CN101664552B - Stabilization of hypoxia inducible factor (HIF) alpha - Google Patents

Abstract

The present invention relates to methods of stabilizing the alpha subunit of hypoxia inducible factor (HIF). The invention further relates to methods of preventing, pretreating, or treating conditions associated with HIF, including ischemic and hypoxic conditions. Compounds for use in these methods are also provided.

Description

The stabilisation of hypoxia inducible factor (HIF) α

The application is to be dividing an application of December in 2002 6 days, application number are 02824098.7, denomination of invention is " stabilisation of hypoxia inducible factor (HIF) α " application for a patent for invention the applying date.

The present invention requires to be registered in the calendar year 2001 December U.S. Provisional Patent Application of 6 days 60/337082, be registered in the U.S. Provisional Patent Application 60/359683 on February 25th, 2002, be registered in the U.S. Provisional Patent Application 60/349659 on January 16th, 2002 and be registered in the U.S. Provisional Patent Application 60/386488 on June 5th, 2002, and the full content of every patent application draws for reference at this.

Invention field

The present invention relates to the stabilization method of the α subunit of hypoxia inducible factor (HIF), also relate to the compound that can be used for these methods.

Background of invention

The early reaction of tissue hypoxia is to produce hypoxia inducible factor (HIF), described HIF is a kind of alkaline helix-loop-helix (bHLH) PAS (Per/Arnt/Sim) activating transcription factor, the variation that it can be expressed for the variation buffering gene of oxygen concentration in cell.HIF is the heterodimer of the β subunit (HIF β) that comprises the α subunit (HIF α) of regulating oxygen and expression structure, also referred to as aromatic hydrocarbons, is subject to part nuclear translocation (ARNT).In normal oxygen (normoxic) cell, HIF α subunit fast degradation under a kind of effect of mechanism, this mechanism relates to the ubiquitin (ubiquitination) of von Hippel-Lindau tumor inhibitor (pVHL) E3 ligase complex.Under hypoxia condition, HIF α is not degraded, active HIF α/β complex is enriched in nucleus, excites the expression of several genes that comprise glycolytic ferment, glucose transporter (GLUT)-1, erythropoietin (EPO) and vascular endothelial cell growth factor (VEGF).(Jiang etc. (1996) J.Biol.Chem.271:17771-17778; Iliopoulus etc. (1996) Proc.Natl.Acad.Sci.USA 93:10595-10599; Maxwell etc. (1999) Nature 399:271-275; Sutter etc. (2000) Proc.Natl.Acad.Sci.USA 97:4748-4753; Cockman etc. (2000) J.Biol.Chem.275:25733-25741; Tanimoto etc. (2000) EMBO J 19:4298-4309.)

In most cells, the content of HIF α albumen can raise because of hypoxia, if animal anemia or hypoxia will produce HIF α in its body.Once hypoxia, the HIF alpha content will rise within a few hours, under the Constant hypoxia condition, will return to baseline.Relate to HIF in countless cellular activities and growth course, comprise that cell proliferation, angiogenesis and cell cycle stop.The cardiac muscle severe ischemic is also relevant to HIF α with early stage infarction, Arterial Hypertention and inflammation.Although had the people that HIF α and growth and metastasis of tumours are connected, do not had ample evidence to show that HIF directly occurs relevant with tumor.Research shows, makes the hypoxia preconditioned method of destination organization short-term hypoxia can protect cardiac muscle and brain to avoid hypoxia-ischemia injury.Stabilisation and the ischemia of HIF α are closely related, can produce by the preconditioned method.(Wang and Semenza (1993) Proc.Natl.Acad.Sci.USA 90:4304-4308; Stroka etc. (2001) FASEB J 15:2445-2453; Semenza etc. (1997) Kidney Int 51:553-555; Carmeliet etc. (1998) Nature 394:485-490; Zhong etc. (1999) Cancer Res.59:5830-5835; Lee etc. (2000) N Engl J Med 343:148-149; Sharp etc. (2000) J Cereb Blood Flow Metab20:1011-1032; Semenza etc. (2000) Adv Exp Med Biol 475:123-130; Thornton etc. (2000) Biochem J 350:307-312; Deindl and Schaper (1998) Mol Cell Biochem 186:43-51; Bergeron etc. (2000) Ann Neurol 48:285-296)

Some research worker have been studied the mechanism of action between HIF α and pVHL.Think at first in HIF-1 α from being enough to pass to protein chimera structure according to the oxygen unstability according to oxygen degraded territory (ODD) residue 401 to 603.The research discovery, the degraded that depends on pVHL need relate to the part ODD zone from 526 to 652.In addition, in the reserve area in HIF α homologous protein (the residue 556-574 in HIF-1 α), P ₅₆₄YI sports aspartic acid or K ₅₃₂Sport sugared propylhomoserin and make whole section HIF α albumen there is stability under normal oxygen condition, and can resist the degraded that pVHL regulates.(Huang etc. (1998) Proc.Natl.Acad.Sci.USA 95:7987-7992; Tanimoto etc. (2000) EMBO J 19:4298-4309)

The HIF alpha content can raise under many factor effects that are similar to hypoxia, comprise the ferrum chimera as desferrioxamine (DFO) and divalent metal salt as CoCl ₂.Angiotensin II, thrombin and PDGF improve the content of HIF α under normal oxygen condition by the mechanism that relates to active oxygen species.Also have report to propose, HIF α is regulated by phosphorylation, and this approach relates to phosphatidylinositol-3 kinase (PI3K), the hepatocyte growth factor of oxidated nitrogen activation or the protein kinase that activated by mitogen.The glycogen synthase kinase is the downstream target of PI3K, directly makes HIF α ODD territory that phosphorylation occurs.(Richard etc. (2000) J Biol Chem 275:26765-26771; Sandau etc. (2000) Biochen Biophys Res Commun 278:263-267; Tacchini etc. (2001) Carcinogenesis22:1363-1371; Sodhi etc. (2001) Biochem Biophys Res Commun 287:292-300)

When impaired lung function or blood flow decline, just there will be the hypoxia phenomenon---the state that a kind of oxygen-supplying amount reduces.With the blood flow corresponding ischemia phenomenon that descends, can be caused by tremulous pulse or vein obstruction, as blood clot (thrombosis) or external recycled material (embolus), or aberrant angiogenesis, as arteriosclerosis.Blood flow descends and can break out and the persistent period short (acute ischemia), also may slowly show effect and lasting long period or outbreak (chronic ischemia) repeatedly.Acute ischemia is usually relevant with local irreversible tissue necrosis (blocking), and the common and of short duration hypoxia of chronic ischemia tissue injury is relevant.But, if perfusion descends the persistent period oversize or too severe, chronic ischemia also may with block relevant.Block and usually appear in spleen, kidney, lung, brain and heart, produce such as intestinal obstruction, pulmonary block, the disease such as Ischemic Apoplexy and cardiac muscle block.

Time and the order of severity and the length of patient's time-to-live that ischemia is lasting is depended in the abnormal pathological change of ischemia.Just there will be tissue necrosis in initial 24 hours of blocking, near meeting in the visible tissue of region of obstruction, serious inflammatory reaction is occurring, leukocyte can move to the slough zone simultaneously.In several days subsequently, have a liking for the cell that region of obstruction is decomposed and eliminates in bacterium effect meeting gradually, in generation, is with collagen or colloid crust.

Low perfusion state in organ or block and tend to involve other organs.For example, the pulmonary's ischemia caused by pulmonary infarction etc. not only affects lung, and makes heart and other organs (as brain) under hypoxia stress.Cardiac muscle blocks the coronary occlusion, coronary arterial wall spasm or the Cardiovirus that usually relate to thrombosis and cause to be infected, and can cause congestive heart failure and systemic hypotension.If heart stops normal operation for a long time because continuing hypotension, can cause the second complication, as the completeness Ischemic Encephalopathy.The most general situation of cerebrum ischemia is to be caused by the angiemphraxis occurred because of arteriosclerosis, and its order of severity can gently arrive transient cerebral ischemia (TIA), heavily arrives cerebral infarction or apoplexy.Although the symptom of TIA is temporary transient, and can recover, TIA easily recurs, and usually causes apoplexy.

Obstructive arterial disease comprises coronary artery disease, can cause that cardiac muscle blocks and peripheral arterial disease, can affect abdominal aorta, it mainly props up tremulous pulse and lower limb tremulous pulse.Peripheral arterial disease comprises Buerger disease, Raynaud disease and acrocyanosis.Although peripheral arterial disease causes by arteriosclerosis usually, other causes also comprise diabetes etc.The complication of peripheral arterial disease comprises serious shank spasm, angor, arrhythmia, heart failure, heart disease, apoplexy and renal failure.

Ischemia and hypoxia problem are the key factors that affects prevalence and mortality rate.Cardiovascular disease at least causes 1,500 ten thousand people's death every year, accounts for 30% of global death toll.In various cardiovascular disease, ischemic heart desease and cerebrovascular disease cause dead number to account for 17% of sum.The serious cardiac muscle of non-lethal that is seen in report every year blocks case 1,300,000, in about 100,000 people, has 600 people to suffer from this type of disease.In addition, estimate at 5,000,000 Americans every year and be subject to the phlebothrombosis puzzlement, wherein approximately 600,000 people finally suffer from pulmonary infarction.Approximately 1/3 Pulmonary Embolism Patients is finally dead, makes pulmonary infarction become the U.S. and causes dead the third-largest main cause.

At present, the treatment of ischemic and hypoxia disease concentrated on relief of symptoms and treatment inducement disease.The treatment of for example cardiac muscle being blocked comprises uses nitroglycerine and analgesic to control slight illness, alleviates cardiac load.Stablize the state of an illness with other drug, as digoxin, diuretic, amrinone, beta blocker, lipid lowering agent and angiotensin converting enzyme inhibitor, be the tissue injury that directly causes for ischemia and hypoxia but there is no a kind of Therapeutic Method simultaneously.

Because there is defect in current Therapeutic Method, still need effectively to treat the method for the disease that ischemia and hypoxia cause, as obstructive arterial disease, angina pectoris, intestinal obstruction, lung block, cerebral ischemia and cardiac muscle block.In addition, also need effectively to prevent ischemia to cause the method for tissue injury, for instance, the cause of described ischemia has arteriosclerosis, diabetes and pulmonary disease, as pulmonary infarction etc.Generally speaking, this area needs to be used for stablizing HIF, treatment and prevention HIF disease, comprises method and the compound of the disease that ischemia, hypoxia cause.

Summary of the invention

Presented here is the method for the α subunit (HIF α) of stablizing hypoxia inducible factor.It is interior or external that these methods can be used for organism.

Present invention relates in general to stablize the method for the α subunit of hypoxia inducible factor (HIF).In one embodiment, the method for stablizing the α subunit (HIF α) of HIF comprises that providing a kind of to treatment target can suppress the compound that hydroxylating occurs HIF α.In some embodiments of the present invention, HIF α is selected from HIF-1 α, HIF-2 α, HIF-3 α and their any fragment.In another embodiment, described method comprises to treatment target provides a kind of compound that can suppress 2-oxoglutaric acid dioxygenase activity.In various embodiments, described 2-oxoglutaric acid dioxygenase is selected from EGLN1, EGLN2, EGLN3, procollagen prolyl 4-hydroxyl enzyme, procollagen prolyl 3-hydroxyl enzyme, procollagen lysyl hydroxyl enzyme, PHD4, FIH-1, their any fragment or subunit.

Some ad hoc approach that the present invention stablizes HIF α comprise inhibition HIF prolyl hydroxylase activity.In other embodiments, the HIF prolyl hydroxylase is selected from EGLN1, EGLN2, EGLN3 and their any fragment or subunit.

One aspect of the present invention provides the method for the stable interior HIF of giving birth to α.Therefore, in a specific implementations, HIF α is born on treatment target in being.Embodiments of the present invention comprise the method for a kind of so stable HIF α, and the compound of stablizing HIF α is provided in the treatment target body.Treatment target comprises animal, is preferably mammal, more is preferably the people.This has also expected the method for treated in vitro.In this method, treatment target can be cell, tissue or organ etc.In some embodiments, treatment target can be intrasystem cell, tissue or organs such as kidney, heart, liver, lung, hemopoietic system, the intestines and stomach, nervous system or skeletal musculature.

The present invention also provides the method for the treatment of, prevention or the pretreat disease relevant with HIF.Especially, the invention provides the method for a kind for the treatment of, prevention or the pretreat disease relevant with HIF, the method comprises stablizes HIF α.Particularly, the present invention is directed to patient's disease relevant with HIF, a kind for the treatment of, prevention or pretreat/preconditioned method is provided, the method comprises stablizes HIF α.Minute and opinion, the disease relevant with HIF comprises and ischemia or the relevant disease of hypoxia.A preferred aspect of the present invention comprises to treatment target takes the compound that can stablize HIF α.

In various embodiments, the prodrug that described compound is selected from heterocyclic carboxamide, phenanthroline, hydroxamic acid and physiologically active salt and is made by them.In specific implementations, this compound is heterocyclic carboxamide, is selected from pyridine carboxamides, quinolinecarboxamideas, isoquinolin carboxylic acid amides, cinnoline carboxylic acid amides and B-carboline carboxylic acid amides.In a preferred embodiment of the present invention, described compound provides with the form of oral agents.In another preferred implementation, described compound provides in the mode of skin transdermal agent.

In the present invention, stablize in a kind of method of HIF α, described compound is stablized HIF α by least one amino acid residue generation hydroxylating in special inhibition HIF α.On the other hand, amino acid residue is selected from proline and agedoite.

The method for the treatment of, prevention or the pretreat disease relevant with HIF is provided, and described method comprises inhibition 2-oxoglutaric acid dioxygenase activity, and the present invention also comprises the method that wherein said HIF disease is relevant with ischemia or hypoxia.One aspect of the present invention provides the method for a kind for the treatment of, prevention or the pretreat disease relevant with HIF, and the method comprises to treatment target provides the compound that can suppress 2-oxoglutaric acid dioxygenase activity.

In a preferred implementation, the present invention is directed to patient's disease relevant with HIF, a kind for the treatment of, prevention or pretreat/preconditioned method is provided, the method comprises the activity that suppresses the HIF prolyl hydroxylase.Equally, the described disease relevant with HIF comprises the disease relevant with hypoxia or ischemia etc.In a specific implementations, described method comprises to treatment target provides the compound that can suppress HIF prolyl hydroxylase activity.

In another embodiment, the method also comprises and takes the second compound.In specific implementations, described the second compound can suppress the activity of 2-oxoglutaric acid dioxygenase, perhaps aforesaid compound and the second compound can suppress the activity of different 2-oxoglutaric acid dioxygenases, or the second compound is selected from ACE inhibitor (ACEI), angiotensin II receptor blocking agent (ARB), diuretic, digoxin, statine or carnitine etc.

In the specific embodiment, the disease relevant with HIF comprises pulmonary disease (such as pulmonary infarction etc.), heart disease (such as myocardial infarction, congestive heart failure etc.), sacred disease etc.Therefore the present invention clearly proposes to be used for the method for the treatment of, prevention or the pretreat/preconditioned ischemic diseases relevant with HIF, no matter is acute or temporary or chronic disease.The acute ischemia situation comprises with surgical operation, organ transplantation, block the relevant situations such as (blocking such as cerebral infarction, intestinal obstruction, myocardial infarction, lung etc.), wound, damage or wound.The chronic ischemia situation comprises hypertension, diabetes, closed arterial disease, chronic venous insufficiency of function, Raynaud's disease (Raynaud ' sdisease), sclerosis, congestive heart failure, systemic sclerosis etc.

The present invention has considered the method for preconditioned or pretreat especially.In one embodiment, the invention provides before the situation with the HIF disease association is as appearance such as ischemias, or, before the disease progression relevant to HIF, carry out the method for pretreat or preconditioned by stablizing HIF α.Ischemia can be caused by acute events, as surgical operation (such as angiopoiesis, organ transplantation etc.) and associated treatment process as implemented anesthesia etc.In addition, in chronic situation, the method of pretreat or preconditioned is applicable to the situation that treatment target suffers from the HIF disease that can predict degradating trend, such as short time ischemia or angina pectoris, early stage apoplexy and myocardial infarction etc., purpose is the deterioration degree that prevents the deterioration of HIF disease or alleviate the HIF disease.In a specific implementations, can provide a kind of compound to treatment target, to increase the ischemic preconditioning reason factor, as EPO etc.

Here proposed especially to increase the method for the expression of various HIF correlation factors.On the one hand, the invention provides a kind of method that patient vessel of increasing generates the expression of the factor, the method comprises stablizes HIF α.On the other hand, the invention provides a kind of method of expression of the patient's of increasing glycolysis factor, the method comprises stablizes HIF α.Again on the one hand, the invention provides the method for the expression that increases patient's factor relevant with response to oxidative stress, the method comprises stablizes HIF α.The invention allows for a kind of patient for the treatment of disease relevant with ischemical reperfusion injury, the method comprises stablizes HIF α.

The method of differentiating the compound that can stablize HIF α also is provided here.For example, the invention provides a kind of a kind of like this method of differentiating the compound that can stablize HIF α, it comprises: (a) for the patient or from patient's sample, provide target compound; (b) measure the HIF alpha content in patient or sample; (c) HIF alpha content and the standard content in patient or sample relatively, if in patient or sample, the HIF alpha content increases, show that this compound can stablize HIF α.

On the other hand, the tissue injury that available the inventive method prevention is caused by the HIF relevant disease, include, but is not limited to ischemia and hypoxia disease.In one embodiment, the state of an illness of diagnosis in advance, such as hypertension, diabetes, closed arterial disease, chronic venous insufficiency of function, Raynaud's disease, sclerosis, congestive heart failure, systemic sclerosis etc. are depended in treatment.

On the other hand, the inventive method can be used as the pretreat method, is used for reducing or preventing the tissue injury caused by the HIF disease, includes, but is not limited to ischemia and hypoxia disease.Whether in one embodiment, need pretreat to depend on ischemia diseases in patient's medical history, as the recurrence period of myocardial infarction or temporary ischemia, or the patient has the symptom of continuous ischemia, such as angina pectoris etc.In another embodiment, whether need pretreat to depend on to express possibility the physical parameter of ischemia or hypoxia, for example individual through general anesthesia or interim in the situation of high latitude work.In another embodiment, the inventive method can be used in organ transplantation, be used for organ donor is carried out to pretreatment, and at organ armour from shifting out in body but before being transplanted to the receiver.

On the other hand, the invention provides the compound of stablizing HIF α and by described compound prevention, pretreat or quality HIF disease as mentioned above.In one embodiment, the compound or pharmaceutically acceptable salt thereof of effective dose can be separately or with grant the treatment target of suffering from the HIF disease together with pharmaceutical excipient.In a specific embodiment, once diagnose out severe ischemic, just should use immediately described compound.In another embodiment, compound is granted the patient in the process of suffering from the chronic ischemia disease.In another specific embodiment, ischemia causes by temporary transient or severe trauma, damage or wound, for example spinal cord injury.In a specific embodiment, diagnosing patient go out pulmonary disease as COPD etc. after, as required described compound is granted to the patient.

Described compound can be granted the patient as chronic disease for susceptible disease on the one hand, or the tissue injury that is used for reduction or prevents the HIF disease to cause as the pretreat medicine.Particularly, described compound can have been granted ischemia diseases, the patient of the recurrence medical history of myocardial infarction or temporary ischemia for example, or suffer from the symptom of continuous ischemia, such as the patient of angina pectoris etc.In another embodiment, described compound use the physical parameter that depends on express possibility ischemia or hypoxia, for example individual through general anesthesia or interim in the situation of high latitude work.In another embodiment, described compound can be used in organ transplantation, be used for organ donor is carried out to pretreatment, and at organ armour from shifting out in body but before being transplanted to the receiver.

On the one hand, the compounds of this invention is stablized HIF α by amino acid residue generation hydroxylating in concrete inhibition HIF α albumen.In one embodiment, described reagent suppresses the hydroxylating of HIF α proline residue.In a specific embodiment, described reagent suppresses HIF-1 α P ₅₆₄The hydroxylating of the proline of the same clan in residue or another HIF αisomer.In another embodiment, described reagent suppresses HIF-1 α P ₄₀₂The hydroxylating of the proline of the same clan in residue or another HIF αisomer.In another embodiment, described compound can additionally suppress the hydroxylating of HIF α asparagine residue.In a specific embodiment, described reagent suppresses HIF-1 α N ₈₀₃The hydroxylating of the asparagine residue of the same clan in residue or another HIF αisomer.

In specific implementations, the inventive method compound used therefor is selected from compound shown in formula (I)

Wherein,

A is 1,2-arlydene, 1,3-arlydene, Isosorbide-5-Nitrae-arlydene; Or (C ₁-C ₄)-alkylene (being optionally one or more halogen atoms, cyano group, nitro, trifluoromethyl replacement), (C ₁-C ₆)-alkyl, (C ₁-C ₆)-hydroxyalkyl, (C ₁-C ₆)-alkoxyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-halogen _g, (C ₁-C ₆)-fluoroalkyl, (C ₁-C ₈The inferior oxyl of)-fluoro, (C ₁-C ₈The inferior alkene oxygen of)-fluoro base ,-OCF ₂Cl ,-O-CF ₂-CHFCl; (C ₁-C ₆)-alkyl thiol, (C ₁-C ₆)-alkyl sulfinyl, (C ₁-C ₆)-alkyl sulfide acyl group, (C ₁-C ₆)-alkyl-carbonyl, (C ₁-C ₆)-alkoxy carbonyl, carbamoyl, N-(C ₁-C ₄)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₆)-alkyl carbonyl oxy, (C ₃-C ₈)-cycloalkyl, phenyl, benzyl, phenoxy group, phenoxy group, anilino-, methylphenylamine base, phenyl sulfydryl, phenyl sulfonyl, phenyl sulfinyl, sulfamoyl, N-(C ₁-C ₄)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₄)-alkylsulfamoyl group replaces; Be perhaps (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₁)-aralkoxy, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl (have 1-5 identical or different substituent group in aryl moiety, described substituent group is selected from halogen, cyano group, nitro, trifluoromethyl), (C ₁-C ₆)-alkyl, (C ₁-C ₆)-alkoxyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-halogen _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₆)-alkyl thiol, (C ₁-C ₆)-alkyl sulfinyl, (C ₁-C ₆)-alkyl sulfide acyl group, (C ₁-C ₆)-alkyl-carbonyl, carbamoyl, N-(C ₁-C ₄)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₆)-alkyl-carbonyl oxygen base, (C ₃-C ₈)-cycloalkyl, sulfamoyl, N-(C ₁-C ₄)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₄)-alkylsulfamoyl group replaces; Perhaps A is CR ⁵R ⁶, R ⁵And R ⁶Independently be selected from separately hydrogen, (C ₁-C ₆)-alkyl, (C ₃-C ₇)-cycloalkyl, aryl, or the substituent group of the alpha-carbon atom of alpha amino acid, wherein aminoacid is natural L aminoacid or its D type isomer.

B is-COH ₂,-NH ₂,-NHSO ₂CF ₃, tetrazole radical, imidazole radicals, the different azoles base of 3-hydroxyl ,-CONHCOR " ' ,-CONHSOR " ', wherein R " ' be aryl, heteroaryl, (C ₃-C ₇)-cycloalkyl, (C ₁-C ₄)-alkyl, optional quilt (C ₁-C ₄)-alkylthio, (C ₁-C ₄)-sulfinyl, (C ₁-C ₄)-sulfonyl, CF3, Cl, Br, F, I, NO2 ,-COOH, (C ₂-C ₅)-alkoxy carbonyl, NH ₂, list-(C ₁-C ₄-alkyl)-amino, two-(C ₁-C ₄-alkyl)-amino or (C ₁-C ₄)-perfluoroalkyl replaces; Perhaps B is CO ₂-G carboxyl, wherein G is the group in pure G-OH, G is selected from (C ₁-C ₂₀)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₂-C ₂₀)-thiazolinyl, (C ₃-C ₈)-cycloalkenyl group, retinyl, (C ₂-C ₂₀)-alkynyl, (C ₄-C ₂₀)-alkapolyenyl (alkenynyl), wherein thiazolinyl, cycloalkenyl group, alkynyl and alkapolyenyl comprise one or more multikeys; (C ₆-C ₁₆)-isocyclic aryl, (C ₇-C ₁₆)-aralkyl, heteroaryl or heteroarylalkyl, wherein the heteroaryl in heteroarylalkyl or assorted virtue part comprise 5-6 annular atoms; The defined group of G can be one or more other groups and replaces, and these groups comprise hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₅-C ₈)-cycloalkenyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₂-C ₁₂)-thiazolinyl, (C ₂-C ₁₂)-alkynyl, (C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₁-C ₈)-hydroxyalkyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, cinnamoyl, (C ₂-C ₁₂)-alkenyl carbonyl, (C ₂-C ₁₂)-alkynyl carbonyl, (C ₁-C ₁₂)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl-carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cyclo alkoxy carbonyl, (C ₂-C ₁₂)-allyloxycarbonyl, (C ₂-C ₁₂)-alkynyloxy group carbonyl, acyloxy, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₆-C ₁₆)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆₎-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, (C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl, (C ₇-C ₁₂)-aryl sulfonyl, sulfamoyl, N-(C ₁-C ₁₀)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₁₀)-alkylsulfamoyl group, (C ₃-C ₈)-cycloalkyl sulfamoyl, N-(C ₆-C ₁₂)-alkylsulfamoyl group, N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-ammonia aryl sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, (C ₁-C ₁₀)-amino-alkyl sulfinyl, N-((C ₁-C ₁₀)-alkyl)-(C ₁-C ₁₀)-amino-alkyl sulfinyl, (C ₇-C ₁₆)-aralkyl sulfonamido or N-((C ₁-C ₁₀)-alkyl)-(C ₇-C ₁₆)-aralkyl sulfonamido; Aryl or can be 1-5 identical or different group containing the aryl in aromatic yl group and replace wherein, they comprise hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₁-C ₈)-hydroxyalkyl, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, (C ₁-C ₁₂)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxy carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, (C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₃-C ₈)-cycloalkyl carbonyl oxygen base, (C ₆-C ₁₂)-aryl-carbonyl oxygen, (C ₇-C ₁₆)-aralkyl carbonyl oxygen base, cinnamoyloxy group, (C ₂-C ₁₂)-thiazolinyl carbonyl oxygen base, (C ₂-C ₁₂)-alkynyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N-(C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl or (C ₇-C ₁₂)-aryl sulfonyl;

X is O or S;

Q is O, S, NR ' or chemical bond;

Wherein, if Q is chemical bond, R ⁴Halogen, nitrile or trifluoromethyl;

Perhaps, if Q is O, S, NR ', R ⁴Hydrogen, (C ₁-C ₁₀)-alkyl, (C ₂-C ₁₀)-thiazolinyl, (C ₂-C ₁₀)-alkynyl, wherein alkenyl or alkynyl comprises one or more C-C multikeys; There is chemical formula-O-[CH ₂] _x-C _fH _(2f+1-g)-F _gNot replacement fluoroalkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₁-C ₆)-alkoxyl-(C ₁-C ₄)-alkoxyl-(C ₁-C ₄)-alkyl, aryl, heteroaryl, (C ₇-C ₁₁)-aralkyl or there is the group of chemical formula shown in formula (Z)

-[CH ₂] _v-[O] _w-[CH ₂] _t-E (Z)

Wherein, E is heteroaryl, (C ₃-C ₈)-cycloalkyl or there is the phenyl of chemical formula shown in formula F

V is 0-6,

W is 0 or 1,

T is 0-3, and R ⁷, R ⁸, R ⁹, R ¹⁰And R ¹¹Can be identical or different, can be hydrogen, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₁-C ₆)-aralkyl ,-O-[CH ₂] _x-C _fH _(f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₆)-alkyl thiol, (C ₁-C ₆)-hydroxy alkyl, (C ₁-C ₆)-alkoxyl-(C ₁-C ₆)-alkoxyl, (C ₁-C ₆)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₁-C ₆)-alkyl sulfinyl, (C ₁-C ₆)-alkyl sulfide acyl group, (C ₁-C ₆)-alkyl-carbonyl, (C ₁-C ₈)-alkoxy carbonyl, carbamoyl, N-(C ₁-C ₈)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₈)-alkyl-carbamoyl or (C ₇-C ₁₁)-aryl-alkyl amino formoxyl, optionally replaced by following group, i.e. fluorine, chlorine, bromine, trifluoromethyl, (C ₁-C ₆)-alkoxyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₆)-alkyl carbonyl oxy, phenyl, benzyl, phenoxy group, benzyloxy, NR ^YR ^Z, R wherein ^YAnd R ^ZIndependently be selected from hydrogen, (C ₁-C ₁₂)-alkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₃-C ₁₀)-cycloalkyl, (C ₃-C ₁₂)-thiazolinyl, (C ₃-C ₁₂)-alkynyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl, (C ₁-C ₁₂)-alkoxyl, (C ₇-C ₁₂)-aryloxy group, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl; Perhaps R ^YAnd R ^ZBe all-[CH ₂] _h, CH wherein ₂Can be O, S, N-(C ₁-C ₄)-alkyl-carbonyl imino group or N-(C ₁-C ₄)-alkoxy carbonyl imino group; Phenyl sulfydryl, benzene sulfonyl, benzene sulfinyl, phenylamino sulfonyl, N-(C ₁-C ₈)-alkylsulfamoyl group or N, N-bis--(C ₁-C ₈)-alkylsulfamoyl group; Perhaps R ⁷And R ⁸, R ⁸And R ⁹, R ⁹And R ¹⁰Or R ¹⁰And R ¹¹All be selected from-[CH ₂] _nOr-chain of CH=CH-CH=CH-, wherein CH ₂Optional is O, S, SO, SO ₂Or NR ^YReplace; N is 3,4 or 5; If E is heteroaryl, described group can be R containing 1-3 ⁷-R ¹¹Defined substituent group, if or E be cycloalkyl, described group can containing one be R ⁷-R ¹¹Defined substituent group;

Perhaps when Q is NR ', R ⁴R ", wherein R ' and R " can be identical or different, can be specifically hydrogen, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl, (C ₁-C ₈)-alkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₁-C ₁₀)-alkyl-carbonyl, the optional (C replaced ₇-C ₁₆)-aromatic alkyl carbonyl or the optional (C replaced ₆-C ₁₂)-aryl carbonyl; Perhaps R ' and R " be-[CH ₂] _h, CH wherein ₂For O, S, N-acylimino or N-(C ₁-C ₁₀)-alkoxy carbonyl imino group, h is 3-7.

Y is N or CR ³

R ¹, R ²And R ³Can be identical or different, be specially hydrogen, hydroxyl, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₂₀)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyloxy, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₁₂)-alkoxyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₁₂)-alkoxyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₈)-alkyl-(C ₁-C ₆)-alkoxyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkoxyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₇-C ₁₆)-arylalkenyl, (C ₇-C ₁₆)-sweet-smelling alkynyl, (C ₂-C ₂₀)-thiazolinyl, (C ₂-C ₂₀)-alkynyl, (C ₁-C ₂₀)-alkoxyl, (C ₂-C ₂₀)-alkene oxygen base, (C ₂-C ₂₀)-alkynyloxy group, look yellow oxygen base, (C ₁-C ₂₀)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₆)-alkoxyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₆)-alkoxyl, (C ₁-C ₁₆)-hydroxyalkyl, (C ₆-C ₁₅)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₂-C ₂₀)-alkene oxygen base-(C ₁-C ₆)-alkyl, (C ₂-C ₂₀)-alkynyloxy group-(C ₁-C ₆)-alkyl, look yellow oxygen base-(C ₁-C ₆)-alkyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₂₀)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, cinnamoyl, (C ₂-C ₂₀)-alkenyl carbonyl, (C ₂-C ₂₀)-alkynyl carbonyl, (C ₁-C ₂₀)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxy carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cyclo alkoxy carbonyl, (C ₂-C ₂₀)-allyloxycarbonyl, look yellow oxygen base carbonyl, (C ₂-C ₂₀)-alkynyloxy group carbonyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₆)-alkoxy carbonyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₆)-alkoxy carbonyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkoxy carbonyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₆)-alkoxy carbonyl, (C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₃-C ₈)-cycloalkyl carbonyl oxygen base, (C ₆-C ₁₂)-aryl-carbonyl oxygen, (C ₇-C ₁₆)-aralkyl carbonyl oxygen base, cinnamoyloxy group, (C ₂-C ₁₂)-thiazolinyl carbonyl oxygen base, (C ₂-C ₁₂)-alkynyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N, N-bis-ring-(C ₃-C ₈)-alkyl-carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-((C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkyl) carbamoyl, N-(C ₁-C ₆)-alkyl-N-((C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkyl) carbamoyl, N-(+)-dehydrogenation fir base carbamoyl, N-(C ₁-C ₆)-alkyl-N-(+)-dehydrogenation fir base carbamoyl, N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl; CON (CH ₂) _h, CH wherein ₂Can be O, S, N-(C ₁-C ₈)-alkyl imino, N-(C ₃-C ₈)-cycloalkyl imino group, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₄)-alkyl imino, N-(C ₆-C ₁₂)-aryl imino group, N-(C ₇-C ₁₂)-aralkyl imino group, N-(C ₁-C ₄)-alkoxyl-(C ₁-C ₆)-alkyl imino replaces, and h is 3-7; Carbamoyloxy with chemical formula R

Wherein, R ^xAnd R ^vBe selected from independently of one another hydrogen, (C ₁-C ₆)-alkyl, (C ₃-C ₇The substituent group of the alpha-carbon atom of)-cycloalkyl, aryl or alpha amino acid, wherein aminoacid is natural L aminoacid or its D type isomer,

S is 1-5,

T is OH or NR ^*R ^*, R ^*, R ^*And R ^{* *}Can be identical or different, be selected from hydrogen, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl, (C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl, (+)-dehydrogenation fir base, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₁-C ₁₀)-alkanoyl, the optional (C that replaces ₇-C ₁₆)-aralkanoyl, the optional (C that replaces ₆-C ₁₂)-aroyl; Perhaps R ^*And R ^*Be CH ₂, CH wherein ₂Can be O, S, SO, SO ₂, N-acylamino-, N-(C ₁-C ₁₀)-alkoxy carbonyl imino group, N-(C ₁-C ₈)-alkyl imino, N-(C ₃-C ₈)-cycloalkyl imino group, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₄)-alkyl imino, N-(C ₆-C ₁₂)-aryl imino group, N-(C ₇-C ₁₆)-aralkyl imino group, N-(C ₁-C ₄)-alkoxyl-(C ₁-C ₆)-alkyl imino, h is 3-7;

Carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N-(C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl, (C ₇-C ₁₂)-aryl sulfonyl, sulfamoyl, N-(C ₁-C ₁₀)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₁₀)-alkylsulfamoyl group, (C ₃-C ₈)-cycloalkyl sulfamoyl, N-(C ₆-C ₁₂)-alkylsulfamoyl group, N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-ammonia aryl sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, (C ₁-C ₁₀)-amino-alkyl sulfinyl, N-((C ₁-C ₁₀)-alkyl)-(C ₁-C ₁₀)-amino-alkyl sulfinyl, (C ₇-C ₁₆)-aralkyl sulfonamido or N-((C ₁-C ₁₀)-alkyl)-(C ₇-C ₁₆)-aralkyl sulfonamido; Aryl or can be 1-5 identical or different group containing the aryl in aromatic yl group and replace wherein, they comprise hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₁-C ₈)-hydroxyalkyl, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, (C ₁-C ₁₂)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxy carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, (C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₃-C ₈)-cycloalkyl carbonyl oxygen base, (C ₆-C ₁₂)-aryl-carbonyl oxygen, (C ₇-C ₁₆)-aralkyl carbonyl oxygen base, cinnamoyloxy group, (C ₂-C ₁₂)-thiazolinyl carbonyl oxygen base, (C ₂-C ₁₂)-alkynyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N-(C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl or (C ₇-C ₁₆)-aryl sulfonyl;

Perhaps R ¹With R ²Or R ²With R ³Form chain [CH ₂] _o, can be saturated chain, can be also the unsaturated chain containing the two keys of C=C, wherein 1 or 2 CH ₂Optional is O, S, SO, SO ₂Or NR ' replacement, wherein R ' is hydrogen, (C ₆-C ₁₂)-aryl, (C ₁-C ₈)-alkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₁-C ₁₀)-alkanoyl, the optional (C that replaces ₇-C ₁₆)-aralkanoyl, the optional (C that replaces ₆-C ₁₂)-aroyl; O is 3,4 or 5;

Perhaps R ¹With R ²Or R ²With R ³Form 5,6,7,8-tetrahydroisoquinoline ring, 5,6,7,8-tetrahydroquinoline ring or 5,6,7,8-tetrahydrochysene cinnoline ring with the pyridine that comprises them or pyridazine;

Perhaps R ¹With R ²Or R ²With R ³Form 5 yuan or 6 yuan of aromatic carbon rings or fragrant heterocycle;

Perhaps R ¹With R ²Or R ²With R ³The heterocyclic system that forms optional replacement with the pyridine that comprises them or pyridazine, described system can be selected from thienopyridine, furo pyridine, pyridopyridine, pyrimido pyridine, imidazopyridine, thiazole pyridine, oxazole pyridine, quinoline, isoquinolin and cinnoline; Wherein, quinoline, isoquinolin and cinnoline should meet formula Ia, Ib and Ic:

R wherein ¹²-R ²³Independent separately, define same R ¹, R ²And R ³

Perhaps radicals R ¹With R ²Form compound shown in formula Id with the pyridine that comprises them:

Wherein V is S, O or NR ^k, R wherein ^kBe selected from hydrogen, (C ₁-C ₆)-alkyl, aryl or benzyl; Wherein aryl can be that 1-5 substituent group replaces as mentioned above;

And R ²⁴, R ²⁵, R ²⁶And R ²⁷Independent separately, define same R ¹, R ²And R ³

F is 1-8;

G is 0 or 1 to (2f+1);

X is 0-3;

H is 3-7;

The physiologically active salt and the prodrug that comprise above-claimed cpd.

In some embodiments, include, but is not limited to [(3-methoxyl group-pyridine-2-carbonyl)-amino]-acetic acid with compound shown in above formula (I); Hydrochloric acid 3-Methoxy Pyridine-2-carboxylic acid N-(((hexadecane oxygen base)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((1-octyloxy)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((hexyloxy)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((butoxy)-carbonyl)-methyl)-amide; Racemic tartaric acid 3-Methoxy Pyridine-2-carboxylic acid N-(((2-oxygen in ninth of the ten Heavenly Stems base)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((oxygen base in heptan)-carbonyl)-methyl)-amide; 3-phenoxypyridines-2-carboxylic acid N-(((octyloxy)-carbonyl)-methyl)-amide; 3-benzyloxy pyridine-2-carboxylic acids N-(((butoxy)-carbonyl)-methyl)-amide; 5-(((3-(1-butoxy)-propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-((benzyloxy carbonyl)-methyl)-amide; 5-(((3-(1-butoxy)-propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-(((1-butoxy)-carbonyl)-methyl)-amide; 5-(((3-dodecyloxy propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-((benzyloxy carbonyl)-methyl)-amide; [(3-hydroxyl-pyridine-2-carbonyl)-amino] acetic acid; [(3-methoxyl group-pyridine-2-carbonyl)-amino)] acetic acid.In other embodiments, with compound shown in above formula (Ia), include, but is not limited to N-((3-hydroxyl-6-isopropoxy-quinoline-2-carbonyl)-amino) acetic acid, N-((6-(1-butoxy)-3-hydroxyquinoline-2-yl)-carbonyl) glycine, [(3-hydroxyl-6-trifluoromethoxy-quinoline-2-carbonyl) amino] acetic acid, N-((6-chloro-3-hydroxyl quinoline-2-yl)-carbonyl) glycine, N-((7-chloro-3-hydroxyl quinoline-2-yl)-carbonyl) glycine and [(6-chloro-3-hydroxyl-quinoline-2-carbonyl) amino] acetic acid.In some other embodiment, with compound shown in above formula (1b), include, but is not limited to N-((1-chloro-4-hydroxyl-7-(2-propoxyl group) isoquinolin-3-yl) carbonyl) glycine, N-((1-chloro-4-hydroxyl-6-(2-propoxyl group) isoquinolin-3-yl) carbonyl) glycine, N-((1-chloro-4-hydroxyl-isoquinolin-3-carbonyl) amino) acetic acid, N-((1-chloro-4-hydroxyl-7-methoxyl group isoquinolin-3-yl) carbonyl) glycine, N-((1-chloro-4-hydroxyl-6-methoxyl group isoquinolin-3-yl) carbonyl) glycine, N-((7-butoxy)-1-chloro-4-hydroxyl isoquinolin-3-yl) carbonyl) glycine, N-((6-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl) amino) acetic acid, ((7-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl) amino) methyl acetate, N-((7-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl) amino) acetic acid, N-((8-chloro-4-hydroxyl isoquinolin-3-yl) carbonyl) glycine, N-((7-butoxy-4-hydroxyl isoquinolin-3-carbonyl) amino) acetic acid.

In other embodiments, be selected from for the compound of the inventive method the there is chemical formula compound of (II)

Wherein, R ²⁸Hydrogen, nitro, amino, cyano group, halogen, (C ₁-C ₄)-alkyl, carboxyl or their easy metabolism ester derivant; (C ₁-C ₄)-alkyl amino, two-(C ₁-C ₄)-alkyl amino, (C ₁-C ₆)-alkoxy carbonyl, (C ₂-C ₄)-alkanoyl, hydroxyl-(C ₁-C ₄)-alkyl, carbamoyl, N-(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₄)-alkylthio group, (C ₁-C ₄)-alkyl sulfinyl, (C ₁-C ₄)-alkyl sulfide acyl group, thiophenyl, phenyl sulfinyl, phenyl sulfonyl, described phenyl may optionally be 1-4 identical or different halogen replacement, (C ₁-C ₄)-alkoxyl, (C ₁-C ₄)-alkyl, cyano group, hydroxyl, trifluoromethyl, fluoro-(C ₁-C ₄)-alkylthio group, fluoro-(C ₁-C ₄)-alkyl sulfinyl, fluoro-(C ₁-C ₄)-alkyl sulfide acyl group, (C ₁-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxy carbonyl, N, N-bis--[(C ₁-C ₄)-alkyl] carbamoyl-(C ₁-C ₄)-alkoxy carbonyl, (C ₁-C ₄)-alkyl amino-(C ₂-C ₄)-alkoxy carbonyl, two-(C ₁-C ₄)-alkyl amino-(C ₂-C ₄)-alkoxy carbonyl, (C ₁-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxy carbonyl, (C ₂-C ₄)-alkanoyloxy-(C ₁-C ₄)-alkyl or N-[amino-(C ₂-C ₈)-alkyl]-carbamoyl;

R ²⁹Hydrogen, hydroxyl, amino, cyano group, halogen, (C ₁-C ₄)-alkyl, carboxyl or their easy metabolism ester derivant; (C ₁-C ₄)-alkyl amino, two-(C ₁-C ₄)-alkyl amino, (C ₁-C ₆)-alkoxy carbonyl, (C ₂-C ₄)-alkanoyl, (C ₁-C ₄)-alkoxyl, carboxyl-(C ₁-C ₄)-alkoxyl, (C ₁-C ₄)-alkoxy carbonyl-(C ₂-C ₄)-alkoxyl, carbamoyl, N-(C ₁-C ₈)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₈)-alkyl-carbamoyl, N-[amino-(C ₂-C ₈)-alkyl] carbamoyl, N-[(C ₁-C ₄)-alkyl amino-(C ₁-C ₈)-alkyl] carbamoyl, N-[bis--(C ₁-C ₄)-alkyl amino-(C ₁-C ₈)-alkyl] carbamoyl, N-cyclohexyl carboxyamide base, N-cyclopenta carbamoyl, N-(C ₁-C ₄)-alkyl-cyclohexyl carbamoyl, N-(C ₁-C ₄)-alkyl cyclopenta carbamoyl, N-phenyl amino formoxyl, N-(C ₁-C ₄)-alkyl-N-phenyl amino formoxyl, N, N-diphenyl amino formoxyl, N-[phenyl-(C ₁-C ₄)-alkyl] carbamoyl, N-(C ₁-C ₄)-alkyl-N-[phenyl-(C ₁-C ₄)-alkyl] carbamoyl or N, N-bis-[phenyl-(C ₁-C ₄)-alkyl] carbamoyl, described phenyl may optionally be 1-4 identical or different halogen replacement, (C ₁-C ₄)-alkoxyl, (C ₁-C ₄)-alkyl, cyano group, hydroxyl, trifluoromethyl, N-[(C ₂-C ₄)-alkanoyl] carbamoyl, N-[(C ₁-C ₄)-alkoxy carbonyl] carbamoyl, the fluoro-(C of N-[ ₂-C ₆)-alkyl] carbamoyl, N, the fluoro-(C of N-[ ₂-C ₆)-alkyl]-N-(C ₁-C ₄)-alkyl-carbamoyl, N, the fluoro-(C of N-[bis- ₂-C ₆)-alkyl] carbamoyl, 1-pyrrole radicals-carbonyl, piperidino carbonyl, 1-piperazinyl-carbonyl, morpholinyl carbonyl, wherein heterocyclic group is optionally 1-4 following group replacement: (C ₁-C ₄)-alkyl, benzyl, 1,2,3,4-tetrahydroisoquinoline-2-base-carbonyl, N, N-[bis--(C ₁-C ₄)-alkyl] thiocarbamoyl, N-(C ₂-C ₄)-alkanoylamino or N-[(C ₁-C ₄)-alkoxy carbonyl] amino;

R ³⁰Hydrogen, (C ₁-C ₄)-alkyl, (C ₂-C ₄)-alkoxyl, halogen, nitro, hydroxyl, fluoro-(1-4C) alkyl or pyridine radicals;

R ³¹Hydrogen, (C ₁-C ₄)-alkyl, (C ₂-C ₄)-alkoxyl, halogen, nitro, hydroxyl, fluoro-(C ₁-C ₄) alkyl, pyridine radicals or methoxyl group;

R ³²Hydrogen, hydroxyl, amino, (C ₁-C ₄)-alkyl amino, two (C ₁-C ₄)-alkyl amino, halogen, (C ₁-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxyl, fluoro-(C ₁-C ₆)-alkoxyl, pyrroles-1-base, piperidyl, piperazine-1-base or morpholinyl, wherein heterocyclic radical is optionally 1-4 identical or different (C ₁-C ₄)-alkyl or phenyl replaces;

R ³³And R ³⁴Be selected from separately separately hydrogen, (C ₁-C ₄)-alkyl and (C ₁-C ₄)-alkoxyl;

The pharmaceutical salts and the prodrug that also comprise them.

In some embodiments, shown in chemical formula (II), compound includes, but is not limited to 4-oxygen-1,4-dihydro-[1,10] phenanthroline-3-carboxylic acid, 3-carboxyl-5-hydroxyl-4-oxygen-3,4-dihydro-1,10-phenanthroline, 3-carboxyl-5-methoxyl group-4-oxygen-3,4-dihydro-1,10-phenanthroline, 5-methoxyl group-4-oxygen-Isosorbide-5-Nitrae-dihydro [1,10] phenanthroline-3-carboxylic acid, ethyl ester, 5-methoxyl group-4-oxygen-1,4-dihydro [1,10] phenanthroline-3-carboxylic acid and 3-carboxyl-8-hydroxyl-4-oxygen-3,4-dihydro-1,10-phenanthroline.

Described compound can be used separately, also can use in conjunction with other various treatment meanss.In one embodiment, described compound is used together with another kind of 2-oxoglutaric acid dioxygenase inhibitor, and for the independent 2-oxoglutaric acid dioxygenase member of family, these two kinds of compounds have different specificitys.These two kinds of compounds can be used by certain proportion simultaneously, also can in therapeutic process, use successively, for example, for myocardial infarction.In a specific embodiment, a kind of compound suppresses the activity of HIF prolyl hydroxylase specially, and another kind of compound suppresses the activity of collagen prolyl 4 hydroxylase specially.In another embodiment, described compound is used together from the medical reagent that another has different binding modes, for example ACE inhibitor (ACEI), angiotensin-II receptor blocking agent (ARB), diuretic and/or digoxin.In another embodiment, described compound is used together with carnitine.

On the one hand, the compounds of this invention can suppress one or more 2-oxoglutaric acid dioxygenases.In one embodiment, described compound can suppress at least two kinds of 2-oxoglutaric acid dioxygenase members of family, for example HIF prolyl hydroxylase and collagen prolyl 4 hydroxylase, and their specificity can be identical or different.In another embodiment, described compound can have specificity to a 2-oxoglutaric acid dioxygenase, HIF prolyl hydroxylase for example, and the member of other families is shown very little specificity or there is no specificity.

The preferred embodiment of the present invention comprises the method for utilizing oral cavity drug delivery or transdermal drug delivery mechanism.Therefore, the present invention also provides a kind of medicinal preparation for oral administration that comprises the compounds of this invention.In another preferred implementation, the inventive method relates to by transdermal mechanism uses the compounds of this invention.Therefore, the present invention also provides the transdermal that comprises the compounds of this invention ointment.

According to the introduction here, those skilled in the art are readily appreciated that all these embodiments of above-mentioned and other embodiment of the present invention are all custom-designed.

Brief description

Figure 1A and 1B are depicted as the stabilisation of HIF-1 α in the cell of processing with the compounds of this invention.Figure 1A is depicted as the stable and enrichment with HIF-1 α in people's clad fibre archeocyte (HFF) of the various compound treatment of the present invention.Figure 1B is depicted as the stable dose response with enrichment of HIF-1 α in the different people cell of processing with the compounds of this invention and replys.Cell line shown in figure comprises HFF, human microvascular endothelial cell (HMEC), venous endothelial cell (AG7), mankind's umbilical vein endotheliocyte (HUVEC), squamous epithelium cancer cell (SCC), people's lung fibroblast (HLF), breast epithelium cancerous cell (MCF7), degeneration fetal kidney cell (293A) and cervical cancer cell (HeLa).

Fig. 2 A and 2B are depicted as the stable and enrichment of HIF-1 α in people's cell of processing with the compounds of this invention.Fig. 2 A is depicted as 293A and the human liver cancer cell (Hep3B) by the various compound treatment of the present invention.Fig. 2 B is depicted as the dose response of HIF-1 α stabilisation in the Hep3B cell of processing by exemplary compounds of the present invention.

Fig. 3 A and 3B are depicted as oxygen consumption and the cell survival ability in the human body cell of processing with the compounds of this invention.Fig. 3 A is depicted as by single dose and dose response oxygen consumption in the cell of the various compound treatment of the present invention.Fig. 3 B is depicted as with cell proliferation and survival ability in the cell of compound treatment selected in Fig. 3 A, mensuration be the division situation (Roche Diagnostics Corp., Indianapolis IN) of WST-1 tetrazolium salts.

Fig. 4 A and 4B are depicted as the human body cell of processing with the compounds of this invention and are improved in the expression of HIF-response gene.After Fig. 4 A is depicted as and processes with the compounds of this invention, the level of human body cell culture medium vascular endothelial growth factor (VEGF), the wherein said factor is the key gene in the vascularization process.Cell line shown in figure is 293A, Hep3B and HFF.Fig. 4 B is depicted as the process that in the cell of processing with the compounds of this invention, aldolase increases in time, and wherein aldolase is the key enzyme in the glycolysis path.

After Fig. 5 A and 5B are depicted as and process with the compounds of this invention, the expression of animal lung angiogenic growth albumen is improved.Fig. 5 A is depicted as the complex chart of angiogenic growth gene expression.Gene shown in figure comprises vascular endothelial cell growth factor (VEGF)-C, Flt-1/VEGF receptor-1, medullin, endothelium quality-1, plasminogen activity factor inhibitor (PAI)-1 and Cyr61.Fig. 5 B is depicted as endothelium quality-1 and the gene expression of medullin in cataloged procedure of selecting from Fig. 5 A.

The expression that Fig. 6 A and 6B are depicted as HIF response gene in body is improved.After Fig. 6 A is depicted as and processes with the compounds of this invention, the liver of rat and the transcriptional level of the VEGF in kidney in cataloged procedure are improved.Fig. 6 B is depicted as with undressed matched group and compares, after doing final processing with the compounds of this invention, and the level of the VEGF in rat plasma when 2 hours, 5 hours and 20 hours.

After Fig. 7 A and 7B are depicted as and process with the compounds of this invention, in animal kidney, the expression of glycolytic ferment is improved.Fig. 7 A is depicted as the complex chart of glycolysis gene expression.Gene shown in figure comprise aldolase-A, ENO1, Glut1, Glut3, GAPDH, hexokinase-1 and-2, lactic acid dehydrogenase-A, phosphofructokinase-L and-C, phosphoglyceric kinase-1 and pyruvate kinase-M.Fig. 7 B is depicted as aldolase-A and the gene expression of phosphofructokinase-L in cataloged procedure of selecting from Fig. 7 A.

Figure 8 shows that and induce in the myocardial infarction sample, the group (n=34) of processing with the compounds of this invention and undressed matched group (n=34) the survival percent when the different time interval.

Fig. 9 A and 9B are depicted as the animal of trouble myocardial infarction with after the compounds of this invention treatment, with respect to undressed matched group, and the improvement on cardiac structure.Fig. 9 A is depicted as the group processed with the compounds of this invention in the sample of suffering from myocardial infarction with respect to undressed matched group, in the variation of the diameter (LVESD) in different interval left ventricular contraction in period latter stage.Fig. 9 B is depicted as the group processed with the compounds of this invention in the sample of suffering from myocardial infarction with respect to undressed matched group, in the variation of different interval left ventricular end diastolic diameter in period (LVEDD).

Figure 10 A and 10B are depicted as the animal of trouble myocardial infarction with after the compounds of this invention treatment, and with respect to undressed matched group, its cardiac function improves.Figure 10 A is depicted as with respect to untreated matched group, and that with the compounds of this invention, treated induces myocardial infarction sample group in the different interval variation of period on the left ventricle ejection fraction.Figure 10 B is depicted as with respect to untreated matched group, the situation that the myocardial infarction sample group for the treatment of with the compounds of this invention reduces at different interval ejection fraction in period.

Figure 11 shows that with respect to undressed matched group, the sample group of processing with the compounds of this invention 4 weeks is the contractile response of heart afterwards, and described sample comprises the sample that is subject to isoproterenol to excite and not excited by it.

Figure 12 A and 12B are depicted as the animal of trouble myocardial infarction with after the compounds of this invention pretreat, with respect to undressed matched group, on cardiac structure, improve.Figure 12 A is depicted as to induce and produces myocardial infarction after 1 week, and treated animal is compared with undressed control animal, and ejection fraction shortens on statistics and obviously improves (p<0.05).Figure 12 B is depicted as to induce and produces myocardial infarction after 1 week, and treated animal is compared with undressed control animal, left ventricular end diastolic diameter (LVEDD; P<0.05) and the diameter (LVESD in left ventricular contraction latter stage; P<0.01) on statistics, obviously improve.

Figure 13 shows that first animal is carried out to pretreat, then make it to be subject to ischemia-reperfusion injury of kidney, process with the compounds of this invention subsequently, compare with the sample that carries out sham-operation with unprocessed, the survival rate of animal improves.

Figure 14 A and 14B are depicted as first animal are carried out to pretreat, then make it to be subject to ischemia-reperfusion injury of kidney, with the compounds of this invention, process subsequently, with undressed check sample, compare, and its renal function improves.After Figure 14 A is depicted as and is subject to inducing ischemical reperfusion injury 3 days and 7 days, the animal of being treated is compared with untreated animal, the content of nitrogen in low hematuria.After Figure 14 B is depicted as and is subject to inducing ischemical reperfusion injury 3 days, 7 days and 14 days, the animal of being treated is compared with untreated animal, the content of low cholesterolemia.

Figure 15 A is depicted as with undressed matched group and compares with 15B, and the animal of processing with the present invention improves on the therapeutic effect of chronic trauma.After Figure 15 A is depicted as injured 7 days and 10 days, treated animal is compared with undressed matched group, and the growing state of epithelial cell and granulation tissue improves.Figure 15 B is depicted as peak-peak distance in treated animal and undressed animal scar does not have difference.

Detailed Description Of The Invention

Before introducing the present composition and method, be to be understood that the present invention is not subject to the restriction of concrete grammar, principle, cell line, chemical examination and reagent, because these all can change.It is also understood that term used, also just for specific implementations of the present invention is described, limits the scope set in appended claims of the present invention anything but here.

Must be pointed out, in description and appended claims, singulative used " " comprises plural situation, unless explicitly pointed out in addition in literary composition.Therefore, for instance, " fragment " comprises many such fragments; " a kind of antibody " can refer to one or more antibody, and the antibody of equal value that those skilled in the art were familiar with; The like.

Except as otherwise noted, the implication that the implication of all technology used and scientific terminology is understood usually with this area here is identical.Although being similar to or being equivalent to any method as described herein and material all can be used for, in the present invention's practice or experiment, below still providing method for optimizing, device and material.Here all with its integral form as a reference, purpose is to introduce method related to the present invention in these documents, reagent and instrument to all documents of quoting.This never can be misinterpreted as us and admit that the present invention haves no right to do similarly to introduce in previous invention.

Except as otherwise noted, the present invention will adopt conventional chemical, biochemistry, molecular biology, cytobiology, genetics, immunology and method of pharmacy in the art in practice.For example, can be referring to Gennaro, " the Remington materia medica " that A.R. compiles (Remington ' s Pharmaceutical Sciences) (1990), the 18th edition, Mack publishing company; Hardam, J.G., Limbird, L.E. and Gilman, " pharmacological basis of Therapeutic Method " (The Pharmaceutical Basis of Therapeutics) (2001) that A.G. compiles, the 10th edition, mcgraw-hill, inc; Colowick, S. etc. compile " immunological method " (Methods In Enzymology), Academic publishing company; Weir, D.M. and Blackwell, C.C. compiles " experiment immunization handbook (Handbook ofExperimental Immunology) (1986), Vo1.I-IV, Blackwell Scientific publishing house; Maniatis, T. etc. compile " molecular cloning: laboratory manual " (Molecualr Cloning:A Laboratory Manual) (1989), the 2nd edition, Vols.I-III, Cold Spring Harbor Laboratory publishing house; Ausubel, F.M. etc. compile " the simple and clear agreement of molecular biology " (Short Protocols in Molecular Biology) (1999), the 4th edition, John Wiley & Sons Ream etc. compile " molecular biotechnology: fine works laboratory course " (Molecualr Biology Techniques:An Intensive Laboratory Course) (1998), Academic publishing house; Newton, C.R. and Graham, A. compiles " PCR " (biotechnology enters gate series) (Introduction to Biotechniques Series) (1997), the 2nd edition, Springer Verlag.

Definition

Term " ischemia " refers to that blood flow descends.Ischemia makes to be supplied to the nutrient substance of tissue to descend, and comprises oxygen.Like this under certain situation, can cause ischemia, as formed thrombosis, tremulous pulse or vein in arteriosclerosis, tremulous pulse or vein, sealed by the blood vessel that embolus stops up, other reasons causes, such as being caused by vasospasm etc.These situations can reduce blood flow, produce the low perfusion state to organ or tissue, or complete plug flow.Other situations that cause ischemia comprise the injured tissue injury caused, for example spinal cord injury; Toadstool infects, and for example, it can cause congestive heart failure etc.Term " ischemic diseases " and " ischemic imbalance " comprise severe ischemic disease and chronic ischemic disease, the severe ischemic disease includes, but is not limited to myocardial infarction, cerebral infarction, pulmonary infarction, enclose the product hypoxia, the blood circulation shock (for example comprises and losing blood, deteriorated blood, the former ischemia of the heart etc.), altitude sickness, severe respiratory failure etc., the chronic ischemic disease comprises arteriosclerosis, chronic venous insufficiency of function, chronic heart failure, the heart sclerosis, diabetes, the speckle shape worsens, sleep apnea, Raynaud's disease, the system sclerosis, non-bacterial becomes the bolt endocarditis, the arterial occlusion disease, angina pectoris, TIAs, Chronic Alcohol hepatopathy etc.When individuality is placed in general anesthesia state lower time, also can cause ischemic diseases, and cause and prepare the tissue damage of transplanting.

Term " hypoxia " and " hypoxia " finger ring border oxygen content are lower than conventional levels.In low-oxygen environment, cultured cell can make the cell hypoxia, or with hypoxia-mimicking compound treatment cell.The oxygen content that is used for determining hypoxia in cell culture can be with the technical measurement in this area.

Term " hypoxia disease " and " hypoxia sexual maladjustment " include, but is not limited to above-mentioned ischemic diseases (ischemic hypoxia), and hypoxia is reduced and caused by circulating load here; Pulmonary disease (hypoxia hypoxia), as COPD, serious pneumonia, pulmonary edema, pulmonary's hypertension, hyaline membrane disease etc., hypoxia is that oxygen exchange capacity in lung reduces and causes here; Anemia disease (anemia hypoxia), as gastric ulcer or duodenal ulcer, hepatopathy or nephropathy, thrombocytopenia or coagulopathy, cancer or other chronic diseases, the anemia that cancer chemotherapy and other treatment intervention produce etc., hypoxia is descended and is caused by hematochrome or red blood cell concentration here; High altitude disease etc.

Term " imbalance " and " disease " are used for making a general reference the disease of any deviation from the norm state.Term " ischemic diseases " refers to any disease relevant with ischemia or discomfort with " ischemic imbalance ".Term " hypoxia disease " refers to any disease relevant with hypoxia or discomfort with " hypoxia sexual maladjustment ".This ischemia and hypoxia disease include, but is not limited to above-mentioned disease.

Term " HIF α " refers to the α subunit of hypoxia inducible factor albumen.HI α can be anyone or mammalian proteins or protein fragments, includes, but is not limited to people HIF-1 α (the gene bank number of sequeling No.Q16665), HIF-2 α (the gene bank number of sequeling No.AAB41495) and HIF-3 α (the gene bank number of sequeling No.AAD22668); Muridae HIF-1 α (the gene bank number of sequeling No.Q61221), HIF-2 α (the gene bank number of sequeling No.BAA20130 and AAB41496) and HIF-3 α (the gene bank number of sequeling No.AAC72734); Rat HIF-1 α (the gene bank number of sequeling No.CAA70701), HIF-2 α (the gene bank number of sequeling No.CAB96612) and HIF-3 α (the gene bank number of sequeling No.CAB96611); Cattle HIF-1 α (the gene bank number of sequeling No.BAA78675).HIF α can be also any nonmammalian albumen or protein fragments, comprises Xenopus laevis H[F-1 α (the gene bank number of sequeling No.CAB96628), Drosophila melanogaster HIF-1 α (the gene bank number of sequeling No.JC4851) and chicken HIF-1 α (the gene bank number of sequeling No.BAA34234).HIF α gene order also can obtain by conventional clone technology, for example uses above-mentioned all or part of HIF α gene order as probe, measures the HIF α sequence in another fragment.

HIF α fragment comprises the zone of human HIF-1-1 α defined, comprise aminoacid 401-603 (Huang etc., the same), aminoacid 531-575 (Jiang etc. (1997) J Biol Chem 272:19253-19260), aminoacid 556-575 (Tanimoto etc., the same), aminoacid 557-571 (Srinivas etc. (1999) Biochem Biophys ResCommun 260:557-561) and aminoacid 556-575 (Ivan and Kaelin (2001) Science 292:464-468).In addition, the fragment of HIF α comprises at least appearance motif LXXLAP once, for example appears at the L in HIF-1 α chief series ₃₉₇TLAP and L ₅₅₉EMLAP.In addition, the fragment of HIF α comprises any at least one function of HIF α or fragment of architectural feature of having retained.For example, the HIF polypeptide for embodiment 7 screening chemical examinations can comprise [methoxy coumarin]-DLDLEALAPYIPADDDFQL-amide (SE1ID NO:5).

Term " HIF prolyl hydroxylase " and " HIF PH " refer to anyly can make the proline residue in HIF albumen that hydroxylated enzyme occurs.Proline residue by HIF PH hydroxylation comprises the proline in motif LXXLAP, for example appears at the L in HIF-1 α chief series ₃₉₇TLAP and L ₅₅₉EMLAP.HIF PH comprises the member in Egl-Nine (EGLN) gene cluster, as Taylor (2001, Gene 275:125-132) described, Aravind and Koonin (2001, Genome Biol 2:RESEARCH0007), Epstein etc. (2001, Cell 107:43-54), Bruick and McKnight (2001, Science 294:1337-1340) characterize it.The example of HIF PH enzyme has people SM-20 (EGLN1) (the gene bank number of sequeling No.AAG33965; Dupuy etc. (2000) Genomics 69:348-54), EGLN2 isomer 1 (the gene bank number of sequeling No.AAG33965; Taylor, the same), EGLN2 isomer 2 (the gene bank number of sequeling No.NP_060025) and EGLN3 (the gene bank number of sequeling No.CAC42511; Taylor, the same); Mice EGLN1 (the gene bank number of sequeling No.CAC42515), EGLN2 (the gene bank number of sequeling No.CAA42511) and EGLN3 (SM-20) (the gene bank number of sequeling No.CAC42517); Rat SM-20 (the gene bank number of sequeling No.AAA19321).In addition, HIF PH can comprise Caenorhabditis elegans EGL-3 (the gene bank number of sequeling No.AAD56365) and Drosophilamelanogaster CG1114 gene prod (the gene bank number of sequeling No.AAF52050).HIF PH also comprises structure at least having retained aforementioned full-length proteins or any fragment of functional character, comprises the fragment with hydroxylase activity.

Here term used " aminoacid sequence " or " polypeptide " refer to HIF α and fragment thereof, or HIF PH and fragment thereof, comprise low peptide, peptide or protein sequence, or refer to their fragment and natural or synthetic molecules." fragment " can refer to an any part in sequence, and it has at least comprised structure or the functional character of protein.Exempt from service former fragment or antigen fragment is polypeptide fragment, is preferably and is about 5-15 aminoacid, retained at least one biological or active fragment of exempting from service.While with " aminoacid sequence ", censuring the peptide sequence of native protein molecule, " aminoacid sequence " not is limited to aminoacid sequence on the complete native sequences relevant to described protein molecular with similar terms.

Here term used " related protein " refers to the protein relevant with HIF α prolyl hydroxylase, and it comprises other 2-oxoglutaric acid dioxygenases, particularly those similar Fe that need ²⁺, 2-oxoglutaric acid and oxygen maintains the member of family of hydroxylase activity.This kind of enzyme includes, but is not limited to collagen lysyl hydroxylase, collagen prolyl 4 hydroxylase and the factor and suppresses HIF (FIH), and the latter is responsible for regulating the agedoite hydroxylase that HIF α intersection activates.(the gene bank number of sequeling No.AAL27308; Mahon etc. (2001) Genes Dev 15:2675-2686; (2002) Genes Dev 16:1466-1471 such as Lando etc. (2002) Science 295:858-861:Lando.Separately see (2002) J Biol Chem C200644200 such as Elkins.)

Term " gaonist " refers to increase or extends specific molecular as enzyme or albumen, or specific environment is as the molecule of the lasting effect of low-oxygen environment.Gaonist comprises that protein, nucleic acid, carbohydrate or other can regulate any molecule of the effect of target molecule.

Term " antagonist " refers to reduce biological or exempt from service active effect or the molecule of persistent period of specific molecular.Antagonist comprises protein, nucleic acid, carbohydrate, antibody or other any molecules that can reduce the target molecule effect.

Term " microarray " refers to that nucleic acid, aminoacid, antibody etc. are at suprabasil any arrangement mode.Substrate can be any suitable carrier, such as pearl, glass, paper, nitrocellulose, nylon or any suitable film etc.Substrate can be any rigidity or semi-rigid carrier, includes, but is not limited to film, filter, wafer, chip, microscope slide, fiber, pearl (comprising magnetic or non magnetic) pearl, gel, tubing, sheet material, polymer, microgranule, capillary tube etc.Described substrate can provide surface to carry out coat operations, and/or has various format surfaces, and as well, pin, ditch, groove and hole, nucleic acid, aminoacid etc. can be attached to above them.

Here term used " excipient " refers to inertia used in the production of medicine or other tablets or inert matter, includes, but is not limited to as binding agent, disintegrating agent, coating, compression/be encapsulated any material of auxiliary agent, cream or lotion, lubricant, injection, sweeting agent or flavoring agent, suspension/gel, wet granulation agent.Binding agent comprises carbopol (carbopol), polyvidone, xanthan gum etc.; Coating comprises that cellulose ethanoate is to dibenzoate, ethyl cellulose, gellan gum, maltodextrin etc.; Compress/be encapsulated auxiliary agent and comprise calcium carbonate, dextrose, fructose dc, Mel dc, a lactose (anhydrous or water; Optional with Aspartame, cellulose or microcrystalline Cellulose, combine), starch dc, sucrose etc.; Disintegrating agent comprises cross-linked carboxymethyl cellulose sodium, gellan gum, primojel etc.; Cream and lotion comprise maltodextrin, carrageenan etc.; Lubricant comprises magnesium stearate, stearic acid, stearyl fumarate; Material for chewable tablet comprises dextrose, fructose dc, lactose (monohydrate optionally mixes with Aspartame or cellulose) etc.; Injection comprises mannitol, polyvidone etc.; Plasticizer comprises dibutyl sebacate, the adjacent dibenzoate of polyethylene acetic acid etc.; Suspension/gel comprises carrageenan, primojel, xanthan gum etc.; Sweeting agent comprises Aspartame, dextrose, fructose dc, sorbitol, sucrose dc etc.; The wet granulation agent comprises calcium carbonate, maltodextrin, microcrystalline Cellulose etc.

Term " sample " is used its broad sense here.Sample can be taken from any source, the cell in body fluid, secretions, tissue, cell or culture fluid for example, described culture fluid includes, but is not limited to saliva, blood, urine, serum, blood plasma, vitreous humour, synovial fluid, brains liquid, amniotic fluid and organ-tissue (for example biopsy); Genomic DNA, cDNA, RNA, mRNA etc.; The cell or tissue of extracing, or the speckle on this cell or tissue or the marking.Sample also can be taken from some sources like this, such as people or people mammal in addition etc.Sample can be also the animal model of any disease.Sample can be placed in solution, also can fix or be attached in substrate.Sample can refer to any material, as long as it is applicable to for measuring the existence of HIF α or HIF α fragment, or is applicable to being attached on the molecule on HIF α or HIF α fragment for screening.The method of obtaining this sample is that the technical merit of this area possesses.

Term " processing object " is used its broad sense here.Process object and can comprise the tissue of growing in isolated cell (prokaryotic cell or eukaryotic cell) or culture fluid.Process object and should comprise animal, particularly mammal, comprise Mus, rabbit, cattle, sheep, pig, muroid, horse and primate, especially the people.

Invention

The invention provides the method for stablizing HIF α, be used in compound and the application of these methods in prevention or treatment HIF disease in described method, include, but is not limited to above-mentioned hypoxia and/or ischemic diseases.The invention still further relates to such discovery, to the stable of hypoxia inducible factor (HIF α) α subunit, be an effective treatment approach, when being used for treatment or prevention hypoxia and/or ischemic diseases, during such as myocardial infarction, apoplexy, closed arterial disease, angina pectoris, heart sclerosis, arteriosclerosis etc., can obtain pennies from heaven.

The present invention proposes the method for stablizing HIF, in order to promoting angiogenic growth, strengthen the reaction of severe hypoxia and promote the adaptation to Chronic hypoxia.Because tissue ischemia is morbidity and dead main inducing, so find the treatment that the method that can stablize HIF α will be of value to the hypoxia disease.In addition, use described method to have good result for preconditioned hypoxia reaction etc., it is to stablize HIF α before ischemia or hypoxia event occur in normal oxygen environment.Described method also can be used to induce and produces the proprietary effect of HIF α, as described below, comprises the therapeutic angiogenic growth, in order to recover the blood flow of damaged tissues; Neuroprotective, in order to prevent neuronatrophy that neurodegenerative disease causes etc.; To the protection of the oxidative damage of reactivity speciation, described oxide species is derived from pouring into again in ischemia or hypoxia event.

When by the inventive method treatment ischemic and/or hypoxia disease, described disease can be the acute ischemia disease, as lung infraction, intestinal obstruction, cerebral infarction and/or myocardial infarction, can be also the chronic ischemia disease, as blood vessel graft disease, liver cirrhosis, congestive heart failure etc.In addition, the inventive method can be used for treatment due to short-term or the acute injured ischemia caused, spinal cord injury for example, or be used for the treatment of the patient who suffers from pulmonary disease etc., as pulmonary infarction etc.

When preventing by the HIF disease by the inventive method, while including, but is not limited to tissue injury that ischemic and hypoxia disease cause, the disease of pre-diagnosis, such as hypertension, diabetes, arterial occlusion disease, chronic venous insufficiency of function, Raynaud's disease, system sclerosis, sclerosis, congestive heart failure etc. are depended in treatment.Similarly, the inventive method can be used for pretreat, with the tissue injury of alleviating or prevention HIF disease causes, includes, but is not limited to ischemic and hypoxia disease.Whether need pretreat to depend on the recurrent history of ischemic diseases, for example myocardial infarction or provisional ischemia; The symptom that depends on continuous ischemia, for example angina pectoris; Perhaps depend on that indication may ischemia or the physical parameter of hypoxia, as in the situation that individuality is subject to general anesthesia or in the high latitude area short-term job.Described method also can be used for organ transplantation, and organ donor is carried out to pretreat, and taking out in body but transplanting is safeguarded organ before to the receiver.

Discovery presented here is, HIF α stable is subject to the hydroxylated adjusting of proline, stablizes HIF α and can effectively treat or prevent ischemic diseases, prevents that it from renewing or developing, and described ischemic diseases is as DVT, angina pectoris, pulmonary infarction, apoplexy, myocardial infarction etc.Especially, research shows, with rabbit skein cell lysate (RRL) preculture together and with residue 556-575[HIF (556-575)] can be attached to specially von Hippel Lindau protein (pVHL) upper for corresponding HIF-1 α and HIF-1 α peptide, this combination can cause ubiquitin and the degraded of HIF-1 α.Research also shows, after the synteny sequence M56LAPYIPM that HIF (556-575) camber retains is mutated into 8 continuous alanine under condition of normal pressure, can stablize HIF (556-575).(Srinivas etc., the same) show the scanning of this zone alanine, P in total length HIF-1 α or in glutathione S-transferase (GST)-HIF-1 α oxygen degraded district (ODD) fusion rotein (Gal4-ODD) ₅₆₄Sport alanine and eliminated pVHL in conjunction with activity.The thin layer chromatography of EFI ion trap tandem mass spectrography (MS/MS) and Gal4-HIF (555-575) is determined P ₅₆₄Degenerative process be the hydroxylating process, wherein Gal4-HIF (555-575) is at [H ³] proline translates in vivo with RRL under existing.The importance of proline hydroxylating on function is, P ₅₆₄It is upper that hydroxylating HIF α is attached to pVHL, and comprise P ₅₆₄The HIF-1 α mutation factor that simple point mutation is alanine is stable in the COS7 cell, hypoxia imitative is desferrioxamined insensitive.(see Ivan and Kaelin, the same; Jaakkola etc. (2001) Science292:468-472)

Because HIF α is subject to proline hydroxylating (a kind of oxygen and Fe of needing ²⁺Reaction) modification, one aspect of the present invention proposes.Enzyme in HIF α hydroxylating is the member in 2-oxoglutaric acid dioxygenase family.This kind of enzyme includes, but is not limited to collagen lysyl hydroxylase, collagen prolyl 3-hydroxylase, collagen prolyl 4 hydroxylase α (I) and α (II), Thymine 7-hydroxylase, aspartic acid (agedoite) β hydroxylase, ε-N-trimethyl lysyl hydroxylase and γ-butyrobetaine hydroxylase etc.These enzyme require oxygen, Fe ²⁺, 2-oxoglutaric acid and vitamin C acid obtains hydroxylase activity.(referring to, such as (1985) Biochem J 229:127-133 such as Majamaa; Myllyharju and Kivirikko (1997) EMBO J 16:1173-1180; Thornburg etc. (1993) 32:14023-14033; Jia etc. (1994) Proc Natl Acad Sci USA 91:7227-7231)

Some micromolecular inhibitors of prolyl 4 hydroxylase are identified.(referring to, such as Majamaa etc., the same; Kivirikko and Myllyharju (1998) Matrix Biol 16:357-368; Bickel etc. (1998) Hepatology 28:404-411; Friedman etc. (2000) Proc Natl Acad Sci USA 97:4736-4741; Franklin etc. (2001) Biochem J 353:333-338; All documents are this complete quoting) use these compounds in the present invention's method of proposing to provide here.

The compound that can be used for the inventive method comprises the analog of 2-oxoglutaric acid.These compounds can suppress the target member in 2-oxoglutaric acid dioxygenase family, have competitive advantage with respect to 2-oxoglutaric acid, and there is no competitive advantage with respect to iron rule.(Majamaa etc. (1984) Eur J Biochem 138:239-245; Majamaa etc., the same)

In some embodiments, the inventive method compound used therefor is selected from compound shown in chemical formula (I)

Wherein A is 1,2-arlydene, 1,3-arlydene, Isosorbide-5-Nitrae-arlydene; Or (C ₁-C ₄)-alkylene (being optionally one or more halogen atoms, cyano group, nitro, trifluoromethyl replacement), (C ₁-C ₆)-alkyl, (C ₁-C ₆)-hydroxyalkyl, (C ₁-C ₆)-alkoxyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)Halogen _g, (C ₁-C ₆)-fluoroalkyl, (C ₁-C ₈The inferior oxyl of)-fluoro, (C ₁-C ₈The inferior alkene oxygen of)-fluoro base ,-OCF ₂Cl ,-O-CF ₂-CHFCl; (C ₁-C ₆)-alkyl thiol, (C ₁-C ₆)-alkyl sulfinyl, (C ₁-C ₆)-alkyl sulfide acyl group, (C ₁-C ₆)-alkyl-carbonyl, (C ₁-C ₆)-alkoxy carbonyl, carbamoyl, N-(C ₁-C ₄)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₆)-alkyl-carbonyl oxygen base, (C ₃-C ₈)-cycloalkyl, phenyl, benzyl, phenoxy group, benzyloxy, anilino-, methylphenylamine base, phenyl sulfydryl, phenyl sulfonyl, phenyl sulfinyl, sulfamoyl, N-(C ₁-C ₄)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₄)-alkylsulfamoyl group replaces; Be perhaps (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₁)-aralkoxy, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl (have 1-5 identical or different substituent group in aryl moiety, described substituent group is selected from halogen, cyano group, nitro, trifluoromethyl), (C ₁-C ₆)-alkyl, (C ₁-C ₆)-alkoxyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)Halogen _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₆)-alkyl thiol, (C ₁-C ₆)-alkyl sulfinyl, (C ₁-C ₆)-alkyl sulfide acyl group, (C ₁-C ₆)-alkyl-carbonyl, carbamoyl, N-(C ₁-C ₄)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₆)-alkyl-carbonyl oxygen base, (C ₃-C ₈)-cycloalkyl, sulfamoyl, N-(C ₁-C ₄)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₄)-alkylsulfamoyl group replaces; Perhaps A is CR ⁵R ⁶, R ⁵And R ⁶Independently be selected from separately hydrogen, (C ₁-C ₆)-alkyl, (C ₃-C ₇)-cycloalkyl, aryl, or the substituent group of the alpha-carbon atom of alpha amino acid, wherein aminoacid is natural L aminoacid or its D type isomer.

B is-COH ₂,-NH ₂,-NHSO ₂CF ₃, tetrazole radical, imidazole radicals, the different azoles base of 3-hydroxyl ,-CONHCOR " ' ,-CONHSOR " ', wherein R " ' be aryl, heteroaryl, (C ₃-C ₇)-cycloalkyl, (C ₁-C ₄)-alkyl, optional quilt (C ₁-C ₄)-alkylthio, (C ₁-C ₄)-sulfinyl, (C ₁-C ₄)-sulfonyl, CF3, Cl, Br, F, I, NO2 ,-COOH, (C ₂-C ₅)-alkoxy carbonyl, NH ₂, list-(C ₁-C ₄-alkyl)-amino, two-(C ₁-C ₄-alkyl)-amino or (C ₁-C ₄)-perfluoroalkyl replaces; Perhaps B is CO ₂-G carboxyl, wherein G is the group in pure G-OH, G is selected from (C ₁-C ₂₀)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₂-C ₂₀)-thiazolinyl, (C ₃-C ₈)-cycloalkenyl group, retinyl, (C ₂-C ₂₀)-alkynyl, (C ₄-C ₂₀)-alkapolyenyl, wherein thiazolinyl, cycloalkenyl group, alkynyl and alkapolyenyl comprise one or more multikeys; (C ₆-C ₁₆)-isocyclic aryl, (C ₇-C ₁₆)-aralkyl, heteroaryl or heteroarylalkyl, wherein the heteroaryl in heteroarylalkyl or assorted virtue part comprise 5-6 annular atoms; The defined group of G can be one or more other groups and replaces, and these groups comprise hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₅-C ₈)-cycloalkenyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₂-C ₁₂)-thiazolinyl, (C ₂-C ₁₂)-alkynyl, (C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₁-C ₈)-hydroxyalkyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, cinnamoyl, (C ₂-C ₁₂)-alkenyl carbonyl, (C ₂-C ₁₂)-alkynyl carbonyl, (C ₁-C ₁₂)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl-carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cyclo alkoxy carbonyl, (C ₂-C ₁₂)-allyloxycarbonyl, (C ₂-C ₁₂)-alkynyloxy group carbonyl, acyloxy, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₆-C ₁₆)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, (C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl, (C ₇-C ₁₂)-aryl sulfonyl, sulfamoyl, N-(C ₁-C ₁₀)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₁₀)-alkylsulfamoyl group, (C ₃-C ₈)-cycloalkyl sulfamoyl, N-(C ₆-C ₁₂)-alkylsulfamoyl group, N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-ammonia aryl sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, (C ₁-C ₁₀)-amino-alkyl sulfinyl, N-((C ₁-C ₁₀)-alkyl)-(C ₁-C ₁₀)-amino-alkyl sulfinyl, (C ₇-C ₁₆)-aralkyl sulfonamido or N-((C ₁-C ₁₀)-alkyl)-(C ₇-C ₁₆)-aralkyl sulfonamido; Aryl or can be 1-5 identical or different group containing the aryl in aromatic yl group and replace wherein, they comprise hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₁-C ₈)-hydroxyalkyl, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, (C ₁-C ₁₂)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxy carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, (C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₃-C ₈)-cycloalkyl carbonyl oxygen base, (C ₆-C ₁₂)-aryl-carbonyl oxygen, (C ₇-C ₁₆) aralkyl carbonyl oxygen base, cinnamoyloxy group, (C ₂-C ₁₂)-thiazolinyl carbonyl oxygen base, (C ₂-C ₁₂)-alkynyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N-(C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl or (C ₇-C ₁₂)-aryl sulfonyl;

X is O or S;

Q is O, S, NR ' or chemical bond;

Wherein, if Q is chemical bond, R ⁴Halogen, nitrile or trifluoromethyl;

Perhaps, if Q is O, S, NR ', R ⁴Hydrogen, (C ₁-C ₁₀)-alkyl, (C ₂-C ₁₀)-thiazolinyl, (C ₂-C ₁₀)-alkynyl, wherein alkenyl or alkynyl comprises one or more C-C multikeys; There is chemical formula-O-[CH ₂] _x-C _fH _(2f+1-g)-F _gNot replacement fluoroalkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₁-C ₆)-alkoxyl-(C ₁-C ₄)-alkoxyl-(C ₁-C ₄)-alkyl, aryl, heteroaryl, (C ₇-C ₁₁)-aralkyl or there is the group of chemical formula shown in formula (Z)

-[CH ₂] _v-[O] _w-[CH ₂] _t-E (Z)

Wherein, E is heteroaryl, (C ₃-C ₈)-cycloalkyl or there is the phenyl of chemical formula shown in formula F

V is 0-6,

W is 0 or 1,

T is 0-3, and R ⁷, R ⁸, R ⁹, R ¹⁰And R ¹¹Can be identical or different, can be hydrogen, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₁-C ₆)-aralkyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₆)-alkyl thiol, (C ₁-C ₆)-hydroxy alkyl, (C ₁-C ₆)-alkoxyl-(C ₁-C ₆)-alkoxyl, (C ₁-C ₆)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₁-C ₆)-alkyl sulfinyl, (C ₁-C ₆)-alkyl sulfide acyl group, (C ₁-C ₆)-alkyl-carbonyl, (C ₁-C ₈)-alkoxy carbonyl, carbamoyl, N-(C ₁-C ₈)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₈)-alkyl-carbamoyl or (C ₇-C ₁₁)-aryl-alkyl amino formoxyl, optionally replaced by following group, i.e. fluorine, chlorine, bromine, trifluoromethyl, (C ₁-C ₆)-alkoxyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₆)-alkyl carbonyl oxy, phenyl, benzyl, phenoxy group, benzyloxy, NR ^YR ^Z, R wherein ^YAnd R ^ZIndependently be selected from hydrogen, (C ₁-C ₁₂)-alkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₃-C ₁₀)-cycloalkyl, (C ₃-C ₁₂)-thiazolinyl, (C ₃-C ₁₂)-alkynyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl, (C ₁-C ₁₂)-alkoxyl, (C ₇-C ₁₂)-aryloxy group, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl; Perhaps R ^YAnd R ^ZBe all-[CH ₂] _h, CH wherein ₂Can be O, S, N-(C ₁-C ₄)-alkyl-carbonyl imino group or N-(C ¹-C ⁴)-alkoxy carbonyl imino group; Phenyl sulfydryl, benzene sulfonyl, benzene sulfinyl, phenylamino sulfonyl, N-(C ₁-C ₈)-alkylsulfamoyl group or N, N-bis--(C ₁-C ₈)-alkylsulfamoyl group; Perhaps R ⁷And R ⁸, R ⁸And R ⁹, R ⁹And R ¹⁰Or R ¹⁰And R ¹¹All be selected from-[CH ₂] _nOr-chain of CH=CH-CH=CH-, wherein CH ₂Optional is O, S, SO, SO ₂Or NR ^YReplace; N is 3,4 or 5; If E is heteroaryl, described group can be R containing 1-3 ⁷-R ¹¹Defined substituent group, if or E be cycloalkyl, described group can containing one be R ⁷-R ¹¹Defined substituent group;

Perhaps when Q is NR ', R ⁴R ", wherein R ' and R " can be identical or different, can be specifically hydrogen, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl, (C ₁-C ₈)-alkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₁-C ₁₀)-alkyl-carbonyl, the optional (C replaced ₇-C ₁₆)-aromatic alkyl carbonyl or the optional (C replaced ₆-C ₁₂)-aryl carbonyl; Perhaps R ' and R " be-[CH ₂] _h, CH wherein ₂For O, S, N-acylimino or N-(C ₁-C ₁₀)-alkoxy carbonyl imino group, h is 3-7.

Y is N or CR ³

R ¹, R ²And R ³Can be identical or different, be specially hydrogen, hydroxyl, cyano group, trifluoromethyl, nitro, carboxyl, (C ₁-C ₂₀)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyloxy, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₁₂)-alkoxyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₁₂)-alkoxyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₈)-alkyl-(C ₁-C ₆)-alkoxyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkoxyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₇-C ₁₆)-arylalkenyl, (C ₇-C ₁₆)-sweet-smelling alkynyl, (C ₂-C ₂₀)-thiazolinyl, (C ₂-C ₂₀)-alkynyl, (C ₁-C ₂₀)-alkoxyl, (C ₂-C ₂₀)-alkene oxygen base, (C ₂-C ₂₀)-alkynyloxy group, look yellow oxygen base, (C ₁-C ₂₀)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₆)-alkoxyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₆)-alkoxyl, (C ₁-C ₁₆)-hydroxyalkyl, (C ₆-C ₁₆)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₂-C ₂₀)-alkene oxygen base-(C ₁-C ₆)-alkyl, (C ₂-C ₂₀)-alkynyloxy group-(C ₁-C ₆)-alkyl, look yellow oxygen base-(C ₁-C ₆)-alkyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₂₀)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, cinnamoyl, (C ₂-C ₂₀)-alkenyl carbonyl, (C ₂-C ₂₀)-alkynyl carbonyl, (C ₁-C ₂₀)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxy carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cyclo alkoxy carbonyl, (C ₂-C ₂₀)-allyloxycarbonyl, look yellow oxygen base carbonyl, (C ₂-C ₂₀)-alkynyloxy group carbonyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₆)-alkoxy carbonyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₆)-alkoxy carbonyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkoxy carbonyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₆)-alkoxy carbonyl, (C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₃-C ₈)-cycloalkyl carbonyl oxygen base, (C ₆-C ₁₂)-aryl-carbonyl oxygen, (C ₇-C ₁₆)-aralkyl carbonyl oxygen base, cinnamoyloxy group, (C ₂-C ₁₂)-thiazolinyl carbonyl oxygen base, (C ₂-C ₁₂)-alkynyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N, N-bis-ring-(C ₃-C ₈)-alkyl-carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-((C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkyl) carbamoyl, N-(C ₁-C ₆)-alkyl-N-((C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkyl) carbamoyl, N-(+)-dehydrogenation fir base carbamoyl, N-(C ₁-C ₆)-alkyl-N-(+)-dehydrogenation fir base carbamoyl, N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl; CON (CH ₂) _h, CH wherein ₂Can be O, S, N-(C ₁-C ₈)-alkyl imino, N-(C ₃-C ₈)-cycloalkyl imino group, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₄)-alkyl imino, N-(C ₆-C ₁₂)-aryl imino group, N-(C ₇-C ₁₂)-aralkyl imino group, N-(C ₁-C ₄)-alkoxyl-(C ₁-C ₆)-alkyl imino replaces, and h is 3-7; Carbamoyloxy with chemical formula R

Wherein, R ^xAnd R ^vBe selected from independently of one another hydrogen, (C ₁-C ₆)-alkyl, (C ₃-C ₇The substituent group of the alpha-carbon atom of)-cycloalkyl, aryl or alpha amino acid, wherein aminoacid is natural L aminoacid or its D type isomer,

S is 1-5,

T is OH or NR ^*R ^*, R ^*, R ^*And R ^{* *}Can be identical or different, be selected from hydrogen, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₁)-aralkyl, (C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl, (+)-dehydrogenation fir base, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₁-C ₁₀)-alkanoyl, the optional (C that replaces ₇-C ₁₆)-aralkanoyl, the optional (C that replaces ₆-C ₁₂)-aroyl; Perhaps R ^*And R ^*Be CH ₂, CH wherein ₂Can be O, S, SO, SO ₂, N-acylamino-, N-(C ₁-C ₁₀)-alkoxy carbonyl imino group, N-(C ₁-C ₈)-alkyl imino, N-(C ₃-C ₈)-cycloalkyl imino group, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₄)-alkyl imino, N-(C ₆-C ₁₂)-aryl imino group, N-(C ₇-C ₁₆)-aralkyl imino group, N-(C ₁-C ₄)-alkoxyl-(C ₁-C ₆)-alkyl imino, h is 3-7;

Carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C ₇-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxy amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)--alkyl-carbonyl (alkanoyl) is amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N-(C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₂₀)-alkyl thiol, (C ₁-C ₂₀)-alkyl sulfinyl, (C ₁-C ₂₀)-alkyl sulfide acyl group, (C ₆-C ₁₂)-aryl sulfydryl, (C ₆-C ₁₂)-aryl sulfinyl, (C ₆-C ₁₂)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aralkyl sulfinyl, (C ₇-C ₁₆)-aralkyl sulfonyl, (C ₁-C ₁₂)-alkyl thiol-(C ₁-C ₆)-alkyl, (C ₁-C ₁₂)-alkyl sulfinyl-(C ₁-C ₆)-alkyl, (C ₁-C ₁₂)-alkyl sulfide acyl group-(C ₁-C ₆)-alkyl, (C ₆-C ₁₂)-aryl sulfydryl-(C ₁-C ₆)-alkyl, (C ₆-C ₁₂)-aryl sulfinyl-(C ₁-C ₆)-alkyl, (C ₆-C ₁₂)-aryl sulfonyl-(C ₁-C ₆)-alkyl, (C ₇-C ₁₆)-aralkyl sulfydryl-(C ₁-C ₆)-alkyl, (C ₇-C ₁₆)-aralkyl sulfinyl-(C ₁-C ₆)-alkyl, (C ₇-C ₁₆)-aralkyl sulfonyl-(C ₁-C ₆)-alkyl, sulfamoyl, N-(C ₁-C ₁₀)-alkylsulfamoyl group, N, N-bis--(C ₁-C ₁₀)-alkylsulfamoyl group, (C ₃-C ₈)-cycloalkyl sulfamoyl, N-(C ₆-C ₁₂)-alkylsulfamoyl group, N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-ammonia aryl sulfonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-alkyl aryl ammonium sulfonyl, (C ₁-C ₁₀)-amino-alkyl sulfinyl, N-((C ₁-C ₁₀)-alkyl)-(C ₁-C ₁₀)-amino-alkyl sulfinyl, (C ₇-C ₁₆)-aralkyl sulfonamido or N-((C ₁-C ₁₀)-alkyl)-(C ₇-C ₁₆)-aralkyl sulfonamido; Aryl or can be 1-5 identical or different group containing the aryl in aromatic yl group and replace wherein, they comprise hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C ₂-C ₁₆)-alkyl, (C ₃-C ₈)-cycloalkyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyloxy, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₁₂)-alkoxyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₁₂)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₁₂)-alkoxyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₈)-alkyl-(C ₁-C ₆)-alkoxyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkoxyl, (C ₆-C ₁₂)-aryl, (C ₇-C ₁₆)-aralkyl, (C ₂-C ₁₆)-thiazolinyl, (C ₂-C ₁₂)-alkynyl, (C ₁-C ₁₆)-alkoxyl, (C ₁-C ₁₆)-alkene oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group, (C ₇-C ₁₆)-aralkoxy, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₆)-alkoxyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₆)-alkoxyl, (C ₁-C ₈)-hydroxyalkyl, (C ₆-C ₁₆)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkoxyl-(C ₁-C ₆)-alkyl ,-O-[CH ₂] _x-C _fH _(2f+1-g)-F _g,-OCF ₂Cl ,-O-CF ₂-CHFCl, (C ₁-C ₁₂)-alkyl-carbonyl, (C ₃-C ₈)-naphthene base carbonyl, (C ₆-C ₁₂)-aryl carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl, (C ₁-C ₁₂)-alkoxy carbonyl, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkoxy carbonyl, (C ₆-C ₁₂)-aryloxycarbonyl, (C ₇-C ₁₆)-aromatic alkoxy carbonyl, (C ₃-C ₈)-cyclo alkoxy carbonyl, (C ₂-C ₁₂)-allyloxycarbonyl, (C ₂-C ₁₂)-alkynyloxy group carbonyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₆)-alkoxy carbonyl, (C ₇-C ₁₆)-aralkoxy-(C ₁-C ₆)-alkoxy carbonyl, (C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkoxy carbonyl, (C ₃-C ₈)-cycloalkyloxy-(C ₁-C ₆)-alkoxy carbonyl, (C ₁-C ₁₂)-alkynyl carbonyl oxygen base, (C ₂-C ₈)-cycloalkyl carbonyl oxygen base, (C ₆-C ₁₂)-aryl-carbonyl oxygen, (C ₇-C ₁₆)-aralkyl carbonyl oxygen base, cinnamoyloxy group, (C ₂-C ₁₂)-thiazolinyl carbonyl oxygen base, (C ₂-C ₁₂)-alkynyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl carbonyl oxygen base, (C ₁-C ₁₂)-alkoxyl-(C ₁-C ₁₂)-alkyl carbonyl oxy, (C ₆-C ₁₂)-aryloxy group carbonyl oxygen base, (C ₇-C ₁₆)-aralkoxy carbonyl oxygen base, (C ₃-C ₈)-cycloalkyloxy carbonyl oxygen base, (C ₂-C ₁₂)-alkene oxygen base carbonyl oxygen base, (C ₂-C ₁₂)-alkynyloxy group carbonyl oxygen base, carbamoyl, N-(C ₁-C ₁₂)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₁₂)-alkyl-carbamoyl, N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N, N-bis-ring-(C ₃-C ₈)-alkyl-carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₃-C ₈)-cycloalkyl amino formoxyl, N-((C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkyl) carbamoyl, N-(C ₁-C ₆)-alkyl-N-((C ₃-C ₈)-cycloalkyl-(C ₁-C ₆)-alkyl) carbamoyl, N-(+)-dehydrogenation fir base carbamoyl, N-(C ₁-C ₆)-alkyl-N-(+)-dehydrogenation fir base carbamoyl, N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₂)-aryl-amino-carbonyl, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formoxyl, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyl, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyl, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyl; CON (CH ₂) _h, CH wherein ₂Can be O, S, N-(C ₁-C ₈)-alkyl imino, N-(C ₃-C ₈)-cycloalkyl imino group, N-(C ₃-C ₈)-cycloalkyl-(C ₁-C ₇)-alkyl imino, N-(C ₆-C ₁₂)-aryl imino group, N-(C ₇-C ₁₂)-aralkyl imino group, N-(C ₁-C ₄)-alkoxyl-(C ₁-C ₆)-alkyl imino replaces, and h is 3-7; Carbamoyloxy, N-(C ₁-C ₁₂)-alkyl carbamoyloxy base, N, N-bis--(C ₁-C ₁₂)-alkyl carbamoyloxy base, N-(C ₃-C ₈)-cycloalkyl amino formyloxy, N-(C ₆-C ₁₂)-arylamino formyloxy, N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₆-C ₁₆)-arylamino formyloxy, N-(C ₁-C ₁₀)-alkyl-N-(C ₇-C ₁₆)-aryl alkyl amino formyloxy, N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-((C ₆-C ₁₂)-aryloxy group-(C ₁-C ₁₀) alkyl) carbamoyloxy, N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₁-C ₁₀)-alkoxyl-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₆-C ₁₆)-aryloxy group-(C ₁-C ₁₀)-alkyl) carbamoyloxy, N-(C ₁-C ₁₀)-alkyl-N-((C ₇-C ₁₆)-aralkoxy-(C ₁-C ₁₀)-alkyl) carbamoyloxy, amino, (C ₁-C ₁₂)-alkyl amino, two (C ₁-C ₁₂)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino, (C ₂-C ₁₂)-alkenyl amino, (C ₂-C ₁₂)-alkynyl is amino, N-(C ₆-C ₁₂)-arylamino, N-(C ₇-C ₁₁)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-Fang amino, (C ₁-C ₁₂)-alkoxyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-cycloalkyl amino carbonyl, (C ₆-C ₁₂)-aryl-amino-carbonyl, (C ₇-C ₁₆)-aromatic alkyl carbonyl is amino, (C ₁-C ₁₂)-alkyl-carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₃-C ₈)-naphthene base carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₆-C ₁₂)-aryl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₇-C ₁₁)-aromatic alkyl carbonyl-N-(C ₁-C ₁₀)-alkyl amino, (C ₁-C ₁₂)-alkyl-carbonyl-amino-(C ₁-C ₈)-alkyl, (C ₃-C ₈)-cycloalkyl amino carbonyl-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryl-amino-carbonyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aromatic alkyl carbonyl amino-(C ₁-C ₈)-alkyl, amino-(C ₁-C ₁₀)-alkyl, N-(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N.N-bis--(C ₁-C ₁₀)-alkyl amino-(C ₁-C ₁₀)-alkyl, N-(C ₃-C ₈)-cycloalkyl amino-(C ₁-C ₁₀)-alkyl, (C ₁-C ₁₂)-alkyl thiol, (C ₁-C ₁₂)-alkyl sulfinyl, (C ₁-C ₁₂)-alkyl sulfide acyl group, (C ₆-C ₁₆)-aryl sulfydryl, (C ₆-C ₁₆)-aryl sulfinyl, (C ₆-C ₁₆)-aryl sulfonyl, (C ₇-C ₁₆)-aralkyl sulfydryl, (C ₇-C ₁₆)-aryl sulfinyl or (C ₇-C ₁₆)-aryl sulfonyl;

Perhaps R ¹With R ²Or R ²With R ³Form chain [CH ₂] _o, can be saturated chain, can be also the unsaturated chain containing the two keys of C=C, wherein 1 or 2 CH ₂Optional is O, S, SO, SO ₂Or NR ' replacement, wherein R ' is hydrogen, (C ₆-C ₁₂)-aryl, (C ₁-C ₈)-alkyl, (C ₁-C ₈)-alkoxyl-(C ₁-C ₈)-alkyl, (C ₇-C ₁₂)-aralkoxy-(C ₁-C ₈)-alkyl, (C ₆-C ₁₂)-aryloxy group-(C ₁-C ₈)-alkyl, (C ₁-C ₁₀)-alkanoyl, the optional (C that replaces ₇-C ₁₆)-aralkanoyl, the optional (C that replaces ₆-C ₁₂)-aroyl; O is 3,4 or 5;

Perhaps R ¹With R ²Or R ²With R ³Form 5,6,7,8-tetrahydroisoquinoline ring, 5,6,7,8-tetrahydroquinoline ring or 5,6,7,8-tetrahydrochysene cinnoline ring with the pyridine that comprises them or pyridazine;

Perhaps R ¹With R ²Or R ²With R ³Form 5 yuan or 6 yuan of aromatic carbon rings or fragrant heterocycle;

Perhaps R ¹With R ²Or R ²With R ³The heterocyclic system that forms optional replacement with the pyridine that comprises them or pyridazine, described system can be selected from thienopyridine, furo pyridine, pyridopyridine, pyrimido pyridine, imidazopyridine, thiazole pyridine, oxazole pyridine, quinoline, isoquinolin and cinnoline; Wherein quinoline, isoquinolin and cinnoline should meet formula Ia, Ib and Ic:

R wherein ¹²-R ²³Independent separately, define same R ¹, R ²And R ³

Perhaps radicals R ¹With R ²Form compound shown in formula Id with the pyridine that comprises them:

Wherein V is S, O or NR ^k, R wherein ^kBe selected from hydrogen, (C ₁-C ₆)-alkyl, aryl or benzyl; Wherein aryl can be that 1-5 substituent group replaces as mentioned above;

And R ²⁴, R ²⁵, R ²⁶And R ²⁷Independent separately, define same R ¹, R ²And R ³

F is 1-8;

G is 0 or 1 to (2f+1);

X is 0-3;

H is 3-7;

The physiologically active salt and the prodrug that comprise above-claimed cpd.

The example of compound shown in formula (I) is shown in and is set forth in European patent EP 065096 and EP0650961.In the listed all compounds of EP065096 and EP0650961, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.The example of compound shown in formula (I) includes, but is not limited to [(3-hydroxyl-pyridine-2-carbonyl)-amino]-acetic acid (compound G) and [(3-methoxyl group-pyridine-2-carbonyl)-amino]-acetic acid (compound P).

In addition, the example of compound shown in formula (I) is shown in and is set forth in United States Patent (USP) 5658933.In the listed all compounds of United States Patent (USP) 5658933, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.The example of compound shown in formula (I) includes, but is not limited to hydrochloric acid 3-Methoxy Pyridine-2-carboxylic acid N-(((hexadecane oxygen base)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((1-octyloxy)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((hexyloxy)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((butoxy)-carbonyl)-methyl)-amide; Racemic tartaric acid 3-Methoxy Pyridine-2-carboxylic acid N-(((2-oxygen in ninth of the ten Heavenly Stems base)-carbonyl)-methyl)-amide; 3-Methoxy Pyridine-2-carboxylic acid N-(((oxygen base in heptan)-carbonyl)-methyl)-amide; 3-phenoxypyridines-2-carboxylic acid N-(((octyloxy)-carbonyl)-methyl)-amide; 3-benzyloxy pyridine-2-carboxylic acids N-(((butoxy)-carbonyl)-methyl)-amide; 5-(((3-(1-butoxy)-propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-((benzyloxy carbonyl)-methyl)-amide; 5-(((3-(1-butoxy)-propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-(((1-butoxy)-carbonyl)-methyl)-amide; 5-(((3-dodecyloxy propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-((benzyloxy carbonyl)-methyl)-amide.

Shown in formula (I), other compounds have substituted heterocycle Carboxylamide described in United States Patent (USP) 5620995; The pyridone of 3-described in United States Patent (USP) 6020350-2-Carboxylamide ester; Sulfonyl-amino-carbnyl-pyridine described in United States Patent (USP) 5607954-2-Carboxylamide; Sulfonyl-amino-carbnyl-pyridine described in United States Patent (USP) 5610172 and 5620996-2-Carboxylamide and sulfonyl-amino-carbnyl-pyridine-2-Carboxylamide ester.In listed all compounds in these patents, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.

The example of compound shown in formula (Ia) is shown in and is set forth in United States Patent (USP) 5719164 and 5726305.In listed all compounds in aforementioned patent, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.The example of compound shown in formula (I) includes, but is not limited to N-((3-hydroxyl-6-isopropoxy-quinoline-2-carbonyl)-amino) acetic acid (compound H), N-((6-(1-butoxy)-3-hydroxyquinoline-2-yl)-carbonyl) glycine, [(3-hydroxyl-6-trifluoromethoxy-quinoline-2-carbonyl) amino] acetic acid (Compound I), N-((6-chloro-3-hydroxyl quinoline-2-yl)-carbonyl) glycine, N-((7-chloro-3-hydroxyl quinoline-2-yl)-carbonyl) glycine and [(6-chloro-3-hydroxyl-quinoline-2-carbonyl) amino] acetic acid (compound O).

The example of compound shown in formula (Ib) is shown in and is set forth in United States Patent (USP) 6093730.In listed all compounds in United States Patent (USP) 6093730, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.The example of compound shown in formula (Ib) includes, but is not limited to N-((1-chloro-4-hydroxyl-7-(2-propoxyl group) isoquinolin-3-yl) carbonyl) glycine, N-((1-chloro-4-hydroxyl-6-(2-propoxyl group) isoquinolin-3-yl) carbonyl) glycine, N-((1-chloro-4-hydroxyl-isoquinolin-3-carbonyl) amino) acetic acid (compd B), N-((1-chloro-4-hydroxyl-7-methoxyl group isoquinolin-3-yl) carbonyl) glycine, N-((1-chloro-4-hydroxyl-6-methoxyl group isoquinolin-3-yl) carbonyl) glycine, N-((7-butoxy)-1-chloro-4-hydroxyl isoquinolin-3-yl) carbonyl) glycine, N-((6-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl) amino) acetic acid (compound J), ((7-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl) amino) methyl acetate (compound K), N-((7-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl) amino) acetic acid (compound L), N-((8-chloro-4-hydroxyl isoquinolin-3-yl) carbonyl) glycine, N-((7-butoxy-4-hydroxyl isoquinolin-3-carbonyl) amino) acetic acid (compound M).

The compound relevant to formula (I) that can be used for equally the inventive method includes, but is not limited to 6-cyclohexyl-1-hydroxy-4-methyl-1H-pyridin-2-ones (compound N), 7-(4-methyl-piperazine-1-methyl)-5-phenyl sulfanylmethyl-quinoline-8-hydroxyl (Compound D), 4-nitro-quinoline-8-hydroxyl (compd E) and 5-butoxymethyl-quinoline-8-hydroxyl (compound F 17-hydroxy-corticosterone).In addition, the invention provides other examples of compounds, wherein position A and B can together with form caproic acid, cyanogen methyl, 2-amino-ethyl, benzoic acid, 1H-benzimidazolyl-2 radicals-methyl etc.

In other embodiments, the inventive method compound used therefor is selected from compound shown in formula (II)

R wherein ²⁸Hydrogen, nitro, amino, cyano group, halogen, (C ₁-C ₄)-alkyl, carboxyl or their easy metabolism ester; (C ₁-C ₄)-alkyl amino, two-(C ₁-C ₄)-alkyl amino, (C ₁-C ₆)-alkoxy carbonyl, (C ₂-C ₄)-alkanoyl, hydroxyl-(C ₁-C ₄)-alkyl, carbamoyl, N-(C ₁-C ₄)-alkyl-carbamoyl, (C ₁-C ₄)-alkylthio group, (C ₁-C ₄)-alkyl sulfinyl, (C ₁-C ₄)-alkyl sulfide acyl group, thiophenyl, phenyl sulfinyl, phenyl sulfonyl, described phenyl may optionally be 1-4 identical or different halogen, (C ₁-C ₄)-alkoxyl, (C ₁-C ₄)-alkyl, cyano group, hydroxyl, trifluoromethyl, fluoro-(C ₁-C ₄)-alkylthio group, fluoro-(C ₁-C ₄)-alkyl sulfinyl, fluoro-(C ₁-C ₄)-alkyl sulfide acyl group, (C ₁-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxy carbonyl, N, N-bis--[(C ₁-C ₄)-alkyl] carbamoyl-(C ₁-C ₄)-alkoxy carbonyl, (C ₁-C ₄)-alkyl amino-(C ₂-C ₄)-alkoxy carbonyl, two-(C ₁-C ₄)-alkyl amino-(C ₂-C ₄)-alkoxy carbonyl, (C ₁-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxy carbonyl, (C ₂-C ₄)-alkanoyloxy-(C ₁-C ₄)-alkyl or N-[amino-(C ₂-C ₈)-alkyl]-carbamoyl;

R ²⁹Hydrogen, hydroxyl, amino, cyano group, halogen, (C ₁-C ₄)-alkyl, carboxyl or their easy metabolism ester derivant; (C ₁-C ₄)-alkyl amino, two-(C ₁-C ₄)-alkyl amino, (C ₁-C ₆)-alkoxy carbonyl, (C ₂-C ₄)-alkanoyl, (C ₁-C ₄)-alkoxyl, carboxyl-(C ₁-C ₄)-alkoxyl, (C ₁-C ₄)-alkoxy carbonyl-(C ₂-C ₄)-alkoxyl, carbamoyl, N-(C ₁-C ₈)-alkyl-carbamoyl, N, N-bis--(C ₁-C ₈)-alkyl-carbamoyl, N-[amino-(C ₂-C ₈)-alkyl] carbamoyl, N-[(C ₁-C ₄)-alkyl amino-(C ₁-C ₈)-alkyl] carbamoyl, N-[bis--(C ₁-C ₄)-alkyl amino-(C ₁-C ₈)-alkyl] carbamoyl, N-cyclohexyl carboxyamide base, N-cyclopenta carbamoyl, N-(C ₁-C ₄)-alkyl-cyclohexyl carbamoyl, N-(C ₁-C ₄)-alkyl cyclopenta carbamoyl, N-phenyl amino formoxyl, N-(C ₁-C ₄)-alkyl-N-phenyl amino formoxyl, N, N-diphenyl amino formoxyl, N-[phenyl-(C ₁-C ₄)-alkyl] carbamoyl, N-(C ₁-C ₄)-alkyl-N-[phenyl-(C ₁-C ₄)-alkyl] carbamoyl or N, N-bis-[phenyl-(C ₁-C ₄)-alkyl] carbamoyl, described phenyl may optionally be 1-4 identical or different halogen, (C ₁-C ₄)-alkoxyl, (C ₁-C ₄)-alkyl, cyano group, hydroxyl, trifluoromethyl, N-[(C ₂-C ₄)-alkanoyl] carbamoyl, N-[(C ₁-C ₄)-alkoxy carbonyl] carbamoyl, the fluoro-(C of N-[ ₂-C ₆)-alkyl] carbamoyl, N, the fluoro-(C of N-[ ₂-C ₆)-alkyl]-N-(C ₁-C ₄)-alkyl-carbamoyl, N, the fluoro-(C of N-[bis- ₂-C ₆)-alkyl] carbamoyl, 1-pyrrole radicals-carbonyl, piperidino carbonyl, 1-piperazinyl-carbonyl, morpholinyl carbonyl, wherein heterocyclic group is optionally 1-4 following group replacement, i.e. (C ₁-C ₄)-alkyl, benzyl, 1,2,3,4-tetrahydroisoquinoline-2-base-carbonyl, N, N-[bis--(C ₁-C ₄)-alkyl] thiocarbamoyl, N-(C ₂-C ₄)-alkanoylamino or N-[(C ₁-C ₄)-alkoxy carbonyl] amino;

R ³⁰Hydrogen, (C ₁-C ₄)-alkyl, (C ₂-C ₄)-alkoxyl, halogen, nitro, hydroxyl, fluoro-(1-4C) alkyl or pyridine radicals;

R ³¹Hydrogen, (C ₁-C ₄)-alkyl, (C ₂-C ₄)-alkoxyl, halogen, nitro, hydroxyl, fluoro-(C ₁-C ₄) alkyl, pyridine radicals or methoxyl group;

R ³²Hydrogen, hydroxyl, amino, (C ₁-C ₄)-alkyl amino, two (C ₁-C ₄)-alkyl amino, halogen, (C ₁-C ₄)-alkoxyl-(C ₂-C ₄)-alkoxyl, fluoro-(C ₁-C ₆)-alkoxyl, pyrroles-1-base, piperidyl, piperazine-1-base or morpholinyl, wherein heterocyclic radical is optionally 1-4 identical or different (C ₁-C ₄)-alkyl or phenyl replaces;

R ³³And R ³⁴Be selected from independently of one another hydrogen, (C ₁-C ₄)-alkyl and (C ₁-C ₄)-alkoxyl;

The pharmaceutical salts and the prodrug that also comprise them.

The example of compound shown in formula (II) is shown in and is set forth in United States Patent (USP) 5916898 and 6200974 and international monopoly bulletin WO99/21860.In listed all compounds in aforementioned patent and bulletin, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.Shown in formula (II), the example of compound comprises 4-oxygen-1, 4-dihydro-[1, 10] phenanthroline-3-carboxylic acid (compd A) (referring to, such as (1974) Chem Abstracts 81:424 such as Seki, No.21), 3-carboxyl-5-hydroxyl-4-oxygen-3, 4-dihydro-1, the 10-phenanthroline, 3-carboxyl-5-methoxyl group-4-oxygen-3, 4-dihydro-1, the 10-phenanthroline, 5-methoxyl group-4-oxygen-1, 4-dihydro [1, 10] phenanthroline-3-carboxylic acid, ethyl ester, 5-methoxyl group-4-oxygen-1, 4-dihydro [1, 10] phenanthroline-3-carboxylic acid (compound Q) and 3-carboxyl-8-hydroxyl-4-oxygen-3, 4-dihydro-1, the 10-phenanthroline.

In other embodiments, the compound for the inventive method is selected from compound or their pharmaceutical salts shown in formula (III)

Wherein a is the integer between 1-4;

B is the integer between 0-4;

C is the integer between 0-4;

Z is selected from (C ₃-C ₁₀) cycloalkyl, by one or more Y ¹Independent (the C replaced ₃-C ₁₀) cycloalkyl, 3-10 unit's Heterocyclylalkyl and by one or more Y ¹The independent 3-10 unit Heterocyclylalkyl replaced; (C ₅-C ₂₀) aryl, by one or more Y ¹Independent (the C replaced ₅-C ₂₀) aryl, 5-20 unit's heteroaryl and by one or more Y ¹The independent 5-20 unit heteroaryl replaced;

Ar1 is selected from (C ₅-C ₂₀) aryl, by one or more Y ²Independent (the C replaced ₅-C ₂₀) aryl, 5-20 unit's heteroaryl and by one or more Y ²The independent 5-20 unit heteroaryl replaced;

Each Y ¹Independently be selected from lipophilic function base, (C ₅-C ₂₀) aryl, (C ₆-C ₂₆) alkylaryl, 5-20 unit's heteroaryl and the assorted virtue level of 6-26 unit alkyl;

Each Y <sup TranNum="4273"> 2 </ sup> is independently selected from-R ',-OR',-OR ",-SR ',-SR",-NR'R',-NO <sub TranNum = "4274"> 2 </ sub>,-CN, - halogen, - trihalomethyl, - trihalomethoxy,-C (O) R ',-C (O) OR',-C (O) NR'R ',-C (O) NR'OR',-C (NR'R ') = NO R',-NR'-C (O) R ',-SO2R',-SO2R ",-NR'-SO2-R'-,-NR'-C (O)-NR'R ', tetrazol-5 - yl -,-NR'-C (O)-OR',-C (NR 'R ' ) = N R',-S (O)-R ',-S (O)-R ", and-NR'-C (S) NR'R';

Independently be selected from-H of each R ', (C ₁-C ₈) alkyl, (C ₂-C ₈) thiazolinyl and (C ₂-C ₈) alkynyl;

Each R " independently be selected from (C ₅-C ₂₀) aryl and by one or more-OR ' ,-SR ' ,-NR ' R ' ,-NO ₂,-CN ,-halogen ,-(C that trihalomethyl group independently replaces ₅-C ₂₀) aryl;

Or wherein c is 0, Ar ¹Be that N ' replaces urea-aryl, described compound has structure shown in formula (IIIa):

Perhaps its pharmaceutical salts;

In formula, a, b and Z definition are the same;

R ³⁵And R ³⁶Be selected from independently of one another hydrogen, (C ₁-C ₈) alkyl, (C ₂-C ₈) thiazolinyl, (C ₂-C ₈) alkynyl, (C ₃-C ₁₀) cycloalkyl, (C ₅-C ₂₀) aryl, (C ₅-C ₂₀) substituted aryl, (C ₆-C ₂₆) alkylaryl, (C ₆-C ₂₆) substituted alkyl aryl, 5-20 unit heteroaryl, 5-20 unit substituted heteroaryl, 6-26 unit's miscellaneous alkyl aryl and 6-26 unit substituted alkyl heteroaryl;

R ³⁷Be selected from independently of one another hydrogen, (C ₁-C ₈) alkyl, (C ₂-C ₈) thiazolinyl and (C ₂-C ₈) alkynyl.

The example of compound shown in formula (III) is shown in that being set forth in international monopoly announces WO00/50390.In listed all compounds in WO00/50390, the particularly final products of compound claim part and working example, listed compound draws for reference in the present patent application.Shown in formula (III), the example of compound comprises 3-{[4-(3; 3-dibenzyl-urea groups)-benzene sulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide. (Compound C), 3-{{4-[3-(the chloro-phenyl of 4-)-urea groups]-the benzene sulfonyl }-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide. and 3-{{4-[3-(1,2-diphenyl-ethyl)-urea groups]-the benzene sulfonyl }-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..

General action mechanism based on the 2-oxoglutaric acid dioxygenase member of family, as activity to Fe ²⁺With the dependency of 2-oxoglutaric acid, the present invention relates in some aspects and uses compound, comprises that above-claimed cpd suppresses the HIFa hydroxylating, thereby stablizes HIF α in the mode that does not rely on oxygen.In addition, embodiments of the invention and data show, utilize these compounds can stablize HIF α, thus in vivo with external generation HIF regulator gene product.In the specific embodiment, these compounds have unique benefit aspect prevention and treatment ischemic and hypoxia disease.

During HIF α in the cell that the inventive method is grown in stablizing normal oxygen environment, dosage is had to dependency.Although, under the compounds of this invention exists, dissimilar cell shows the HIF α of varying level, the cell line of measuring all demonstrates the HIF α Stabilization of certain level.In undressed cell, the level of HIF α is generally very low, can't detect.

HIF α stable causes be the HIF dependent gene in vivo with external expression, comprise the gene code angiogenesis factor, as VEGF, Flt-1, EG-VEGF, PAI-1, medullin and Cyr61.Therefore, in induction of vascular growth with prevent that aspect tissue injury that ischemia and hypoxia cause, the ability of stablizing HIF α has potential benefit.For example, in epithelium, the transgenic mouse of the active HIF-1 α of expression structure is strengthened to the expression of each VEGF isomer, and the epidermal hair tubule significantly increases.After expressing unlike single VEGF isomer is undue, HIF α induces the super vascularity of generation edema, inflammation or angiorrhexis not to occur.(see (2001) Genes Dev 15:2520-2532 such as Elson; Detmar etc. (1998) J Invest Derm 111:1-6; Larcher etc. (1998) Oncogene 17:303-311; Thurston etc. (1999) Science 286:2511-2514) therefore, in some aspects, the inventive method can be used to induce and produces the medical treatment angiogenic growth, and it relates to the growth of collateral blood vessels, makes ischemic tissue again form blood vessel.

In addition, the inventive method makes the oxygen consumption in cell reduce according to dosage, and can not produce any impact to cell viability.Stable HIF complex can activate with the absorption of glucose and utilize relevant protein expression, as glucose transporter (GluT)-1 and GluT-3; Aldolase-A, ENO1, hexokinase-1 and-2, phosphofructokinase-L and-C.After HIF α is stable, oxygen consumption may reduce, because cellular metabolism is from generating and turn to the anaerobism energy to generate according to oxygen energy.Therefore, the inventive method can be used to produce power under hypoxia condition, and this is conducive to ischemic and hypoxia disease, as peripheral arterial disease, DVT, angina pectoris, pulmonary infarction, apoplexy and myocardial infarction.The present invention also provides increases the method that somatic cell absorbs and utilize glucose, usually can be used for the quality other diseases.

The present invention also provides the method that improves oxygen carrying capacity, the transportation and the utilization that for example induce and produce erythrocyte, promote ferrum.Particularly, the inventive method has promoted the expression of erythropoietin (EPO), and it is naturally occurring hormone, can excite erythrocytic generation.(referring to, the pending trial U.S. Patent application of for example owning together _ _ _ _ _, exercise question is " method of giving birth to erythropoietin (EPO) in increasing ", registration, quote in full at this on the same day.) method that strengthens enzyme and protein expression is specifically related to absorption, transportation and the processing of ferrum.These enzymes and albumen include, but is not limited to siderophillin and TfR, and they promote Railway transportation to be passed in erythrocytic tissue and ceruloplasmin together, and are absorbed; Ceruloplasmin is that ferrous ion is oxidized to the needed Ferroxidase of iron ion.Due to siderophillin can only in conjunction with and transport iron ion, ceruloplasmin has important function in the process that ferrum is provided to tissue.Give birth to transportation and the utilization that erythropoietin promotes ferrum simultaneously in the inventive method can increase, it has special benefit for delivery of oxygen in normal oxygen and low-oxygen environment.

One aspect of the present invention provides the method for neuroprotective, for example, by stablizing HIF α.For example, VEGF and EPO can neuroprotectives.(referring to, such as (2000) Proc Natl Acad Sci USA 97:10242-10247 such as Jin; Bocker-Meffert etc. (2002) Invest Ophthalmol Vis Sci 43:2021-2026; Buemi etc. (2002) Clin Sci (Lond) 103:275-282; Siren etc. (2001) Proc Natl Acad Sci USA98:4044-4049).If introduce EPO before the ischemia event occurs, it can also accelerate the recovery of spinal cord injury, and can neuroprotective.(referring to, such as (2002) Proc Natl Acad Sci USA 99:9450-9455 such as Gorio; Dawson (2002) Lancet 359:96-97) because the inventive method can promote the neuroprotective factor; expression as VEGF and EPO; described method can provide neuroprotective; thereby can be used for the treatment of, pretreat is lived the prevention various diseases; such as comprising diabetic neuropathy, apoplexy, neurodegenerative disease, injured (such as concussion, spinal cord impaired etc.); perhaps for before operation, because operation likely causes cerebral ischemia reperfusion injury etc.

For the protection severe ischemic, damage has good result to the hypoxia preconditioned.Because the initial reaction of hypoxia is the stabilisation of HIF α and subsequently to the activation of HIF regulator gene, the inventive method will be in normal oxygen environment the simulated ischemia preconditioned.For example, described method can be applied before operation, because the ischemical reperfusion injury in operation likely produces harmful result to the patient.This prophylactic treatment is application before the ischemia event occurs, and can apply in any moment before the ischemia event occurs, and can apply a course for the treatment of, also can apply a plurality of courses for the treatment of.

The inventive method can also be worked in coordination with and be regulated the gene related in oxidation emergency and blood vessel quality.These genes comprise induced oxidation one nitrogen synzyme (iNOS) and heme oxidation kinases 1.In several animal specimen, producing iNOS is also the beneficial effect that the hypoxia preconditioned is brought.(referring to, such as (2002) BMCGastroenterol 2:22-27 such as Serracino-Inglott; Kuntscher etc. (2002) Br J Plast Surg 55:430-433) especially, the activity of blocking-up iNOS can reduce but can not eliminate the beneficial effect that preconditioned is brought, but the generation of non-specific blocking protein can be eliminated the benefit that preconditioned is brought fully.(Wang etc. (2002) Cardiovasc Res 56:33-42) shows this, and iNOS is the important component part of the physiological reaction of preconditioned generation, but is not unique factor.Because the inventive method can be worked in coordination with the various factors of adjusting, comprise the iNOS related in the hypoxia reaction, the inventive method can be reproduced the benefit that the hypoxia preconditioned has more accurately.

Use the method for the compounds of this invention

The invention provides the method that suppresses HIF α hydroxylation, stablize thus HIF and activate the expression of HIF regulator gene.Described method can be used for prevention, pretreat or treatment HIF disease, comprises ischemic and hypoxia disease.For instance, these diseases comprise myocardial infarction, hepatic ischemia, renal ischaemia and apoplexy; The periphery angiopathy, ulcer, burn and chronic trauma; Pulmonary infarction; Ischemical reperfusion injury, the ischemical reperfusion injury for example occurred in operation and organ transplantation.In a kind of minority mode, the invention provides before ischemia or hypoxia occur, central or stablize afterwards the method, particularly ischemia or the hypoxia relevant with myocardial infarction, apoplexy or renal ischemic reperfusion injury of HIF α.

One aspect of the present invention provides the method for the treatment of various ischemics and hypoxia disease, the method for particularly being treated with above-claimed cpd.In one embodiment, the inventive method application after ischemia or hypoxia generation can bring therapeutic effect preferably.For example, when the inventive method is used for myocardial infarction, can significantly reduces M & M, and can significantly improve cardiac structure and function.In addition, to liver poisoning ischemic injuries application the inventive method, can improve liver function.Hypoxia is the important behaviour of hepatopathy, the chronic hepatopathy particularly caused as ethanol by the liver poisoning compound.In addition, HIF α, for example nitric oxide synzyme and glucose transporter-1 induces the gene expression of generation to be strengthened in hepatopathy due to ethanol.(referring to, such as (1997) Hepatology25:920-926 such as Areel; Strubelt (1984) Fundam Appl Toxicol 4:144-151; Sato (1983) PharmacolBiochem Behav 18 (Suppl 1): 443-447; Nanji etc. (1995) Am J Pathol 146:329-334; Morio etc. (2001) Toxicol Appl Pharmacol 172:44-51)

Therefore, the invention provides the method for the treatment of ischemic or hypoxia disease, described method comprises the compound or pharmaceutically acceptable salt thereof of using effective dose to treatment target, use separately or with the pharmaceutical excipient combined administration.In one embodiment, compound is used immediately after the acute ischemic disease occurs, for example myocardial infarction, pulmonary infarction, intestinal obstruction, cerebral infarction and renal ischemic reperfusion injury.In another embodiment, compound can be applied to the patient who suffers from after diagnosing the chronic ischemia disease, for example heart sclerosis, the degeneration of speckle shape, pulmonary infarction, the exhaustion of acute respiration system, newborn respiratory depression syndrome and congestive heart failure.In another embodiment, described compound is used immediately after injured.

The potential patient who another aspect of the present invention is ischemic or hypoxia disease provides the method for being treated with above-claimed cpd, for example very likely suffers from arteriosclerotic individuality.Cause that arteriosclerotic latency comprises hyperlipidemia, smoking, hypertension, diabetes, insulin is too high and abdominal obesity.Therefore, the invention provides the method for prevention ischemic tissue damage, described method comprises the compound or pharmaceutically acceptable salt thereof of using effective dose to treatment target, use separately or with the pharmaceutical excipient combined administration.In one embodiment, described compound can be used according to the prior disease of diagnosing out, for example hypertension, diabetes, arterial occlusion disease, chronic venous insufficiency of function, Raynaud's disease, chronic skin ulcer, sclerosis, congestive heart failure and system sclerosis.

In a specific embodiment, described method can be used to promote the formation of wounded tissue, wound and ulcer medium vessels and/or granulation tissue.For example, research shows, the compounds of this invention can effectively promote the formation of granulation tissue in trauma care.Granulation tissue comprises new life but still leaks the blood vessel of blood and the interim substrate of plasma protein, as Fibrinogen and blood plasma Fibronectin.Inflammatory cells, platelet and be excited in endothelium the somatomedin energy excitation fiber archeocyte and migration and the hypertrophy of endotheliocyte in granulation tissue that discharge.If angiogenic growth or nerve excitation are impaired, ulcer can occur.The inventive method can effectively promote the formation of granulation tissue.Therefore, Therapeutic Method provided by the invention can be used for treating tissue injury that the patient causes because infraction waits, the wound that causes because of wound etc. or the chronic trauma or the ulcer that cause because of diseases such as diabetes.Described method comprises the compound or pharmaceutically acceptable salt thereof of using as required effective dose to treatment target, use separately or with the pharmaceutical excipient combined administration.

The present invention provides on the other hand and has used described compound the patient to be carried out to the method for pretreat, to alleviate or to prevent the deterioration of ischemic or hypoxia tissue injury.The inventive method was applied and can be produced therapeutic effect preferably before suffering from ischemic or hypoxia disease.For example, from statistical result, can find out, before inducing the generation myocardial infarction, application the inventive method has obviously been improved the 26S Proteasome Structure and Function of heart.In addition, the inventive method is applied and can be produced therapeutic effect preferably immediately before ischemic damage and reperfusion damage occurs and in generating process, has obviously reduced the Diagnostic parameters relevant with renal failure.

Therefore, the invention provides the patient is launched to pretreat, to alleviate or prevention ischemic or hypoxia tissue injury, described method for example comprises, for example, to ischemic medical history (myocardial infarction) being arranged or having the patient of chronic ischemia symptom (angina pectoris) to use the compound or pharmaceutically acceptable salt thereof of effective dose, use separately or with the pharmaceutical excipient combined administration.In another embodiment, according to the indicative physical parameter of possibility ischemia or hypoxia, use described compound, for example individual through general anesthesia or temporarily in high latitude work.In another embodiment, the inventive method can be used in organ transplantation, be used for organ donor is carried out to pretreatment, and at organ armour from shifting out in body but before being transplanted to the receiver.

The research of front shows, for some compound of the inventive method, can effectively suppress the collagen prolyl 4 hydroxylase.Although people recognize from initial infraction or wound, recover to deposit connective tissue in necrotic area, with regard to forming cicatrix, the present invention does not show negative effect in treatment.Therefore, in view of some compound of the present invention has advantages of aspect treatment and prevention hypoxia tissue injury and cystic fibrosis, the present invention proposes a kind of " two curative effect " method, be used for the treatment of or prevent ischemic or hypoxia disease, comprise the concurrent ischemia of reactive fibreization or hypoxia, for example myocardial infarction and the congestive heart failure that causes thus.Described method can suppress with a kind of compound more than one 2-oxoglutaric acid dioxygenase, for example HIF prolyl hydroxylase and collagen prolyl 4 hydroxylase, and they have identical or different specificity.Perhaps, described method application compound capable of being combined, wherein every kind of compound suppresses a kind of 2-oxoglutaric acid dioxygenase specially, and for example a kind of compound suppresses the HIF prolyl hydroxylase specially, and the second compound suppresses the collagen prolyl 4 hydroxylase specially.

The compounds of this invention can suppress one or more 2-oxoglutaric acid dioxygenases on the one hand.In one embodiment, described compound can suppress at least two 2-oxoglutaric acid dioxygenase members of family, for example HIF prolyl hydroxylase and HIF agedoite hydroxylase (FIH-1), and their specificity can be identical or different.In another embodiment, described compound has specificity to a kind of 2-oxoglutaric acid dioxygenase, HIF prolyl hydroxylase for example, and other members of the same clan are had seldom and even are not had specificity.

Described compound can be used in conjunction with other various treatment meanss.In one embodiment, described compound can be used with another kind of 2-oxoglutaric acid dioxygenase inhibitor simultaneously, and these two kinds of compounds have different specificitys to the single 2-oxoglutaric acid dioxygenase member of family.These two kinds of compounds can be used by certain proportion simultaneously.The applicable ratio of TA process or particular patient can utilize this area prior art to measure.Perhaps, two compounds can be used successively in therapeutic process, for example, for myocardial infarction.In a specific implementations, a kind of compound suppresses HIF prolyl hydroxylase activity specially, and the second compound suppresses collagen prolyl 4 hydroxylase activity specially.In another embodiment, a kind of compound suppresses HIF prolyl hydroxylase activity specially, and the second compound suppresses HIF agedoite hydroxylase activity specially.In another embodiment, described compound is used from the therapeutic agent that another has different binding modes simultaneously, such as ACE inhibitor (ACEI), angiotensin-II receptor blocking agent (ARB), inhibin, diuretic, digoxin, carnitine etc.

Medicine ingredients and route of administration

The present composition can directly be inputted, and also can together with suitable carrier or excipient, form Pharmaceutical composition, as known in the art.Therapeutic Method of the present invention comprises to suffering from ischemic diseases, uses the compounds of this invention of effective dose such as the patient of congestive heart failure, arteriosclerosis etc.In a preferred implementation, treatment target is mammal, and in most preferred implementations, treatment target is the people.Preferred route of administering comprises oral and transdermal infusion mechanism.

Utilize the normal experiment method can determine easily the effective dose of this reagent, also can determine the most effectively and route of administration and the most suitable ingredients of most convenient.Various ingredients and medicine input system are all known, and the selection of involutory adaptive agent is also within art technology is grasped.(referring to, for example Gennaro compiles (1995) " Remington pharmacy ", the same; Hardman, Limbird and Gilman compile (2001) " pharmacological basis for the treatment of ", the same.)

That suitable route of administration can comprise is oral, rectally, wall, snuffing or little enteral administration and parenteral thoroughly, comprise intramuscular injection, subcutaneous injection, intraspinal injection, and injection in film, the directly interior injection of ventricle, intravenous injection, peritoneal injection, nasal injection or intraocular injection.Described reagent or compositions can be at topicals, rather than are administered systemically.For example, suitable reagent can or be sent into by injection in target drug delivery system, as the slow release ingredients.

Pharmaceutical composition of the present invention can pass through the method production of this area maturation, as routine mixing, dissolving, pelletize, sugaring ball, efflorescence, emulsifying, be encapsulated or dry freeze.As mentioned above, the present composition can comprise one or more physiologically acceptable carriers, as excipient and adjuvant, to help bioactive molecule, makes medicine.

Suitable ingredients depends on selected route of administration.For example, for injecting method, compositions can form aqueous solution, is preferably physiological buffer, as Hanks solution, Ringer's mixture physiologically active brine buffer solution alive.For saturating wall or nasal-cavity administration, can use the penetrating agent that is applicable to the screener infiltration in ingredients.This penetrating agent is normally known in this area.For oral administration, by making reactive compound, with pharmaceutical carrier well known in the art, to be combined, described compound can be formulated easily.Such carrier can make the compounds of this invention form tablet, pill, sugar pill, capsule, medicinal liquid, gel, syrup, serosity, suspension etc., so that the patient is oral.Described compound also can be made into rectal compositions, and as suppository or slow release enema, they can comprise traditional suppository substrate, as cupu oil or other glyceride.

Medicinal preparation for oral administration can be such acquisition, adds solid excipient in compound, optionally grinds the gained mixture, and the processing particulate mixtures, if need to add suitable adjuvant, then obtains tablet or sugar pill core.Suitable excipient, particularly including filler, as sugar, comprises fructose, sucrose, mannitol or sorbitol; The cellulose ingredients, as Semen Maydis powder, wheat flour, rice flour, mealy potato, gel, Tragacanth, methylcellulose, HYDROXY PROPYL METHYLCELLULOSE, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).If necessary, can add distintegrant, as crospolyvinylpyrrolidone, agar or alginic acid or their salt, as Brown algae salt.

To wrap up suitable coating on sugar pill core.For this reason, concentrated sugar solution can be used, wherein arabic gum, Talcum, polyvinylpyrrolidone, carbopol glue, Polyethylene Glycol and/or titanium dioxide, varnish solution and organic solvent or solvent mixture can be optionally comprised.Can on tablet or sugar pill Bao Nong, add dyestuff or pigment, to mark or to characterize different active compound doses combinations.

Medicinal preparation for oral administration comprises the grafting capsule of being made by gel, and the soft seal capsule of being made by gel and plasticizer, as glycerol or sorbitol.The grafting capsule can comprise the mixture that active component and filler form as Talcum or magnesium stearate and optional stabilizer as starch and/or lubricant as lactose, binding agent.In soft capsule, reactive compound solubilized or be suspended in suitable liquid, as fatty oil, liquid paraffin or liquid macrogol.In addition, can add stabilizing agent.

In one embodiment, but the compounds of this invention transdermal administration (as passed through plaster) or topical.On the one hand, transdermal of the present invention or local ingredients can comprise one or more penetration enhancers or other synergists in addition, comprise the reagent that can promote compound migration to be sent.Under the occasions such as needs site-specific delivery of drugs, preferred transdermal or topical.

For inhalation, form that can aerosol spray for compound of the present invention is carried easily from compressed package or aerosol apparatus, can be by suitable propellant, for example dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other any suitable gas.For compresed gas aerosol, suitable dosage can be controlled by valve, to realize dosed administration.Can be made into the form of capsule or cylinder for the gel of medicine absorber or insufflator.

For parenteral injection, for example the compositions of contrast-medium injection or continous pouring can be made into the form of unit dose, for example is contained in ampoule or multi-dose container, can add antiseptic.The form of described compositions can be to take suspension, solution or the emulsion that oil or water is medium, can comprise the reagent that makes up a prescription, as suspending agent, stabilizing agent and/or dispersant.The parenteral injection ingredients comprises aqueous solution or other water-soluble composition forms.

The suspension of reactive compound also can be made into suitable oily injectable suspensions.Suitable lipophilic solvent or medium comprise fatty oil (as Oleum sesami) and Acrawax (as ethyl oleate or triglyceride), or liposome.The water solublity injectable suspensions can comprise the material that can increase suspension viscosity, as sodium carboxymethyl cellulose, sorbitol or dextrose.Optionally, suspension also can comprise suitable stabilizing agent or reagent, to increase the dissolubility of these compounds, thus the highly concentrated solution of convenient preparation.Perhaps, active component can be powder type, in order to mix without thermal source matter water as sterilization with suitable medium before use.

As mentioned above, the present composition also can be made into the deposit medicament.This long-acting ingredients can be for example, by implantation (subcutaneous or muscle is implanted) or by the intramuscular injection drug delivery.Therefore, for instance, the compounds of this invention can make up a prescription with suitable polymeric material or hydrophobic material (for example in usable oils, forming emulsion), or makes up a prescription with ion exchange resin, or is made into the slightly soluble derivant, for example is made into slightly soluble salt.

The carrier that is suitable as hydrophobic molecule of the present invention is widely known by the people in this area, comprises cosolvent system and water, and the former comprises benzyl alcohol, non-polar surfactant, organic polymer soluble in water etc.Cosolvent system can be the VPD cosolvent system.VPD is that 3% (w/v) benzylalcohol, 8% (w/v) non-polar surfactant's polysorbate80 and 65% (w/v) Liquid Macrogol form, and joins the solution of sufficient volume with dehydrated alcohol.VPD in VPD cosolvent system (VPD:5W) be in aqueous solution with 5% dextrose with the dilution proportion of 1: 1.This cosolvent system is the solubilizing hydrophobic compound effectively, and the toxicity produced in the process of being administered systemically is low.Certainly, the proportioning of cosolvent system can be done sizable change, only otherwise destroy its dissolubility and toxic characteristic.In addition, cosolvent component liquid can change.For example, available other low toxicities non-polar surfactant replaces polysorbate80, and the ratio of Polyethylene Glycol can change, and the other biological compatible polymeric can replace Polyethylene Glycol, polyvinylpyrrolidone for example, and other sugar and polysaccharide can replace dextrose.

In addition, for hydrophobic molecule, also can adopt other drug delivery systems.Conveying medium or carrier that liposome and emulsion can be used as hydrophobic drug are the examples be widely known by the people.The liposome drug delivery system was discussed in conjunction with the gene delivery system in the above.Some organic solvent, as dimethyl sulfoxide also can adopt, although tend to bring larger toxicity.In addition, described compound can be carried with slow-releasing system, and as the semipermeability substrate of solid hydrophobic polymer, described polymer comprises the compositions that effective dose will be inputted.Various slow-release materials are all known, and those skilled in the art are not difficult to obtain.According to their chemical characteristic, slow releasing capsule can discharge compound in the time more than several weeks to 100 day.According to chemical property and the biological stability of medical reagent, can adopt other strategies to increase the stability of protein.

For any compositions used in Therapeutic Method of the present invention, the familiar medicable dosage of technical measurement in available this area at the beginning.For example, the information that chemical examination obtains according to cell culture, can be made into a dosage, reaches in animal specimen and comprise IC ₅₀The circulation composition scope.Can determine the dosage range that is suitable for human body similarly, for example utilize the information obtained in cell culture chemical examination and other zooscopies.

The medicable dosage of a kind of reagent refers to this reagent energy relief of symptoms or the amount of extended treatment object time-to-live.The toxicity of this molecule and curative effect can be measured at cultured cell or laboratory animal by the standard pharmaceutical program, for example measure LD ₅₀(making 50% fatal dosage of colony) and ED ₅₀(to 50% medicable dosage of colony).The dose ratio of toxicity and curative effect is therapeutic index, can be expressed as LD ₅₀/ ED ₅₀Ratio.The therapeutic index of reagent is more high better.

Within dosage should drop on the circulation composition scope, this concentration range comprises ED ₅₀But have very little or there is no toxicity.Dosage can change according to medicine type used and route of administration in this scope.According to means known in the art, actual ingredients, route of administration and the dosage adopted will be selected according to the concrete condition of disease that the patient takes a disease.

Dosage and interval can make the active component content that offers blood plasma be enough to the gene induced process of regulating the stabilization procedures of HIF α and regulated by HIF according to the individual instances adjustment, obtain as desired minimum effective drug concentration (MEC).MEC can change with each compound, but can estimate from vitro data.Reach personal feature and route of administration that the required dosage of MEC depends on compound.Reagent or compositions should be controlled carrier by one and carry, and make blood plasma level be kept above MEC, are about 10-90% during treating, and are preferably about 30-90%, preferably are about 50-90%.In the situation of topical or selection absorption, effective local concentration of medicine is not necessarily relevant to plasma concentration.

Certainly, the dosage of reagent or compositions depends on many factors, comprises sex, age and the body weight for the treatment of target, the ailing order of severity, administering mode and prescriber's judgement.

If necessary, the present composition can be contained in medicated bag or minute medicine device, can contain the medicine that contains active component of one or more dosage.For instance, this medicated bag or device can comprise metal or plastic foil, as rigid foam liner bag.But note medication instruction on described medicated bag or minute medicine device.The compositions of preparing in pharmaceutical carrier, contain the compounds of this invention also can be contained in suitable container, and sticks the label that disease is specified in the explanation treatment.On label, specified corresponding disease can comprise that take ischemia or hypoxia is main disease or imbalance.

The screening and identification of compound

The present invention also provides screening and identification can suppress other compounds of HIF α hydroxylating or stable HIF α etc.

Various chemical examinations and triage techniques, comprise described belowly, can be used to identify to regulate (for example increase or reduce) HIF alpha levels or active micromolecule.Chemical examination provides the detectable signal relevant to the generation of the consumption of reaction substrate or product usually.For instance, detection can relate to fluorogen, radiosiotope, conjugation enzyme and other detectable labels known in the art.Acquired results can be qualitative or quantitative.Can help to follow the tracks of the separation case of product such as biotin or the such labelling of histidine, this separation can by precipitation or affinity chromatography, by a kind of product, from other reactive components, purification be out.

To the chemical examination of HIF α hydroxylating process can relate to hydroxylation proline in HIF α or its fragment or lysine residue mensuration (referring to, such as (1985) J Chromatogr 339:285-292 such as Palmerini), or relate under enzyme and HIF α or its fragment exist by 2-oxoglutaric acid form succinate mensuration (referring to, such as (1986) Biochem J 240:617-619 such as Cunliffe).Measure the exemplary process of HIF α hydroxylating process and see (the same) and embodiment 10 such as being set forth in Ivan.The exemplary process that mensuration forms succinate by 2-oxoglutaric acid is shown in and is set forth in Kaule and Gunzler (1990; Anal Biochem 184:291-297.) substrate molecule can comprise HIF α or its fragment, HIF (556-575) for example; For example, the exemplary substrate adopted in the described chemical examination of embodiment 10 is [methoxy coumarin]=DLDLEALAPYIPADDDFQL-amide (SEQ ID NO:5).Enzyme can comprise, for example HIF α prolyl hydroxylase (referring to, such as the gene bank number of sequeling No.AAG33965 etc.), can be from any source.Enzyme also can be present in crude cell lysate or with partially purified form and exist.Stablize HIF α or suppress HIF α hydroxylation compound can by measure and relatively this compound exist with non-existent situation under the activity of enzyme.

In addition, compound can be by various triage techniqueses evaluations known in the art, and these technology also can be used in combination with said method.These screening techniques can make in solution not contain target polypeptides or compound, also can make them be attached on solid carrier, are created on cell surface, or are positioned at cell interior.For example, experimental compound can be similar to the existing array approach array in this area from the teeth outwards, then analyze its activity (referring to, such as (1995) International Publication patent WO95/35505 such as Shalon; Baldeschweiler etc. (1995) International Publication patent WO95/251116; Brennan etc. (1995) United States Patent (USP) 5474796; Heller etc. (1997) United States Patent (USP) 5605662.)

Those of ordinary skills, after having seen this description, are understood that above-mentioned and other embodiment of the present invention, and these embodiments have passed through specially to be deliberated repeatedly.

Embodiment

Can better understand the present invention with the following Examples, but these embodiment are only examples of the present invention.Scope of the present invention is not limited by the embodiment in example, and these embodiments have just illustrated single side of the present invention.Within any method of equal value in effect all is included in the scope of the invention.According to description and the accompanying drawing of front, those skilled in the art are not difficult outside embodiment described here, the present invention to be made to various modifications.These are revised also within the appended claims scope.

In embodiment 1 cell in vitro, HIF α's is stable

Will from adenovirus degeneration fetal kidney epithelial cell (293A), cervix uteri epithelial gland cancerous cell (HeLa), hepatoma carcinoma cell (Hep3B), clad fibre archeocyte (HFF), breast epithelium cancerous cell (MCF7), band venous endothelial cell (HUVEC), capillary endothelium (HMEC-1), umbilicus squamous epithelium cancer cell (SCC-25), lung fibroblast (HLF), venous endothelial cell (AG10774B) tissue (referring to, for example the US mode strain is collected, ManassaVA; Qbiogene, Carlsbad CA) human body cell is sowed respectively in the 35mm culture dish, at 37 ℃, 20%O ₂, 5%CO ₂In environment, cultivate, medium is as follows: the HeLa cell is in the improved Eagle medium of Dulbecco (DMEM), 2% solcoseryl (FBS); HFF and HLF cell in DMEM, 10%FBS; The 293A cell in DMEM, 5%FBS; HUVEC and AG10774B cell (EGM-2 in the endothelial growth medium; BioWhittaker, Inc., Walkersville MD); HMEC-1 in RPMI 1640,10%FBS; The Hep3B cell in Minimal Essential medium (MEM), Earle BSS (Mediatech Inc., HerndonVA), 2mM Pidolidone, the less important aminoacid of 0.1mM, 1mM Sodium Pyruvate, 10%FBS.After cellular layer converges, medium is replaced (Invitrogen Life Technologies, Carlsbad CA) with the OPTI-MEM medium, and cellular layer is at the 20%O of 37 ℃ ₂, 5%CO ₂Middle cultivation approximately 24 hours.Then add the compounds of this invention (one of compd A-O) or DMSO (0.5-1%) in existing medium, continue overnight incubation.

Cultivate and finish, remove medium, centrifugal, preservation, in order to analyzing (seeing below).In cold phosphate buffer (PBS), washed cell is 2 times, then in freezing situation, allow cell dissolve 15 minutes in 1ml 10mM Tris (pH 7.4), 1mMEDTA, 150mM NaCl, 0.5%IGEPAL (Sigma-Aldrich, St.Louis MO) and protease inhibitor cocktail (Roche Molecular Biochemcals).Cytolysis thing under 4 ℃ under 3000xg centrifugal 5 minutes, collecting cell dissolves part (supernatant).Make core (bead) Eddy diffusion, and 100 μ l 20mM HEPES (pH=7.2), 400mM NaCl dissolve (Roche Molecular Biochemicals) in 1mM EDTA, 1mM dithiothreitol, DTT and proteinase mixture, rotating speed with 13000xg under 4 ℃ is centrifugal, collects the nucleoprotein part.

Proofread and correct the core part according to protein concentration, load in the 4-12%TG gel, and fractional distillation under reducing condition.Under the 500mA electric current, in 1.5 hours, protein transduction is moved on to pvdf membrane in (Invitrogen Corp., Carlsbad CA).Film shields 1 hour in T-TBS, 2% milk under room temperature, and, by the anti-human body HIF-1 of rat Alpha antibodies (BD Bioscience, Bedford MA) overnight incubation, described antibody is pressed the dilution proportion of 1: 250 in T-TBS, 2% milk.Cultivate speckle with SUPERSIGNAL WEST chemiluminescence substrate (Pierce, Rockford IL).As shown in Figure 1A, various compound of the present invention (A-F) is stablized HIF α in the dose dependent mode in normal oxygen environment.As shown in Figure 1B, when processing with the compounds of this invention in normal oxygen environment, various cell types, comprise fibroblast, epithelial cell, endotheliocyte and hepatocyte from various sources, all demonstrates the dose dependent Stabilization of HIF α.

Perhaps, utilize the QUANTIKINE determinator (R&amp that exempts from service; D Systems, Inc., MinneapolisMN), according to the HIF-1 α of businessman's description analysis of cells core part and bag liquid part.As shown in Figure 2 A, with the compared with control cells with mordanting, compare, the epithelial cell (293A) of processing with the various compounds of the present invention (compd B and G-O) and the HIF α in hepatocyte (Hep3B) are stablized and enrichment.As shown in Fig. 2 B, the cell of processing with the compounds of this invention has the Stabilization that depends on dosage to HIF α.

Embodiment 2: the oxygen consumption effect

Oxygen sensing Tissue Culture Dish (BD Bioscience, Bedford MA) contains ruthenium complex, and this complex has better fluorescence in the hypoxia situation.Therefore, if there is the oxygen consumption cell in culture dish, the fluorescence read output signal strengthens, and it makes balanced deflection hypoxia saturability and high fluorescence state.Can estimate, the compound of stablizing HIF by suppressing hydroxylation can reduce oxygen consumption, and the oxygen consumption of hydroxylation itself reduces, and/or makes cellular metabolism turn to the anaerobic energy supply from Aerobic energy supply.

Cultivate adenovirus degeneration fetal kidney epithelial cell (293A) or cervix uteri epithelial gland cancerous cell (HeLa) (collection of US mode strain), make them at 37 ℃ and 10%CO ₂Under condition, in medium (high glucose DMEM (Mediatech, Inc., Herndon VA), 1% penicillin/streptomycin mixture (Mediatech), 1% solcoseryl), converge.Collecting cell, and again be suspended in medium, density is 500000 cells/ml.Suspension is distributed to each Zhong，Mei cave, cave 0.2ml of Oxygen Biosensor 96 cave Tissue Culture Dishs (BD Bioscience).With 10 μ l volumes, reagent adds in three cave groups during by following processing: (1) 0.5%DMSO; (2) 200 μ M sodium lauryl sulphates; Or (3) 1,10 or 50 μ M compound (one of prodrug [pV] of compd B, G or compound V).

Culture fluid is at 37 ℃ of 10%CO ₂Middle cultivation 72 hours, then read the culture dish data by FL600 luminoscope (BiotekInstruments, Inc., Winooski VT) at excitation wavelength 485nm and emission wavelength 590nm.Utilize the data obtained to draw phase antithetical phrase DMSO object of reference or at numeral multipling function relation (the consumption O of the absorbance at 450nm (WST-1) wavelength place ₂Amount), and with EXCEL software (Microsoft, Bellevue WA) being described property statistical analysis.

Fig. 3 A is depicted as the cell variation of the multiple on oxygen consumption with respect to object of reference by described compound treatment.As can be seen from the figure, all compounds have all reduced oxygen consumption to a certain extent.In addition, the reduction of oxygen consumption depends on dosage (Fig. 3 A), even under maximum dose level, the loss (Figure 1B) of cell survival rate also seldom or not detected.Other experiment (not shown) that carry out in various cell culture experiments systems, comprise and adding ³H-thymidine and all amino acid, all confirm that the decline of oxygen consumption and cytotoxicity are irrelevant.

Embodiment 3: the expression of external HIF regulator gene

Utilize QUANTIKINE immunoassay apparatus (R& D), the conditioning medium analysis VEGF (VEGF) to collecting the cell culture fluid from as embodiment 1 preparation according to businessman's description.As shown in Figure 4 A, clad fibre archeocyte (HFF), adenovirus degeneration fetal kidney epithelial cell (293A) and the hepatoma carcinoma cell (Hep3B) of processing with the various compounds of the present invention (one of prodrug [pV] of compd A, B, C, H, K, L, Q and compound V) be all decline (Fig. 4 A) to some extent in the expression of VEGF.Value representative on Y-axis is with respect to the log that induces multiple of object of reference ₂Value, numerical value 1 represents that 2 times are induced like this.

Perhaps, at 37 ℃ and 10%CO ₂Cultivate the human body cell from adenovirus degeneration fetal kidney epithelial cell (293A) in DMEM under environment, 5%FBS, 1% penicillin-streptomycin.After 48 hours, collecting cell, collect in the 35mm culture dish, and used medium is conventional media, after 1 day, medium changed into to Opti-Mem I.After 18-24 hour, add compd B in medium, then cultivate 18 hours.Remove the supernatant in culture dish, culture dish is placed on ice, add cytolysis buffer (LB)-1, scrape cell.By the cell harvesting scraped, cultivate then under 4 ℃ with 3000xg centrifugal 5 minutes 15 minutes on ice.Collection represents the supernatant of Cell sap, uses SDS polyacrylic acid amide gel isolated cell liquid eggs white matter, the protein of every passage load equivalent on the turn with under reduced pressure.

Carry out 2 hours gel electrophoresiss under 150V voltage, after SDS-PAGE, at 400mA electric current and 4 ℃ of temperature, in 1.5 hours, protein transduction is moved on on pvdf membrane.Then culture membrane in the shielding buffer, with the T-TBS washing once, be added in the anti-al ketone antibody that is diluted to working concentration in the shielding buffer, under 4 ℃, stirs and spend the night gently, cultivates speckle.Then wash film 4 times with T-TBS, then at room temperature cultivate 1 hour, wherein shield buffer and contain the labelling secondary antibodies.Then wash film 4 times with T-TBS.Exposure X-ray film, developed according to explanation with ECL SUPERSIGNAL WEST FEMTO or PICO chemiluminescence substrate (Pierce, Rockford IL), can see original antibody is had to narrow spectrum antigen.

Fig. 4 B is depicted as the increase situation that described compound is expressed aldolase at different time, and aldolase is the enzyme related in glycolysis.Therefore, the compounds of this invention is to the subsidiary increase that causes that the HIF regulator gene is expressed of the stable meeting of HIF α.

Embodiment 4: in cells in vivo, HIF α's is stable

From Charles River Laboratories, Inc. (Wilmington MA) or Simonsen, Inc. units such as (Gilroy, CA) obtains the public Mus (30-32g) of Switzerland's Robert Webster, by feeding 2ml/kg volume 0.5% carboxymethyl cellulose (CMC; Sigma-Aldrich) (contrast) or 5.0% compound (0.5%CMC), at least feed one day, once a day or repeatedly.In the end, on one or more time points of a dosage, for example 2-5 hour, implement anesthesia with isoflurane to animal, extracts 0.1ml blood, for example from the eye hole, extracts, and puts into the heparinization test tube.After all time points are all chosen, impose the CO of sublethal dose to animal ₂, from the epigastric vein blood drawing, put into the heparinization test tube.All blood sample are kept under-80 ℃.

Fabric analysis HIF α protein level to separating with it from above-mentioned animal of processing through the compounds of this invention by the following method.With POLYTRON PT-1200 homogenizer (Brinkmann Instruments, Inc., Westbury NY) in 3ml 10mM Tris (pH 7.4), 1mM EDTA, 150mM NaCl, 0.5%IGEPAL (Sigma-Aldrich), to described, organize 15 seconds of homogenize.Under 4 ℃ to cytolysate with 3000xg centrifugal 5 minutes, collecting cell liquid (supernatant).Nucleus (bead) is suspended again and be dissolved in 100 μ l20mM HEPES (pH 7.2), 400mM NaCl, 1mM EDTA, 1mM dithiothreitol, DTT and proteinase mixture (Roche Molecular Biochemicals), to cytolysate with 3000xg centrifugal 5 minutes, collect nucleoprotein (supernatant) under 4 ℃.

According to protein concentration, nucleus is partly carried out to normalization, load under reduced pressure on the 4-12%TG gel.Under the 500mA electric current, protein transduction is moved on to (Invitrogen LifeTechnologies) on pvdf membrane in 1.5 hours.T-TBS at room temperature, in 2% milk, screened film is 1 hour, is used in T-TBS, the anti-HIF Alpha antibodies overnight incubation of diluting in 2% milk.Cultivate speckle with SUPERSIGNAL WEST chemiluminescence substrate (Pierce, Rockford IL).

Perhaps, utilize the QUANTIKINE determinator (R&amp that exempts from service; D Systems, Inc., MinneapolisMN), the HIF-1 α of the nucleus part as above prepared according to businessman's description analysis and bag liquid part.

Embodiment 5: the expression of HIF regulator gene in body

Experiment I

From Charles River Laboratories, Inc. (Wilmington MA) or Simonsen, Inc. units such as (Gilroy, CA) obtains 24 public Mus (30-32g) of Switzerland's Robert Webster, feeding 4ml/kg volume 0.5% carboxymethyl cellulose (CMC; Sigma-Aldrich) (0mg/kg/ days) or 1.25% compd A (25mg/ml, in 0.5%CMC) are (100mg/kg).Feed last dosage after 4,8,16,24,48 or 72 hours, with isoflurane, animal is implemented to anesthesia, take a blood sample from epigastric vein.By blood sample collection to (Becton-Dickinson in the MICROTAINER serum separation test tube, Franklin Lakes NJ), at room temperature cultivate 30 minutes, under 4 ℃ with the rotating speed of 8000rpm centrifugal 10 minutes, cell plates are resuspended in to (Ambion) in RNALATER solution, are stored under-80 ℃.Then kill these rats, separate the tissue samples in kidney, liver, brain, lung and heart, and be kept under-80 ℃ in RNALATER solution (Ambion).

Carry out the RNA separation by following method.Cut the tissue of a 50mg from each organ, add 875 μ lRLT buffer (RNEASY workboxes; Qiagen Inc., Valencia CA), with rotor-stator POLYTRON homogenizer (Kinematica, Inc., Cincinnati OH) to every block organization homogenize approximately 20 seconds.By homogenate carry out 3 minutes micro-centrifugal, make the insoluble matter granulating, supernatant is transferred in new test tube, with RNEASY workbox (Qiagen) isolation of RNA to specifications.The RNA elution, in 80 μ l water, is measured to its amount with RIBOGREEN reagent (MolecularProbes, Eugene OR).With DNA-FREE workbox (Ambion Inc., Austin TX), to specifications genomic DNA is removed from RNA.260 and the 280nm place measure absorptance, thereby determine purity and the concentration of RNA.

Perhaps, after cutting tissue samples, use rotor-stator POLYTRON homogenizer (Kinematica) homogenize in TRIZOL reagent (Invitrogen Life Technologies, CarlsbadCA).Homogenate is placed under room temperature, adds the chloroform of 0.2 part of volume, the vigorous stirring sample.Culture mix number minute under room temperature, then 4 ℃ with 12000g centrifugal 15 minutes.Collect water, add the isopropyl alcohol of 0.5 part of volume.Mixing sample, cultivate under room temperature 10 minutes, 4 ℃ with 12000g centrifugal 10 minutes.Remove supernatant, with 75%EtOH washed cell plate, 4 ℃ with 7500g centrifugal 5 minutes.Then use DNA-FREE workbox (Ambion Inc., Austin TX) to specifications genomic DNA to be removed from RNA.260 and the 280nm place measure absorptance, thereby determine purity and the concentration of RNA.

Under-20 ℃, in 0.3M sodium acetate (pH 5.2), 50ng/ml glycogen and 2.5 parts of volume ethanol, precipitated rna is 1 hour.Centrifugal sample, by 80% cold washing with alcohol cell plates, drying, water suspends again.Utilize T7-(dT) 24 as first strand of primer (Affymetrix, Inc., Santa Clara CA), by SUPERSCRIPTCHOICE system (Invitrogen), according to operation instruction, synthesize bifilar cDNA.Utilize PHASE LOCK GEL plug-in unit (Brinkman, Inc., Westbury NY), with 25: 24: 1 phenol: chloroform: isoamyl alcohol extracted last cDNA.Collect water, with the 7.5M ammonium acetate of 0.5 volume and the ethanol precipitation cDNA of 2.5 volumes.Perhaps, purify cDNA with GENECHIP sample cleaning module (Affymetrix) according to operation instruction.

Utilize BIOARRAY High Yield rna transcription marking tool case (Enzo Diagnostices, Inc., Farmingdale NY), according to operation instruction translate in vitro (IVT) reaction in by the cDNA complex sign cRNA of biotin.According to operation instruction, final marked product is carried out to purification and burst with GENECHIP sample cleaning module (Affymetrix).

5 μ g are surveyed to sample and add 100 μ l 1x hydridization buffer (100mM MES, 1M[Na ⁺], 20mMEDTA, 0.01%Tween 20), 100 μ g/ml Mylopharyngodon piceus sperm DNAs, the low B of 500 μ g/ml acid esterification BSA, 0.03nM contrast ₂(Affymetrix) and 1x GENECHIP eucaryon hydridization contrast (Affymetrix), prepare thus hydridization " cocktail ".Described cocktail is cultivated 5 minutes and cultivates 5 minutes then centrifugal 5 minutes at 45 ℃ at 99 ℃ successively.By Murine genome U74AV2 array (MG-U74AV2; Affymetrix) be placed under room temperature, then add 1x hydridization buffer, 45 ℃ of rotations 10 minutes, carry out pre-hydridization.Then replace buffer with 80 μ l hydridization cocktail, under 45 ℃ with the rotating speed reverse rotation of 60rpm, by this array hydridization 16 hours.After hydridization, use 6x SSPE, 0.1%Tween 20 pair array washings once, then use R-PE-conjugation N,N'''-(2,4,5,6-tetrahydroxy-1,3-cyclohexanediyl)bisguanidine (molecular probe, Eugene OR), anti-N,N'''-(2,4,5,6-tetrahydroxy-1,3-cyclohexanediyl)bisguanidine antibody (the Vector Laboratories of goat, Burlingame CA) and GENECHIP Fluidices Station 400 instruments (Affymetrix), according to businessman micro--1vl agreement (Affymetrix) washed and stain.Analyze array with GENEARRY scanner (Affymetrix) and Microarray Suite software (Affymetrix).

Murine Genome U74AV2 array (Affymetrix) represent in Mouse UniGene data base 74 (NCBI, Bethesda MD) all sequences (～6000) (they are all characterized on function) and approximately 6000 do not mark expressed sequence tag (EST) bunch.

As can be seen from Figure 5A, with lung, for representing organ, after processing with the compounds of this invention, in equal mode, increase to the gene expression of angiogenic growth protein coding.The mode of transcribing shown in figure comprises VEGF-C, Flt-1/VEGF receptor-1, medullin, endothelium quality-1, plasminogen activation inhibitor (PAI)-1 and Cyr61.Along with the variation of time, mRNA leaves peak early, after 24 hours, returns to control level.Fig. 5 B is depicted as the embody-time relationship of two genes, and these two genes are endothelium quality-1 and medullin, the gene cluster in representative graph 5A.In similar experiment; the expression of other HIF regulator gene also can be seen obvious increase, and these genes comprise phosphofructokinase, Enolase 1, lactic acid dehydrogenase, glucose transporter 1, acyl-CoA sulfo-esterase, Heme oxygenase, TfR, IGFBP-1, nip3, nix and cyclin G3.

From Fig. 7 A, can find out, with kidney, for representing organ, after processing with the compounds of this invention, the gene expression of encoding to glycolytic ferment increases in equal mode.The mode of transcribing shown in figure comprise aldolase A, Enolase 1, glucose transporter (GluT)-1 and-3, GAPDH, hexokinase-1 and-2, lactate dehydrogenase A, phosphofructokinase-L and-C, PGK-1 and pyruvate kinases-M.Along with the variation of time, mRNA leaves peak early, after 24 hours, returns to control level.Fig. 7 B is depicted as the specifically embody-time relationship of two genes, and these two genes are aldolase and phosphofructokinase, the gene cluster in representative graph 7A.

Experiment II

From Simonsen, Inc. obtains 12 public Mus (30-32g) of Switzerland's Robert Webster, feeding 4ml/kg volume 0.5% carboxymethyl cellulose (CMC; Sigma-Aldrich) (0mg/kg/ days) or 2.5% compd B or E (25mg/ml, in 0.5%CMC) (200mg/kg), every day 2 times, feed 2.5 days (5 dosage) altogether.After feeding last dosage 4, with isoflurane, animal is implemented to anesthesia, take a blood sample from epigastric vein.By blood sample collection (Becton-Dickinson) in the MICROTAINER serum separation test tube, at room temperature cultivate 30 minutes, under 4 ℃ with the rotating speed of 8000rpm centrifugal 10 minutes, utilize the QUANTIKINE determinator (R&amp that exempts from service; DSystems), process the also expression of the VEGF (VEGF) of serum analysis part according to businessman's description.Then kill these rats, separate about 150mg liver and kidney, and be kept under-20 ℃ in RNALATER solution (Ambion).

Carry out the RNA separation by following method.Tissue slice is cut into small pieces, adds 1.75ml RLT buffer (RNEASY workbox; Qiagen), with rotor-stator POLYTRON homogenizer (Kinematica, Inc., Cincinnati OH) to every block organization homogenize approximately 20 seconds.To 350 μ l homogenate carry out 3 minutes micro-centrifugal, make the insoluble matter granulating, supernatant is transferred in new test tube, with RNEASY workbox (Qiagen) isolation of RNA to specifications.The RNA elution, in 80 μ l water, is measured to its amount with RIBOGREEN reagent (Molecular Probes, EugeneOR).With DNA-FREE workbox (Ambion), to specifications genomic DNA is removed from RNA.260 and the 280nm place measure absorptance, thereby determine purity and the concentration of RNA.

Synthesize to specifications cDNA by the poly-primer of 1 μ M random six, the full RNA of 1 μ g and OMNISCRIPT reverse transcriptase (Qiagen).Gained cDNA dilutes with the water of four times, and making final volume is 100 μ l.With FASTSTART DNA MASTER SYBR GREEN I workbox (Roche Molecular Biochemicals) and VEGF Auele Specific Primer, utilize LIGHTCYCLE system (Roche Molecular Biochemicals) to pass through the relative level of quantitative PCR analysis VEGF (VEGF) gene expression according to operation instruction.In 6 minutes, sample is heated to 94, then in 15 seconds, forwards 95 to, in 5 seconds, forward 60 to, in 10 seconds, forward 72 to, altogether circulate 42 times.The VEGF Auele Specific Primer is as follows:

m-VEGF-F1 GTTGCAAGGCGAGGCAGCTT (SEQ ID NO：1)

m-VEGF-R1 TGACGATGATGGCATGGTGGT (SEQ ID NO：2)

Measure the relative level that 18S core candy body rna gene is expressed, in contrast.Utilize QUANTITECTSYBR GREEN PCR workbox (Qiagen) and 18S rRNA Auele Specific Primer, use LIGHTCYCLER system (Roche MolecularBiochemicals) according to operation instruction quantitative assay PCR.In 6 minutes, sample is heated to 94, then in 15 seconds, forwards 95 to, in 5 seconds, forward 60 to, in 10 seconds, forward 72 to, altogether circulate 42 times.Core candy body RNA Auele Specific Primer is as follows:

18S-rat-2B TAGGCACGGCGACTACCATCGA (SEQ ID NO:3)

18S-rat-2A CGGCGGCTTTGGTGACTCTAGAT (SEQ ID NO:4)

Each is taken turns PCR and all comprises a standard curve and water blank.In addition, after every PCR of wheel completes, measure melting curve, to estimate the specificity enlarged.VEGF gene expression is proofreaied and correct according to the expression of the 18S ribosomal RNA of this sample.

Fig. 6 A is depicted as the vegf expression with in vegf expression regulating liver-QI and kidney in the kidney of compd E enhancing, compd B strengthens.Can find out from Fig. 6 B, with respect to undressed control animal, use 2,5 and 20 hours after last dosage, obviously increase by VEGF level in the animal blood slurry of described compound treatment.

Embodiment 6: cardiac ischemia

Experiment I

Nwogu etc. (2001; Circulation 104:2216-2221) once reported with the compounds of this invention and treated myocardial infarction.Although the author has explained their result from the angle of described compounds affect cystic fibrosis, the present invention is clear to be shown, what cardiac function was brought to main benefit is the stable of HIF α.Experimental technique, as (the same) as described in Nwogu etc., below also will specifically be introduced.

The male Mus of 70 adult Wistar (200-250g) are implemented to anesthesia, make their left coronary artery closure, thereby produce acute myocardial infarction (AMI).9 animals are carried out to identical operation, but do not tie up coronary artery.Postoperative 24-48 hour, receive the electrode of electrocardiograph (ECG) on claw, makes 15MHz straight line probe (AcusonCorp., Mountain View CA) contact chest, obtains minor axis thoracic cavity echocardiogram picture (2DE) near the mastoid process of middle part.Probe is moved to head or afterbody, and change angle, until the intracardiac image of left ventricle can clearly be detected.Image obtains with Sequoia ultrasonic system (Acuson).The upper classification of 2DE is shortened and is less than 20% and moving abnormal compd A for animal (n=14) or medium (n=12) random process of partial wall.The contrast dummy copy is also used compd A (n=4) or medium (n=5) random process.

At experimental session, separately feed 2 compd As (50mg/kg) or medium to animal every day.The serum levels of period measurement medicine, to determine the medicine that has continued to accept q.s the treatment animal, the level of measuring out is enough to suppress prolyl 4-hydroxyl enzyme, and representative is the 2-oxoglutaric acid dioxygenase.

Collect weekly serial 2DE image.Catch and preserve three minor axis 2DE numeral folders, this numeral clip pack is containing 5 or more oversensitive dirty contraction and diastolic images.Two observers that are unfamiliar with Therapeutic Method carry out determined off-line.During measurement, slow down digital picture, and fix in contraction and diastole end.Measure two that in three numeral folders, each numeral is pressed from both sides and shrink and diastolic images, until obtain consistent results, then be averaging.Shrink antetheca (AWS) and diastole antetheca (AWD), shrink rear wall (PWS) and diastole rear wall (PWD), and left ventricular contraction end (LVESD) and diastole end (LVEDD) draw limit method mensuration according to U.S. heart catacoustics association (ASE).In order to keep coherent, be determined at left ventricle the past mid point and carry out between rear mid point, the random repetition, to guarantee repeatability (replication rate is about 96%).

After treating 4 weeks, by femoral vein, pour into (0.2ml in 1 minute) 10 ⁷With afterwards, carry out in vivo hemodynamics mensuration before the M isoproterenol.Follow exercise as described below weighing heart.

Perhaps, the male Mus of 140 adult Wistar (200-250g) are implemented to anesthesia, make their left coronary artery closure, thereby produce acute myocardial infarction (AMI).Within postoperative 48 hours, obtain the 2DE image, have the animal of remarkable infarct area to use at random compd A (n=34) or medium (n=34).

At experimental session, separately feed 2 compd As (50mg/kg) or medium to animal every day.The serum levels of period measurement medicine, to determine the medicine that has continued to accept q.s the treatment animal, the level of measuring out is enough to suppress prolyl 4-hydroxyl enzyme.

To half zoometry middle part mastoid process muscle in every group and the 2DE digital picture of four Room, top, every other week once, survey full 8 weeks.Carry out determined off-line by two observers that are unfamiliar with Therapeutic Method.During measurement, slow down digital picture, and fix in contraction and diastole end.Follow the tracks of the inner surface of heart two or three at minor axis and four ventricle directions, average.Variation, wall thickness, Bicuspid valve peak E velocity of wave, large artery trunks peak speed and the thromboembolism size of the area while measuring left ventricular contraction and diastole, ejection fraction ratio, each several part area.

After treating 10 weeks, carry out in vivo the hemodynamics test, heart is as described below to perform physical exercise and weighs.

For collection body inner blood dynamic test data, animal is implemented to anesthesia, cut right carotid from surrounding tissue, make the structure of hollow with SPR-671 ultra micro pressure converter (Millar Instruments, Inc., Houston TX).Then catheter is inserted to left ventricle.After the equilibrium establishment state, the maximum rate of pressure (DP), shrinkage index (CI), left ventricular systolic pressure (SBP), diastolic pressure in latter stage (LVEDP) and the pressure lifting that record benchmark heart rate (HR), produces (being respectively+dP/dt and-dP/dt).

In hemodynamics is measured, take exercise and the weighing heart.To cut out small pieces away from incrustation cardiac muscle, right ventricle and the left ventricle muscle at thromboembolism position, weigh.Measure hydroxylation proline and proline by the method for (1985, J Chromatogr 339:285-92) such as Palmerini, different is to replace 3,4-dehydroproline as interior mark with L-azetidine-2-carboxylic acid (Sigma-Aldrich).

If the compounds of this invention is applied in it Patients With Myocardial Infarction, can sees that mortality rate descends immediately.As can be seen from Figure 8, after heart is injured through in the group for the treatment of, not at once not seeing case death, and after 8 weeks treatment group still have surpass 90% case survive.Form contrast, 60% the case of only not having an appointment in treatment group is survived during this period.Statistically see, compare with untreated group, at 2-8, the survival rate within week obviously improves (P<0.05) to treatment group, and mortality rate descends 77% relatively.

With treatment group not, compare, the cardiac parameters for the treatment of group also improves.Table 1 shows, the left ventricular end diastolic diameter (LVEDD) for the treatment of group does not increase, and the diameter (LVESD) in the LVEDD that treatment group is not measured the same period and left ventricular contraction latter stage increases.After treating 1 week, treatment group and the cardiac dilatation statistical result for the treatment of group is obviously not different.

The variation of the left ventricular end diastolic diameter of table 1

All numbers	Treatment-MI (mm)	Treatment-MI (mm) not	Pseudo-sample (mm)
				0	69±1	67±2	43±3
1	68±2	76±2	44±3
				2	69±3	74±4	45±2
3	68±4	75±3	45±2

Numeric representation meansigma methods ± standard deviation in table.

The variation of the diameter in table 2 left ventricular contraction latter stage

All numbers	Treatment-MI (mm)	Treatment-MI (mm) not	Pseudo-sample (mm)
				0	77±2	75±1	67±2
1	82±2	88±1	65±2
				2	85±3	86±3	69±2
3	85±4	86±2	68±4

Numeric representation meansigma methods ± standard deviation in table.

Fig. 9 A and 9B are depicted as LVESD and LVEDD time dependent figure line separately.Left ventricular end diastolic all close at any time with contracted diameter in three groups.In Figure 10 A, at the 2nd week to the 8th week, through the animal for the treatment of, with the untreated matched group, to compare, left ventricle ejection fraction (LVEF) obviously improves in statistical result.At any time, the LVEF of two groups is 33%.At the 4th week to the 6th week, the LVEF that does not treat matched group obviously improved, and this mortality rate that reflects this group membership is high.

The part shortening for the treatment of group in the myocardial contraction process also improves.Table 3 statistically shows, at 1-4 within week, treatment group is compared with treatment group not, and part shortens obviously and improves.

The variation that table 3 part shortens

All numbers	Treatment-MI (%)	Treatment-MI (%) not	Pseudo-sample (%)
				0	10±0.8	12±1	34±3
1	17±1	13±1	33±3
				2	20±2	15±2	33±2
3	21±2	12±1	35±2
				4	21±3	16±2	36±1

Numeric representation meansigma methods ± standard deviation in table.

In addition, from Figure 10 B, can find out, the part for the treatment of group shortens 20% while from 10% of starting point, being increased to the 2nd week, with respect to starting point, has increased by 79%.Treatment group and the pseudo-matched group of processing do not remain unchanged in 4 time-of-weeks.

Because of after heart ischemia causes damage, the heart contraction for the treatment of group and diastole ability also improve.Table 4A statistically shows, after treating 4 weeks, treatment group is compared with treatment group not, and pressure negative variation (dP/dt) in time has notable difference, and wherein-dP/dt weighs is the diastole ability after heart contraction.As show as shown in 4A and Figure 11, treatment group is compared with treatment group not, and after stimulated by isoproterenol, there is notable difference in the just variation (+dP/dt) in time of the pressure of heart on statistics, and what wherein+dP/dt weighed is systaltic ability.

Hemodynamic data after table 4A trouble MI surrounding

	Treatment-MI	Treatment-MI not	Pseudo-sample
				Shrink BP (mmHg) baseline isoproterenol	143±7 130±9	142±3 123±7	144±5 197±3
Pressure development (mmHg) baseline isoproterenol	133±6 121±9	133±3 115±8	135±6 173±3
				+ dP/dt (mmHg/sec) baseline isoproterenol	9477±581 16830±1195	8642±209 13832±1097	9925±1194 21515±1074
-dP/dt (mmHg/sec) baseline isoproterenol	9978±827 9234±703	8009±426 8984±622	11578±622 11549±10742

Numeric representation meansigma methods ± standard deviation in table.

Table 4B statistically shows, after treating 10 weeks, treatment group is compared with treatment group not ,+dP/dt and-there is notable difference in dP/dt.

Hemodynamic data after table 4B trouble MI surrounding

	Treatment-MI	Treatment-MI not	The P-value
				Shrink BP (mmHg)	106±4	92±5	0.053
Pressure development (mmHg)	97±3	69±14	0.031
				+dP/dt(mmHg/sec)	6701±331	4937±828	0.042
-dP/dt(mmHg/sec)	6395±373	3641±737	0.002

Numeric representation meansigma methods ± standard deviation in table.

Treatment group is compared with treatment group not, and improvement pressure and systolic pressure at the 10th week also have clear improvement.

Although people recognize from initial infraction or wound, recover to deposit connective tissue in necrotic area, with regard to forming cicatrix, the present invention does not show negative effect in treatment.On the contrary, from table 5A, statistical result can be found out, cicatrixes and obviously not changing without the deposition of the collagen in infarction tissue after 4 weeks, and this shows that the improvement of cardiac function in a surrounding and the deposition of collagen have nothing to do.

Collagen content after Fig. 5 A trouble MI surrounding in heart

	Treatment-MI	Treatment-MI not	Pseudo-sample
				Without the hydroxyproline/proline in the infraction myocardium of left ventricle	0.12±0.06	0.18±0.05	0.11±0.02
Without the hydroxyproline/proline in the infraction myocardium of right ventricle	0.13±0.02	0.17±0.03	0.15±0.03
				Hydroxyproline/proline in the infarction cicatrix	0.34±0.08	0.45±0.09

Numeric representation meansigma methods ± standard deviation in table.

But, from table 5B, can find out, treatment group is compared after 10 weeks with treatment group not, and in NIM and scar tissue, protoplasm content in suburb has significantly and definitely descends on statistics, and this shows that the inventive method can reduce the fibrosis of active myocardium really in the longer time.

Fig. 5 B trouble MI collagen content in heart after 10 weeks

	Treatment-MI	Treatment-MI not	The P-value
				Without the hydroxyproline/proline in the infraction myocardium of left ventricle	0.099±0.025	0.135±0.036	＜0.05
Without the hydroxyproline/proline in the infraction myocardium of right ventricle	0.152±0.044	0.175±0.042	-
				Hydroxyproline/proline in the infarction cicatrix	0.471±0.024	0.638±0.020	＜0.05

Numeric representation meansigma methods ± standard deviation in table.

Experiment II

The Wistar male Mus (100-110g) large to 4-5 week carries out routine feeding, be placed on 12 hours the daytime-cycle at night in.As follows these animals are divided at random to three treatment groups: (1) sham-operation animal (n=12), the group (n=25) of compd B treatment myocardial infarction for (2) myocardial infarction matched group (n=25) and (3).Before being performed the operation, to treatment of animals two days, after operation, continual cure was one week.In therapeutic process, every day feeds 2 0.5%CMC (Sigma-Aldrich) (contrast) to animal or 50mg/kg is dissolved in the compd B in 0.5%CMC.After animal is cut trunnion, under artificial oxygen supply, left front descending coronary artery is tied up.Postoperative 1 week kill animals, do the test of heart echo.Measure objectively part shortening, ED diameter and end-systolic diameter.

As can be seen from Figure 12A, the part shortening drops to and does not treat 29% of MI reference group from 51% of sham-operation animal.By the statistical result showed after compounds for treating, with treating matched group, not compare, part shortens (p<0.05 that is significantly improved; Unidirectional ANOVA/Turey experiment), reach 41%.Similarly, Figure 12 B statistically shows, through the animal for the treatment of, with the matched group of not treating MI, compares, and left ventricular end diastolic (LVEDD) and end-systolic (LVESD) diameter has clear improvement, and (p is less than respectively 0.005 and 0.001; Unidirectional ANOVA/Turey experiment).Compare with the animal of doing sham-operation, use the animal of compounds for treating in the left ventricular contraction not increase of diameter in latter stage, and ED diameter increases by 18%.But the LYESD of untreated matched group and LVEDD increase respectively 15% and 65%.

Embodiment 7: hepatic ischemia

Bickel etc. (1998; Hepatology 28:404-411) once reported with the compounds of this invention treatment hepatogenotoxicity ischemic injuries.Although the author has explained their result from compound to this angle of Fibrotic effect, they admit, to liver function index, comprise that the good result of bilirubinic serum levels, cholic acid and alkali phosphatase can not be directly owing to Fibrotic minimizing.

Bickel etc. (the same) have described the model of toxic hypoxia hepatic injury.Simply say the male Mus (212-320g) of Wistar or accept the carbon tetrachloride (CCl that 1ml/kg is dissolved in olive oil by feeding ₄) (1: 1), weekly twice, 9 weeks (n=140) altogether, or do not receive treatment (contrast; N=10).In addition, accept CCl for one group ₄(n=60) animal is accepted compound P simultaneously.Described compound is used by peritoneal injection, and every day 2 times, dosage is 60mg compound/2ml saline/kg body weight.After 9 weeks, kill animals, the weight of title liver.Measure bilirubin, alanine aminotransferase, alkali phosphatase, albumin and the total bile acid in serum with the commercial tool case.

As can be seen from Table 6 (Bickel etc., the same, table 2), the impaired rear body weight of liver (BW) obviously descends, but the weight of liver own does not have the significant change (not shown).

The serum parameters of table 6 treatment liver function after 9 weeks

Treatment

N

BW(g)

BR(μmol/L)

tBA(μmol/L)

ALT(U/L )

AP(U/L)

Contrast

10

425±66.9

2.00±0.50

8.48±8.40

27.5±10.9

156±57.5

CCl 4

80

370±43.3

4.34±3.93

81.3±87.9

83.1±51.7

269±117

CCl 4+CPD

60

373±38.9

2.83±2.21

40.8±51.4

59.0±29.5

195±72.7

Numeric representation meansigma methods ± standard deviation in table.

Statistically see, hepatic injury is also to obviously descending liver function, this mensuration from following index can be found out: the serum levels bilirubin (BR), total resistance acid (tBA), alanine aminotransferase (ALT) and alkali phosphatase (AP) increase respectively 117%, 856%, 201% and 72%.But, with the compounds of this invention (CPD) treatment, statistically see and can obviously improve liver function.Treatment group is compared with treatment group not, and serum levels BR, tBA, ALT and AP descend respectively 64%, 65%, 43% and 65%.The improvement of liver function is attributable to the Stabilization of the inventive method to HIF α.

Embodiment 8: renal ischemic reperfusion injury

Nemoto etc. have described the model (2001, Kidney Int 59:246-251) of ischemic acute renal failure.In brief, by feeding 0.5% carboxymethyl cellulose (CMC; Sigma-Aldrich) or 1.5% compd B be suspended in CMC is treated the male Mus of Sprague-Dawley (200-250g), and dosage is 4ml/kg/d.First rat is carried out to 4 days continuous pretreats (the-3 days to the 0th day).Carried out the 4th at the 0th day, namely feeding rear a few hours of last dosage, carry out renal ischemic reperfusion injury (IRI) experiment.

Animal is divided into to four groups: (1) is with the medium pretreatment and carry out sham operated; (2) with the compd B pretreat and carry out sham operated; (3) with the medium pretreatment and carry out IRI hands art; (4) with the compd B pretreat and carry out the IRI operation.With isoflurane, animal is implemented to anesthesia, at the abdominal part center line, cut openning one, directly cut the kidney base of a fruit.With vascular clamp, right renal pedicle clamp is lived 45 minutes, excise left kidney simultaneously.After each closure, clip is unclamped 45 minutes, by the variation of kidney color, can find out again and pour into.It is constant that temperature maintains, and the warm saline that contains the Buprenex analgesic (0.5% body weight) directly pours into abdominal part, then sews up the incision.

Monitoring the weight of animals and mortality rate.From tail vein, take a blood sample, serum chemistry forms and CBC send IDEXX Veterinary Services (West Sacramento CA) to measure.Data record is meansigma methods ± SE, and bracket inner digital is number of animals.Compare the data of four groups of samples on each time point by unidirectional variance analysis (ANOVA, SIGMASTAT) and Student-Newman-Keuls method.P<0.05 means to differ remarkable.

The Deaths that as shown in figure 13, by compound treatment, can prevent ischemical reperfusion injury to cause.In addition, at the 3rd day and the 7th day, kidney IRI significantly improved serum urea nitrogen (BUN), and the BUN that makes IRI cause with compounds for treating increases degree and obviously descends, and wherein BUN is that of renal function quality measures (Figure 14 A).In addition, at the 3rd, 7 and 14 days, kidney IRI can significantly improve serum cholesterol, the increase (Figure 14 B) of the serum cholesterol of having prevented IRI to cause fully with compounds for treating.Although reason is still among exploring, it is the reflection naturally of renal ischemic reperfusion injury that the kidney cholesterol rises.(Zager etc. (2001) Am J Pathol 159:743-752; Appel (1991) Kidney Int 39:169-183; Abdel-Gayoum etc. (1999) Hum Exp Toxicol18:454-459)

Embodiment 9: the promotion that granulation tissue in chronic trauma is formed

The ability employing Morris for the treatment of chronic trauma etc. (1997, Plast Reconstr Surg 100:674-681) and the scale-model investigation that scabs of the rabbit corticohyperplassia introduced of Marcus etc. (2000, Plast Reconstr Surg 105:1591-1599).In brief, to female New Zealand white rabbits (n=12; 3-6 month is large) implement anesthesia, by removing perichondrium, cause the skin ulcer wound of 4 7mm length in the ventral surface of every ear.The treatment wound, and wrap up with the semiclosed polyurethane gauze of TEGADERM (3M Health Care, St.Paul MN).The 1st week, local coating 0.5% or 1% (w/v) is blended in 0.5% (w/v) CARBOPOL 971PNF hydrosol, and (pH 6.5; Noveon Inc., Cleveland OH) the compound V prodrug [pV] in, with the treatment wound, once a day.While carrying out testing in vitro, find that gel discharged 50% medicine in 2 hours, discharged 95% medicine in 4 hours.At the ear for the treatment of or accept low dosage and administer (0.5% compound), or accept high-dose therapy (1% compound), and the contrast ear is only accepted gel.Open a hole on the gauze of wrapping when injured, the medicine for the treatment of use is sent into from hole, avoids taking off every day gauze and causes the infection of wound circumference zone.Then at hole upper cover one fritter gauze, prevent the wound drying.Too dry or infected wound will be got rid of outside research.

After injured 7 days and 12 days, open and cut wound, be infected with the hematoxylin potato is red, with the formation of mensuration granulation tissue and the situation of wound skin.The observer who is unfamiliar with Therapeutic Method collects the trauma care quantitative parameter by the eyepiece cross-hair with scale.By the t-experimental analysis data of Student, relatively treat and do not treat sample.P<0.05 means to differ remarkable.

Wound has been measured to the formation of granulation tissue and the situation of wound skin; Measured trauma care parameter (Corral etc. (1999) the Arch Surg 134:200-205 to ischemia and hypoxia sensitivity; Ahn and Mustoe (1990) AnnPlast Surg 24:17-23).As shown in Figure 15 A, through the wound for the treatment of, with untreated wound, compare, can observe the increase in granulation tissue zone.From Figure 15 B, can find out, in the animal through treatment and not process treatment, the peak-peak distance is as broad as long.Peak-to-peak value is the index that granulation tissue covers wound.Therefore, available the inventive method increases the formation of wound medium vessels and granulation tissue, as chronic trauma and ulcer.

Embodiment 10; Screening experiment

Can suppress HIF specificity prolyl hydroxylase activity, thereby the compound of stablizing HIF α can utilize following experimental technique to identify and characterize.Take out the separatory such as 50 μ l from the reactant mixture that comprises 4mg/ml BSA, 0.1M Tris HCl (pH 7.2), 2mM Ascorbate, 80 μ M ferrous sulfate, 0.2mM 2-oxoglutaric acid, 600 units/ml catalase and optional 100 μ MHIF α peptides, mix with 50 μ l HeLa cell extraction things or purification HIF prolyl hydroxylase, cultivate 1.5 hours at 37 ℃.Then add 50 μ l N,N'''-(2,4,5,6-tetrahydroxy-1,3-cyclohexanediyl)bisguanidine pearls, 4 ℃ and stir under cultivate the gained mixture 1 hour.Mixture is transferred in test tube, and low-speed centrifugal, make pearl form bead.Wash pearl 3 times with 0.5-1ml 20mM Tris HCl (pH 7.2).From pearl, elution is out by peptide to be dissolved in 2mM biotin in 20mM Tris HCl (pH 7.2) with 5 μ l.Centrifuge tube, make the resin formation bead, removes 40-50 μ l supernatant, adds the equivalent acetyl nitrile.Perhaps, peptide is attached on methoxy coumarin, it is to the insensitive fluorogen of pH.Described fluorogen can provide sensitivity and specificity, thereby promotes the detection to the thickness cytosol.Exemplary HIF peptide for screening experiment can comprise [methoxy coumarin]-DLDLEALAPYIPADDDFQL-amide (SEQ ID NO:5).Then separate non-hydroxylated and hydroxylating peptide with reversed-phase HPLC by the C18 chromatographic column, and detect with 214nmUV.

Those skilled in the art are understood that from the introduction of front, except embodiment as described herein, can do various modifications to the present invention.Within these modifications are included in appended claims scope of the present invention.

Here all documents of quoting all with its whole content as a reference.

Sequence table

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T.B. Buddhist nun husband (Neff, Thomas B.)

Q. king (Wang, Qingjian)

M. A Lande (Arend, Michael)

L.A. Fu Liping (Flippin, Lee A.)

A. prunus mume (sieb.) sieb.et zucc. Rec Hough (Melekhov, Alexey)

<120 > stabilisation of hypoxia inducible factor (HIF) α

<130>FP0600 PCT

<140>PCT/US02/38867

<141>2002-12-06

<150>US 60/337,082

<151>2001-12-06

<150>US 60/359,683

<151>2002-02-25

<150>US 60/349,659

<151>2002-01-16

<150>US 60/386,488

<151>2002-06-05

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<170>Patentln version 3.2

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<213 > rat (Rattus norvegicus)

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cggcggcttt ggtgactcta gat 23

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<213 > artificial

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Phe Gln Leu

Claims (29)

1. a compound is for the preparation of the purposes of the medicine of ischemic disease in treatment target, it is characterized in that, described compound is the isoquinolin carboxylic acid amides that hydroxylating is stablized the α subunit (HIF-α) of hypoxia inducible factor to occur by suppressing hypoxia inducible factor (HIF), and described isoquinolin carboxylic acid amides is selected from following chemical formula (Ib)

Wherein,

A is-CH ₂-;

B is-CO ₂H or-CO ₂-G, and wherein G is (C1-C20)-alkyl;

X is O;

Q is O;

R ⁴Hydrogen or methyl;

R ³Hydrogen, halogen or (C1-C20) alkyl

R ¹⁶And R ¹⁹Hydrogen

R ¹⁷And R ¹⁸The group that each forms independently selected from following group: hydrogen, halogen, (C1-C20) alkoxyl, (C7-C16) aralkoxy, (C6-C12) aryloxy group, and-O-[CH ₂] _xC _fH _(2f+1-g)F _g, wherein f be 1, g be 3 and x be 0; Wherein any aryl can be replaced by 1 to 5 substituent group be selected from the group that following group forms: halogen, (C2-C16) alkyl and (C1-C16) alkoxyl;

Or the derivative physiologically active salt of above-claimed cpd.

2. purposes as claimed in claim 1, wherein said compound is the isoquinolin carboxylic acid amides shown in chemical formula (Ib), wherein,

A is-CH ₂-;

B is-CO ₂H;

X is O;

Q is O;

R ⁴Hydrogen;

R ³Hydrogen, halogen or (C1-C20) alkyl;

R ¹⁶And R ¹⁹Hydrogen;

R ¹⁷Hydrogen or (C6-C12) aryloxy group; And

R ¹⁸Hydrogen, (C1-C20) alkoxyl, (C6-C12) aryloxy group or trifluoromethoxy.

3. purposes as claimed in claim 1, wherein said compound is selected from following group:

N-((1-chloro-4-hydroxyl-isoquinolin-3-carboxyl)-amino)-acetic acid;

N-((6-benzyloxy 1-chloro-4-hydroxyl-isoquinolin-3-carboxyl)-amino)-acetic acid;

((7-benzyloxy-1-chloro-4-hydroxyl-isoquinolin-3-carboxyl-amino)-methyl acetate;

N-((7-benzyloxy-1-chloro-4-hydroxyl-isoquinolin-3-carboxyl)-amino)-acetic acid;

N-((7-butoxy-4-hydroxyl-isoquinolin-3-carboxyl)-amino)-acetic acid

4. as the described purposes of claim 1-3 any one, wherein said compound suppresses the activity of 2-oxoglutaric acid dioxygenase.

5. as the described purposes of claim 1-3 any one, wherein said compound suppresses the activity of HIF prolyl hydroxylase.

6. purposes as claimed in claim 5, wherein said HIF prolyl hydroxylase is selected from EGLN1, EGLN2, EGLN3 and their any fragment or subunit.

7. as the described purposes of claim 1-3 any one, wherein said compound suppresses FIH-1 or its any fragment or the enzymatic activity of subunit.

8. as the described purposes of claim 1-3 any one, wherein said treatment target is mammal.

9. as the described purposes of claim 1-3 any one, wherein said treatment target is the people.

10. as the described purposes of claim 1-3 any one, wherein said HIF-α is selected from HIF-1 α, HIF-2 α, HIF-3 α and their any fragment.

11., as the described purposes of claim 1-3 any one, be born on treatment target in wherein said HIF-α.

12., as the described purposes of claim 1-3 any one, wherein said ischemic disease is relevant with hypoxia.

13., as the described purposes of claim 1-3 any one, wherein said ischemic disease is pulmonary disease.

14., as the described purposes of claim 1-3 any one, wherein said ischemic disease is heart disease.

15., as the described purposes of claim 1-3 any one, wherein said ischemic disease is sacred disease.

16., as the described purposes of claim 1-3 any one, wherein said ischemic disease is relevant with the ischemia event.

17. purposes as claimed in claim 16, wherein said ischemia event is acute.

18. purposes as claimed in claim 16, wherein said ischemia event is with operation, organ transplantation, infraction, wound or damage relevant.

19. purposes as claimed in claim 16, wherein said ischemia event is chronic.

20. purposes as claimed in claim 16, wherein said ischemia event is with to be selected from hypertension, diabetes, closed arterial disease, chronic venous insufficiency of function, Raynaud's disease, sclerosis, congestive heart failure relevant with the disease of system sclerosis.

21., as the described purposes of claim 1-3 any one, wherein said medicine comprises the second compound.

22. purposes as claimed in claim 21, wherein said the second compound can suppress the activity of 2-oxoglutaric acid dioxygenase.

23. purposes as claimed in claim 21, wherein said compound can suppress the activity of the first 2-oxoglutaric acid dioxygenase, and described the second compound can suppress the activity of the second 2-oxoglutaric acid dioxygenase.

24. purposes as claimed in claim 21, wherein said the second compound is selected from ACE inhibitor (ACEI), angiotensin II receptor blocking agent (ARB), diuretic, digoxin, statine or carnitine.

25., as the described purposes of claim 1-3 any one, wherein said compound can be stablized HIF-α by least one amino acid residue generation hydroxylating in special inhibition HIF-α.

26. purposes as claimed in claim 25, wherein said amino acid residue is selected from proline and agedoite.

27., as the described purposes of claim 1-3 any one, wherein said compound is for the preparation of the medicine of ischemical reperfusion injury in treatment target.

28., as the described purposes of claim 1-3 any one, wherein said compound provides with the form of oral agents.

29., as the described purposes of claim 1-3 any one, wherein said compound provides with the form of skin transdermal agent.

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