 0.01).
  • Figure 15 Identification of ALOX12 mRNA expression in H9C2 cells transfected with shRNA and shALOX12 lentivirus (** indicates P ⁇ 0.01).
  • Figure 16 Effect of ALOX12 knockdown on H9C2 cell activity after H/R treatment (n.s. indicates P ⁇ 0.05, ** indicates P ⁇ 0.01).
  • mice Male wild-type mice (purchased from Beijing Huakang Biotechnology Co., Ltd.) of 8-10 weeks old, weighing 24 g-27 g, and background C57BL/6 strain were used.
  • mice All experimental mice were housed in the SPF laboratory animal center of Wuhan University. Breeding conditions: room temperature between 22-24 ° C, humidity between 40-70%, alternating light and dark lighting time is 12h, free to drink water.
  • HEK293T human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu43.
  • H9C2 rat cardiomyocytes, purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNR5.
  • the cells were cultured in DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin). Culture environment: 37 ° C, 5% CO 2 .
  • mice were fasted 12 h before surgery and were given free access to water.
  • the mice were anesthetized with 3% pentobarbital sodium before surgery, and the limbs were fixed in a flat position.
  • the abdomen area of the mice was shaved with a shaver, and the surgical area was disinfected with 10% iodine and 75% ethanol.
  • the midline incision was taken into the abdomen to expose the liver pedicle of the left and middle leaves of the liver.
  • the portal vein and hepatic artery of the middle and left lobe were clipped with non-invasive vascular clamps, causing approximately 70% of liver ischemia to prevent severe mesenteric venous congestion.
  • the onset of ischemia was recorded and ischemia was maintained for 60 minutes.
  • the mice in the Sham group were not blocked by hepatic blood flow.
  • mice after the operation were placed in a clean cage and kept alone.
  • the sham operation group (Sham group) and the ischemia-reperfusion group were anesthetized at 0h, 1h, 3h, 6h, 12h, and 24h after operation, 3% pentobarbital sodium was anesthetized, and 1 mL of blood was taken from the orbital venous plexus. Separate serum.
  • the left lobe tissue of the ischemic area was uniformly placed in liquid nitrogen for rapid freezing or fixed in 10% neutral formalin for 24 hours, then dehydrated, embedded, and paraffin sections were prepared.
  • the EP tube collecting blood was allowed to stand at room temperature for 1-2 h to allow the blood to naturally coagulate. Centrifuge at 4 ° C, 4000 rpm / min for 30 min, fully separate the serum, and store in a -80 ° C refrigerator for use.
  • the indicators of hepatic ischemia-reperfusion injury severity include liver necrosis area, liver function index (AST, ALT), inflammatory response, cell death, etc., all of which are positively correlated with the severity of hepatic ischemia-reperfusion injury.
  • the serum ALT and AST contents were determined by an automatic biochemical analyzer (Sysmex, Chemix 180i).
  • the paraffin sections were stained with HE, photographed by microscope, and pathological changes of the liver were observed.
  • TUNEL kit TUNEL kit: Plus In Situ Apoptosis Fluorescein Detection Kit (S7111, Chemicon).
  • the inflammatory cell infiltration of liver after ischemia-reperfusion was detected by Ly6G and MAC1 immunofluorescence staining.
  • the specific steps are as follows:
  • citrate tissue antigen repair solution 100 ⁇ , pH 6.0, Fuzhou Maixin
  • sham operation group (Sham) and ischemia-reperfusion group mice were anesthetized with 3% pentobarbital sodium 1 hour after operation, liver tissue in ischemic area was taken, immediately placed in liquid nitrogen for more than 30 minutes, and then preserved. It was used in RT-PCR and Western blot analysis in a -80 °C refrigerator.
  • Cardiac ischemia is caused by blocking the left anterior descending coronary artery (LAD) of the mouse heart.
  • LAD left anterior descending coronary artery
  • mice were anesthetized with 3% pentobarbital sodium before surgery, and the limbs were fixed in a flat position, and the hair of the mouse was shaved with a shaver. After the trachea was intubated and connected to the ventilator successfully, the next step was performed. The whole body was heated with a heating pad to maintain the body temperature at 37 °C.
  • mice were placed in the right lateral position, and the skin of the operation area was disinfected and cleaned with medical iodine and 75% medical alcohol.
  • the skin was cut along the ribs at 0.5 cm below the left forelimb with ophthalmic scissors, and the fascia, muscles, etc. were separated layer by layer.
  • Tissue use micro-shear to open the thoracic cavity between the three and four intercostals to fully expose the heart, use a microscopic straight sputum to clip a small amount of pericardium and tear a small pericardium under the left atrial appendage to fully expose the left anterior descending coronary artery (LAD) or where region.
  • LAD left anterior descending coronary artery
  • the 6-0 suture completely sutured the thoracic opening to close the chest cavity, and the 5 mL syringe was inserted through the incision into the chest cavity, and 1 mL of gas was withdrawn.
  • mice in the ischemia-reperfusion group and the sham-operated group were anesthetized at 0h, 3h, 6h, 12h, and 24h after operation, 3% sodium pentobarbital anesthesia, 1ml of blood was taken from the orbital venous plexus, and serum was separated.
  • the serum CK and LDH levels were determined by an automated biochemical analyzer (Sysmex, Chemix 180i).
  • mice 24 hours after ischemia-reperfusion, mice were anesthetized with 3% pentobarbital sodium, the jugular vein was isolated, and LAD was re-ligated. Then 2 ml of 2.5% Evans blue solution was slowly injected into the jugular vein with a syringe. After the blue color is no longer faded, stop the injection, quickly remove the heart, clean and squeeze the blood and blood stains of the heart, rinse it with 4°C saline, dry it, and freeze it in the refrigerator at -20 °C for 15 minutes until the heart becomes hard. Remove and cut into 1 mm thick sections from the apex of the heart from the apex of the heart to the center of the sulcus. Cut a total of 5 pieces, and quickly place the sections in 5 ml of TTC phosphate buffer solution at 37 ° C and 1% PH 7.4, and observe after 15 minutes in a water bath.
  • RNA extraction in the tissue 2-5 The cells were collected and washed twice with PBS buffer. After completion, 1 ml of TRizol was added, uniformly blown with a pipette, and inhaled into a 1.5 ml centrifuge tube. The vortex mixer was shaken for 30 s, and allowed to stand at room temperature for 5 min to completely dissociate the nucleoprotein from the nucleic acid. . The remaining steps are the same as RNA extraction in the tissue 2-5.
  • mice were fasted for 12 hours before surgery and were given free access to water. Mice were anesthetized by intraperitoneal injection of 3% pentobarbital sodium. The back of the mouse was depilated and the skin was sterilized. The skin and muscles were cut at 0.5 cm next to the back spine and 0.5 cm at the lower edge of the ribs, and the kidneys were visible. The renal arteries of both kidneys were isolated and the renal arteries were clamped with arterial clips. After 60 minutes of ischemia, the arterial clip was released, the blood flow was restored, and the kidney recovery was observed. Suture the opening.
  • the sham-operated group (Sham) and the ischemia-reperfusion group were anesthetized 24 hours after surgery, 3% sodium pentobarbital anesthesia, 1 ml of blood was taken from the orbital venous plexus, serum was taken after centrifugation, and an automatic biochemical analyzer (Sysmex, Chemix180i) detects serum BUN (urea nitrogen) and Scr (serocreatin) levels.
  • the sample was ground in a -80 ° C pre-cooled grinder adapter with a grinding parameter set to 30 Hz/s for 90 s.
  • the ultrasonic pyrolyzer lysed the sample (5 KHz/time, 1 s each time, interval 1 s, repeated 10 times), and placed on ice for 10 min after completion of the ultrasound.
  • the supernatant was accurately aspirated and protein quantitation was performed using the BCA Protein Assay Kit (PierecTM, 23225).
  • the cells were added to the lysate, and after the completion of the lysis, the supernatant was centrifuged, and the protein sample was quantitatively collected using the BCA Protein Assay Kit.
  • the film transfer tank is connected to the power supply, the voltage is set to 250V, and the current is set to 0.2A. Transfer 1.5h.
  • the extracted plasmid can be directly used for transient transduction of ALOX12 or construction of a lentiviral stable cell line.
  • ALOX12 targeted interference sequence is GCATCGAGAGAAGGAACTGAA, designed for oligonucleotides of pLKO.1 vector; negative control siRNA sequence: CAACAAGATGAAGAGCACCAA; forward oligonucleotide: 5'CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTGTGTGTTTTG 3'; reverse oligonucleo Glycosylate: 5'AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC 3';
  • the resulting plasmid can be used for lentiviral-mediated construction of the ALOX12 knockdown cell line;
  • PEI 1.6 ⁇ g/ ⁇ l
  • the virus-containing supernatant was harvested 48-72 h after transfection, centrifuged at 3000 rpm for 10 min, the precipitate was removed, and filtered through a 0.45 ⁇ m filter;
  • the filtered virus can be used immediately for infection or storage at -80 °C.
  • the cells were divided into normal control group and H/R experimental group.
  • the control group was changed to complete medium, and cultured at 37 ° C, 5% CO 2 .
  • the experimental group was changed to glucose-free and serum-free DMEM medium, and O 2 /CO was placed. 2 cell culture system in the incubator (37 ° C, 5% CO 2 , 5% O 2 ) hypoxia culture, 1 h later, the experimental group was replaced with complete medium reoxygenation culture;
  • LDH cytotoxic colorimetric test kit G1782, Promega, Madison, WI, USA.
  • Cell viability was measured using a non-radioactive CCK-8 kit (CK04; Dojindo, Kumamoto, Japan). Carry out relevant tests according to the instructions.
  • Example 1 Inhibition of hepatic ischemia-reperfusion injury by ML355 in a dose-dependent manner
  • serum and liver tissues were taken for ALT, AST enzyme activity detection and HE staining.
  • ALT and AST were as shown in Fig. 1A and Fig. 1B respectively.
  • the serum ALT and AST levels were significantly decreased after 6 hours of ML355 administration, and the ML355 3mg/kg administration group was significantly lower.
  • the contents of ALT and AST were significantly lower than those of ML355 1mg/kg and ML355 2mg/kg.
  • the efficacy of ML355 was dose-dependent.
  • the results of HE staining are shown in Fig. 2.
  • the liver tissue structure of the solvent group and ML355 1mg/kg group was fuzzy and arranged disorderly, showing a large area of necrotic area. With the increase of ML355 dose, the necrotic area of liver tissue gradually decreased.
  • the liver tissue of ML355 3mg/kg group was basically normal, the liver tissue structure was neat, and there was no obvious necrotic area.
  • Example 2 ML355 effectively alleviates liver function damage caused by ischemia-reperfusion
  • mice were randomly divided into 7 groups, 20 in each group. Ten of the mice in each group were given 3 mg/kg of ML355 dissolved in a solvent by tail vein injection, and the other 10 were given a solvent. After the completion of the administration, 7 groups of mice were subjected to I/R operation (one group was Sham control group and the remaining 6 groups were I/R experimental group), and the Sham control group and 0h, 1h, 3h, 6h, respectively. The serum of mice in the I/R experimental group at 12h and 24h was tested by ALT and AST to evaluate the degree of liver function damage.
  • liver tissue of the Sham control group and the 0h and 6h I/R experimental group were used to make paraffin sections.
  • TUNEL staining, Mac1 immunofluorescence staining and Ly6G immunofluorescence staining were used to evaluate the apoptosis of liver cells and the infiltration of liver inflammatory cells.
  • ALT and AST test are shown in Figures 3A and 3B.
  • the contents of ALT and AST in the Sham sham operation group were lower, and there was no significant difference between the ML355 group and the solvent group.
  • the levels of ALT and AST increased gradually with the prolongation of time, and reached the highest point at 6h after operation. Then the ALT and AST levels decreased slowly.
  • the serum levels of ALT and AST in the ML355 group were significantly lower than those in the solvent group at 1h, 3h, 6h, 12h and 24h after operation.
  • mice were randomly divided into 12 groups (Sham-solvent group, sham-ML355 group, I/R 0h-solvent group, I/R0h-ML355 group, I/R 3h-solvent group, I/R 3h-ML355 group, I/R 6h-solvent group, I/R 6h-ML355 group, I/R 12h-solvent group, I/R 12h-ML355 group, I/R 24h-solvent group, I/R 24h-ML355 group), each Groups of 6-9 mice.
  • Mice in the ML355 group were given 3 mg/kg of ML355 dissolved in a solvent by tail vein injection, and the solvent group was given a blank solvent control.
  • mice in the I/R group were subjected to cardiac ischemia reperfusion.
  • the serum of the experimental group was taken at 0h, 3h, 6h, 12h, and 24h after reperfusion.
  • the sham group was taken for 24 hours after reperfusion, and the CK and LDH were detected to evaluate the degree of cardiac injury.
  • the heart tissue of the 24h I/R experimental group was taken and TTC staining was performed to evaluate the ischemia and necrosis of myocardial tissue.
  • the heart tissue of 24h I/R experimental group was extracted and RNA was extracted for RT-PCR.
  • the mRNA expression levels of inflammatory factors Tnf and Il6, pro-apoptotic gene Bax and anti-apoptotic gene Bcl2 were detected.
  • RT-PCR detection primers are as follows:
  • Serum test results are shown in Figures 6A and 6B. Serum CK and LDH levels were significantly increased after I/R. When ML355 was administered through the tail vein, serum CK levels were lower at all time points after I/R than in the ML355 group. Serum LDH levels were significantly lower at 3h, 6h, and 12h after I/R. In the ML355 group, 24 hours after surgery, there was no significant difference between the ML355-administered group and the solvent control group because LDH had almost returned to the background level.
  • the infarct area was white after TTC staining. As shown in Figure 7, the area of myocardial infarction in the ML355 group was significantly lower than that in the solvent group at 24 h after I/R.
  • the mRNA expression levels of the proapoptotic gene Bax and the apoptosis-inhibiting gene Bcl2 in the heart are shown in Figure 9. Compared with the solvent group, the expression of Bcl2 in the heart tissue of mice in the ML355 group was significantly up-regulated, while the expression level of Bax was significantly lower than that in the solvent group. .
  • ML355 has an inhibitory effect on cardiac damage, inflammation and apoptosis caused by ischemia-reperfusion.
  • ML355 inhibits renal ischemia-reperfusion injury
  • mice were randomly divided into two groups: Sham group and operation group. Liver tissues of mice in operation group and Sham group were taken after ischemia for 1 hour. Western blot and RT-RCR were used to detect ALOX12, ALOX5 and ALOX15 proteins in liver tissue. Content and mRNA content.
  • the primary antibody used for WB was: 12-LO Antibody (C-5) (sc-365194; Santa Cruz), 15-LO Antibody (B-7) (sc-133085; Santa Cruz), 5-Lipoxygenase (C49G1) Rabbit mAb (#3289; CST), secondary antibody: Peroxidase AffiniPure goat anti-rabbit-IgG (H+L) (#111-035-003; Jackson Laboratory) and goat anti-mouse-IgG (H+L) (# 115-035-003; Jackson Laboratory); primer sequences used in RT-RCR are as follows:
  • Example 5 Effect of ALOX12 overexpression on L02 cell injury and inflammatory response induced by H/R treatment
  • L02 cells were divided into 4 groups: GFP overexpression control group, ALOX12 overexpression control group, GFP overexpressing H/R group, and ALOX12 overexpressing H/R group.
  • the corresponding plasmids were transfected with adherent L02 cells (combination degree about 80%), and H/R treatment (anoxia 1 h, reoxygenation 6 h) was performed 24 hours later. After the plasmid transfection was completed, the total protein was extracted and subjected to WB analysis (3 independent replicates, 2 replicates each time) to detect the overexpression of ALOX12.
  • ALOX12 overexpression WB assay results of ALOX12 overexpression WB assay are shown in Figure 12. Compared to the GFP group, the ALOX12 overexpressing histone band was significantly enhanced, ie, ALOX12 overexpression was significant in L02 cells.
  • H9C2 cells were divided into 4 groups: shRNA control group, shALOX12 control group, shRNA H/R group, and shALOX12H/R group.
  • the corresponding recombinant lentiviral virus solution was infected with cultured H9C2 cells, and H/R treatment was performed 24 hours later (anoxia 1 h, reoxygenation 6 h).
  • H/R treatment was performed 24 hours later (anoxia 1 h, reoxygenation 6 h).
  • the mRNA of the cells was extracted, and mRNA analysis was performed (three independent repeated experiments) to detect the knockdown of ALOX12.
  • Cell viability was measured after completion of H/R (6 replicates per group).
  • the results of the shRNA control group were 1, and the ratio of the remaining groups to the group was calculated.
  • the results of the cell activity assay are shown in Figure 16. There was no significant difference in the activity of the shALOX12 control group compared to the shRNA control group. When H/R treatment was performed on the two groups of H/R cells, the cell viability of the shRNA group was significantly lower than that of the control group. When the expression of ALOX12 was knocked down, the degree of cell viability in the shALOX12H/R group was significantly lower than that in the shRNA H/R group. This result indicates that the decrease in ALOX12 expression significantly attenuates H/R-induced cardiomyocyte injury and maintains cardiomyocyte activity. That is, ALOX12 can promote the development of diseases related to myocardial cell injury.

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Abstract

Disclosed is the use of an ALOX12 inhibitor, such as a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, in the preparation of a drug for treating ischemia-reperfusion injuries and related diseases, inflammatory diseases, and diseases related to cell death. Ischemia-reperfusion injuries are those of the liver, heart, and kidneys. The inflammatory diseases are hepatitis, myocarditis, endocarditis, and nephritis.

Description

治疗缺血再灌注损伤的化合物Compounds for the treatment of ischemia-reperfusion injury
本申请要求2017年8月21日向中国国家知识产权局提交的专利申请号为201710719320.6，发明名称为“治疗缺血再灌注损伤的化合物”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。The present application claims priority to the prior application of the patent application No. 201710719320.6, entitled "Compound for the Treatment of Ischemia Reperfusion Injury", filed on August 21, 2017, to the Chinese National Intellectual Property Office. The entire contents of this prior application are incorporated herein by reference.
技术领域Technical field
本发明属于医药化学技术领域，尤其是涉及ALOX12抑制剂，例如式(I)化合物、或其药学上可接受的盐、或其溶剂化物、或其代谢产物，在制备治疗缺血再灌注损伤及相关疾病，炎症疾病，细胞死亡相关疾病的药物中的用途，尤其是治疗肝脏、心脏、肾脏等脏器的缺血再灌注导致的机体损伤的药物。The invention belongs to the technical field of medicinal chemistry, in particular to an ALOX12 inhibitor, for example, a compound of the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, for preparing an ischemia-reperfusion injury and The use of drugs for related diseases, inflammatory diseases, and cell death-related diseases, particularly drugs for treating body damage caused by ischemia-reperfusion of organs such as liver, heart, and kidney.
背景技术Background technique
缺血再灌注损伤(Ischemia-Reperfusion Injury，IRI)是1960年Jennings首先提出的概念，是指组织器官缺血后血液再灌注，不仅不能使组织器官功能恢复，反而加重组织器官的功能障碍和结构损伤。缺血再灌注损伤在许多重要器官包括心、肝、肺、肾、胃肠道等均可发生。Ischemia-Reperfusion Injury (IRI) is the first concept proposed by Jennings in 1960. It refers to blood reperfusion after tissue and organ ischemia, which not only fails to restore the function of tissues and organs, but also increases the dysfunction and structure of tissues and organs. damage. Ischemia-reperfusion injury can occur in many important organs including heart, liver, lung, kidney, gastrointestinal tract, and the like.
肝脏缺血再灌注损伤(Hepatic Ischemia Reperfusion Injury，HIRI)是肝脏外科手术中常见的病理过程，多见于休克、需要阻断肝脏血流的肝外科手术以及肝移植术等病理生理过程中。近年来，随着临床治疗技术的发展，肝移植、溶栓治疗以及肝门阻断术等手术的开展越来越多，尽管肝脏保护、外科技巧及术中监护不断改进，但缺血再灌注所致肝损伤依然是引起术后脏器无功能，移植失败甚至患者死亡的主要原因。肝脏经历缺血再灌注后，肝脏组织细胞发生一系列代谢、结构和功能的损伤，易诱发肝功能衰竭，是影响疾病预后、手术成功率和病人存活率的主要原因之一。Hepatic Ischemia Reperfusion Injury (HIRI) is a common pathological process in liver surgery. It is more common in pathological and physiological processes such as shock, liver surgery requiring liver blood flow, and liver transplantation. In recent years, with the development of clinical treatment technology, liver transplantation, thrombolytic therapy and hepatic occlusion surgery have been carried out more and more, although liver protection, surgical techniques and intraoperative monitoring are improving, but ischemia and reperfusion The liver injury is still the main cause of postoperative organ dysfunction, graft failure and even patient death. After the liver undergoes ischemia-reperfusion, liver tissue cells undergo a series of metabolic, structural and functional damages, which are easy to induce liver failure, which is one of the main factors affecting disease prognosis, surgical success rate and patient survival rate.
急性冠状动脉梗阻性疾病是目前心脑血管疾病的主要致死原因之一。尽管心脏搭桥术、介入及溶栓等治疗有了很大的进步，但急性心梗患者的死亡率依 然较高，其中一个很重要的原因就是尚无有效办法抑制缺血心肌恢复血流时所引起的缺血再灌注损伤。心肌缺血一定时间后，重新恢复血供，会造成机体内炎症因子及氧自由基等大量释放，心肌细胞凋亡率增加，恶性心律失常如室颤、室速等发生增加，心肌能量代谢及结构上出现损伤。Acute coronary artery obstructive disease is one of the main causes of death of cardiovascular and cerebrovascular diseases. Despite significant advances in the treatment of bypass surgery, intervention, and thrombolysis, the mortality rate of patients with acute myocardial infarction is still high. One of the most important reasons is that there is no effective way to inhibit the blood flow of ischemic myocardium. Caused by ischemia-reperfusion injury. After a certain period of myocardial ischemia, the blood supply will be restored, which will cause a large amount of inflammatory factors and oxygen free radicals to be released in the body, increase the apoptotic rate of cardiomyocytes, increase the malignant arrhythmia such as ventricular fibrillation, ventricular tachycardia, myocardial energy metabolism and There is damage in the structure.
肾脏也同样是高灌注器官，对缺血以及缺血再灌注均敏感。肾脏缺血再灌注损伤是缺血性急性肾功能衰竭的重要损伤环节，也是肾移植中影响移植肾早期功能恢复的制约因素。The kidney is also a high perfusion organ, sensitive to ischemia and ischemia-reperfusion. Renal ischemia-reperfusion injury is an important injury link of ischemic acute renal failure, and also a limiting factor affecting early recovery of renal function in kidney transplantation.
因此，如何减轻和消除缺血再灌注损伤以及阐明这种损伤的机制，有重要的临床实用价值。目前认为有多个机制参与了器官的缺血再灌注损伤：如致炎性细胞因子(TNF-α和IL等)、氧自由基、钙超载、微循环障碍、能量代谢紊乱等，还受缺血的时间、组织对氧的需求、侧枝循环的建立及电解质浓度等因素影响。Therefore, how to reduce and eliminate ischemia-reperfusion injury and to clarify the mechanism of this injury has important clinical practical value. It is currently believed that multiple mechanisms are involved in organ ischemia-reperfusion injury: such as inflammatory cytokines (TNF-α and IL), oxygen free radicals, calcium overload, microcirculatory disorders, energy metabolism disorders, etc. Blood time, tissue demand for oxygen, establishment of collateral circulation, and electrolyte concentration.
发明内容Summary of the invention
本发明通过实验研究发现了ALOX12参与器官缺血再灌注损伤的发生和发展，ALOX12抑制剂，如式(I)化合物、或其药学上可接受的盐、或其溶剂化物、或其代谢产物，能够治疗和预防缺血再灌注损伤，特别是对肝脏、心脏、肾脏的缺血再灌注损伤有良好的治疗和预防效果，尤其在肝脏的缺血再灌注损伤中有特别优异的治疗和预防效果，可以显著抑制缺血再灌注过程中的炎症反应和细胞死亡，在此基础上完成了本发明。The present invention has found through experimental studies that ALOX12 is involved in the occurrence and development of organ ischemia-reperfusion injury, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, It can treat and prevent ischemia-reperfusion injury, especially for ischemia, reperfusion injury of liver, heart and kidney. It has excellent therapeutic and preventive effects especially in liver ischemia-reperfusion injury. The present invention can be accomplished by significantly inhibiting the inflammatory reaction and cell death during ischemia-reperfusion.
本发明提供ALOX12抑制剂在制备治疗缺血再灌注损伤及相关疾病，炎症疾病，细胞死亡相关疾病的药物中的用途。The present invention provides the use of an ALOX12 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, and cell death-related diseases.
优选，所述ALOX12抑制剂是如下式(I)所示的化合物、或其药学上可接受的盐、或其溶剂化物、或其代谢产物。Preferably, the ALOX12 inhibitor is a compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof.
本发明提供式(I)所示的化合物、或其药学上可接受的盐、或其溶剂化物、或其代谢产物在制备治疗缺血再灌注损伤及相关疾病，炎症疾病，细胞死亡相关疾病的药物中的用途。The present invention provides a compound represented by the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof, for the preparation of a disease for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, and cell death-related diseases. Use in medicine.
本发明还提供一种治疗缺血再灌注损伤及相关疾病，炎症疾病，或细胞死亡相关疾病的方法，其特征在于，给有需要的患者施用含有ALOX12抑制剂的药 物。The present invention also provides a method for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, or cell death-related diseases, characterized in that a drug containing an ALOX12 inhibitor is administered to a patient in need thereof.
优选，所述ALOX12抑制剂是如下式(I)所示的化合物、或其药学上可接受的盐、或其溶剂化物、或其代谢产物。Preferably, the ALOX12 inhibitor is a compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof.
本发明所述式(I)化合物的结构如下：The structure of the compound of the formula (I) according to the invention is as follows:
Figure PCTCN2018101397-appb-000001
Figure PCTCN2018101397-appb-000001
其中，R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基； Wherein R 1 and R 2 are independently selected from the group consisting of H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, An aryl group, a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl , Br, hydroxy, C 1-6 alkoxy;
R 3选自芳基、杂芳基，上述基团任选被一个或多个选自如下基团被取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、羟基、C 1-6烷氧基、C 1-6烷氧基羰基、环烷基、芳基、哌嗪基、哌啶基、吡啶基、吗啉基、吡咯烷基、吡唑烷基、咪唑烷基和硫代吗啉基；上述基团C 1-6烷基、C 2-6烯基、C 2-6炔基、C 1-6烷氧基、环烷基、芳基、哌嗪基、哌啶基、吡啶基、吗啉基、吡咯烷基、吡唑烷基、咪唑烷基、硫代吗啉基还可以被一个或多个C 1-6烷基、C 1-6烷氧基、或C 1-6烷氧基羰基所取代。 R 3 is selected from aryl and heteroaryl, and the above group is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, hydroxy, C 1-6 alkoxy, C 1-6 alkoxycarbonyl, cycloalkyl, aryl, piperazinyl, piperidinyl, pyridyl, morpholinyl, pyrrole An alkyl group, a pyrazolidinyl group, an imidazolidinyl group, and a thiomorpholinyl group; the above group C 1-6 alkyl group, C 2-6 alkenyl group, C 2-6 alkynyl group, C 1-6 alkoxy group, Cycloalkyl, aryl, piperazinyl, piperidinyl, pyridyl, morpholinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, thiomorpholinyl can also be one or more C 1- Substituted by a 6 alkyl group, a C 1-6 alkoxy group, or a C 1-6 alkoxycarbonyl group.
本发明中，In the present invention,
所述烷基指具有1-6个碳原子的直连或支链烷基，所述烷基例如为甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、仲丁基、戊基、新戊基。The alkyl group means a straight or branched alkyl group having 1 to 6 carbon atoms, and the alkyl group is, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group or a t-butyl group. , sec-butyl, pentyl, neopentyl.
所述烯基指具有2-6个碳原子的直连或支链烯基，所述烯基例如为乙烯基、丙烯基、异丙烯基。The alkenyl group means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as a vinyl group, a propenyl group, an isopropenyl group.
所述炔基指具有2-6个碳原子的直连或支链炔基，所述炔基例如为乙炔基、丙炔基、丁炔基。The alkynyl group refers to a straight or branched alkynyl group having 2 to 6 carbon atoms, such as an ethynyl group, a propynyl group, a butynyl group.
所述烷氧基指具有1-6个碳原子的直连或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、叔丁氧基、仲丁氧基。The alkoxy group means a straight or branched alkoxy group having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, Tert-butoxy, sec-butoxy.
所述卤素为氟、氯、溴、碘，优选为氟、氯、溴。The halogen is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
所述芳基指具有6-20个(优选6-14个)碳原子的单环或多环芳族基团，代表性的芳基包括：苯基、萘基、蒽基等。The aryl group refers to a monocyclic or polycyclic aromatic group having 6 to 20 (preferably 6 to 14) carbon atoms, and representative aryl groups include phenyl, naphthyl, anthryl and the like.
所述的杂芳基是指具有1-20个碳原子和1-4个选自N、O、S的杂原子的单环或多环芳族基团。杂芳基可以是具有3-7个环原子(2-6个碳原子和1-3个选自N、O、S的杂原子)的单环或具有7-10个环原子(4-9个碳原子和1至3个选自N、O、S的杂原子)的双环。杂芳基包括但不限于吡啶基、哒嗪基、嘧啶基、吡嗪基、s-三嗪基、噁唑基、咪唑基、噻唑基、异噁唑基、吡唑基、异噻唑基、呋喃基、噻吩基、吡咯基等。The heteroaryl group means a monocyclic or polycyclic aromatic group having 1 to 20 carbon atoms and 1 to 4 hetero atoms selected from N, O, and S. The heteroaryl group may be a single ring having 3 to 7 ring atoms (2 to 6 carbon atoms and 1 to 3 hetero atoms selected from N, O, S) or having 7 to 10 ring atoms (4-9) A bicyclic ring of one carbon atom and one to three hetero atoms selected from N, O, and S. Heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, Furanyl, thienyl, pyrrolyl and the like.
根据本发明，所述式(I)化合物中，优选的，According to the invention, among the compounds of formula (I), preferred,
R 3选自苯基、萘基、噻唑基、苯并噻唑基、苯并噁唑基、咪唑基、苯并咪唑基、噻吩基、苯并噻吩基、吡啶基、喹啉基、异喹啉基、噁唑基、呋喃基、苯并呋喃基、吡咯基、吡唑基、吡嗪基、嘧啶基、三嗪基、吲哚基、嘌呤基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、羟基、C 1-6烷氧基、C 1-6烷氧基羰基、苯基、环烷基、哌嗪、哌啶、吡啶、吗啉、吡咯烷、吡唑烷、咪唑烷硫代吗啉、和C 1-6烷基氧基羰基哌啶基。 R 3 is selected from the group consisting of phenyl, naphthyl, thiazolyl, benzothiazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thienyl, benzothienyl, pyridyl, quinolyl, isoquinoline a group, an oxazolyl group, a furyl group, a benzofuranyl group, a pyrrolyl group, a pyrazolyl group, a pyrazinyl group, a pyrimidinyl group, a triazinyl group, a fluorenyl group, a fluorenyl group, each of which is optionally one or more Substituted from the group: C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, hydroxy, C 1-6 alkoxy, C 1- 6 alkoxycarbonyl, phenyl, cycloalkyl, piperazine, piperidine, pyridine, morpholine, pyrrolidine, pyrazolidine, imidazolidine thiomorpholine, and C 1-6 alkyloxycarbonylpiperidine base.
在一个优选技术方案中，R 3选自苯基、萘基、噻唑基、苯并噻唑基、苯并噁唑基、苯并咪唑基、噻吩基、喹啉基、异喹啉基、吡啶基，上述基团任选被一个或多个选自如下取代基所取代：C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基羰基、F、Cl、Br、羟基、苯基、哌嗪基、哌啶基、吗啉基、C 1-6烷氧基羰基哌啶基。 In a preferred embodiment, R 3 is selected from the group consisting of phenyl, naphthyl, thiazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, thienyl, quinolyl, isoquinolinyl, pyridyl And the above group is optionally substituted by one or more substituents selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxycarbonyl, F, Cl, Br, hydroxy Phenyl, piperazinyl, piperidinyl, morpholinyl, C1-6 alkoxycarbonylpiperidinyl.
在一个优选方案中，R 3选自噻唑基、2-苯并噻唑基、2-苯并噁唑基、2-苯并咪唑基、4-甲基-2-苯并噻唑基、噻吩基、4-甲基-2-噻唑基、5-甲基-2-噻唑基、4,5-二甲基-2-噻唑基、苯基、1-萘基、2-萘基、1,4-联苯基、3-哌嗪-苯基、4-哌啶-苯基、4-哌嗪-3-吡啶基、4-甲基-3-吡啶基、3-吡啶基、2-吡啶基、3-叔丁基-苯基、6-甲氧基-2-苯并噻唑基、6-氟-2-苯并噻唑基、4-苯基-2-噻唑基、3-吗啉-苯基、4-(N-叔丁氧基羰基)哌啶基-3-苯基、3-哌啶基-苯基、3-异丙基-苯基、3- 喹啉基、8-喹啉基、8-异喹啉。 In a preferred embodiment, R 3 is selected from the group consisting of thiazolyl, 2-benzothiazolyl, 2-benzoxazolyl, 2-benzimidazolyl, 4-methyl-2-benzothiazolyl, thienyl, 4-methyl-2-thiazolyl, 5-methyl-2-thiazolyl, 4,5-dimethyl-2-thiazolyl, phenyl, 1-naphthyl, 2-naphthyl, 1,4- Biphenyl, 3-piperazine-phenyl, 4-piperidine-phenyl, 4-piperazin-3-pyridyl, 4-methyl-3-pyridyl, 3-pyridyl, 2-pyridyl, 3-tert-Butyl-phenyl, 6-methoxy-2-benzothiazolyl, 6-fluoro-2-benzothiazolyl, 4-phenyl-2-thiazolyl, 3-morpholine-phenyl 4-(N-tert-Butoxycarbonyl)piperidinyl-3-phenyl, 3-piperidinyl-phenyl, 3-isopropyl-phenyl, 3-quinolinyl, 8-quinolinyl , 8-isoquinoline.
在一个优选技术方案中，R 1和R 2独立地选自H、F、Cl、Br、C 1-6烷基、C 1-6烷氧基。 In a preferred embodiment, R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
在又一个优选方案中，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl； In still another preferred embodiment, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl;
在一个优选方案中，式(I)化合物为如下式(II)化合物，In a preferred embodiment, the compound of formula (I) is a compound of formula (II) below,
Figure PCTCN2018101397-appb-000002
Figure PCTCN2018101397-appb-000002
其中，X选自O、S、NH或C；R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基；优选地，R 1和R 2独立地选自H、卤素、羟基、C 1-6烷氧基、C 1-6烷基；R 4、R 5、R 6、R 7相同或不同，独立地选自H、卤素、C 1-6烷氧基或C 1-6烷基。 Wherein X is selected from O, S, NH or C; and R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 Alkynyl, F, Cl, Br, amino, aryl, heteroaryl, each of which is optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl , C 2-6 alkynyl, F, Cl, Br, hydroxy, C 1-6 alkoxy; preferably, R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl; R 4 , R 5 , R 6 , R 7 are the same or different and are independently selected from H, halogen, C 1-6 alkoxy or C 1-6 alkyl.
在一个优选技术方案中，R 1和R 2独立地选自H、F、Cl、Br、C 1-6烷基、C 1-6烷氧基。 In a preferred embodiment, R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
在又一个优选方案中，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl。 In still another preferred embodiment, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
在一个优选方案中，式(I)化合物为如下式(III)化合物，In a preferred embodiment, the compound of formula (I) is a compound of formula (III) below,
Figure PCTCN2018101397-appb-000003
Figure PCTCN2018101397-appb-000003
R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基。优选地，R 1和R 2独立地选自H、卤素、羟基、C 1-6烷氧基、C 1-6烷基；R 8、R 9独立地选自H、 卤素、C 1-6烷氧基、C 1-6烷基、苯基、C 1-6烷基苯基、C 1-6烷氧基苯基、卤素取代的苯基。 R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, aryl a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br , hydroxy, C 1-6 alkoxy. Preferably, R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl; R 8 , R 9 are independently selected from H, halo, C 1-6 Alkoxy, C 1-6 alkyl, phenyl, C 1-6 alkylphenyl, C 1-6 alkoxyphenyl, halogen substituted phenyl.
在一个优选技术方案中，R 1和R 2独立地选自H、F、Cl、Br、C 1-6烷基、C 1-6烷氧基。 In a preferred embodiment, R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
在又一个优选方案中，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl。 In still another preferred embodiment, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
在一个优选方案中，所述式(I)化合物为如下式(IV)化合物，In a preferred embodiment, the compound of formula (I) is a compound of formula (IV) below,
Figure PCTCN2018101397-appb-000004
Figure PCTCN2018101397-appb-000004
R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基；优选地，R 1和R 2独立地选自H、卤素、羟基、C 1-6烷氧基、C 1-6烷基；R 10、R 11独立地选自H、卤素、C 1-6烷氧基、C 1-6烷基、任选取代的苯基、任选取代的哌啶基、任选取代的哌嗪基，所述取代基可为C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基羰基、卤素。优选的，R 10、R 11独立地选自H、叔丁基、异丙基、苯基、哌嗪基、4-哌啶基、4-叔丁基氧基羰基哌啶基。 R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, aryl a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br , hydroxy, C 1-6 alkoxy; preferably, R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl; R 10 , R 11 are independently Is selected from H, halogen, C 1-6 alkoxy, C 1-6 alkyl, optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted piperazinyl, said substituent It is a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 alkoxycarbonyl group, or a halogen. Preferably, R 10 and R 11 are independently selected from the group consisting of H, tert-butyl, isopropyl, phenyl, piperazinyl, 4-piperidinyl, 4-tert-butyloxycarbonylpiperidinyl.
在一个优选技术方案中，R 1和R 2独立地选自H、F、Cl、Br、C 1-6烷基、C 1-6烷氧基。 In a preferred embodiment, R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxy.
在又一个优选方案中，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl。 In still another preferred embodiment, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
根据本发明，所述式(I)化合物优选为如下具体化合物：According to the invention, the compound of formula (I) is preferably the following specific compound:
Figure PCTCN2018101397-appb-000005
Figure PCTCN2018101397-appb-000005
Figure PCTCN2018101397-appb-000006
Figure PCTCN2018101397-appb-000006
Figure PCTCN2018101397-appb-000007
Figure PCTCN2018101397-appb-000007
Figure PCTCN2018101397-appb-000008
Figure PCTCN2018101397-appb-000008
Figure PCTCN2018101397-appb-000009
Figure PCTCN2018101397-appb-000009
Figure PCTCN2018101397-appb-000010
Figure PCTCN2018101397-appb-000010
Figure PCTCN2018101397-appb-000011
Figure PCTCN2018101397-appb-000011
Figure PCTCN2018101397-appb-000012
Figure PCTCN2018101397-appb-000012
Figure PCTCN2018101397-appb-000013
Figure PCTCN2018101397-appb-000013
根据本发明，所述式(I)化合物进一步优选为如下具体化合物：According to the invention, the compound of the formula (I) is further preferably a specific compound as follows:
Figure PCTCN2018101397-appb-000014
Figure PCTCN2018101397-appb-000014
Figure PCTCN2018101397-appb-000015
Figure PCTCN2018101397-appb-000015
Figure PCTCN2018101397-appb-000016
Figure PCTCN2018101397-appb-000016
在本发明的一个具体实施方案中，所述式(I)化合物是
Figure PCTCN2018101397-appb-000017
(又简称为ML355)、 In a particular embodiment of the invention, the compound of formula (I) is
Figure PCTCN2018101397-appb-000017
(also referred to as ML355),
Figure PCTCN2018101397-appb-000018
Figure PCTCN2018101397-appb-000018
Figure PCTCN2018101397-appb-000019
Figure PCTCN2018101397-appb-000019
本文所用术语“药学上可接受的盐”是指药物活性化合物的衍生物，其中通过制备其酸式盐或碱式盐来修饰母体化合物。药学上可接受的盐的实例包括但不限于，碱性残基(如胺类)的无机酸盐或有机酸盐，酸性残基(如羧酸)的碱性盐或有机盐，等。药学上可接受的盐包括由诸如无毒性的无机或有机酸形成的母体化合物的常规无毒性的盐或季铵盐。例如，这些常规的无毒性盐包括那些源自无机酸的盐，如盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等；以及由有机酸制备的盐，如，乙酸、丙酸、琥珀酸、羟基乙酸、乳酸、苹果酸、酒石酸、柠檬酸、延胡索酸、甲磺酸、甲苯磺酸、水杨酸、对氨基苯磺酸等。The term "pharmaceutically acceptable salt" as used herein refers to a derivative of a pharmaceutically active compound wherein the parent compound is modified by preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, basic or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from, for example, non-toxic inorganic or organic acids. For example, these conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and salts prepared from organic acids, such as acetic acid, propionic acid, Succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, methanesulfonic acid, toluenesulfonic acid, salicylic acid, p-aminobenzenesulfonic acid and the like.
本发明的药学上可接受的盐可以通过常规的化学方法从含有碱性或酸性部分的母体化合物合成。通常，这种盐可以通过使这些化合物的游离酸或碱形式与化学计量的适当的碱或酸在水或有机溶剂或二者的混合物中反应制备。The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
本发明式(I)化合物可以按照US2017001955A1记载的方法进行合成，该专利文献全文引入参考。The compound of the formula (I) of the present invention can be synthesized according to the method described in US Pat. No. 1,00, 1955, A1, the entire disclosure of which is incorporated herein by reference.
对于ML355，可以商购获得，也可以按照文献记载的方法进行合成(参见US2017001955A1；Journal of medicinal chemistry 2014，57，495-506)。根据本发明，所述缺血再灌注损伤及相关疾病优选为肝脏缺血再灌注损伤及相关疾病、心脏缺血再灌注损伤及相关疾病、肾脏缺血再灌注损伤及相关疾病；更优选为 肝脏缺血再灌注损伤及相关疾病。For ML355, it is commercially available or can be synthesized according to the methods described in the literature (see US2017001955A1; Journal of medicinal chemistry 2014, 57, 495-506). According to the present invention, the ischemia-reperfusion injury and related diseases are preferably liver ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia-reperfusion injury and related diseases; more preferably liver Ischemia-reperfusion injury and related diseases.
根据本发明，所述缺血再灌注损伤优选为肝脏缺血再灌注损伤、心脏缺血再灌注损伤、肾脏缺血再灌注损伤；更优选为肝脏缺血再灌注损伤。According to the present invention, the ischemia-reperfusion injury is preferably liver ischemia-reperfusion injury, cardiac ischemia-reperfusion injury, renal ischemia-reperfusion injury; more preferably liver ischemia-reperfusion injury.
肝脏缺血再灌注损伤及相关疾病的引发因素包括但不限于：肝脏囊肿、肝脏移植、溶栓治疗、肝门阻断术、肝昏迷。Inflammatory factors of hepatic ischemia-reperfusion injury and related diseases include, but are not limited to, liver cysts, liver transplantation, thrombolytic therapy, hepatic occlusion, and hepatic coma.
心脏缺血再灌注损伤及相关疾病的引发因素包括但不限于：心肌梗塞、心梗再通损伤、心脏移植、冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术，冠状动脉旁路术。Cardiac ischemia-reperfusion injury and related factors include, but are not limited to, myocardial infarction, myocardial infarction, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, coronary artery Bypass.
肾脏缺血再灌注损伤及相关疾病的引发因素包括但不限于：肾脏移植、肾脏囊肿、肾脏血管手术。Causes of renal ischemia-reperfusion injury and related diseases include, but are not limited to, kidney transplantation, renal cysts, and renal vascular surgery.
所述的炎症疾病包括但不限于：肝炎、心肌炎、心内膜炎、肾炎。The inflammatory diseases include, but are not limited to, hepatitis, myocarditis, endocarditis, and nephritis.
根据本发明，所述药物进一步包含药学上可接受的辅料。According to the invention, the medicament further comprises a pharmaceutically acceptable adjuvant.
所述药学上可接受的辅料是制药领域中常用或已知的各种辅料，包括但不限于：稀释剂、粘合剂、抗氧化剂、pH调节剂、防腐剂、润滑剂、崩解剂等。The pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrators, etc. .
所述稀释剂例如：乳糖、淀粉、纤维素衍生物、无机钙盐、山梨醇等。所述粘合剂例如：淀粉、明胶、羧甲基纤维素钠、聚乙烯吡咯烷酮等。所述抗氧化剂例如：维生素E、亚硫酸氢钠、亚硫酸钠、丁羟基茴香醚等。所述pH调节剂例如：盐酸、氢氧化钠、柠檬酸、酒石酸、Tris、乙酸、磷酸二氢钠、磷酸氢二钠等。所述防腐剂例如：对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、间甲酚、苯扎氯铵等。所述润滑剂例如：硬脂酸镁、微粉硅胶、滑石粉等。所述崩解剂例如：淀粉、甲基纤维素、黄原胶、交联羧甲基纤维素钠等。The diluent is, for example, lactose, starch, cellulose derivative, inorganic calcium salt, sorbitol or the like. The binder is, for example, starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone or the like. The antioxidant is, for example, vitamin E, sodium hydrogen sulfite, sodium sulfite, butylated hydroxyanisole or the like. The pH adjusting agent is, for example, hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate or the like. The preservative is, for example, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, m-cresol, benzalkonium chloride or the like. The lubricant is, for example, magnesium stearate, finely divided silica gel, talc, or the like. The disintegrant is, for example, starch, methyl cellulose, xanthan gum, croscarmellose sodium or the like.
本发明药物的剂型可以是口服剂的形式，例如片剂、胶囊、丸剂、粉剂、颗粒剂、悬浮剂、糖浆剂等；也可以是注射给药的剂型，例如注射液、粉针剂等，通过静脉内、腹膜内、皮下或肌肉内的途径。所有使用的剂型形式都是药学领域普通技术人员所熟知的。The dosage form of the medicament of the present invention may be in the form of an oral preparation, such as a tablet, a capsule, a pill, a powder, a granule, a suspension, a syrup, etc.; or a dosage form for injection administration, such as an injection solution, a powder injection, etc., Intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage form forms used are well known to those of ordinary skill in the pharmaceutical arts.
本发明的药物可施用于会发生或已经发生缺血再灌注损伤的任何动物。这些动物包括人类和非人类的动物，例如宠物或牲畜等。The medicament of the invention can be administered to any animal that develops or has developed ischemia-reperfusion injury. These animals include human and non-human animals such as pets or livestock.
本发明的药物可以本领域已知的途径施用于受试者，包括但不限于口服，胃肠外，皮下，肌内，静脉内，腹腔内，肝内，心肌内，肾内，阴道，直肠， 颊，舌下，鼻内，透皮方式等。The medicament of the present invention can be administered to a subject by a route known in the art including, but not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal. , buccal, sublingual, intranasal, transdermal, etc.
ALOX12抑制剂的施用剂量将取决于接受者的年龄、健康和体重，联用药物的种类，治疗频率，给药途径等。药物可以单一日剂量施用，或者总日剂量以每天两次，三次或四次的分开剂量施用。药物可以在发生缺血再灌注损伤前给予或者发生了缺血再灌注损伤后及时给予，例如在肝脏、心脏、肾脏等器官的手术前、手术中、手术后施用。剂量可以施用一次或多次，施药时间可以单日至几个月或更长时间。ALOX12抑制剂的单次剂量可以在每个患者每天约0.0001至约10000mg的宽范围内变化。该范围可以更特别地为成人(约60kg)每天约0.001mg/kg至100mg/kg体重/天，例如为0.01mg/kg至10mg/kg，或者为0.1mg/kg至1mg/kg。The dosage of the ALOX12 inhibitor to be administered will depend on the age, health and weight of the recipient, the type of combination, the frequency of treatment, the route of administration, and the like. The drug can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three or four times daily. The drug can be administered before the ischemia-reperfusion injury occurs or after ischemia-reperfusion injury occurs, for example, before, during, and after surgery of organs such as the liver, heart, and kidney. The dose can be administered one or more times, and the administration time can be from one day to several months or longer. A single dose of the ALOX12 inhibitor can vary widely from about 0.0001 to about 10,000 mg per patient per day. The range may more particularly be from about 0.001 mg/kg to 100 mg/kg body weight per day for adults (about 60 kg), such as from 0.01 mg/kg to 10 mg/kg, or from 0.1 mg/kg to 1 mg/kg.
根据本发明，所述药物还可以与其他的治疗缺血再灌注相关损伤、炎症疾病、细胞死亡相关疾病的药物联用给药。本发明的发明人发现式(I)化合物具有所述治疗活性，与其是ALOX12抑制剂有关。本发明的发明人在研究中发现，在肝脏缺血再灌注损伤的组织中ALOX12蛋白表达量和mRNA表达量都显著增加，变化的幅度和显著性远高于ALOX的其他成员(例如ALOX5、ALOX15)，表明ALOX12与缺血再灌注损伤，尤其是肝脏缺血再灌注损伤的关联更大。并且，发明人还发现，过表达ALOX12会促进缺氧和复氧处理后的肝细胞、心脏细胞和肾脏细胞的活性降低以及炎症反应，而低表达ALOX12则会缓解缺氧和复氧处理后的肝细胞、心脏细胞和肾脏细胞的活性降低以及炎症反应，表明ALOX12会促进缺血再灌注损伤的发生发展，以及这些脏器的炎症疾病和细胞死亡性疾病，抑制ALOX12的活性能够治疗缺血再灌注损伤，以及这些脏器的炎症疾病和细胞死亡性疾病。According to the present invention, the medicament can also be administered in combination with other drugs for treating ischemia-reperfusion-related injury, inflammatory diseases, and cell death-related diseases. The inventors of the present invention have found that the compounds of formula (I) have said therapeutic activity and are associated with their ALOX12 inhibitors. The inventors of the present invention found in the study that the expression levels of ALOX12 protein and mRNA expression in the tissues of liver ischemia-reperfusion injury were significantly increased, and the magnitude and significance of the changes were much higher than other members of ALOX (for example, ALOX5, ALOX15). ), indicating that ALOX12 is associated with ischemia-reperfusion injury, especially liver ischemia-reperfusion injury. Moreover, the inventors also found that overexpression of ALOX12 promotes decreased activity and inflammatory response of hepatocytes, heart cells, and kidney cells after hypoxia and reoxygenation, while low expression of ALOX12 attenuates hypoxia and reoxygenation. Reduced activity of liver cells, heart cells, and kidney cells, as well as inflammatory responses, suggest that ALOX12 promotes the development of ischemia-reperfusion injury, as well as inflammatory and cell-dead diseases in these organs, inhibiting the activity of ALOX12 to treat ischemia. Perfusion damage, as well as inflammatory and cell death diseases in these organs.
附图说明DRAWINGS
图1A和1B：肝脏缺血再灌注损伤中，给药ML355 1mg/kg、2mg/kg、3mg/kg后，小鼠血清中ALT、AST的检测结果(n.s.代表P≥0.05，*代表0.01≤P<0.05，**代表P<0.01)。Figure 1A and 1B: In the liver ischemia-reperfusion injury, after ML355 1mg/kg, 2mg/kg, 3mg/kg, the serum ALT, AST test results (ns represent P ≥ 0.05, * represents 0.01 ≤ P < 0.05, ** represents P < 0.01).
图2：肝脏缺血再灌注损伤中，给药ML355 1mg/kg、2mg/kg、3mg/kg后，小鼠肝脏HE染色镜检图。Figure 2: Hepatic HE staining micrographs of mice after administration of ML355 1 mg/kg, 2 mg/kg, 3 mg/kg in hepatic ischemia-reperfusion injury.
图3A和3B：肝脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移小鼠血清中ALT、AST的检测结果(n.s.代表P≥0.05，*代表0.01≤P<0.05，**代表P<0.01)。Figure 3A and 3B: In the liver ischemia-reperfusion injury, after ML355 3 mg/kg, the serum ALT and AST results were detected over time (ns represent P≥0.05, * represents 0.01≤P<0.05,* * represents P < 0.01).
图4：肝脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移肝脏组织TUNEL染色结果，图中白色细胞代表凋亡的细胞。Figure 4: TUNEL staining of liver tissue over time after administration of ML355 3 mg/kg in hepatic ischemia-reperfusion injury, in which white cells represent apoptotic cells.
图5A和5B：肝脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移肝脏Mac1和Ly6G阳性炎症细胞免疫荧光染色结果，图中浅灰色细胞代表炎症细胞。Fig. 5A and Fig. 5B: Immunofluorescence staining of liver Mac1 and Ly6G positive inflammatory cells over time in liver hepatic ischemia-reperfusion injury after administration of ML355 3 mg/kg, in which light gray cells represent inflammatory cells.
图6A和6B：心脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移CK、LDH的检测结果(n.s.代表P≥0.05，*代表0.01≤P<0.05，**代表P<0.01)。Fig. 6A and Fig. 6B are the results of detection of CK and LDH over time after administration of ML355 3 mg/kg in cardiac ischemia-reperfusion injury (ns represent P ≥ 0.05, * represents 0.01 ≤ P < 0.05, ** represents P < 0.01).
图7：心脏缺血再灌注损伤中，给药ML355 3mg/kg后，再灌注24小时TTC染色结果，图中白色区域代表组织梗死区域。Figure 7: TTC staining results of reperfusion for 24 hours after administration of ML355 3 mg/kg in cardiac ischemia-reperfusion injury. The white area in the figure represents the tissue infarct area.
图8：心脏缺血再灌注损伤中，给药ML355 3mg/kg后，心脏组织中再灌注24小时，Tnf和Il6的mRNA表达量RT-PCR的检测结果图(**代表P<0.01)Figure 8: RT-PCR results of Tnf and Il6 mRNA expression in heart tissue after reperfusion for 24 hours after administration of ML355 3 mg/kg in cardiac ischemia-reperfusion injury (** represents P<0.01)
图9：心脏缺血再灌注损伤中，给药ML355 3mg/kg后，心脏组织中再灌注24小时，Bcl2和Bax的mRNA表达量RT-PCR的检测结果图(*代表0.01≤P<0.05)Figure 9: RT-PCR results of Bcl2 and Bax mRNA expression in heart tissue after reperfusion for 24 hours after administration of ML355 3mg/kg in cardiac ischemia-reperfusion injury (* represents 0.01≤P<0.05)
图10A和10B：肾脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移BUN、Scr的检测结果(n.s.代表P≥0.05，*代表0.01≤P<0.05)。Fig. 10A and Fig. 10B are the results of detection of BUN and Scr over time after administration of ML355 3 mg/kg in renal ischemia-reperfusion injury (n.s. represents P ≥ 0.05, * represents 0.01 ≤ P < 0.05).
图11A：肝脏缺血再灌注损伤中，ALOX12、ALOX5、ALOX15的mRNA表达量RT-PCR的检测结果图(n.s.表示P≥0.05，**表示P<0.01)。Fig. 11A is a graph showing the results of RT-PCR detection of mRNA expression levels of ALOX12, ALOX5, and ALOX15 in hepatic ischemia-reperfusion injury (n.s. indicates P≥0.05, ** indicates P<0.01).
图11B：肝脏缺血再灌注损伤中，ALOX12、ALOX5、ALOX15的蛋白表达量western-blot的检测结果图。图中GAPDH为对照标准品。Fig. 11B is a graph showing the results of Western-blot expression of protein expression of ALOX12, ALOX5 and ALOX15 in hepatic ischemia-reperfusion injury. In the figure, GAPDH is a control standard.
图12：L02细胞经GFP及ALOX12过表达慢病毒转染后ALOX12蛋白表达情况鉴定图。图中GAPDH为对照标准品。Figure 12: Identification of ALOX12 protein expression after L02 cells were transfected with GFP and ALOX12 overexpression lentivirus. In the figure, GAPDH is a control standard.
图13：ALOX12过表达对H/R处理诱导的L02细胞损伤中，LDH释放检测结果(n.s.表示P≥0.05，*表示0.01≤P<0.05，**表示P<0.01)。Figure 13: LDH release detection results in L02 cell injury induced by H/R treatment by ALOX12 overexpression (n.s. indicates P ≥ 0.05, * indicates 0.01 ≤ P < 0.05, ** indicates P < 0.01).
图14：ALOX12过表达对H/R处理诱导的L02细胞损伤中，炎症因子Il-6、Tnf-α及趋化因子Ccl2、Cxcl10的mRNA的检测结果(n.s.表示P≥0.05，*表示0.01≤P<0.05，**表示P<0.01)。Figure 14: Detection results of inflammatory factor Il-6, Tnf-α and chemokine Ccl2, Cxcl10 mRNA in L02 cell injury induced by H/R treatment by ALOX12 overexpression (ns indicates P≥0.05, * indicates 0.01≤ P < 0.05, ** indicates P < 0.01).
图15：H9C2细胞经shRNA及shALOX12慢病毒转染后ALOX12mRNA表达情况鉴定图(**表示P<0.01)。Figure 15: Identification of ALOX12 mRNA expression in H9C2 cells transfected with shRNA and shALOX12 lentivirus (** indicates P < 0.01).
图16：ALOX12敲低对H/R处理后H9C2细胞活性的影响(n.s.表示P≥0.05，**表示P<0.01)。Figure 16: Effect of ALOX12 knockdown on H9C2 cell activity after H/R treatment (n.s. indicates P ≥ 0.05, ** indicates P < 0.01).
具体实施方式Detailed ways
以下结合实施例对本发明做进一步描述。需要说明的是，实施例不能作为对本发明保护范围的限制，本领域的技术人员理解，任何在本发明基础上所作的改进和变化都在本发明的保护范围之内。The invention will be further described below in conjunction with the embodiments. It should be noted that the embodiments are not intended to limit the scope of the invention, and those skilled in the art understand that any modifications and variations made on the basis of the invention are within the scope of the invention.
以下实施例所用化学试剂都是常规试剂，均可商购获得。未做特殊说明的实验方法都是采用本领域已知的常规方法。The chemical reagents used in the following examples are all conventional reagents and are commercially available. Experimental methods not specifically described are by conventional methods known in the art.
在以下实施例中所采用的动物模型及各研究指标的检测方法：The animal model used in the following examples and the detection methods of each research index:
实验动物：选用8-10周龄、体重在24g-27g、背景为C57BL/6品系的雄性野生型小鼠(购自北京华阜康生物科技股份有限公司)。Experimental animals: Male wild-type mice (purchased from Beijing Huakang Biotechnology Co., Ltd.) of 8-10 weeks old, weighing 24 g-27 g, and background C57BL/6 strain were used.
动物饲养:所有实验小鼠均饲养在武汉大学SPF级实验动物中心。饲养条件：室温在22-24℃之间，湿度在40-70％之间，明暗交替照明时间为12h，自由饮水摄食。Animal feeding: All experimental mice were housed in the SPF laboratory animal center of Wuhan University. Breeding conditions: room temperature between 22-24 ° C, humidity between 40-70%, alternating light and dark lighting time is 12h, free to drink water.
HEK293T，人胚肾细胞，购自中国科学院细胞库，目录号GNHu43。HEK293T, human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu43.
L02，人肝细胞系，购自中国科学院细胞库，目录号GNHu6。L02, human hepatocyte cell line, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu6.
H9C2，大鼠心肌细胞，购自中国科学院细胞库，目录号GNR5。H9C2, rat cardiomyocytes, purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNR5.
细胞均培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中。培养环境：37℃，5％CO 2。 The cells were cultured in DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin). Culture environment: 37 ° C, 5% CO 2 .
1、小鼠肝缺血再灌注(ischemia/reperfusion，I/R)损伤模型构建及相关检测：1. Construction of mouse ischemic/reperfusion (I/R) injury model and related detection:
手术前12h给小鼠禁食，可自由饮水。术前用3％戊巴比妥钠麻醉小鼠后，将其平卧固定肢体，用剃毛器将小鼠腹部术区毛刮净，用10％碘酒和75％乙醇对手术区消毒。The mice were fasted 12 h before surgery and were given free access to water. The mice were anesthetized with 3% pentobarbital sodium before surgery, and the limbs were fixed in a flat position. The abdomen area of the mice was shaved with a shaver, and the surgical area was disinfected with 10% iodine and 75% ethanol.
取腹正中切口进腹，暴露肝脏左、中叶之肝蒂。用无创血管夹夹闭中叶和左叶的门静脉和肝动脉，使约70％的肝脏缺血，以防止发生严重肠系膜静脉淤血。0.5min后，与非阻断的右叶相比，可见阻断叶变白，说明阻断成功。记录缺血开始时间，维持缺血60分钟，Sham组的小鼠不进行肝脏血流阻断。The midline incision was taken into the abdomen to expose the liver pedicle of the left and middle leaves of the liver. The portal vein and hepatic artery of the middle and left lobe were clipped with non-invasive vascular clamps, causing approximately 70% of liver ischemia to prevent severe mesenteric venous congestion. After 0.5 min, compared with the non-blocked right lobe, it was seen that the blocking leaves turned white, indicating that the blocking was successful. The onset of ischemia was recorded and ischemia was maintained for 60 minutes. The mice in the Sham group were not blocked by hepatic blood flow.
缺血60min后去除血管夹，恢复缺血的肝脏血流，然后关闭腹腔缝合后，将手术后的小鼠置于干净的笼子中单独饲养，观察。After 60 minutes of ischemia, the blood vessel clip was removed, the ischemic liver blood flow was restored, and then the abdominal cavity was sutured. The mice after the operation were placed in a clean cage and kept alone.
取材：分别于术后0h、1h、3h、6h、12h、24h取假手术组(Sham组)以及缺血再灌注组小鼠，3％戊巴比妥钠麻醉，眼眶静脉丛取血1mL，分离血清。同时统一取缺血区肝左叶组织分别置于液氮中进行速冻或于10％中性福尔马林中固定24h后脱水，包埋，制作石蜡切片。Materials: The sham operation group (Sham group) and the ischemia-reperfusion group were anesthetized at 0h, 1h, 3h, 6h, 12h, and 24h after operation, 3% pentobarbital sodium was anesthetized, and 1 mL of blood was taken from the orbital venous plexus. Separate serum. At the same time, the left lobe tissue of the ischemic area was uniformly placed in liquid nitrogen for rapid freezing or fixed in 10% neutral formalin for 24 hours, then dehydrated, embedded, and paraffin sections were prepared.
分离血清：收集血液的EP管于室温下静置1-2h使血液自然凝固。4℃、4000rpm/min离心30min，充分分离血清，保存于-80℃冰箱备用。Separation of serum: The EP tube collecting blood was allowed to stand at room temperature for 1-2 h to allow the blood to naturally coagulate. Centrifuge at 4 ° C, 4000 rpm / min for 30 min, fully separate the serum, and store in a -80 ° C refrigerator for use.
肝脏缺血再灌注损伤严重程度的评估指标主要包括肝脏坏死面积、肝功能指标(AST、ALT)、炎症反应、细胞死亡等，均与肝脏缺血再灌注损伤严重程度正相关。The indicators of hepatic ischemia-reperfusion injury severity include liver necrosis area, liver function index (AST, ALT), inflammatory response, cell death, etc., all of which are positively correlated with the severity of hepatic ischemia-reperfusion injury.
采用全自动生化分析仪(Sysmex，Chemix 180i)测定小鼠血清的ALT、AST含量。The serum ALT and AST contents were determined by an automatic biochemical analyzer (Sysmex, Chemix 180i).
对石蜡切片采用HE染色，显微镜拍照，观察肝脏病理改变。The paraffin sections were stained with HE, photographed by microscope, and pathological changes of the liver were observed.
采用TUNEL试剂盒染色石蜡切片检测肝细胞凋亡情况：TUNEL试剂盒：
Figure PCTCN2018101397-appb-000020
Plus In Situ Apoptosis Fluorescein Detection Kit(S7111，Chemicon)。按照试剂盒说明书进行操作。 Hepatocyte apoptosis was detected by staining paraffin sections with TUNEL kit: TUNEL kit:
Figure PCTCN2018101397-appb-000020
Plus In Situ Apoptosis Fluorescein Detection Kit (S7111, Chemicon). Follow the kit instructions.
用Ly6G和MAC1免疫荧光染色检测缺血再灌注后肝脏炎症细胞浸润情况，具体步骤如下：The inflammatory cell infiltration of liver after ischemia-reperfusion was detected by Ly6G and MAC1 immunofluorescence staining. The specific steps are as follows:
1)将石蜡切片置于烤箱中，60℃烤片30分钟；1) Place the paraffin section in an oven and bake at 60 ° C for 30 minutes;
2)二甲苯，5分钟×3；2) xylene, 5 minutes × 3;
3)100％乙醇，5分钟×2次；95％乙醇，5分钟；70％乙醇，5分钟；3) 100% ethanol, 5 minutes × 2 times; 95% ethanol, 5 minutes; 70% ethanol, 5 minutes;
4)ddH 2O漂洗，5分钟×2次； 4) ddH 2 O rinse, 5 minutes × 2 times;
5)柠檬酸盐组织抗原修复液(100×，pH6.0，福州迈新)高压修复5min；5) citrate tissue antigen repair solution (100 ×, pH 6.0, Fuzhou Maixin) high pressure repair 5min;
6)ddH 2O漂洗5分钟×2次，PBS漂洗5分钟×2次； 6) ddH 2 O rinse for 5 minutes × 2 times, PBS rinse for 5 minutes × 2 times;
7)组化笔划圈，滴加10％羊血清(GTX27481，GeneTex)封闭，于湿盒中37℃封闭60min；7) Tissue stroke circle, 10% sheep serum (GTX27481, GeneTex) was added dropwise, and sealed in a wet box at 37 ° C for 60 min;
8)弃封闭液，滴加适当比例稀释的一抗：兔抗Mac1(CD11b，1:100稀释，ab75476，Abcam)；大鼠抗Ly6G(1:100稀释，551459，BD Biosciences)，4℃孵育过夜；8) Discard the blocking solution and add the appropriate dilution of the primary antibody: rabbit anti-Mac1 (CD11b, 1:100 dilution, ab75476, Abcam); rat anti-Ly6G (1:100 dilution, 551459, BD Biosciences), incubate at 4 °C overnight;
9)37℃复温30min；9) Rewarming at 37 ° C for 30 min;
10)弃去一抗，PBS洗10min×3；10) Discard the primary antibody and wash the PBS for 10 min × 3;
11)滴加二抗(羊抗兔IgG，Invitrogen；羊抗大鼠IgG，Carlsbad)，于湿盒中37℃孵育60min；11) Add secondary antibody (goat anti-rabbit IgG, Invitrogen; goat anti-rat IgG, Carlsbad), and incubate in a humid box at 37 ° C for 60 min;
12)弃去二抗，PBS浸洗5min×3；12) Discard the secondary antibody and PBS for 5 min × 3;
13)SlowFade Gold antifade reagent with DAPI封片13) SlowFade Gold antifade reagent with DAPI
14)荧光镜(OLMPUS DX51)下观察，拍照，用Image Pro Plus(version 6.0)软件进行图片分析。14) Observed under fluoroscope (OLMPUS DX51), photographed, and analyzed with Image Pro Plus (version 6.0) software.
此外，于术后1h取假手术组(Sham)以及缺血再灌注组小鼠，3％戊巴比妥钠过量麻醉，取缺血区肝组织，立即放入液氮中30min以上，之后保存于-80℃冰箱中，用于RT-PCR及Western blot分析。In addition, sham operation group (Sham) and ischemia-reperfusion group mice were anesthetized with 3% pentobarbital sodium 1 hour after operation, liver tissue in ischemic area was taken, immediately placed in liquid nitrogen for more than 30 minutes, and then preserved. It was used in RT-PCR and Western blot analysis in a -80 °C refrigerator.
2、小鼠心脏缺血再灌注(ischemia/reperfusion，I/R)损伤模型构建及相关检测：2. Construction of mouse ischemic/reperfusion (I/R) injury model and related detection:
采用阻断小鼠心脏左冠状动脉前降支(LAD)造成心脏缺血，具体步骤如下：Cardiac ischemia is caused by blocking the left anterior descending coronary artery (LAD) of the mouse heart. The specific steps are as follows:
术前用3％戊巴比妥钠麻醉小鼠后，将其平卧固定肢体，用剃毛器将小鼠术区毛刮净。气管插管并连接呼吸机成功后，进行下步手术，整个手术过程用加热垫维持小鼠体温在37℃左右。The mice were anesthetized with 3% pentobarbital sodium before surgery, and the limbs were fixed in a flat position, and the hair of the mouse was shaved with a shaver. After the trachea was intubated and connected to the ventilator successfully, the next step was performed. The whole body was heated with a heating pad to maintain the body temperature at 37 °C.
小鼠采用右侧卧位，用医用碘酊及75％医用酒精对手术区皮肤进行消毒清洁处理，用眼科剪在左前肢下0.5cm处沿肋骨走向剪开皮肤，逐层分离筋膜、肌肉等组织，用显微剪于三、四肋间打开胸腔充分暴露心脏，用显微直镊夹起少量心包并于左心耳下撕开少许心包，充分暴露左冠状动脉前降支(LAD)或所在区域。The mice were placed in the right lateral position, and the skin of the operation area was disinfected and cleaned with medical iodine and 75% medical alcohol. The skin was cut along the ribs at 0.5 cm below the left forelimb with ophthalmic scissors, and the fascia, muscles, etc. were separated layer by layer. Tissue, use micro-shear to open the thoracic cavity between the three and four intercostals to fully expose the heart, use a microscopic straight sputum to clip a small amount of pericardium and tear a small pericardium under the left atrial appendage to fully expose the left anterior descending coronary artery (LAD) or where region.
结扎冠状动脉：用无齿持针器持取7-0带针缝合线，于左心耳下缘1mm处进针，肺动脉圆锥旁出针，缝线从LAD下方穿过，稳定5s后，结扎LAD。结扎成功后，可见左室前壁明显由鲜红色变为苍白而不再恢复，同时心电图显示sT段抬高和(或)T波高耸或者倒置呈弓背向上单向曲线。6-0缝线完全缝合胸腔开口关闭胸腔，5mL注射器接套管经切口插入胸腔，抽取1mL气体，平整各层肌肉，合拢皮肤切口暂不缝合，Sham组不结扎LAD，直接关胸。维持血流阻断60min。Ligation of the coronary artery: 7-0 needle suture was taken with a needleless needle holder, needle was inserted at 1 mm from the lower edge of the left atrial appendage, needle was inserted next to the pulmonary artery cone, suture was passed under the LAD, and LAD was ligated after 5 s of stabilization. . After successful ligation, it can be seen that the anterior wall of the left ventricle is obviously changed from bright red to pale and no longer recovers. At the same time, the electrocardiogram shows that the sT segment elevation and/or T wave is towering or inverted and the unidirectional curve is upward. 6-0 suture completely sutured the thoracic cavity to close the thoracic cavity, 5mL syringe was inserted into the thoracic cavity through the incision, 1mL gas was taken, the muscles of each layer were leveled, and the skin incision was not sutured. The Sham group did not ligature the LAD and directly closed the chest. Maintain blood flow block for 60 min.
结扎完成后，6-0缝线完全缝合胸腔开口关闭胸腔，5mL注射器接套管经切口插入胸腔，抽取1mL气体。After the ligation was completed, the 6-0 suture completely sutured the thoracic opening to close the chest cavity, and the 5 mL syringe was inserted through the incision into the chest cavity, and 1 mL of gas was withdrawn.
术后密切关注小鼠状态，有无呼吸异常等。待小鼠自然苏醒后将小鼠从呼吸机上取下并取下气管插管，放入干净的饲养笼。After the operation, the state of the mouse was closely monitored, and there was no abnormality in breathing. After the mice were naturally awakened, the mice were removed from the ventilator and the tracheal intubation was removed and placed in a clean feeding cage.
血清生化指标检测：Serum biochemical indicators test:
分别于术后0h、3h、6h、12h、24h取缺血再灌注组以及假手术组(Sham组)小鼠，3％戊巴比妥钠麻醉，眼眶静脉丛取血1ml，分离血清，用全自动生化分析仪(Sysmex，Chemix 180i)测定血清中CK及LDH含量。The mice in the ischemia-reperfusion group and the sham-operated group (Sham group) were anesthetized at 0h, 3h, 6h, 12h, and 24h after operation, 3% sodium pentobarbital anesthesia, 1ml of blood was taken from the orbital venous plexus, and serum was separated. The serum CK and LDH levels were determined by an automated biochemical analyzer (Sysmex, Chemix 180i).
TTC染色：TTC staining:
缺血再灌注24h后，取小鼠，3％戊巴比妥钠麻醉，分离出颈静脉，同时重新结扎LAD后，用注射器向颈静脉缓慢注入2ml 2.5％的伊文思蓝溶液，见心肌变为蓝色且不再褪色后停止注入，迅速取下心脏，清洁并挤压心脏蘸干血渍和染液，4℃生理盐水冲洗干净后蘸干，于-20℃冰箱冷冻15min至心脏变硬，取出并用刀片自心尖向心底沿房室沟方向切成1mm厚切片，共切5片，迅速将切片置于5ml 37℃ 1％PH为7.4的TTC磷酸缓冲液中，水浴15min后观察。24 hours after ischemia-reperfusion, mice were anesthetized with 3% pentobarbital sodium, the jugular vein was isolated, and LAD was re-ligated. Then 2 ml of 2.5% Evans blue solution was slowly injected into the jugular vein with a syringe. After the blue color is no longer faded, stop the injection, quickly remove the heart, clean and squeeze the blood and blood stains of the heart, rinse it with 4°C saline, dry it, and freeze it in the refrigerator at -20 °C for 15 minutes until the heart becomes hard. Remove and cut into 1 mm thick sections from the apex of the heart from the apex of the heart to the center of the sulcus. Cut a total of 5 pieces, and quickly place the sections in 5 ml of TTC phosphate buffer solution at 37 ° C and 1% PH 7.4, and observe after 15 minutes in a water bath.
RT-PCR检测：RT-PCR detection:
组织中RNA的提取RNA extraction from tissues
①取100mg组织，放入1ml玻璃匀浆器中，加入1ml TRizol，在冰浴中研磨，悬液转入1.5ml离心管中，室温静置5min，使核蛋白完全从核酸上解离；1 Take 100mg tissue, put it into 1ml glass homogenizer, add 1ml TRizol, grind in ice bath, transfer the suspension into 1.5ml centrifuge tube, let stand for 5min at room temperature, completely dissociate nucleoprotein from nucleic acid;
②4℃12000r/min离心5min，取上清液，加氯仿200μl，漩涡混匀器震荡30s，冰盒上静置10min；Centrifuge at 12000r/min for 24min at 24°C, take the supernatant, add 200μl of chloroform, vortex the mixer for 30s, and let stand on the ice box for 10min;
③4℃12000r/min离心15min，取上清液，加入0.5ml异丙醇，充分混匀，冰盒上静置10min，使RNA充分沉淀；Centrifuge at 34 ° C 12000r / min for 15min, take the supernatant, add 0.5ml of isopropanol, mix well, let stand on the ice box for 10min, so that the RNA is fully precipitated;
④4℃12000r/min离心15min，弃去上清，加入1ml预冷的75％乙醇，漩涡混匀器震荡30s洗涤RNA沉淀；Centrifuge at 44 ° C 12000 r / min for 15 min, discard the supernatant, add 1 ml of pre-cooled 75% ethanol, vortex mixer for 30s to wash the RNA precipitate;
⑤4℃12000r/min离心5min，弃去上清液，沉淀快速风干。提取RNA加适量的DEPC去离子水溶解。Centrifuge at 54 ° C 12000 r / min for 5 min, discard the supernatant, and precipitate quickly and air dry. The extracted RNA was dissolved in an appropriate amount of DEPC deionized water.
细胞中RNA的提取RNA extraction from cells
收集细胞并用PBS缓冲液洗涤2次，完成后加入1mlTRizol，用加样器吹打均匀，吸入1.5ml离心管中，漩涡混匀器震荡30s，室温静置5min，使核蛋白完全从核酸上解离。其余操作步骤同组织中RNA提取②-⑤。The cells were collected and washed twice with PBS buffer. After completion, 1 ml of TRizol was added, uniformly blown with a pipette, and inhaled into a 1.5 ml centrifuge tube. The vortex mixer was shaken for 30 s, and allowed to stand at room temperature for 5 min to completely dissociate the nucleoprotein from the nucleic acid. . The remaining steps are the same as RNA extraction in the tissue 2-5.
反转录Reverse Transcription
使用Transcriptor First Strand cDNA Synthesis Kit(04896866001,Roche,Basel,Switzerland)反转录试剂盒根据试剂盒说明书进行反转录实验。Reverse transcription experiments were performed using the Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche, Basel, Switzerland) reverse transcription kit according to the kit instructions.
3、小鼠肾脏缺血再灌注(ischemia/reperfusion，I/R)损伤模型构建及相关检测：3. Construction of mouse ischemia/reperfusion (I/R) injury model and related detection:
模型构建：Model construction:
小鼠术前禁食12h，自由饮水。3％戊巴比妥钠腹腔注射麻醉小鼠。小鼠背部去毛，消毒备皮。在背部脊椎旁0.5cm、肋骨下缘0.5cm处剪开皮肤及肌肉，可见到肾脏。分离出两侧肾脏的肾动脉，迅速用动脉夹夹闭两侧肾动脉。缺血60min后松开动脉夹，恢复血流，观察肾脏恢复情况。缝合开口。The mice were fasted for 12 hours before surgery and were given free access to water. Mice were anesthetized by intraperitoneal injection of 3% pentobarbital sodium. The back of the mouse was depilated and the skin was sterilized. The skin and muscles were cut at 0.5 cm next to the back spine and 0.5 cm at the lower edge of the ribs, and the kidneys were visible. The renal arteries of both kidneys were isolated and the renal arteries were clamped with arterial clips. After 60 minutes of ischemia, the arterial clip was released, the blood flow was restored, and the kidney recovery was observed. Suture the opening.
血清生化指标检测：Serum biochemical indicators test:
于术后24h取假手术组(Sham)以及缺血再灌注组小鼠，3％戊巴比妥钠麻醉，眼眶静脉丛取血1ml，离心后取血清，用全自动生化分析仪(Sysmex，Chemix180i)检测血清BUN(尿素氮)和Scr(血肌酐)水平。The sham-operated group (Sham) and the ischemia-reperfusion group were anesthetized 24 hours after surgery, 3% sodium pentobarbital anesthesia, 1 ml of blood was taken from the orbital venous plexus, serum was taken after centrifugation, and an automatic biochemical analyzer (Sysmex, Chemix180i) detects serum BUN (urea nitrogen) and Scr (serocreatin) levels.
Western blot：Western blot:
1)组织蛋白提取1) Tissue protein extraction
①向于干冰中预冷的EP管中放入3-4颗钢珠，并加入称重定量之后的组织样本。1 Add 3-4 steel balls to the EP tube pre-cooled in dry ice, and add the tissue sample after weighing and quantification.
②向裂解液中加入PMSF，混匀后加至样品中，迅速摇匀。2 Add PMSF to the lysate, mix and add to the sample, and shake quickly.
③于-80℃预冷研磨仪适配器中研磨样品，研磨参数设置为30Hz/s，90s。3 The sample was ground in a -80 ° C pre-cooled grinder adapter with a grinding parameter set to 30 Hz/s for 90 s.
④研磨结束后，冰上放置10min，取出钢珠。4 After the completion of the grinding, the ice was placed on the ice for 10 minutes, and the steel balls were taken out.
⑤超声裂解仪裂解样本(5KHz/次，每次1s，间隔1s，重复10次)，超声完成后冰上放置10min。5 The ultrasonic pyrolyzer lysed the sample (5 KHz/time, 1 s each time, interval 1 s, repeated 10 times), and placed on ice for 10 min after completion of the ultrasound.
⑥样本放入4℃预冷的离心机中，12000rpm/min离心30min。6 samples were placed in a 4 ° C pre-cooled centrifuge and centrifuged at 12000 rpm / min for 30 min.
⑦吸取上清转移到新的EP管中，4℃，14000rpm/min离心10min。7 The supernatant was transferred to a new EP tube and centrifuged at 14,000 rpm/min for 10 min at 4 °C.
⑧吸取上清转移到新的EP管中继续离心，4℃，14000rpm/min离心5min。8 Pipette the supernatant and transfer to a new EP tube and continue centrifugation. Centrifuge at 14000 rpm/min for 5 min at 4 °C.
准确吸取上清液并利用BCA Protein Assay Kit(PierecTM，23225)进行蛋白定量。The supernatant was accurately aspirated and protein quantitation was performed using the BCA Protein Assay Kit (PierecTM, 23225).
2)细胞中蛋白提取2) Protein extraction in cells
细胞加入裂解液，裂解完成后离心取上清，运用BCA Protein Assay Kit定量收集蛋白样品。The cells were added to the lysate, and after the completion of the lysis, the supernatant was centrifuged, and the protein sample was quantitatively collected using the BCA Protein Assay Kit.
3)上样与电泳3) Loading and electrophoresis
配置好电泳凝胶，并在电泳槽内加入电泳液。把蛋白样品上样到SDS-PAGE胶加样孔内，点样完成后开始电泳。Configure the electrophoresis gel and add the electrophoresis solution to the electrophoresis tank. The protein sample was loaded into the SDS-PAGE gel well and the electrophoresis was started after the spotting was completed.
4)转膜4) Transfer film
①配制转膜液，于4℃预冷。1 Prepare the transfer solution and pre-cool at 4 °C.
②将PVDF在甲醇中浸泡15s后放入转膜液中备用。2 Soak the PVDF in methanol for 15 s and put it in the transfer solution for use.
③取出凝胶板中的凝胶，用转膜液洗涤凝胶，将凝胶平铺在负极的滤纸上，将PVDF膜覆盖其上，夹上夹板。3 The gel in the gel plate was taken out, the gel was washed with a transfer liquid, the gel was spread on the filter paper of the negative electrode, the PVDF film was covered thereon, and the splint was sandwiched.
④将夹板放入转膜槽中，灌满转膜液以淹没凝胶。4 Place the splint in the transfer tank and fill the transfer liquid to submerge the gel.
⑤转膜槽接通电源，电压设为250V，电流设为0.2A。转移1.5h。5 The film transfer tank is connected to the power supply, the voltage is set to 250V, and the current is set to 0.2A. Transfer 1.5h.
⑥转移结束后，取出PVDF膜。6 After the transfer is completed, the PVDF membrane is taken out.
5)封闭5) Closed
把蛋白膜放置到预先准备好的TBST中，洗去膜上的转膜液。蛋白膜放入封闭液中，在摇床上缓慢摇动，室温封闭1-4h。Place the protein film in the pre-prepared TBST and wash off the transfer solution on the membrane. The protein membrane was placed in a blocking solution and shaken slowly on a shaker at room temperature for 1-4 h.
6)一抗孵育6) Primary antibody incubation
①用TBST洗涤蛋白膜3次，每次5min。1 Wash the protein membrane 3 times with TBST for 5 min each time.
②封口机将薄膜封入杂交袋中，加上一抗。2 Sealer seals the film into the hybrid bag and adds the primary antibody.
③将杂交袋放入4℃摇床中，过夜。3 Place the hybrid bag in a 4 ° C shaker overnight.
7)二抗孵育7) Secondary antibody incubation
①将薄膜取出用TBST洗涤3次，每次5min，回收一抗。1 The film was taken out and washed three times with TBST for 5 minutes each time, and the primary antibody was recovered.
②将膜放入对应的加有二抗的二抗稀释液中，避光孵育1h。2 Place the membrane in the corresponding secondary antibody dilution with secondary antibody and incubate for 1 h in the dark.
8)蛋白检测8) Protein detection
孵育后用TBST洗涤3次，每次5min。利用Bio-Rad Chemi Doc XRS+凝胶成像系统检测目的条带After incubation, wash 3 times with TBST for 5 min each time. Detection of target bands using the Bio-Rad Chemi Doc XRS+ gel imaging system
ALOX12过表达质粒构建：Construction of ALOX12 overexpression plasmid:
1)PCR扩增ALOX12基因，引物为：1) PCR amplification of the ALOX12 gene, the primers are:
正向：5’-TCGGGTTTAAACGGATCCATGGGCCGCTACCGCATCCG-3’；Forward: 5'-TCGGGTTTAAACGGATCCATGGGCCGCTACCGCATCCG-3';
反向；5’-GGGCCCTCTAGACTCGAGTCAGATGGTGACACTGTTCT-3’；Reverse; 5'-GGGCCCTCTAGACTCGAGTCAGATGGTGACACTGTTCT-3';
2)PCR产物进行琼脂糖凝胶电泳，随后使用DNA凝胶回收试剂盒(天根)进行DNA片段的回收；2) The PCR product is subjected to agarose gel electrophoresis, followed by recovery of the DNA fragment using a DNA gel recovery kit (Tiangen);
3)将所得DNA产物和限制性内切核酸酶FastDigest restriction enzymes(Thermo)、
Figure PCTCN2018101397-appb-000021
buffer or
Figure PCTCN2018101397-appb-000022
Green buffer、ddH 2O混合均匀(50μl体系)，置于37℃条件下反应。使用
Figure PCTCN2018101397-appb-000023
AxyPrep TM PCR Clean-Up Kit(Axygen)回收酶切产物； 3) the resulting DNA product and restriction endonuclease FastDigest restriction enzymes (Thermo),
Figure PCTCN2018101397-appb-000021
Buffer or
Figure PCTCN2018101397-appb-000022
The Green buffer and ddH 2 O were uniformly mixed (50 μl system) and placed at 37 ° C for reaction. use
Figure PCTCN2018101397-appb-000023
AxyPrep TM PCR Clean-Up Kit ( Axygen) recovering the digestion products;
4)使用
Figure PCTCN2018101397-appb-000024
PCR一步定向克隆试剂盒(Novoprotein)按照试剂盒说明书进行重组反应； 4) use
Figure PCTCN2018101397-appb-000024
PCR one-step cloning kit (Novoprotein) according to the kit instructions for recombination reaction;
5)制作大肠杆菌感受态细胞，将上述连接产物进行转化实验，涂板，置于37℃培养箱，过夜培养；5) Making E. coli competent cells, performing the transformation test on the above-mentioned ligation products, plating the plates, placing them in a 37 ° C incubator, and culturing overnight;
6)从37℃培养箱中取出过夜培养的平板，挑克隆摇菌，并检测菌落PCR阳性克隆；6) Take the overnight cultured plate from the 37 ° C incubator, pick up the cloning bacteria, and detect the colony PCR positive clone;
7)将PCR鉴定为阳性的菌液吸取5-10μl接种至5ml LB(含抗性)培养基中，在220rpm，37℃摇床中过夜培养；7) 5-10 μl of the bacterial solution identified as positive by PCR was inoculated into 5 ml of LB (containing resistant) medium, and cultured overnight at 220 rpm, 37 ° C in a shaker;
8)取出过夜培养的菌液，对已经混浊的菌液进行质粒提取(天根质粒DNA小提试剂盒)；8) taking out the culture liquid of the overnight culture, and performing plasmid extraction on the turbid bacterial liquid (Tiangen Plasmid DNA Mini Kit);
9)提取后的质粒可直接用于ALOX12瞬转或构建慢病毒稳转细胞系。9) The extracted plasmid can be directly used for transient transduction of ALOX12 or construction of a lentiviral stable cell line.
ALOX12干扰质粒构建：ALOX12 interference plasmid construction:
1)ALOX12靶向干扰序列为GCATCGAGAGAAGGAACTGAA，设计适合pLKO.1载体的寡聚核苷酸；阴性对照siRNA序列为：CAACAAGATGAAGAGCACCAA；正向寡聚核苷酸:5’CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG 3’；反向寡聚核苷酸:5’AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC 3’；1) ALOX12 targeted interference sequence is GCATCGAGAGAAGGAACTGAA, designed for oligonucleotides of pLKO.1 vector; negative control siRNA sequence: CAACAAGATGAAGAGCACCAA; forward oligonucleotide: 5'CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTGTGTGTTTTG 3'; reverse oligonucleo Glycosylate: 5'AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC 3';
2)将上述两条寡聚核苷酸分半加无菌水溶解，终浓度为100mM，进行融合；2) dissolving the above two oligonucleotides in half by adding sterile water to a final concentration of 100 mM for fusion;
3)根据“ALOX12表达质粒构建”步骤进行酶切反应、酶切产物的回收、连接反应、转化、挑单克隆、测序和提取质粒；3) according to the "ALOX12 expression plasmid construction" step of enzymatic cleavage reaction, enzymatic cleavage product recovery, ligation reaction, transformation, single selection, sequencing and extraction of plasmids;
4)所得质粒可用于慢病毒介导的ALOX12敲低细胞系构建；4) The resulting plasmid can be used for lentiviral-mediated construction of the ALOX12 knockdown cell line;
慢病毒载体构建和包装：Lentiviral vector construction and packaging:
1)用胰酶消化并记数293T细胞，按1×10 6个293T/孔传至6孔板中； 1) trypsinize and count 293T cells, transfer 1 × 10 6 293T / well to a 6-well plate;
2)第二天细胞汇合度至80％时开始转染；2) Transfection is started when the cell confluence reaches 80% on the second day;
3)取1.5ml灭菌EP管，加入2个包装质粒(pSpax和pMD2G)和过表达或干扰质粒各1μg溶于100μl的无血清培养基。轻柔混匀，室温孵育5min。3) Take 1.5 ml of sterilized EP tube, add 2 packaging plasmids (pSpax and pMD2G) and 1 μg of each of the overexpressing or interference plasmids in 100 μl of serum-free medium. Gently mix and incubate for 5 min at room temperature.
4)取1.5ml灭菌EP管，取3μl PEI(1.6μg/μl)溶于100μl无血清培养基中。轻柔混匀，室温孵育5min。4) A 1.5 ml sterile EP tube was taken, and 3 μl of PEI (1.6 μg/μl) was dissolved in 100 μl of serum-free medium. Gently mix and incubate for 5 min at room temperature.
5)将DNA溶液和PEI溶液轻柔混匀，室温孵育15min；5) gently mix the DNA solution and the PEI solution, and incubate for 15 min at room temperature;
6)将上述DNA-PEI混合液，逐滴加入6孔板中；6) adding the above DNA-PEI mixture to a 6-well plate dropwise;
7)转染6h后，换新鲜培养基；7) After 6 hours of transfection, change the fresh medium;
8)转染后48-72h收获含病毒的上清，3000rpm离心10min，去除沉淀，并用0.45μm的滤膜过滤；8) The virus-containing supernatant was harvested 48-72 h after transfection, centrifuged at 3000 rpm for 10 min, the precipitate was removed, and filtered through a 0.45 μm filter;
9)过滤后的病毒可立即用于感染或-80℃贮存。9) The filtered virus can be used immediately for infection or storage at -80 °C.
细胞缺氧复氧(H/R)：Cell hypoxia reoxygenation (H/R):
1)细胞培养至对数期，预温PBS洗2次，弃去；1) The cells were cultured to log phase, washed twice with pre-warmed PBS, and discarded;
2)将细胞分成正常对照组和H/R实验组，对照组换完全培养基，放37℃，5％CO 2培养，实验组换无糖无血清的DMEM培养基，并放O 2/CO 2细胞培养系统的培养箱内(37℃，5％CO 2，5％O 2)缺氧培养，1h后，实验组换完全培养基复氧培养； 2) The cells were divided into normal control group and H/R experimental group. The control group was changed to complete medium, and cultured at 37 ° C, 5% CO 2 . The experimental group was changed to glucose-free and serum-free DMEM medium, and O 2 /CO was placed. 2 cell culture system in the incubator (37 ° C, 5% CO 2 , 5% O 2 ) hypoxia culture, 1 h later, the experimental group was replaced with complete medium reoxygenation culture;
3)复氧至预定的复氧培养时间后，弃去上清，用PBS洗2次，保存；3) After reoxygenation to a predetermined reoxygenation culture time, the supernatant is discarded, washed twice with PBS, and stored;
LDH释放及细胞活性(cell viability)检测：LDH release and cell viability assay:
使用LDH细胞毒性比色测试试剂盒(G1782,Promega,Madison,WI,USA)检测LDH的释放量。使用非放射性CCK-8试剂盒(CK04；Dojindo,Kumamoto,Japan)检测细胞活性。按照说明书进行相关检测。The amount of LDH released was measured using an LDH cytotoxic colorimetric test kit (G1782, Promega, Madison, WI, USA). Cell viability was measured using a non-radioactive CCK-8 kit (CK04; Dojindo, Kumamoto, Japan). Carry out relevant tests according to the instructions.
实施例1 ML355对肝脏缺血再灌注损伤的抑制作用呈剂量依赖性Example 1 Inhibition of hepatic ischemia-reperfusion injury by ML355 in a dose-dependent manner
为验证不同剂量ML355对肝脏缺血再灌注损伤的抑制作用，C57小鼠随机分为4组，分别通过尾静脉注射1mg/kg ML355(HY-12341，MCE公司)、2mg/kg ML355、3mg/kg ML355以及溶剂(对照组，DMSO:Solutol:PEG400:水＝5:10:20:65(v:v:v:v))，之后按照前述肝脏I/R动物模型的方法进行I/R手术。术后不同时间点，取血清以及肝脏组织进行ALT、AST酶活检测以及HE染色。To verify the inhibitory effect of different doses of ML355 on hepatic ischemia-reperfusion injury, C57 mice were randomly divided into 4 groups, respectively, by tail vein injection of 1mg/kg ML355 (HY-12341, MCE company), 2mg/kg ML355, 3mg/ Kg ML355 and solvent (control group, DMSO: Solutol: PEG400: water = 5:10:20:65 (v:v:v:v)), followed by I/R surgery according to the aforementioned liver I/R animal model . At different time points after operation, serum and liver tissues were taken for ALT, AST enzyme activity detection and HE staining.
ALT、AST检测结果分别如图1A、图1B所示，相比于溶剂对照组，ML355给药后，术后6h，小鼠血清中ALT、AST含量显著降低，且ML355 3mg/kg给药组ALT、AST含量显著低于ML355 1mg/kg及ML355 2mg/kg给药组，ML355药效呈剂量依赖性。The results of ALT and AST were as shown in Fig. 1A and Fig. 1B respectively. Compared with the solvent control group, the serum ALT and AST levels were significantly decreased after 6 hours of ML355 administration, and the ML355 3mg/kg administration group was significantly lower. The contents of ALT and AST were significantly lower than those of ML355 1mg/kg and ML355 2mg/kg. The efficacy of ML355 was dose-dependent.
HE染色结果如图2所示，溶剂组及ML355 1mg/kg组肝组织结构模糊，排列紊乱，可见大面积坏死区，而随着ML355剂量的增加，肝脏组织坏死面积逐渐降低。ML355 3mg/kg组肝组织基本正常，肝组织结构整齐，无明显的坏死区域。上述结果说明ML355对肝脏缺血再灌注损伤的抑制作用呈剂量依赖性，2mg/kg和3mg/kg的ML355可有效抑制肝脏缺血再灌注损伤。The results of HE staining are shown in Fig. 2. The liver tissue structure of the solvent group and ML355 1mg/kg group was fuzzy and arranged disorderly, showing a large area of necrotic area. With the increase of ML355 dose, the necrotic area of liver tissue gradually decreased. The liver tissue of ML355 3mg/kg group was basically normal, the liver tissue structure was neat, and there was no obvious necrotic area. These results indicate that ML355 inhibits hepatic ischemia-reperfusion injury in a dose-dependent manner, and ML355 at 2 mg/kg and 3 mg/kg can effectively inhibit hepatic ischemia-reperfusion injury.
实施例2 ML355有效减轻缺血再灌注引起的肝功能损伤Example 2 ML355 effectively alleviates liver function damage caused by ischemia-reperfusion
为研究ML355对肝脏缺血再灌注后不同时间的保护作用，C57小鼠随机分为 7组，每组20只。每组的其中10只小鼠通过尾静脉注射给以3mg/kg的溶于溶剂中的ML355，另外10只给以溶剂。给药完成后对7组小鼠进行I/R手术(其中一组为Sham对照组，剩余6组为I/R实验组)，分别取Sham对照组以及术后0h、1h、3h、6h、12h、24h I/R实验组小鼠血清，进行ALT、AST检测，以评价肝功能损伤程度；取Sham对照组以及术后0h、6h I/R实验组小鼠肝脏组织，制作石蜡切片，进行TUNEL染色、Mac1免疫荧光染色和Ly6G免疫荧光染色，以评价肝脏细胞的凋亡情况以及肝脏炎症细胞浸润情况。To study the protective effects of ML355 at different times after hepatic ischemia-reperfusion, C57 mice were randomly divided into 7 groups, 20 in each group. Ten of the mice in each group were given 3 mg/kg of ML355 dissolved in a solvent by tail vein injection, and the other 10 were given a solvent. After the completion of the administration, 7 groups of mice were subjected to I/R operation (one group was Sham control group and the remaining 6 groups were I/R experimental group), and the Sham control group and 0h, 1h, 3h, 6h, respectively. The serum of mice in the I/R experimental group at 12h and 24h was tested by ALT and AST to evaluate the degree of liver function damage. The liver tissue of the Sham control group and the 0h and 6h I/R experimental group were used to make paraffin sections. TUNEL staining, Mac1 immunofluorescence staining and Ly6G immunofluorescence staining were used to evaluate the apoptosis of liver cells and the infiltration of liver inflammatory cells.
ALT、AST检测结果如图3A和3B所示，Sham假手术组ALT、AST含量均较低，且ML355组与溶剂组之间无显著性差异。ML355组与溶剂组在I/R术后，随时间点的延长，ALT、AST含量逐渐升高，并在术后6h达到最高点，随后ALT、AST含量缓慢降低。而ML355组在术后1h、3h、6h、12h、24h，小鼠血清中ALT、AST含量均较溶剂组显著降低。The results of ALT and AST test are shown in Figures 3A and 3B. The contents of ALT and AST in the Sham sham operation group were lower, and there was no significant difference between the ML355 group and the solvent group. After I/R in the ML355 group and the solvent group, the levels of ALT and AST increased gradually with the prolongation of time, and reached the highest point at 6h after operation. Then the ALT and AST levels decreased slowly. The serum levels of ALT and AST in the ML355 group were significantly lower than those in the solvent group at 1h, 3h, 6h, 12h and 24h after operation.
TUNEL染色结果如图4所示，随I/R术后时间的延长，肝脏细胞凋亡数量逐渐增多，在同一时间点，ML355组凋亡肝细胞数量显著低于溶剂组。The results of TUNEL staining are shown in Figure 4. The number of liver cell apoptosis increased gradually with the prolongation of I/R time. At the same time point, the number of apoptotic hepatocytes in ML355 group was significantly lower than that in the solvent group.
Mac1以及Ly6G免疫荧光染色结果如图5A和5B所示，I/R术后6h，相比于Sham组，Mac1阳性细胞数以及Ly6G阳性细胞数显著增多，说明I/R术后6h，小鼠肝脏中出现了明显的炎症细胞浸润情况；而相比于溶剂组，ML355组小鼠炎症细胞浸润情况显著降低。上述结果说明ML355可显著抑制缺血再灌注导致的肝脏损伤，减轻肝细胞凋亡，抑制炎症细胞浸润，保护肝功能。The results of immunofluorescence staining of Mac1 and Ly6G are shown in Figures 5A and 5B. Compared with the Sham group, the number of Mac1 positive cells and the number of Ly6G positive cells increased significantly at 6 h after I/R, indicating that mice were 6 h after I/R. Significant inflammatory cell infiltration occurred in the liver; whereas in the ML355 group, inflammatory cell infiltration was significantly reduced compared to the solvent group. The above results indicate that ML355 can significantly inhibit liver damage caused by ischemia-reperfusion, reduce hepatocyte apoptosis, inhibit inflammatory cell infiltration, and protect liver function.
由上述实验可见，ML355对肝脏缺血再灌注损伤的保护作用不仅起效快，并在足够长的时间内均有显著作用。It can be seen from the above experiments that the protective effect of ML355 on hepatic ischemia-reperfusion injury is not only effective, but also has a significant effect for a long enough time.
实施例3 ML355对不同组织缺血再灌注损伤的抑制Example 3 Inhibition of ML355 on ischemia-reperfusion injury in different tissues
1、ML355抑制心脏缺血再灌注损伤1, ML355 inhibits cardiac ischemia-reperfusion injury
C57小鼠随机分为12组(Sham-溶剂组、sham-ML355组、I/R 0h-溶剂组、I/R0h-ML355组、I/R 3h-溶剂组、I/R 3h-ML355组、I/R 6h-溶剂组、I/R 6h-ML355组、I/R 12h-溶剂组、I/R 12h-ML355组、I/R 24h-溶剂组、I/R 24h-ML355组)，每组6-9只小鼠。ML355组小鼠通过尾静脉注射给以3mg/kg的溶于溶剂中的ML355，溶剂组给以空白溶剂对照。给药完成后对I/R组小鼠进行心脏缺血再灌 注手术。分别于再灌注后0h、3h、6h、12h、24h取实验组小鼠血清，sham组于再灌注24小时取小鼠血清，进行CK及LDH检测，以评价心脏损伤程度。取24h I/R实验组小鼠心脏组织，制作TTC染色，以评价心肌组织的缺血、坏死情况。取24h I/R实验组小鼠心脏组织，提取RNA，进行RT-PCR，检测心脏中炎症因子Tnf和Il6、促凋亡基因Bax和抑制凋亡基因Bcl2的mRNA表达水平。C57 mice were randomly divided into 12 groups (Sham-solvent group, sham-ML355 group, I/R 0h-solvent group, I/R0h-ML355 group, I/R 3h-solvent group, I/R 3h-ML355 group, I/R 6h-solvent group, I/R 6h-ML355 group, I/R 12h-solvent group, I/R 12h-ML355 group, I/R 24h-solvent group, I/R 24h-ML355 group), each Groups of 6-9 mice. Mice in the ML355 group were given 3 mg/kg of ML355 dissolved in a solvent by tail vein injection, and the solvent group was given a blank solvent control. After the administration, the mice in the I/R group were subjected to cardiac ischemia reperfusion. The serum of the experimental group was taken at 0h, 3h, 6h, 12h, and 24h after reperfusion. The sham group was taken for 24 hours after reperfusion, and the CK and LDH were detected to evaluate the degree of cardiac injury. The heart tissue of the 24h I/R experimental group was taken and TTC staining was performed to evaluate the ischemia and necrosis of myocardial tissue. The heart tissue of 24h I/R experimental group was extracted and RNA was extracted for RT-PCR. The mRNA expression levels of inflammatory factors Tnf and Il6, pro-apoptotic gene Bax and anti-apoptotic gene Bcl2 were detected.
RT-PCR检测引物如下：RT-PCR detection primers are as follows:
基因gene 正向引物Forward primer 反向引物Reverse primer
TnfTnf AGCCGATGGGTTGTACCTTGAGCCGATGGGTTGTACCTTG ATAGCAAATCGGCTGACGGTATAGCAAATCGGCTGACGGT
Il6Il6 AGGATACCACTCCCAACAGACCTAGGATACCACTCCCAACAGACCT CAAGTGCATCATCGTTGTTCATACCAAGTGCATCATCGTTGTTCATAC
BaxBax AACCATCATGGGCTGGACACTAACCATCATGGGCTGGACACT AAAGATGGTCACTGTCTGCCAAAAGATGGTCACTGTCTGCCA
Bcl2Bcl2 TGGTGGACAACATCGCCCTGTGTGGTGGACAACATCGCCCTGTG GGTCGCATGCTGGGGCCATATAGGTCGCATGCTGGGGCCATATA
血清检测结果如图6A和6B所示，I/R术后血清中CK及LDH含量显著增加。当通过尾静脉给以ML355后，I/R术后各时间点，血清中CK含量较未给ML355组降低；同时血清中LDH含量在I/R术后3h、6h和12h明显低于未给ML355组，而术后24h，由于LDH基本已恢复到本底水平，未见ML355给药组和溶剂对照组有显著差异。Serum test results are shown in Figures 6A and 6B. Serum CK and LDH levels were significantly increased after I/R. When ML355 was administered through the tail vein, serum CK levels were lower at all time points after I/R than in the ML355 group. Serum LDH levels were significantly lower at 3h, 6h, and 12h after I/R. In the ML355 group, 24 hours after surgery, there was no significant difference between the ML355-administered group and the solvent control group because LDH had almost returned to the background level.
TTC染色后梗死区为白色。如图7所示，I/R术后24h，相比于溶剂组，ML355组小鼠心肌梗死区面积显著降低。The infarct area was white after TTC staining. As shown in Figure 7, the area of myocardial infarction in the ML355 group was significantly lower than that in the solvent group at 24 h after I/R.
心脏中炎症因子mRNA表达水平如图8所示，再灌注24h，相比于溶剂组，ML355组小鼠心脏组织中Tnf和Il6的表达水平显著下调。The expression level of inflammatory factor mRNA in the heart was as shown in Fig. 8. After reperfusion for 24 hours, the expression levels of Tnf and Il6 in the heart tissue of mice in the ML355 group were significantly down-regulated compared with the solvent group.
心脏中促凋亡基因Bax和抑制凋亡基因Bcl2的mRNA表达水平如图9所示，与溶剂组相比，ML355组小鼠心脏组织中Bcl2表达显著上调，而Bax表达水平明显低于溶剂组。The mRNA expression levels of the proapoptotic gene Bax and the apoptosis-inhibiting gene Bcl2 in the heart are shown in Figure 9. Compared with the solvent group, the expression of Bcl2 in the heart tissue of mice in the ML355 group was significantly up-regulated, while the expression level of Bax was significantly lower than that in the solvent group. .
上述结果说明ML355对缺血再灌注造成的心脏损伤、炎症、凋亡具有抑制作用。The above results indicate that ML355 has an inhibitory effect on cardiac damage, inflammation and apoptosis caused by ischemia-reperfusion.
2、ML355抑制肾脏缺血再灌注损伤2, ML355 inhibits renal ischemia-reperfusion injury
C57小鼠随机分为2组(Sham组、I/R 24h组)，每组14只。每组的其中7只 小鼠通过尾静脉注射给以3mg/kg的溶于溶剂中的ML355，另外7只给以溶剂。给药完成后对小鼠进行I/R手术，分别取Sham对照组以及术后24h I/R实验组小鼠血清，进行血清BUN(尿素氮)和Scr(血肌酐)检测，以评价肾脏损伤程度。C57 mice were randomly divided into 2 groups (Sham group, I/R 24h group), 14 in each group. Seven of the mice in each group were given 3 mg/kg of ML355 dissolved in a solvent by tail vein injection, and the other 7 were given a solvent. After the completion of the administration, the mice were subjected to I/R surgery, and the Sham control group and the serum of the I/R experimental group 24 h after surgery were taken for serum BUN (urea nitrogen) and Scr (serum creatinine) to evaluate renal damage. degree.
实验结果如图10A和10B所示，I/R术后血清中BUN及Scr含量较Sham组显著增加，当通过尾静脉给以ML355后，I/R术后24h血清中BUN及Scr含量低于未给ML355组，并出现统计学的显著性差异。上述结果说明ML355对缺血再灌注造成的肾脏损伤也具有抑制作用。The results of the experiment are shown in Figures 10A and 10B. The levels of BUN and Scr in the serum after I/R were significantly increased compared with the Sham group. When the ML355 was administered through the tail vein, the serum BUN and Scr levels were lower after 24 hours of I/R. The ML355 group was not given and a statistically significant difference occurred. The above results indicate that ML355 also has an inhibitory effect on renal damage caused by ischemia-reperfusion.
实施例4 不同ALOX在缺血肝脏组织中表达变化Example 4 Expression changes of different ALOX in ischemic liver tissue
C57小鼠随机分为2组，分别为Sham组及手术组，取缺血1h后手术组小鼠以及Sham组小鼠肝脏组织，Western blot及RT-RCR检测肝脏组织中ALOX12、ALOX5、ALOX15蛋白含量以及mRNA含量。其中WB所用一抗为：12-LO Antibody(C-5)(sc-365194；Santa Cruz),15-LO Antibody(B-7)(sc-133085；Santa Cruz),5-Lipoxygenase(C49G1)Rabbit mAb(#3289；CST)，二抗为：Peroxidase AffiniPure goat anti-rabbit-IgG(H+L)(#111-035-003；Jackson Laboratory)和goat anti-mouse-IgG(H+L)(#115-035-003；Jackson Laboratory)；RT-RCR所用引物序列如下：C57 mice were randomly divided into two groups: Sham group and operation group. Liver tissues of mice in operation group and Sham group were taken after ischemia for 1 hour. Western blot and RT-RCR were used to detect ALOX12, ALOX5 and ALOX15 proteins in liver tissue. Content and mRNA content. The primary antibody used for WB was: 12-LO Antibody (C-5) (sc-365194; Santa Cruz), 15-LO Antibody (B-7) (sc-133085; Santa Cruz), 5-Lipoxygenase (C49G1) Rabbit mAb (#3289; CST), secondary antibody: Peroxidase AffiniPure goat anti-rabbit-IgG (H+L) (#111-035-003; Jackson Laboratory) and goat anti-mouse-IgG (H+L) (# 115-035-003; Jackson Laboratory); primer sequences used in RT-RCR are as follows:
基因gene 正向引物Forward primer 反向引物Reverse primer
ALOX12ALOX12 TCCCTCAACCTAGTGCGTTTGTCCCTCAACCTAGTGCGTTTG GTTGCAGCTCCAGTTTCGCGTTGCAGCTCCAGTTTCGC
ALOX5ALOX5 AACGATCACCCACCTTCTGCAACGATCACCCACCTTCTGC TCGCAGATAAGCTGTTCCCGTCGCAGATAAGCTGTTCCCG
ALOX15ALOX15 GCTGCCCAATCCTAATCAGTCGCTGCCCAATCCTAATCAGTC TTCCTTATCCAAGGCAGCCAGTTCCTTATCCAAGGCAGCCAG
结果如图11A和11B所示，肝脏缺血1h后，肝脏组织中ALOX12基因mRNA含量相对于Sham组显著上升，约为Sham组的2.6倍，而ALOX5及ALOX15的mRNA含量相比于Sham组则上升不明显(图11A)。与RT-PCR结果相似，WB分析三种不同ALOX蛋白表达量，结果显示ALOX12蛋白在缺血1h后表达量相比于Sham组显著增加，而ALOX5及ALOX15蛋白表达量则增加不明显(图11B)。The results are shown in Figures 11A and 11B. After 1 h of hepatic ischemia, the mRNA level of ALOX12 mRNA in liver tissue increased significantly compared with Sham group, which was 2.6 times higher than that in Sham group, while the mRNA content of ALOX5 and ALOX15 was higher than that in Sham group. The rise is not obvious (Figure 11A). Similar to the results of RT-PCR, WB analyzed the expression levels of three different ALOX proteins. The results showed that the expression of ALOX12 protein was significantly increased after ischemia for 1 h compared with the Sham group, while the expression of ALOX5 and ALOX15 protein was not significantly increased (Fig. 11B). ).
上述RT-PCR及WB结果一致地表明了，在肝脏缺血后，肝脏组织中不同ALOX表达量变化间存在差异，且ALOX12表达量增加最明显。表明相比于ALOX的其他主要成员，肝脏组织缺血再灌注损伤和ALOX12之间关联更明显。The above RT-PCR and WB results consistently showed that there was a difference in the expression of different ALOX in liver tissue after hepatic ischemia, and the expression of ALOX12 was the most obvious. This indicates that the association between liver tissue ischemia-reperfusion injury and ALOX12 is more pronounced than other major members of ALOX.
实施例5 ALOX12过表达对H/R处理诱导的L02细胞损伤及炎症反应的影响Example 5 Effect of ALOX12 overexpression on L02 cell injury and inflammatory response induced by H/R treatment
L02细胞分为4组：GFP过表达对照组、ALOX12过表达对照组、GFP过表达H/R组、ALOX12过表达H/R组。对应质粒分别转染贴壁L02细胞(融合度约80％)，24h后进行H/R处理(缺氧1h，复氧6h)。质粒转染完成后提取细胞总蛋白，进行WB分析(3次独立重复实验，每次2个重复)，检测ALOX12的过表达情况。H/R处理完成后，检测培养基中LDH的释放量(每组6个重复)，以评价ALOX12过表达对H/R诱导的肝细胞损伤的影响；提取RNA进行RT-PCR分析(2次独立重复实验，每次3个技术重复)，检测炎症相关细胞因子及趋化因子mRNA含量变化，以评价ALOX12过表达对H/R诱导的肝细胞炎症反应的影响。以GFP过表达对照组LDH释放检测结果以及炎症相关因子mRNA含量为1，计算其余各组相比于该组的比值。L02 cells were divided into 4 groups: GFP overexpression control group, ALOX12 overexpression control group, GFP overexpressing H/R group, and ALOX12 overexpressing H/R group. The corresponding plasmids were transfected with adherent L02 cells (combination degree about 80%), and H/R treatment (anoxia 1 h, reoxygenation 6 h) was performed 24 hours later. After the plasmid transfection was completed, the total protein was extracted and subjected to WB analysis (3 independent replicates, 2 replicates each time) to detect the overexpression of ALOX12. After H/R treatment, the release of LDH in the culture medium (6 replicates per group) was examined to evaluate the effect of ALOX12 overexpression on H/R-induced hepatocyte injury; RNA was extracted for RT-PCR analysis (2 times) Independently repeat the experiment, each time 3 technical replicates), to detect changes in inflammation-related cytokines and chemokine mRNA levels to evaluate the effect of ALOX12 overexpression on H/R-induced hepatocyte inflammatory response. The LDH release test results and the inflammation-related factor mRNA content of the GFP overexpression control group were 1, and the ratios of the remaining groups compared to the group were calculated.
RT-RCR所用引物序列如下：The primer sequences used in RT-RCR are as follows:
基因gene 正向引物Forward primer 反向引物Reverse primer
Il6Il6 TCTGGATTCAATGAGGAGACTTGTCTGGATTCAATGAGGAGACTTG GTTGGGTCAGGGGTGGTTATGTTGGGTCAGGGGTGGTTAT
TnfαTnfα TACTCCCAGGTCCTCTTCAAGGTACTCCCAGGTCCTCTTCAAGG TTGATGGCAGAGAGGAGGTTGTTGATGGCAGAGAGGAGGTTG
Ccl2Ccl2 GTCTCTGCCGCCCTTCTGGTCTCTGCCGCCCTTCTG ACTTGCTGCTGGTGATTCTTCTACTTGCTGCTGGTGATTCTTCT
Cxcl10Cxcl10 GTGGCATTCAAGGAGTACCTCGTGGCATTCAAGGAGTACCTC TGATGGCCTTCGATTCTGGATTTGATGGCCTTCGATTCTGGATT
ALOX12过表达WB检测结果如图12所示，相比于GFP组，ALOX12过表达组蛋白条带显著增强，即L02细胞中ALOX12过表达显著。The results of ALOX12 overexpression WB assay are shown in Figure 12. Compared to the GFP group, the ALOX12 overexpressing histone band was significantly enhanced, ie, ALOX12 overexpression was significant in L02 cells.
LDH释放检测结果如图13所示，ALOX12过表达对照组LDH释放与GFP过表达对照组相比无显著差异，表明ALOX12的过表达对正常培养的L02细胞无影响。当进行H/R处理后，LDH的释放量显著增加，且ALOX12过表达组LDH的释放量的增加程度显著高于GFP组。这一结果表明，ALOX12过表达加重H/R处理引起的肝细胞损伤及肝细胞毒性。The results of LDH release assay are shown in Figure 13. There was no significant difference in LDH release between ALOX12 overexpressing control group and GFP overexpression control group, indicating that overexpression of ALOX12 had no effect on normal cultured L02 cells. When H/R treatment was performed, the release amount of LDH was significantly increased, and the release amount of LDH in the ALOX12 overexpression group was significantly higher than that in the GFP group. This result indicates that overexpression of ALOX12 aggravates hepatocyte injury and hepatotoxicity caused by H/R treatment.
炎症因子及趋化因子mRNA检测结果如图14所示，与LDH释放检测结果相同，ALOX12过表达对照组炎症因子Il-6、Tnf-α，趋化因子Ccl2、Cxcl10的mRNA含量与GFP过表达对照组相比无显著差异，表明ALOX12的过表达对正常培养的L02细胞炎症反应无影响。当进行H/R处理后，各因子mRNA含量显著增加，且 ALOX12组的增加量显著大于GFP组。这一结果表明，ALOX12过表达加重H/R处理引起的肝细胞炎症反应。The results of inflammatory factor and chemokine mRNA assay are shown in Figure 14. Compared with LDH release assay, ALOX12 overexpressed inflammatory factors Il-6, Tnf-α, chemokine Ccl2, Cxcl10 mRNA and GFP overexpression in control group. There was no significant difference between the control groups, indicating that the overexpression of ALOX12 had no effect on the inflammatory response of normal cultured L02 cells. When H/R treatment was performed, the mRNA content of each factor increased significantly, and the increase in the ALOX12 group was significantly greater than that in the GFP group. This result indicates that overexpression of ALOX12 aggravates the hepatocyte inflammatory response caused by H/R treatment.
实施例6 ALOX12敲低(shALOX12)对H/R处理后H9C2细胞活性的影响Example 6 Effect of ALOX12 knockdown (shALOX12) on H9C2 cell activity after H/R treatment
H9C2细胞分为4组：shRNA对照组、shALOX12对照组、shRNA H/R组、shALOX12H/R组。对应的重组慢病毒病毒液分别感染培养的H9C2细胞，24h后进行H/R处理(缺氧1h，复氧6h)。质粒转染完成后提取细胞mRNA，进行mRNA分析(3次独立重复实验)，检测ALOX12的敲低情况。H/R完成后检测细胞活性(每组6个重复)。以shRNA对照组检测结果为1，计算其余各组相比于该组的比值。H9C2 cells were divided into 4 groups: shRNA control group, shALOX12 control group, shRNA H/R group, and shALOX12H/R group. The corresponding recombinant lentiviral virus solution was infected with cultured H9C2 cells, and H/R treatment was performed 24 hours later (anoxia 1 h, reoxygenation 6 h). After the plasmid transfection was completed, the mRNA of the cells was extracted, and mRNA analysis was performed (three independent repeated experiments) to detect the knockdown of ALOX12. Cell viability was measured after completion of H/R (6 replicates per group). The results of the shRNA control group were 1, and the ratio of the remaining groups to the group was calculated.
ALOX12敲低mRNA检测结果如图15所示，相比于shRNA组，shALOX12组mRNA水平显著减弱，即H9C2细胞中ALOX12表达被敲低。The ALOX12 knockdown mRNA assay results are shown in Figure 15. Compared to the shRNA group, the shALOX12 group mRNA levels were significantly attenuated, i.e., ALOX12 expression was knocked down in H9C2 cells.
细胞活性检测结果如图16所示，shALOX12对照组细胞活性相比于shRNA对照组无显著差异。当对H/R的两组细胞进行H/R处理后，shRNA组细胞活性相比于对照组显著降低。而当ALOX12的表达被敲低后，shALOX12H/R组细胞活性的降低程度显著低于shRNA H/R组。这一结果表明ALOX12表达的降低可显著缓解H/R诱导的心肌细胞损伤，维持心肌细胞的活性。即ALOX12可促进心肌细胞损伤相关疾病的发生发展。The results of the cell activity assay are shown in Figure 16. There was no significant difference in the activity of the shALOX12 control group compared to the shRNA control group. When H/R treatment was performed on the two groups of H/R cells, the cell viability of the shRNA group was significantly lower than that of the control group. When the expression of ALOX12 was knocked down, the degree of cell viability in the shALOX12H/R group was significantly lower than that in the shRNA H/R group. This result indicates that the decrease in ALOX12 expression significantly attenuates H/R-induced cardiomyocyte injury and maintains cardiomyocyte activity. That is, ALOX12 can promote the development of diseases related to myocardial cell injury.

Claims (12)

  1. ALXO12抑制剂在制备治疗缺血再灌注损伤及相关疾病，炎症疾病，细胞死亡相关疾病的药物中的用途。The use of an ALXO12 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, and cell death-related diseases.
  2. 一种治疗缺血再灌注损伤及相关疾病，炎症疾病，或细胞死亡相关疾病的方法，其特征在于，给予有需要的患者含有ALOX12抑制剂的药物。A method for treating ischemia-reperfusion injury and related diseases, inflammatory diseases, or cell death-related diseases, which comprises administering a drug containing an ALOX12 inhibitor to a patient in need thereof.
  3. 如权利要求1所述的用途或权利要求2所述的方法，其特征在于，所述ALOX12抑制剂是式(I)所示的化合物、或其药学上可接受的盐、或其溶剂化物、或其代谢产物，所述式(I)化合物的结构如下：The use according to claim 1 or the method according to claim 2, wherein the ALOX12 inhibitor is a compound of the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, Or a metabolite thereof, the structure of the compound of formula (I) is as follows:
    Figure PCTCN2018101397-appb-100001
    Figure PCTCN2018101397-appb-100001
    其中，R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基； Wherein R 1 and R 2 are independently selected from the group consisting of H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, An aryl group, a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl , Br, hydroxy, C 1-6 alkoxy;
    R 3选自芳基、杂芳基，上述基团任选被一个或多个选自如下基团被取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、羟基、C 1-6烷氧基、C 1-6烷氧基羰基、环烷基、芳基、哌嗪基、哌啶基、吡啶基、吗啉基、吡咯烷基、吡唑烷基、咪唑烷基和硫代吗啉基；上述基团C 1-6烷基、C 2-6烯基、C 2-6炔基、C 1-6烷氧基、环烷基、芳基、哌嗪基、哌啶基、吡啶基、吗啉基、吡咯烷基、吡唑烷基、咪唑烷基、硫代吗啉基还可以被一个或多个C 1-6烷基、C 1-6烷氧基、或C 1-6烷氧基羰基所取代； R 3 is selected from aryl and heteroaryl, and the above group is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, hydroxy, C 1-6 alkoxy, C 1-6 alkoxycarbonyl, cycloalkyl, aryl, piperazinyl, piperidinyl, pyridyl, morpholinyl, pyrrole An alkyl group, a pyrazolidinyl group, an imidazolidinyl group, and a thiomorpholinyl group; the above group C 1-6 alkyl group, C 2-6 alkenyl group, C 2-6 alkynyl group, C 1-6 alkoxy group, Cycloalkyl, aryl, piperazinyl, piperidinyl, pyridyl, morpholinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, thiomorpholinyl can also be one or more C 1- Substituted by a 6 alkyl group, a C 1-6 alkoxy group, or a C 1-6 alkoxycarbonyl group;
    优选的，Preferably,
    R 3选自苯基、萘基、噻唑基、苯并噻唑基、苯并噁唑基、咪唑基、苯并咪唑基、噻吩基、苯并噻吩基、吡啶基、喹啉基、异喹啉基、噁唑基、呋喃基、 苯并呋喃基、吡咯基、吡唑基、吡嗪基、嘧啶基、三嗪基、吲哚基、嘌呤基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、羟基、C 1-6烷氧基、C 1-6烷氧基羰基、苯基、环烷基、哌嗪、哌啶、吡啶、吗啉、吡咯烷、吡唑烷、咪唑烷硫代吗啉、和C 1-6烷基氧基羰基哌啶基； R 3 is selected from the group consisting of phenyl, naphthyl, thiazolyl, benzothiazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thienyl, benzothienyl, pyridyl, quinolyl, isoquinoline a group, an oxazolyl group, a furyl group, a benzofuranyl group, a pyrrolyl group, a pyrazolyl group, a pyrazinyl group, a pyrimidinyl group, a triazinyl group, a fluorenyl group, a fluorenyl group, each of which is optionally one or more Substituted from the group: C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, hydroxy, C 1-6 alkoxy, C 1- 6 alkoxycarbonyl, phenyl, cycloalkyl, piperazine, piperidine, pyridine, morpholine, pyrrolidine, pyrazolidine, imidazolidine thiomorpholine, and C 1-6 alkyloxycarbonylpiperidine base;
    更优选，R 3选自苯基、萘基、噻唑基、苯并噻唑基、苯并噁唑基、苯并咪唑基、噻吩基、喹啉基、异喹啉基、吡啶基，上述基团任选被一个或多个选自如下取代基所取代：C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基羰基、F、Cl、Br、羟基、苯基、哌嗪基、哌啶基、吗啉基、C 1-6烷氧基羰基哌啶基； More preferably, R 3 is selected from the group consisting of phenyl, naphthyl, thiazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, thienyl, quinolyl, isoquinolyl, pyridyl, the above groups Optionally substituted with one or more substituents selected from C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxycarbonyl, F, Cl, Br, hydroxy, phenyl, Piperazinyl, piperidinyl, morpholinyl, C 1-6 alkoxycarbonylpiperidinyl;
    进一步优选，R 3选自噻唑基、2-苯并噻唑基、2-苯并噁唑基、2-苯并咪唑基、4-甲基-2-苯并噻唑基、噻吩基、4-甲基-2-噻唑基、5-甲基-2-噻唑基、4,5-二甲基-2-噻唑基、苯基、1-萘基、2-萘基、1,4-联苯基、3-哌嗪-苯基、4-哌啶-苯基、4-哌嗪-3-吡啶基、4-甲基-3-吡啶基、3-吡啶基、2-吡啶基、3-叔丁基-苯基、6-甲氧基-2-苯并噻唑基、6-氟-2-苯并噻唑基、4-苯基-2-噻唑基、3-吗啉-苯基、4-(N-叔丁氧基羰基)哌啶基-3-苯基、3-哌啶基-苯基、3-异丙基-苯基、3-喹啉基、8-喹啉基、8-异喹啉。 Further preferably, R 3 is selected from the group consisting of thiazolyl, 2-benzothiazolyl, 2-benzoxazolyl, 2-benzimidazolyl, 4-methyl-2-benzothiazolyl, thienyl, 4-methyl 2-thiazolyl, 5-methyl-2-thiazolyl, 4,5-dimethyl-2-thiazolyl, phenyl, 1-naphthyl, 2-naphthyl, 1,4-biphenyl , 3-piperazine-phenyl, 4-piperidine-phenyl, 4-piperazin-3-pyridyl, 4-methyl-3-pyridyl, 3-pyridyl, 2-pyridyl, 3-tert Butyl-phenyl, 6-methoxy-2-benzothiazolyl, 6-fluoro-2-benzothiazolyl, 4-phenyl-2-thiazolyl, 3-morpholine-phenyl, 4- (N-tert-Butoxycarbonyl)piperidinyl-3-phenyl, 3-piperidinyl-phenyl, 3-isopropyl-phenyl, 3-quinolyl, 8-quinolinyl, 8- Isoquinoline.
  4. 如权利要求3所述的用途或方法，所述式(I)化合物中，R 1和R 2独立地选自H、F、Cl、Br、C 1-6烷基、C 1-6烷氧基； The use or method according to claim 3, wherein in the compound of the formula (I), R 1 and R 2 are independently selected from the group consisting of H, F, Cl, Br, C 1-6 alkyl, C 1-6 alkoxylate. base;
    优选的，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、Cl；当R 1为H时、R 2选自Br和Cl。 Preferably, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, and Cl; when R 1 is H, R 2 is selected from Br and Cl.
  5. 如权利要求3所述的用途或方法，式(I)化合物为如下式(II)化合物，The use or method of claim 3, the compound of formula (I) is a compound of formula (II) below,
    Figure PCTCN2018101397-appb-100002
    Figure PCTCN2018101397-appb-100002
    其中，X选自O、S、NH或C；R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟 基、C 1-6烷氧基；优选地，R 1和R 2独立地选自H、卤素、羟基、C 1-6烷氧基、C 1-6烷基； Wherein X is selected from O, S, NH or C; and R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 Alkynyl, F, Cl, Br, amino, aryl, heteroaryl, each of which is optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl , C 2-6 alkynyl, F, Cl, Br, hydroxy, C 1-6 alkoxy; preferably, R 1 and R 2 are independently selected from H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl;
    R 4、R 5、R 6、R 7相同或不同，独立地选自H、卤素、C 1-6烷氧基或C 1-6烷基； R 4 , R 5 , R 6 , R 7 are the same or different and are independently selected from H, halogen, C 1-6 alkoxy or C 1-6 alkyl;
    优选的，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl。 Preferably, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
  6. 如权利要求3所述的用途或方法，式(I)化合物为如下式(III)化合物，The use or method of claim 3, the compound of formula (I) is a compound of formula (III) below,
    Figure PCTCN2018101397-appb-100003
    Figure PCTCN2018101397-appb-100003
    R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基；优选地，R 1和R 2独立地选自H、卤素、羟基、C 1-6烷氧基、C 1-6烷基； R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, aryl a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br a hydroxy group, a C 1-6 alkoxy group; preferably, R 1 and R 2 are independently selected from the group consisting of H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl;
    R 8、R 9独立地选自H、卤素、C 1-6烷氧基、C 1-6烷基、苯基、C 1-6烷基苯基、C 1-6烷氧基苯基、卤素取代的苯基。 R 8 and R 9 are independently selected from the group consisting of H, halogen, C 1-6 alkoxy, C 1-6 alkyl, phenyl, C 1-6 alkylphenyl, C 1-6 alkoxyphenyl, Halogen substituted phenyl.
    优选的，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl。 Preferably, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
  7. 如权利要求3所述的用途或方法，所述式(I)化合物为如下式(IV)化合物，The use or method of claim 3, wherein the compound of formula (I) is a compound of formula (IV):
    Figure PCTCN2018101397-appb-100004
    Figure PCTCN2018101397-appb-100004
    R 1和R 2独立地选自H、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基、F、Cl、Br、氨基、芳基、杂芳基，上述基团各自任选被一个或多个选自如下基团所取代：C 1-6烷基、C 2-6烯基、C 2-6炔基、F、Cl、Br、羟基、C 1-6烷氧基；优选地，R 1和R 2独立地选自H、卤素、羟基、C 1-6烷氧基、C 1-6烷基； R 1 and R 2 are independently selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br, amino, aryl a heteroaryl group, each of which is optionally substituted by one or more groups selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, F, Cl, Br a hydroxy group, a C 1-6 alkoxy group; preferably, R 1 and R 2 are independently selected from the group consisting of H, halogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl;
    R 10、R 11独立地选自H、卤素、C 1-6烷氧基、C 1-6烷基、任选取代的苯基、任选取代的哌啶基、任选取代的哌嗪基，所述取代基可为C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基羰基、卤素；优选的，R 10、R 11独立地选自H、叔丁基、异丙基、苯基、哌嗪基、4-哌啶基、4-叔丁基氧基羰基哌啶基。 R 10 and R 11 are independently selected from H, halogen, C 1-6 alkoxy, C 1-6 alkyl, optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted piperazinyl The substituent may be a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 alkoxycarbonyl group, or a halogen; preferably, R 10 and R 11 are independently selected from H, t-butyl group. , isopropyl, phenyl, piperazinyl, 4-piperidinyl, 4-tert-butyloxycarbonylpiperidinyl.
    优选的，当R 2为H时、R 1选自甲氧基、乙氧基、丙氧基、C1；当R 1为H时、R 2选自Br和Cl。 Preferably, when R 2 is H, R 1 is selected from the group consisting of methoxy, ethoxy, propoxy, C1; when R 1 is H, R 2 is selected from Br and Cl.
  8. 如权利要求3所述的用途或方法，所述式(I)化合物选自如下具体化合物：The use or method of claim 3, wherein the compound of formula (I) is selected from the group consisting of the following specific compounds:
    Figure PCTCN2018101397-appb-100005
    Figure PCTCN2018101397-appb-100005
    Figure PCTCN2018101397-appb-100006
    Figure PCTCN2018101397-appb-100006
    Figure PCTCN2018101397-appb-100007
    Figure PCTCN2018101397-appb-100007
    Figure PCTCN2018101397-appb-100008
    Figure PCTCN2018101397-appb-100008
    Figure PCTCN2018101397-appb-100009
    Figure PCTCN2018101397-appb-100009
    Figure PCTCN2018101397-appb-100010
    Figure PCTCN2018101397-appb-100010
    Figure PCTCN2018101397-appb-100011
    Figure PCTCN2018101397-appb-100011
    Figure PCTCN2018101397-appb-100012
    Figure PCTCN2018101397-appb-100012
    Figure PCTCN2018101397-appb-100013
    Figure PCTCN2018101397-appb-100013
  9. 如权利要求1-8任一项所述的用途或方法，所述缺血再灌注损伤及相关疾病为肝脏缺血再灌注损伤及相关疾病、心脏缺血再灌注损伤及相关疾病、肾脏缺血再灌注损伤及相关疾病；更优选为肝脏缺血再灌注损伤及相关疾病；The use or method according to any one of claims 1-8, wherein the ischemia-reperfusion injury and related diseases are hepatic ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia Reperfusion injury and related diseases; more preferably hepatic ischemia-reperfusion injury and related diseases;
    优选所述缺血再灌注损伤为肝脏缺血再灌注损伤、心脏缺血再灌注损伤、肾脏缺血再灌注损伤；优选为肝脏缺血再灌注损伤；Preferably, the ischemia-reperfusion injury is hepatic ischemia-reperfusion injury, cardiac ischemia-reperfusion injury, renal ischemia-reperfusion injury; preferably liver ischemia-reperfusion injury;
  10. 如权利要求1-8任一项所述的用途或方法，所述的炎症疾病为肝炎、心肌炎、心内膜炎、肾炎。The use or method of any of claims 1-8, wherein the inflammatory disease is hepatitis, myocarditis, endocarditis, nephritis.
  11. 如权利要求1-10任一项所述的用途或方法，所述药物还进一步包含药学上可接受的辅料。The use or method of any of claims 1-10, further comprising a pharmaceutically acceptable excipient.
  12. 如权利要求1-11任一项所述的用途或方法，所述药物是口服剂或注射给药的剂型；优选为片剂、胶囊、丸剂、粉剂、颗粒剂、悬浮剂、糖浆剂、注射液、粉针剂。The use or method according to any one of claims 1 to 11, which is a dosage form for oral administration or injection; preferably a tablet, a capsule, a pill, a powder, a granule, a suspension, a syrup, an injection Liquid, powder injection.
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