CN1138472C

CN1138472C - Dipeptide apoptosis inhibitors and the use thereof

Info

Publication number: CN1138472C
Application number: CNB988100223A
Authority: CN
Inventors: J・F・W・克阿纳; J·F·W·克阿纳; ⑺固乩; 蔡遂雄; 德里威; J·古阿斯特拉; 杨武; J·A·德里威
Original assignee: Cytovia Inc
Current assignee: Cytovia Therapeutics LLC
Priority date: 1997-10-10
Filing date: 1998-10-09
Publication date: 2004-02-18
Anticipated expiration: 2018-10-09
Also published as: NO20001323D0; EP1033910A1; KR100580333B1; KR20010031053A; CN1301131A; EP1033910A4; AU9793098A; AU741203B2; EA200000409A1; NO20001323L; CA2306692A1; WO1999018781A1; JP4439111B2; BR9814817A; JP2001519358A; CA2306692C

Abstract

The present invention is directed to novel dipeptides thereof, represented by the general Formula I: where R1-R3 and AA are defined herein. The present invention relates to the discovery that compounds having Formula I are potent inhibitors of apoptotic cell death. Therefore, the inhibitors of this invention can retard or block cell death in a variety of clinical conditions in which the loss of cells, tissues or entire organs occurs.

Description

Dipeptides type apoptosis inhibiting agent and uses thereof

Background of invention

Invention field

The present invention is the medical chemistry field.Specifically, the present invention relates to dipeptides into the potent inhibitor of programmed cell death.The invention still further relates to the application of these dipeptides in reduction or treatment programmed cell death.

The description of background technology

Organism is removed unwanted cells by the process that differently is called controlled cell death, program cell death or programmed cell death.This cell death is animal development and organizes homeostasis and aging normal condition (Glucksmann, A., biology comment (Biol.Rev.Cambridge Philos.Soc.) 26:59-86 (1951) of Cambridge philosophy association; Glucksmann, A., biology document 76:419-437 (1965); Ellis etc. grow (Dev.) 112:591-603 (1991); Vaux etc., cell 76:777-779 (1994)).Programmed cell death is regulated cell number, promotes form to form, and removes harmful or abnormal cell and remove the cell that those have finished its function.In addition, programmed cell death can respond various physical stress, (the application WO96/20721 that PCT announces) takes place such as hypoxgia or ischaemic.

The cell of experience controlled cell death has many total morphological change, comprise that endochylema and nuclear membrane bubble, cell shrinkage (caryoplasm and kytoplasm concentrate), organelle is reorientated and is compressed, chromatin concentrates and produces apoptotic cell body (the membrane closure particle that contains intracellular organic matter) (Orrenius, S., international medical journal 237:529-536 (1995)).

Programmed cell death is (Wyllie, A.H., the cell death in biology and the pathology, Bowen and Lockshin volume, Chapman and Hall publication (1981), the 9-34 page or leaf) by the endogenous mechanism realization of cell suicide.Because of the effect of inside or external signal, the suicide programme of its in-line coding of cell-stimulating.This suicide programme is (Wylie etc., the Int.Rev.Cyt.68:251 (1980) that finishes by the activation of the gene program of finely regulating; Ellis etc. comment 7:663 (1991) cell biological academic year).Apoptotic cell and cyton are discerned and are removed by adjacent cells or macrophage before cracking usually.Because this removing mechanism although there is a large amount of cells to be eliminated, can't be induced inflammation (Orrenius, S., international medical journal 237:529-536 (1995)).

Mammal il-1 β (IL-1 β) plays an important role in various pathological processes, and described pathological process comprises chronic and acute inflammation and autoimmune disease (Oppenheim, J.H. etc., immunology 7:45-56 today (1986)).IL-1 β is synthetic as cell associated precursors polypeptide (IL-1 β is former), and this IL-1 β is former can not be in conjunction with the IL-1 acceptor, and abiology activity (Mosley etc., journal of biological chemistry 262:2941-2944 (1987)).By suppressing of the conversion of IL-1 β precursor, just can suppress the activity of il-1 to ripe IL-1 β.Il-1 'beta ' converting emzyme (ICE) be protease that the activation of il-1 β (IL-1 β) is worked (Thornberry, N.A. is etc., natural 356:768 (1992); Yuan, J. etc., cell 75:641 (1993)).ICE is that a kind of energy cracking non-activity il-1 originates in the substrate specificity cysteine proteinase that generates ripe IL-1.The gene of coding ICE and CPP32 is the member in the mammal ICE/Ced-3 gene family, this gene family comprises at least 12 members: ICE, CPP32/Yama/Apopain, mICE2 at present, ICE4, ICH1, TX/ICH-2, MCH2, MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICErel III.It is very crucial that the proteolytic activity of this cysteine proteinase family seems in the mediation of cell death, and their avtive spot cysteine residues is the programmed cell death essential (Miura etc., cell 75:653-660 (1993)) of ICE mediation.Recently, this gene family has been named as caspases (Alnernri, E.S. etc., cell 87:171 (1996)).

IL-1 also is a kind of cell factor that participates in mediating extensive biological answer-reply, described biological response comprises inflammation, septic shock, wound healing, hemopoietic and some leukemia growth (Dinarello, C.A., blood 77:1627-1652 (1991); DiGiovine etc., immunology 11:13 today (1990)).

Now many strong caspases inhibitor have been prepared based on the peptide substrates structure of caspases.Yet opposite with their external potent energy, also report does not have pair programmed cell death intact cell model to have good efficacy (IC ₅₀＜1 μ M) inhibitor (Thornberry, N.A., chemical biology (Chem.Biol.) 5:R97-103 (1998)).Therefore, need and in the intact cell model of programmed cell death, to demonstrate effect (IC ₅₀＜1 μ M) effective cell death inhibitor and in the animal model of programmed cell death.These inhibitor can be used as therapeutic agent, are used for the treatment of the morbid state that the cytokine activity dead and IL-1 of controlled cell wherein works.

WO93/05071 discloses peptide ICE inhibitor, and its structural formula is:

Z-Q ₂-Asp-Q ₁Wherein Z is the terminal protecting group of N-; Q ₂For 0-4 amino acid, make calling sequence Q ₂-Asp is equivalent at least a portion of sequence A la-Tyr-Val-His-Asp; Q ₁Comprise the elecrtonegativity leaving group.Exemplary dipeptides is Boc-His-Asp-CH ₂F, Boc-Tyr-Asp-CH ₂F, Boc-Phe-Asp-CH ₂F, Ac-His-Asp-CH ₂F, Ac-Tyr-Asp-CH ₂F, Ac-Phe-Asp-CH ₂F, Cbz-His-Asp-CH ₂F, Cbz-Tyr-Asp-CH ₂F and Cbz-Phe-Asp-CH ₂F.

WO96/03982 discloses the aspartic acid analog as the ICE inhibitor, and its structural formula is:

R wherein ₂Be H or alkyl; R ₃Be that leaving group is such as halogen; R ₁Be heteroaryl-CO or amino acid residue.

United States Patent (USP) 5,585,357 disclose the peptide ketone as the ICE inhibitor, and its structural formula is: Wherein n is 0-2; AA is L-valine or L-alanine independently of one another; R ₁Be selected from N-benzyloxycarbonyl and other groups; R ₈, R ₉, R ₁₀Be hydrogen, low alkyl group and other groups independently of one another.

Revesz etc. (tetrahedron wall bulletin 35:9693-9696,1994) have reported the preparation as the ethyl ester tripeptides of the prodrug of respective acids:

Described respective acids is a kind of strong ICE inhibitor.

Summary of the invention

The present invention relates to the dipeptides of formula I:

R wherein ₁It is the terminal protecting group of N-; AA is the residue or the beta-amino acids of any natural a-amino acid; R ₂Be H or CH ₂R ₄, R wherein ₄It is the elecrtonegativity leaving group; R ₃Be alkyl or H, condition is that AA is not His, Tyr, Pro or Phe.

The present invention relates to a discovery, although that is: two peptidyl caspase inhibitor of formula I representative show the effectiveness of enzyme lowlyer than tripeptides and tetrapeptide inhibitor, in based on the system of cell, be shown as the potent inhibitor of programmed cell death astoundingly in enzyme test.These compounds show in the body system activity and are that anti-Fas induces deadly potent inhibitor in Mouse Liver programmed cell death model, in the ishemic stroke rat model, demonstrate strong neuroprotection.

The invention still further relates to dipeptides of the present invention is application in paathogenic factor or its result's the disease alleviating, prevent or treating programmed cell death wherein.The example that the present invention uses comprises: neuroprotective system behind focus ischaemic and general ischemic; The treatment neurodegenerative disease is such as alzheimer's disease, Huntington, prion disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, incoordination, capillarectasia and spinobulbar atrophy; The treatment cardiopathy comprises miocardial infarction, congestive heart failure and cardiomyopathy; The treatment retinal disease; The treatment autoimmune disease comprises lupus erythematosus, rheumatoid arthritis, type i diabetes, siogren's syndrome and glomerulonephritis; Treatment MCKD and anaemia/erythropoiesis; The treatment disease of immune system comprises AIDS and SCIDS; In migration process, reduce or prevention cell, tissue and organ damage; In the industrial biological engineering, reduce or the death of prevention cell-line; Reduce or prevention alopecia (trichomadesis); And the premature dead that reduces Skin Cell.

The invention provides a kind of pharmaceutical composition, it comprises the formula I compound of the effective dose of the programmed cell death that can reduce animal.

The present invention also provides the solution that is used for the anticorrosion of mammalian organs or tissue or stores, or be used for the somatomedin of mammal or yeast cells, the formula I compound that has wherein comprised effective dose in described solution or somatomedin is to reduce the programmed cell death of described organ, tissue or cell.

Brief description of the drawings

Figure 1A-1G shown respectively with Cyclohexamide (CHX) and DMSO stimulate (Figure 1A), with tumor necrosis factor-alpha (TNF-α)/CHX and DMSO stimulate (Figure 1B), with 50 μ MBOC-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 1 C), with 50 μ M Cbz-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 1 D), with 50 μ M Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 1 E), with 50 μ MCbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 1 F) and stimulate the photo of the Hela cell of (Fig. 1 G) with DMSO.

Fig. 2 A-2G shown respectively with Cyclohexamide (CHX) and DMSO stimulate (Fig. 2 A), with TNF-α/CHX and DMSO stimulate (Fig. 2 B), with 5 μ M BOC-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 2 C), with 5 μ M Cbz-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 2 D), with 5 μ M Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 2 E), with 5 μ M Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 2 F) and stimulate the photo of the Hela cell of (Fig. 2 G) with DMSO.

Fig. 3 A-3G shown respectively with Cyclohexamide (CHX) and DMSO stimulate (Fig. 3 A), with TNF-α/CHX and DMSO stimulate (Fig. 3 B), with 0.5 μ M BOC-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 3 C), with 0.5 μ M Cbz-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 3 D), with 0.5 μ M Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 3 E), with 0.5 μ M Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F, TNF-α/CHX stimulate (Fig. 3 F) and stimulate the photo of the Hela cell of (Fig. 3 G) with DMSO.

Fig. 4 is a bar chart, has shown Cbz-Val-Asp (the OMe)-CH of various concentration ₂Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (the OMe)-CH of F (a) and various concentration ₂F (b) compares, and protection Hela cell is avoided the situation of TNF-α/CHX infringement.

Fig. 5 is a bar chart, has shown Cbz-Val-Asp (the OMe)-CH of various concentration ₂F (a) and BOC-Asp (OMe)-CH ₂F (b) protection Hela cell is avoided the situation of TNF-α/CHX infringement.

Fig. 6 has shown Cbz-Val-Asp (the OMe)-CH of various low dosages ₂Cbz-Val-Ala-Asp (the OMe)-CH of F (a) and various low dosages ₂F (b) compares, and protection Hela cell is avoided the situation of TNF-α/CHX infringement.

Fig. 7 A-7E has shown the PARP breaking test result of Jurkat cell.Compound 1=Cbz-Val-Asp (OMe)-CH ₂F, compound 2=Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F, compound 3=Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F, compound 4=Cbz-Ile-Glu (OMe)-Thr-Asp (OMe)-CH ₂F, compound 5=BOC-Asp (OMe)-CH ₂F, compound 6=Cbz-Asp-α-([2,6-dichloro-benzoyl oxygen base] methyl ketone), compound 7=Cbz-Val-Ala-Asp (OMe)-CH ₂F.

Fig. 8 A and 8B are the photos of PARP cracking, have shown Z-VD-fmk and the Z-VAD-fmk inhibition to the PARP cracking of the Jurkat cell handled with anti-Fas.

Fig. 9 be survivaling cell than Z-VD-fmk concentration map, shown the inhibition of the cell death that Z-VD-fmk induces TNF-α.

Figure 10 is the photo of dna ladder shapeization, has shown the inhibition of Z-VD-fmk to the dna ladder shapeization of the Jurkat cell handled with anti-Fas.

Figure 11 A and 11B have shown the Cbz-Val-Asp-CH that is administered systemically ₂F is to the neuroprotection of instantaneous focus ischaemic rat model.Moment focus ischaemic 2.25 hours and pour into 22 hours again after, the volume of quantitative measurment cortex infraction.

The detailed description of invention

The inhibitor of programmed cell death of the present invention is the compound of general formula I representative:

Or its pharmaceutically acceptable salt or prodrug, wherein:

R ₁Be the terminal protecting group of N-, comprise tert-butoxycarbonyl, acetyl group and benzyloxycarbonyl; AA is residue or the beta-amino acids of any natural a-amino acid, for example: Gly, Thr, Glu, Lys, Arg, Ser, Asn, Gln, Val, Ala, Leu, Ile, Met, and beta-amino acids is such as β-Ala, but they are not His, Tyr, Pro or Phe; R₂H or CH₂R ₄, Rx is elecrtonegativity leaving group such as F, Cl, TsO-, MeO-, ArO-, ArCOO-, ArN-and ArS-; R₃Alkyl or H.

About R₃, preferred alkyl is C_1-6Alkyl, for example: methyl, ethyl, propyl group, isopropyl, isobutyl group, amyl group and hexyl.

The present invention relates to a discovery, although that is: two peptidyl caspase inhibitor of formula I representative are lower than tripeptides and tetrapeptide inhibitor to the effectiveness of enzyme, be based on astoundingly the potent inhibitor of the programmed cell death in the system of cell. These compounds show in the body system activity and are that anti-Fas-induces deadly potent inhibitor in Mouse Liver programmed cell death model, in the ishemic stroke rat model, demonstrate strong neuroprotection. These inhibitor will occur in the relevant various clinical disease of loss with cell, tissue or whole organ and slow down in the commercial Application or block cell death. Therefore, the invention still further relates to treatment, prevention or the methods to reduce noises of the disease that programmed cell death wherein works. These diseases will be described hereinafter more comprehensively.

Described method comprises that the animal capable that needs this treatment suppresses the inhibitor of the present invention of the effective dose of programmed cell death, or its pharmaceutically acceptable salt or prodrug.

The preferred embodiment that can be used as the compound of apoptosis inhibiting agent is the compound of formula II representative:

Or its pharmaceutically acceptable salt or prodrug, wherein AA, R₁And R₃Such as about formula I above definition.

Preferred R ₁Be tert-butoxycarbonyl, acetyl group and benzyloxycarbonyl.Preferred R ₃Be H, Me, Et or t-Bu.Preferred AA is that Val, Ala, Leu, Ile, Met and beta-amino acids are such as β-Ala.

The apoptosis inhibiting agent that the preferred formula I that exemplifies represents includes but not limited to:

Boc-Ala-Asp-CH ₂F，

Boc-Val-Asp-CH ₂F，

Boc-Leu-Asp-CH ₂F，

Ac-Val-Asp-CH ₂F，

Ac-Ile-Asp-CH ₂F，

Ac-Met-Asp-CH ₂F，

Cbz-Val-Asp-CH ₂F，

Cbz-β-Ala-Asp-CH ₂F，

Cbz-Leu-Asp-CH ₂F，

Cbz-Ile-Asp-CH ₂F，

Boc-Ala-Asp(OMe)-CH ₂F，

Boc-Val-Asp(OMe)-CH ₂F，

Boc-Leu-Asp(OMe)-CH ₂F，

Ac-Val-Asp(OMe)-CH ₂F，

Ac-Ile-Asp(OMe)-CH ₂F，

Ac-Met-Asp(OMe)-CH ₂F，

Cbz-Val-Asp(OMe)-CH ₂F，

Cbz-β-Ala-Asp(OMe)-CH ₂F，

Cbz-Leu-Asp (OMe)-CH ₂F and

Cbz-Ile-Asp(OMe)-CH ₂F.

Some compound of the present invention can be used as stereoisomer and comprises that optical isomer exists.The present invention includes the racemic mixture of all stereoisomers and these stereoisomers and the independent enantiomer that can separate according to the method that those of ordinary skills know.

The example of pharmaceutically acceptable addition salt comprises inorganic and organic acid addition salt, such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate.

The example of prodrug comprises wherein R ₃Be that alkyl or substituted alkyl are (such as CH ₂OCH ₃) formula I-II compound.In addition, when AA contains hydroxy-acid group, R wherein ₃For the example of the prodrug of the formula I-II of H comprises that one of them carboxyl is esterified or two carboxyls are all esterified (for example by C _1-6Alcohol esterification) or the corresponding amide form (for example with C _1-6Amine formation) compound.

The invention still further relates to the treatment of diseases method that response is arranged for the inhibition of programmed cell death in the animal of the hardship that suffers programmed cell death.The embodiment that is used for the particularly preferred compound of the inventive method is the compound of the defined formula II representative in front.

Compound of the present invention can utilize and well known to a person skilled in the art the method preparation.Particularly, formula I-II compound can be prepared according to the reaction of scheme 1 illustrated.Method (tetrahedron wall bulletin 35:9693-9696,1994) preparation intermediate product 1 according to Revesz etc.1 obtains acid amides 2 with the amino acid such as the Z-Val-OH coupling of N-protected, with Dess-Martin reagent 2 oxidations is obtained 3 according to the method (tetrahedron wall bulletin 35:9693-9696,1994) of Revesz etc.This ester obtains free acid 4 by acid-catalyzed cleavage, is converted into ester 5 with 4 again.

Scheme 1

An importance of the present invention has been to find that formula I-II compound is the potent inhibitor of programmed cell death.Therefore, expect that these inhibitor can slow down or block cell death in the various clinical diseases of loss take place for cell, tissue or whole organ.

Cell death inhibitor of the present invention can be used for reducing in various ischaemics and exitotoxicity disease or the cell death of prevention nervous system (brain, spinal cord and peripheral nervous system), the general ischemic that described disease includes but not limited to the focus ischaemic that causes because of apoplexy and stops to cause because of heartbeat.A specific usage is to be used for the treatment of the anoxic that may occur in neonate's birth process in the high-risk childbirth.The neural cell death that these cell death inhibitors can also be used for reducing or prevention causes because of traumatic injury (such as head trauma), virus infections or radiation-induced the nerve cell death side effect of cancer radiation (for example, as) etc.These cell death inhibitors can also be used to reduce or prevent the interior cell death of neurodegenerative disease scope, and described disease includes but not limited to alzheimer's disease, Huntington, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis and spinobulbar atrophy.The neuroprotective performance can be checked (Xue etc., apoplexy 21:166 (1990)) with instantaneous focus ischaemic rat model in the body of cell death inhibitor of the present invention.

Cell death inhibitor of the present invention can be used for preventing to cause the cell death of the dead any disease of cardiac muscle.These diseases comprise miocardial infarction, congestive heart failure and cardiomyopathy.Specific application is the cardiomyocyte cell death that is used to reduce or takes place when preventing heart because of some virus infections.

" Mouse Liver programmed cell death " model that the activity in vivo of cell death inhibitor of the present invention can utilize Rodriguez etc. (Rodriguez etc., The Journal of Experimental Medicine 184:2067-2072 (1996)) to describe is tested.In this model, give mouse mainline (IV) anti-Fas antibody, this antibody capable is induced large-area programmed cell death in liver and other organs, thereby causes generality organ failure and death.Can be with the systemic bioavailability of this model indirectly testing cell death inhibitor of the present invention, and anti-programmed cell death performance in their body.

Cell death inhibitor of the present invention can be used for preventing the cell death of intraocular pressure rising disease (such as glaucoma) or retinal disease (such as AMD) the contingent retinal neurons relevant with ageing process.These inhibitor also can be used for treating the hereditary retinal dystrophy disease, such as retinitis pigmentosa.

Cell death inhibitor of the present invention also can be used for reducing or the premature dead of epidemic prevention system cells, especially for the treatment immune deficiency disorder, such as acquired immunodeficiency syndrome (AIDS), severe combined immunodeficiency syndrome (severe combined immunedeficiency syndrome, SCIDS) and relevant disease.These cell death inhibitors also can be used for treating radiation-induced immunosupress.

People's organ and tissue transplantation are methods of treatments commonly used in the organ failure.Yet in transplanting disposal process, donor organ or tissue have the danger that cell death takes place, because they had lost normal blood supply before implanting the host.This ischaemic state can perhaps be treated by cell death inhibitor is directly joined in the organ-/ tissue storage medium by giving donor organ or organizing the infusion cell death inhibitor to treat.Cell death inhibitor also can be used to reduce or prevent to transplant the cell death of back donor organ/tissue, is not subjected to by triggering the killing effect that programmed cell death kills the host immune cell of its target to protect them.The cytoprotection of cell death inhibitor also can be used for preventing the human or animal's seminal fluid used in the inseminatio externalis process and the death of ovum.These inhibitor can use in the results process, also can be included in the storage medium.

Mammal cell line and yeast cells are usually used in producing a large amount of industry or medicinal recombinant protein (such as antibody, enzyme or hormone).Because the character (some are poisonous) of growth conditions, expressed recombinant molecule and the influence of other X factors, the valid expiration date that some such cell-lines are arranged is limited.Can prolong the valid expiration date of industrial cell-line by the cell death inhibitor that in growth substrate, adds 10-200mM concentration.

The factor of regulating hair growth and loss is not clear mostly.Yet, there are some evidences to show, hair follicle degeneration (being called catagen) to small part is attributable to programmed cell death.Therefore, expect that cell death inhibitor of the present invention can be used for treating the alopecia that causes because of various diseases, the alopecia that described disease includes but not limited to male pattern baldness, radiation-induced or alopecia that chemotherapy is induced and causes because of emotional stress.Show on evidence that also programmed cell death works in the going down of color development.Therefore, expection cell death inhibitor of the present invention also can be used for treating or prevents hair grizzled situation too early.

The death of skin epithelial cell can take place after contacting high-caliber radiation, heat or chemical substance.Expect that cell death inhibitor of the present invention can be used for treatment, reduces or prevent such skin lesion.In a specific application, these cell death inhibitors can be used for the treatment of the serious solar radiation of skin excessively and prevent skin to blister and peel with ointment form.

Goldberg etc. (natural genetics 13:442-449 (1996)) are report recently, and the protein product-Hang Ting of Huntington (HD) gene pauses element (huntingtin) can be by CPP32 rather than ICE cracking.Sudden change among the HD is the expansion of the CAG trinucleotide of HD gene 5 ' end.This trinucleotide expansion repetition above 36 times is relevant with the clinical manifestation of HD.It is plain by the CPP32 cracking that CAG expansion has promoted that Hang Ting pauses, and therefore in HD effect and the programmed cell death of CPP32 connected.The CPP32 of having of the present invention suppresses active compound will can be used for blocking the programmed cell death that CPP32 induces, prevent and treat HD and other diseases that expands to feature thus, such as myotonia atrophica, fragile X mental retardation, spinal cord oblongata amyotrophia, the spinocebellar ataxia of I type and dentate nucleus-rubrum globus pallidus corpus hypothalamicum atrophy with the trinucleotide duplicate block.

Composition in the scope of the invention comprises all compositions that wherein comprise The compounds of this invention, and The compounds of this invention amount therein is enough to reach its intended purposes.Although individual needs are inequality, determining of the best effective dose scope of every kind of component is routine techniques in this area.In general, to mammal for example the oral dose of the compound of people's administration be the 0.0025-50mg/kg body weight/day, or its pharmaceutically acceptable salt of a great deal of, body weight is meant the mammiferous body weight of the disease of suffering from programmed cell death mediation for the treatment of, and described disease is for example nerve cell death, cardiopathy, retinal disease, MCKD and disease of immune system.Preferably, be used for prevention or treat these diseases with the amount oral administration of the about 10mg/kg of about 0.01-.For intramuscular injection, dosage generally is about 1/2nd of oral dose.For example, when treatment or prevention nerve cell death, suitable intramuscular injection dosage should be the about 15mg/kg of about 0.0025-, most preferably is the about 10mg/kg of about 0.01-.

The unit oral dose can comprise the about 50mg of about 0.01-, the preferred about 10mg compound of about 0.1-.Unit dose can be used as that one or more tablet is administered once every day or repeatedly, contain in every the 0.1-that has an appointment about 10, be generally about 0.25-50mg compound or its solvate.

Except as this compound of crude drug administration, The compounds of this invention also can be used as a part of administration of the pharmaceutical preparation that contains suitable pharmaceutically acceptable carrier, and described carrier includes and helps excipient and the auxiliary agent that these compounds are processed into pharmaceutically acceptable preparation.Preferably, these preparations, but particularly Orally-administrable and can be used for preferred administration type those preparations such as tablet, dragee and capsule rectally preparation such as suppository and by the injection or the suitable solution of oral administration, they contain the 0.01-99% that has an appointment, the preferably active substance of about 0.25-75%, and excipient.

The nontoxic pharmaceutically acceptable salt that also comprises The compounds of this invention in the scope of the invention.The solution of specific cells death inhibitor of the present invention mixed with the solution of pharmaceutically acceptable non-toxic acid can form acid-addition salts, described non-toxic acid is all example hydrochloric acids, fumaric acid, maleic acid, succinic acid, acetate, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, or the like.The solution of specific cells death inhibitor of the present invention mixed can form basic salt with the solution of pharmaceutically acceptable nontoxic alkali, described nontoxic alkali is such as sodium hydroxide, potassium hydroxide, bursine, sodium carbonate etc.

Pharmaceutical composition of the present invention can be to experiencing any animals administer of The compounds of this invention beneficial effect.The most important thing is mammal in these animals, people for example is not though the present invention desires to be limited in this.

Pharmaceutical composition of the present invention can be with any way administration that can reach its intended purposes.For example, can be interior by outer, subcutaneous, the intravenous of stomach and intestine, intramuscular, peritonaeum, through skin, in the cheek, sheath or the encephalic administration.In addition, perhaps concurrently, can the by oral route administration.Dosage will depend on recipient's age, health status and body weight, the kind (if any) of concurrent treatment, the frequency of treatment and the character of Expected Results.

Pharmaceutical preparation of the present invention prepares in a known manner, for example, utilizes conventional mixing, granulation, sugar coating, dissolving or freeze-drying method.Therefore, oral pharmaceutical preparation can obtain with following method: reactive compound is mixed with solid excipient, grind this gained mixture alternatively and process this compound particles, if expectation afterwards or needs, add proper auxiliary agent, make tablet or dragee core thus.

Suitable excipient is filler especially, such as carbohydrate, and for example lactose or sucrose, mannitol or sorbitol, cellulose preparation and/or calcium phosphate, for example tricalcium phosphate or calcium monohydrogen phosphate, and adhesive is such as gelatinized corn starch, for example use corn starch, wheaten starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone.If desired, also can add disintegrant, all starch as mentioned above and carboxymethyl starch, crospolyvinylpyrrolidone, agar, or alginic acid or its salt are such as mosanom.Auxiliary agent is flow velocity conditioning agent and lubricant especially, silica for example, and talcum, stearic acid or its salt, such as dolomol or calcium stearate, and/or polyethylene glycol.The dragee core is wrapped suitable dressing, and if desired, this dressing is anti-gastric juice.For this purpose, can use priming, it can contain gum Arabic, talcum, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture alternatively.In order to prepare the dressing of anti-gastric juice, can use the solution of suitable cellulose preparation such as acetylcellulose phthalic acid ester or hydroxypropylmethyl cellulose phthalate.For example, in order to discern or, can in tablet or dragee dressing, to add dyestuff or pigment in order to describe the combined characteristic of active compound doses.

Other pharmaceutical preparations that can orally use comprise the sucking fit formula capsule that gelatin is made, and the sealing soft capsule of being made by gelatin and plasticizer such as glycerine and sorbierite.Sucking fit formula capsule can contain the reactive compound of particle form, and these particles can and mix with stabilizing agent alternatively with filler such as lactose, adhesive such as starch and/or lubricant such as talcum or dolomol.In soft capsule, reactive compound preferred dissolution or be suspended in the suitable liquid is such as fat oil or atoleine.Can add stabilizing agent in addition.

But the possible pharmaceutical preparation that per rectum uses comprises, suppository for example, and it is made up of the composition and the suppository base of one or more reactive compounds.Suitable suppository base is for example natural or synthetic glycerine three esters, or paraffin hydrocarbon.In addition, also might use the gelatin rectal capsule of forming by the mixture of reactive compound and matrix.Possible host material comprises, for example liquid triglycerides, polyethylene glycol or paraffin hydrocarbon.

The preparation of suitable parenteral comprises the aqueous solution of the reactive compound of water-soluble form, for example, and water soluble salt and aqueous slkali.Can add for example Tris of buffer solution.In addition, also can be used as of the reactive compound suspension administration of suitable oily injection with suspensions.Suitable lipophilic solvent or carrier comprise fat oil, for example sesame oil, or Acrawax, for example ethyl oleate or triglycerides or polyethylene glycol-400 (compound dissolves in PEG-400).Moisture injection suspension can contain the material that can improve this suspension viscosity, comprises for example sodium carboxymethylcellulose, sorbierite and/or glucan.Alternatively, this suspension also can contain stabilizing agent.

According to an aspect of the present invention, The compounds of this invention uses with local and parenteral formulation form, and is used for the treatment of skin injury, such as because of contacting high-caliber radiation, comprises what ultraviolet irradiation, heat or chemical substance caused.

Also can add in the said composition medicative one or more added substances of skin.Therefore, said composition also can contain the compound of cyclic adenosine monophosphate concentration in one or more skins that can raise.Suitable compound comprises the adenosine of about 0.1-1% or the papaverine of nucleic acid hydrolysis product and about 0.5-5%, and the two all is based on the percetage by weight of composition weight.The beta-Adrenergic agonists of about 0.1-2% is such as isoprel, or the cyclic adenosine monophosphate of about 0.1-1% also is suitable, and these two also is based on the percetage by weight of composition weight.The type that can join other the suitable additional activity compositions in the present composition comprises the known any compound that skin is had beneficial effect.Such compound comprises the chromanol of A1 vitamin group material (retinoid) such as the vitamin A of about 0.003-0.3 weight % and about 0.1-10 weight % such as the vitamin E or derivatives thereof, and the two is all based on composition weight.In addition, in cosmetic composition, can add antiinflammatory and cutin binder (keratoplastic agent).Typical antiinflammatory is the corticosteroid of the hydrocortisone of all weight of 0.25-5 according to appointment % or its acetate and so on, or the corticosteroid of the dexamethasone of all weight of 0.025-0.5 according to appointment % and so on, and the two is all based on the weight of composition.Typical cutin binder is the coal tar of about 0.1-20 weight % or the dithranol of about 0.05-2 weight %, and the two is all based on composition weight.

By the carrier of selecting to suit, topical compositions of the present invention preferably is mixed with finish, cream frost, washing lotion, ointment etc.Suitable carriers comprises vegetable oil or mineral oil, albolene (paraffinum molle alba), and chain fatty or oil, animal tallow and high molecular weight alcohol are (greater than C ₁₂).Preferred carrier is those of solubilized active component.If desired, can also comprise emulsifier, stabilizing agent, wetting agent and antioxidant, and the reagent that can produce color or fragrance.In addition, in these topical preparations, can use the transdermal penetration reinforcing agent.At United States Patent (USP) 3,989, can find the example of this reinforcing agent in 816 and 4,444,762.

The preferred mixture preparation with mineral oil, self-emulsifying beeswax and water of cream frost is mixed with in this mixture and is dissolved in a small amount of oil such as the active component in the apricot kernel oil.A representative instance of this cream frost is the cream frost that comprises about 40 parts of water, about 20 parts of beeswaxs, about 40 parts of mineral oil and about 1 portion of apricot kernel oil.

Ointment can be with the preparation of following method: active component is mixed with warm soft paraffin and this mixture is cooled off at vegetable oil such as the solution in the apricot kernel oil.A representative instance of this ointment is the ointment that comprises the white soft paraffin of the apricot kernel oil of about 30 weight % and about 70 weight %.

Washing lotion can prepare with following method is convenient: active component is dissolved in proper polymer amount alcohol such as in propane diols or the polyethylene glycol.

In addition, can comprise other as well known to those skilled in the art or clear medicaments, growth factor, wound sealant, carrier etc. in these compositions.Such as people's administration, the agglutination that its consumption is enough to make the host makes progress sooner during than untreated the present composition to the warm blooded animal that suffers skin injury such as burn.To depend on the patient's that treats the order of severity of skin lesion and this patient's general health state about the effective dose of this purposes.The maintenance dose of prolonged application can be adjusted as required.For veterinary purpose, can be as required with higher concentration administration.

When the hair growth of animal reduced, the present composition was to be enough to improve the amount administration of hair growth speed.Effective dose about this purposes will depend on the degree that the patient's that treats hair growth reduces, and this patient's general health state.The maintenance dose of prolonged application can be adjusted as required.For veterinary purpose, can be as required with higher horizontal administration.

Following examples explanation and do not limit method and composition of the present invention.The various conditions that normally run in the clinical treatment and other suitable improvement of parameter and variation and conspicuous for a person skilled in the art various improvement and variation are all within the spirit and scope of the present invention.

Embodiment 1

The 5-fluoro-4-hydroxyl-3-nitro valeric acid tert-butyl ester

To be present in dry CH ₂Cl ₂(1.9mL, 21.8mmol) solution is cooled to-78 ℃ to oxalyl chloride (100mL), adds while stirring and is present in dry CH ₂Cl ₂DMSO (10mL) (3.0mL, 42.3mmol) solution add speed and should make temperature remain on-50 ℃--and 60 ℃.Stir after 5 minutes, add and be present in dry CH ₂Cl ₂(1.2mL, 18.4mmol) solution continue to stir 15 minutes 2-fluoroethanol (10mL), add dry Et then ₃N (13.5mL).This reactant mixture stirred 15 minutes, made it return to room temperature then.In this reactant mixture, add the 3-nitropropionic acid tert-butyl ester (2.87g, CH 16.38mmol) ₂Cl ₂Solution (20mL).This reactant mixture at room temperature stirred 3 hours, poured into then in the water (100mL).Separate organic layer, water layer CH ₂Cl ₂(2 * 50mL) extractions.This CH ₂Cl ₂Solution also evaporates with salt water washing, drying.Residue obtains 950mg (24.5%) title product with silica gel column chromatography (hexane-EtOAc, 7: 3) purifying twice, is colourless viscous oil. ¹H NMR(CDCl ₃)，1.450(s，9H)，2.80-2.90(m，2H)，3.12-3.20(m，1H)，4.41-4.59(m，2H)，4.57-4.59(m，1H)，4.95-5.01(m，1H).

Embodiment 2

3-amino-5-fluoro-4-hydroxyl-valeric acid tert-butyl ester

(add Raney nickel (about 200mg) among the 950mg, MeOH solution (20mL) 4.0mmol), this mixture is at room temperature and H to 5-fluorine 4-hydroxyl-3-nitro valeric acid tert-butyl ester ₂Jolting is 18 hours (30-35psi).With its filtration, (2 * 10mL) wash catalyzer with MeOH.With the MeOH solution evaporation, residue obtains 840mg (96%) title compound with silica gel column chromatography purifying (EtOAc-MeOH, 10: 1), is faint yellow viscous oil. ¹HNMR (CDCl ₃), 1.450 (s, 9H), 2.12 (bs, 3H, OH and NH ₂), 2.28-2.38 (m, 1H), 2.47-2.57 (m, 1H), 3.24-3.30 (m, 1H), 3.54-3.76 (m, 1H), 4.38-4.48 (m, 1H), 4.54-4.61 (m, 1H).

Embodiment 3

3-(Cbz-Val-amide groups)-5-fluoro-4-hydroxyl-valeric acid tert-butyl ester

To the Cbz-valine (396mg, add in the THF solution (20mL) 1.58mmol) EDCl (300mg, 1.57mmol), HOBT (240mg, 1.57mmol) and DMAP (129mg, 1.06mmol).The gained mixture stirred 5 minutes, added 3-amino-5-fluoro-4-hydroxyl-valeric acid tert-butyl ester (215mg, THF solution (10mL) 1.04mmol), and it was at room temperature stirred 18 hours then.Mixture is filtered, evaporation THF solution, residue obtains 290mg (68%) title compound with silica gel column chromatography purifying (hexane-EtOAc, 3: 2), is white solid. ¹HNMR(CDCl ₃)，0.905(d，3H，J＝7)，0.965(d，3H，J＝7)，1.428(s，9H)，2.07-2.16(m，1H)，2.50-2.57(m，1H)，2.64-2.70(m，1H)，3.52(bs，1H，OH)，3.92-3.96(m，2H)，4.20-4.27(m，1H)，4.40(bs，1H)，4.49(bs，1H)，5.10(s，2H)，5.31-5.4(m，1H，NH)，6.86-6.93(m，1H，NH)，7.350(s，5H).

Embodiment 4

The Z-Val-Asp-Fmk tert-butyl ester

To periodinane (485mg, CH 1.14mmol) ₂Cl ₂Emulsion (imussion) is middle 3-(Cbz-Val-the amide groups)-5-fluoro-4-hydroxyl-valeric acid tert-butyl ester (230mg, CH 0.52mmol) that add (20mL) ₂Cl ₂Solution (12mL), gained white mixture at room temperature stirred 40 minutes, poured 25mL then into and contained 1.26g (8mmol) Na ₂S ₂O ₃Saturated NaHCO ₃In the aqueous solution.The gained mixture stirred 20 minutes, and gained is clarified CH ₂Cl ₂Solution separates, water layer CH ₂Cl ₂(2 * 25mL) extractions.CH ₂Cl ₂Solution also evaporates with the salt water washing, and residue obtains 190mg (83%) title compound with silica gel column chromatography purifying (hexane-EtOAc, 3: 2), is white solid. ¹HNMR(CDCl ₃)，0.91-0.97(m，6H)，1.415(s，9H)，2.10-2.20(m，1H)，2.70-2.77(m，1H)，2.95-3.01(m，1H)，3.98-4.06(m，1H)，4.87-5.28(m，6H)，6.95-7.01(m，1H)，7.350(s，5H).

Embodiment 5

Z-Val-Asp-fmk

To the Z-Val-Asp-fmk tert-butyl ester (180mg, dry CH 0.41mmol) ₂Cl ₂Add F in the solution (5mL) ₃CCO ₂H (1.0mL), it at room temperature stirred 40 minutes, then evaporation.Residue obtains 120mg (76%) title compound with silica gel column chromatography purifying (EtOAc-MeOH, 10: 1), is white solid. ¹HNMR(DMSO-d ₆)，0.81-0.84(m，6H)，1.87-1.96(m，1H)，2.47-2.67(m，2H)，3.77-3.87(m，1H)，4.47-4.59(m，1H)，4.91-5.16(m，4H)，7.25-7.42(s，5H)，8.40-8.49(m，1H).

Utilize the same procedure of describing among the embodiment 3-5 to make following compounds:

Embodiment 6

Z-Leu-Asp-fmk

White solid. ¹H NMR(CDCl ₃)，0.87(m，6H)，1.11(m，1H)，1.47(m，1H)，1.81(m，1H)，2.7(m，1H)，2.95(m，1H)，4.10(m，1H).4.80-5.20(m，6H)，7.31(s，5H).

Embodiment 7

Z-Ile-Asp-fmk

White solid. ¹HNMR(CDCl ₃)，0.85-0.96(m，6H)，1.14-1.26(m，1H)，1.422(s，9H)，1.87-2.04(m，1H)，2.70-2.77(m，2H)，2.93-3.00(m，1H)，4.02-4.13(m，1H)，4.80-5.30(s，6H)，6.96(m，1H)，7.35(s，5H).

Embodiment 8

Z-Ala-Asp-fmk

White solid. ¹H NMR(DMSO-d ₆)，1.160(d，3H，J＝7)，2.54-2.70(m，2H)，4.00(m，1H)，4.54(m，1H)，5.10-5.30(m，3H)，7.235(s，5H)，8.46-8.52(m，1H).

Embodiment 9

Ac-Val-Asp-fmk

White solid. ¹HNMR(DMSO-d ₆)，0.80-0.84(m，6H)，1.85-1.97(m，4H)，2.56-2.75(m，2H)，4.00-4.45(m，4H)，5.00-5.30(m，2H)，7.85-8.00(m，1H)，8.53-8.60(m，1H).

Embodiment 10

Z-N-Me-Val-Asp-fmk

White solid. ¹H NMR(DMSO-d ₆)，0.765(d，3H，J＝7)，0.832(d，3H，J＝7)，2.06(m，1H)，2.57-2.85(m，5H)，4.21(m，1H)，4.63(m，1H)，5.02-5.18(m，4H)，7.337(s，5H)，8.850(m，1H).

Embodiment 11

Z-Ala-Asp-fmk

White solid. ¹H NMR(DMSO-d ₆)，2.30(t，2H，J＝7)，2.48-2.70(m，3H)，3.17(m，2H)，4.41-4.60(m，2H)，4.98-5.30(m，3H)，5.40(m，1H)，6.63(m，1H)，7.32(s，5H)，8.52(m，1H).

Embodiment 12

Z-Gly-Asp-fmk

White solid. ¹H NMR(DMSO-d ₆)，12.50(s，1H)，8.49(m，1H)，7.52(m，1H)，7.33(s，5H)，5.08-5.25(m，1H)，5.01(s，2H)，4.10(m，1H)，3.63(d，J＝6.0Hz，2H)，2.50-2.80(m，2H).

Embodiment 13

Z-Phe-Asp-fmk

White solid. ¹H NMR(DMSO-d ₆)，8.60(m，1H)，7.60(m，1H)，7.24-7.30(m，10H)，4.92(m，3H)，4.60(m，1H)，4.28(m，1H)，2.90(m，2H)，2.70(m，2H).

Embodiment 14

Z-Glu-Asp-fmk

White solid. ¹H NMR(DMSO-d ₆)，12.20(m，1H)，8.48(m，1H)，7.56(m，1H)，7.34(m，5H)，5.12(m，1H)，5.01(s，2H)，4.51(m，1H)，3.95(m，1H)，2.71(m，2H)，2.24(m，2H)，1.72-1.82(m，2H).

Embodiment 15

Z-Pro-Asp-fmk

White solid. ¹H NMR(CD3OD)：7.36-7.33(m，5H)，5.13-5.11(m，2H)，4.30(s，1H)，3.58-3.50(m，2H)，2.77-2.64(m，2H)，2.24(m，1H)，1.94(s，2H).

Embodiment 16

Z-His-Asp-fmk

White solid. ¹H NMR(CD ₃OD)：8.78(s，1H)，7.93(s，1H)，7.36-7.33(m，7H)，5.52(s，2H)，5.10(s，2H)，4.49(s，1H)，3.13-3.05(m，2H)，2.84(s，2H).

Embodiment 17

Z-Tyr-Asp-fmk

Described in embodiment 3 and 4, prepare Z-Tyr (Bu-t)-Asp-fmk tert-butyl ester from Z-Tyr (Bu-t)-OH and 3-amino-5-fluoro-4-hydroxypentanoic acid tert-butyl ester.(add TFA (1mL) among the 15mg, dichloromethane solution 0.027mmol) (1mL) to Z-Tyr (Bu-t)-Asp-fmk tert-butyl ester.This mixture at room temperature stirred 8 hours, stirred 2 days down at 4 ℃ then.With ethyl acetate (30mL) with its dilution, water (4 * 20mL) and the salt water washing, with dried over sodium sulfate and vacuum concentration, obtain yellow solid title compound (10mg, 0.022mmol, 83%). ¹H NMR(DMSO-d ₆)：δ9.20(s，1H)，8.50(br s，1H)，7.50(m，1H)，7.32-7.03(m，7H)，6.63(d，J＝7.5，2H)，4.94(s，2H)，4.55(m，1H)，4.15(m，1H)，2.90-2.60(m，4H).

Embodiment 18

The Z-Val-Asp-fmk methyl esters

(110mg slowly feeds the HCl air-flow in the MeOH solution (20mL) 0.28mmol), become highly acid up to measuring this solution with the pH test paper to the Z-Val-Asp-fmk that cools off in ice bath.This solution at room temperature stirred 4 hours, then evaporation.Residue obtains 63mg (55%) title compound with silica gel column chromatography purifying (hexane-EtOAc, 3: 2), is white solid. ¹HNMR (CDCl ₃), 0.91-0.87 (m, 6H), 2.10-2.20 (m, 1H), 2.81-2.88 (m, 1H), 3.02-3.08 (m, 1H), 3.675 and 3.682 (2S, 3H), 3.97-4.01 (m, 1H), 4.90-5.25 (m, 6H), 6.94-7.02 (m, 1H), 7.354 (s, 5H).

Utilize the same procedure of describing among the embodiment 18 to make following compounds.

Embodiment 19

The Z-Leu-Asp-fmk methyl esters

Colourless viscous oil. ¹H NMR(CDCl ₃)，0.92-0.94(m，6H)，1.25-1.80(m，4H)，2.78-2.82(m，1H)，3.00-3.05(m，1H)，3.675(s，3H)，4.15-4.20(m，1H)，4.85-5.10(m，6H)，7.10-7.20(m，1H)，7.344(s，5H).

Embodiment 20

The Z-Ile-Asp-fmk methyl esters

White solid. ¹H NMR(CDCl ₃)，0.85-0.96(m，6H)，1.14(m，1H)，1.46(s，1H)，1.87(m，1H)，1.91-2.86(m，1H)，2.88-3.02(m，1H)，3.257(s，3H)，4.68-4.06(m，1H)，4.80-5.30(s，6H)，6.99(m，1H)，7.35(s，5H).

Embodiment 21

Test with the cell death that the Hela cell carries out

Utilize Hela cell tests Cbz-Val-Asp (OMe) CH that stimulates with tumor necrosis factor-alpha (TNF-α) and cycloheximide (CHX) ₂The cytoprotection performance of F.This is the cell culture object model of a kind of fine sign of programmed cell death, and it is generally used for analyzing anti-programmed cell death reagent.Carry out two type of experiment: utilize the phase-contrast microscopy observation of cell and the qualitative evaluation cell death; With utilize the cell death of fluorescent dye calcein AM qualitative assessment.

For light microscopic examination, the density of Hela cell with 100000 cells/well is inoculated in the minimal medium in 12 orifice plates, that contain 2mM glutamine and 10% hyclone.After 24 hours, this bed board medium is removed, added the fresh culture of the cytoprotection test compounds that contains variable concentrations.These cells under 37 ℃ at CO ₂Cultivated 2 hours with test compounds is pre-in the insulating box, add TNF-α and CHX then, make final concentration be respectively 25ng/mL and 30 μ g/mL.Cultivate after 24 hours, by these cells of visual inspection, to obtain evidence based on the cell death of cell shape and sticky limit.If cell has become circle, phase place is bright and separated with basic unit, just think that cell is dead.If cell still keeps their normal shape and keep with basic unit and to adhere to, just think that cell still lives.

For quantitative test, utilize the cell survival degree of indicator dye calcein AM quantitative analysis in the presence of test compounds.Living cells is taken in this dyestuff and is translated into fluorescent derivative; Measure the amount of activation dyestuff in every hole then with the fluorescent plate reader, with fluorescence measuring as survivaling cell quantity.For carrying out these tests, the density of Hela cell with 25000 cells/well is inoculated in the 0.4mL minimal medium in 48 orifice plates, that contain 2mM glutamine and 10% hyclone.After 24 hours, remove the bed board medium, add the 0.5mL fresh culture that contains the variable concentrations test compounds.These cells under 37 ℃ at CO ₂Cultivated 2 hours with test compounds is pre-in the insulating box, add TNF-α and CHX then, make final concentration be respectively 25ng/mL and 30 μ g/mL.Cultivate after 24 hours, culture with serum-free, no phenol red Ham ' s F12 washed twice, removing dead cell, and is added Ham ' the s F12 that 125 μ L contain 8 μ M calcein AM.This culture was at room temperature cultivated 1 hour, utilized the BioTek plate reader that uses 485nm (exciting) and 530nm (emission) filter for installation to measure fluorescence signal.Data are with " relative comparison percentage " expression, and it calculates with following formula: The culture that to handle with CHX in contrast, rather than in contrast with untreated culture, to revise the cyto-inhibition of CHX.But, because CHX self also is the slight derivant of the programmed cell death of Hela cell, strong anti-programmed cell death medicine will obtain the relative comparison percent value greater than 100%.

Typical qualitative test result is presented at Figure 1A-1G, among 2A-2G and the 3A-3G.In these experiments, with Cbz-Val-Asp (OMe)-CH ₂The cytoprotection usefulness of F and BOC-Asp (OMe)-CH ₂F, Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F and Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F is at three variable concentrations: 0.5, compare under the 5 and 50 μ M.All these compounds all are methyl ester derivations.Figure 1A-1G shows: under 50 μ M concentration, and Cbz-Val-Asp (OMe)-CH ₂F (Fig. 1 D) has protected the Hela cell to avoid the programmed cell death effect of TNF-α and CHX fully.When 50 μ M, related peptides BOC-Asp (OMe)-CH ₂F (Fig. 1 C) and Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F (Fig. 1 E) also is a protectant.CPP32 inhibitor C bz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F (Fig. 1 F) only is reluctantly effective cell-protecting when 50 μ M.Fig. 2 A-2G demonstration, under 5 μ M concentration, Cbz-Val-Asp (OMe)-CH ₂F (Fig. 2 D) still demonstrates strong cytoprotection astoundingly, and 5 μ M BOC-Asp (OMe)-CH ₂F (Fig. 2 C) and 5 μ M Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂The effect of F (Fig. 2 E) is relatively poor.CPP32 inhibitor C bz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F (Fig. 2 F) does not have the cytoprotection performance when 5 μ M.Fig. 3 A-3G demonstration, even under 0.5 μ M, Cbz-Val-Asp (OMe)-CH ₂F (Fig. 3 D) is still effective cell-protecting, and other compounds (Fig. 3 C, 3E and 3F) have only slight or do not have cytoprotection.These experiment showed, Cbz-Val-Asp (OMe)-CH ₂F can hang down the programmed cell death that protection Hela cell is not induced by TNF-α/CHX under the 10-100 concentration doubly than the anti-programmed cell death agent of other suppositions.

As mentioned above, the quantitative experiment that utilizes calcein AM to carry out has confirmed the result that obtains with microscopic examination.Figure 4 and 5 have illustrated two such experimental results, wherein with Cbz-Val-Asp (OMe)-CH ₂The cytoprotection performance of F (a) and BOC-Asp (OMe)-CH ₂F (b) (Fig. 5) and Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F (c) (Fig. 4) compares under three kinds of concentration (0.5,5 and 50 μ M).Fig. 4 shows, Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F (b) is even use with maximum concentration (50 μ M), and it is not subjected to have only minimal effect in TNF-α/CHX injury at protection Hela cell yet.Fig. 5 shows, the BOC-Asp of 50 and 5 μ M (OMe)-CH ₂F (b) is effective cell-protecting, but when 0.5 μ M, its active significantly reduction.By contrast, the Cbz-Val-Asp of 0.5 μ M (OMe)-CH ₂F (a) is as Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH of cell-protecting and 50 μ M ₂F (b) effect identical (Fig. 4).In addition, the Cbz-Val-Asp of 0.5 μ M (OMe)-CH ₂F (a) is highly active, and the BOC-Asp of 0.5 μ M (OMe)-CH ₂F (b) does not have activity (Fig. 5).

In order to determine Cbz-Val-Asp (the OMe)-CH of low dosage ₂The effect of F is with this compound treatment Hela cell in the 0.05 μ M-1 μ M concentration range.As shown in Figure 6, when being low to moderate 0.25 μ M concentration, Cbz-Val-Asp (OMe)-CH ₂F (a) has produced significant protective effect.By contrast, widely used anti-programmed cell death agent Cbz-Val-Ala-Asp (OMe)-CH in cell death research ₂F (b) shows in this concentration range does not have cytoprotection.

Take together, these experiments of explanation show Cbz-Val-Asp (OMe)-CH from Figure 1A to Fig. 6 ₂F is the wonderful strong anti-programmed cell death agent of intact cell, and it is all stronger than the effect of other any known caspase inhibitor.

Embodiment 22

Suppress the PARP cracking in the Jurkat cell

The cracking of poly-(ADP) ribose polymerase (PARP) may occur in all cells that wherein caspase proteolysis cascade reaction is activated.For this reason, the PARP cracking is widely used as the biochemical marker of the programmed cell death of caspase mediation.The ability of cell-protecting medicines blocking-up PARP cracking is considered to the symbol that medicine suppresses caspase proteolysis cascade reaction ability, particularly suppresses the symbol of the ability of this main PARP protease of CPP32 (caspase-3).In the programmed cell death process of a kind of Fas of human T-cell system-Jurkat cell mediation, check Cbz-Val-Asp (OMe)-CH ₂F suppresses the ability of PARP cracking.The characteristic of the cell culture object model of this programmed cell death is described preferably, and known it is relevant with the activation of at least two kinds of caspase (caspase-3 (CPP32) and caspase-8 (FLICE/MACH)).

Measure for the PARP cracking, the Jurkat cell is inoculated in the RPMI that contains 10%FBS 1640 medium in six orifice plates with the density of 500000 cells/well.These cells under 37 ℃ at CO ₂In the insulating box with Cbz-Val-Asp (OMe)-CH ₂F or other test compounds are pre-cultivated 2 hours, added the monoclone antibody of Fas then, and making final concentration is 500ng/mL.Under 37 ℃ at CO ₂Continue in the insulating box to cultivate again 4 hours.When culture period finishes, by these cells of centrifugal results and containing 50mM Tris-HCl, cracking in the buffer solution of pH7.4,150mM NaCl, 1%NP-40,0.25% NaTDC, 1mM EDTA and protease inhibitor cocktail.An amount of lysate application of sample that will be equivalent to 10-20 μ g protein to the 7.5%SDS polyacrylamide gel, under 25mA electrophoresis 2-2.5 hour.Then protein transduction is moved on on the pvdf membrane, survey, and utilize the chemiluminescence method to observe with the rabbit polyclonal antibody of PARP.

Fig. 7 A-7E has shown three such result of experiment.The Jurkat cell is cultivated in advance with the following compounds of 0.5,5 or 50 μ M: Cbz-Val-Asp (OMe)-CH ₂F (compound 1); BOC-Asp (OMe)-CH ₂F (compound 5); Cbz-Asp-α-([2,6-dichloro-benzoyl oxygen base]-methyl ketone (compound 6); Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F (compound 3); Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F (compound 2); Cbz-Ile-Glu (OMe)-Thr-Asp (OMe)-CH ₂F (compound 4); Or Cbz-Val-Ala-Asp (OMe)-CH ₂F (compound 7).These cells are handled with anti-Fas and are carried out western blot analysis then.Cbz-Val-Asp (OMe)-CH ₂F (compound 1) has suppressed the PARP cracking fully when 50 μ M and 5 μ M, even also significantly suppresses cracking (Fig. 7 A) when 0.5 μ M.By contrast, Cbz-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH ₂F (compound 2) (Fig. 7 A) and Cbz-Ile-Glu (OMe)-Thr-Asp (OMe)-CH ₂F (compound 4) (Fig. 7 C) and Cbz-Asp-DCB (compound 6) (Fig. 7 D) have suppressed the PARP cracking fully when 50 μ M, but only are inadequate effective inhibitors when 5 μ M and 0.5 μ M.BOC-Asp (OMe)-CH ₂F (compound 5) (Fig. 7 D) and Cbz-Val-Ala-Asp (OMe)-CH ₂F (compound 7) (Fig. 7 E) and Cbz-Glu (OMe)-Val-Asp (OMe)-CH ₂F (compound 3) (Fig. 7 B) is effective inhibitor of PARP cracking when 50 μ M and 5 μ M, but only effective reluctantly when 0.5 μ M, and under this concentration, Cbz-Val-Asp (OMe)-CH ₂F (compound 1) still demonstrates obvious suppression effect (Fig. 7 A).These experiment showed, Cbz-Val-Asp (OMe)-CH ₂F can be under the concentration of low at least 10 times of known caspase inhibitor than other caspase proteolysis cascade reaction in blocking-up intact cell.

Embodiment 23

Enzymic activity

With luciferase test determination Cbz-Val-Asp (OMe)-CH ₂F and Cbz-Val-Asp-CH ₂F (free acid) is as the activity of the inhibitor of CPP32, ICE and cathepsin B.By utilizing baculoviral, in insect host cell (sf9 cell), express the dna clone of encoding such enzymes, preparation recombinant C PP32 protein and ICE protein as carrier.Referring to Webb, N.R. etc., " utilizing recombinant baculovirus expression protein ", process (Techniques) 2:173-188 (1990).The preparation of natural tissues protease obtains from commercial source.The enzymic activity utilization is attached to the synthetic peptide substrates of fluorescence leaving group and measures.Enzyme produces fluorescence signal to the cracking of synthetic substrate, and it is with spectrofluorimeter or with fluorescence micro titer plate reader reading.

The CPP32 activity utilizes following buffer condition to measure: 100mM HEPES pH7.5 is added with 10% sucrose, 1%CHAPS, 5mM glutathione and 5 μ M peptide substrates.Peptide substrates is formed by the oligomer with sequence A sp-Glu-Val-Asp with the fluorescent chemicals amino methyl coumarin that the C end is puted together.Enzyme assay was carried out under 37 ℃ 30 minutes usually.

Table I has been listed Cbz-Val-Asp (OMe)-CH ₂F and Cbz-Val-Asp-CH ₂F (free acid) is to the IC of CPP32 and other protease ₅₀

Table I: Cbz-Val-Asp (OMe)-CH ₂F and Cbz-Val-Asp-CH ₂F (free acid)

Usefulness enzyme Cbz-Val-Asp (OMe)-CH as the inhibitor of CPP32 and other protease ₂F Cbz-Val-Asp-CH ₂F (free acid)

IC ₅₀(μM) IC ₅₀(μM)

CPP32 1.1 0.043

ICE 0.9 0.02 cathepsin Bs 0.3＞10

Xa factor＞100＞100

Fibrin ferment＞100＞100

Result displayed shows in the Table I, and The compounds of this invention is the inhibitor of the moderate strength of CPP32 and ICE.The result also shows, Cbz-Val-Asp-CH ₂F is potent inhibitor and the selective depressant of CPP32 and ICE.

Cbz-Val-Asp-CH ₂F is to (reorganization caspase 3,6,7 that CA) obtains and 8 inhibition activity utilize Ac-DEVD-AMC to measure for Becton branch company, SanDiego from PharMington.The consumption of every kind of enzyme in each test is as follows: 1ng caspase3,15ng caspase6,2ng caspase7 and 60ng caspase8.Enzyme reaction utilizes caspase buffer solution (20mM PIPES in 96 orifice plates, 100mM NaCl, 10mM DTT, 1mM EDTA, 0.1%CHAPS and 10% sucrose pH7.2) carry out, and this reaction is by adding 10 μ M Ac-DEVD-AMC (available from Quality Controlled Biochemicals, Inc.Hopkinton MA) causes.The Cbz-Val-Asp-CH of 12 kinds of concentration from 30pM to 10 μ M ₂The F compound is being tested after hatching 30 minutes under 37 ℃ with reorganization caspase.With adopting 355nm to excite the fluorescent plate reader (EC﹠amp of filter/460nm emission filter; G WALLAG, the 1420-002 type) to this plate reading.Data utilize GraphPrism software to analyze.Data are summarized in the Table II.

Table II: Cbz-Val-Asp-CH ₂F is as the usefulness of caspase inhibitor

	Caspase-3	Caspase-6	Caspase-7	Caspase-8
	Caspase-3	Caspase-6	Caspase-7	Caspase-8	IC ₅₀(nM)	19.8	18.4	6.8	7.2

Result displayed shows in the Table II, Cbz-Val-Asp-CH ₂F is the potent inhibitor of all tested caspase.

Table III has shown the activity of various two inhibitor peptides to caspase-3.The result shows, in the compound of being tested, and Z-Val-Asp-CH ₂F is the strongest caspase-3 inhibitor.

Table III. the Caspase-3 activity of two inhibitor peptides

Title	Caspase-3 IC ₅₀(μM)
Title	Caspase-3 IC ₅₀(μM)	Z-L-Val-Asp-fmk	0.04
Z-L-Leu-Asp-fmk	0.2	Z-L-Val-Asp-fmk	0.04
Z-L-Leu-Asp-fmk	0.2	Z-L-Ile-Asp-fmk	0.7
Z-L-Phe-Asp-fmk	0.4	Z-L-Ile-Asp-fmk	0.7
Z-L-Phe-Asp-fmk	0.4	Z-Gly-Asp-fmk	1.9
Z-L-Ala-Asp-fmk	0.6	Z-Gly-Asp-fmk	1.9
Z-L-Ala-Asp-fmk	0.6	Z-β-Ala-Asp-fmk	3.5
Ac-L-Val-Asp-fmk	0.25	Z-β-Ala-Asp-fmk	3.5
Ac-L-Val-Asp-fmk	0.25	Z-L-Glu-Asp-fmk	14.2
Z-L-Lys-Asp-fmk.TFA	1.6	Z-L-Glu-Asp-fmk	14.2
Z-L-Lys-Asp-fmk.TFA	1.6	Z-N-Me-L-Val-Asp-fmk	1.3
Z-L-Pro-Asp-fmk	0.41	Z-N-Me-L-Val-Asp-fmk	1.3
Z-L-Pro-Asp-fmk	0.41	Z-L-His-Asp-fmk	0.77
Z-L-Tyr-Asp-fmk	0.66	Z-L-His-Asp-fmk	0.77

Embodiment 24

Z-VD-fmk is to the effect of PARP cracking

Poly-(ADP) ribose polymerase (PARP) is one of caspase-3 substrate of at first determining, and the cracking of PARP still is considered to the approximate common tags of the programmed cell death of caspase-3 activation and caspase mediation.Therefore, the ability of anti-programmed cell death compounds block PARP cracking is its useful indication that suppresses the programmed cell death ability.The Jurkat cell that utilizes anti-Fas to handle is checked the usefulness of Z-VD-fmk in the PARP breaking test.With 2 * 10 ⁶The Jurkat cell inoculation and was cultivated 30 minutes with test compounds is pre-in each hole of 6 hole wares.These cells stimulated 4 hours with 500ng/mL excitability anti-Fas antibody or PBS then.Gather in the crops these cells afterwards, gently make them agglomerating, use the PBS washed twice, and make its cracking in the RIPA buffer solution.The lysate of five equilibrium is analyzed with SDS-PAGE, protein transduction is moved on to carry out western blot analysis on the pvdf membrane.One anti-be the anti-PARP serum of polyclone that the pyrolysis product that generates with total length PARP and caspase-3 all has cross reaction.

Fig. 8 A shows, Z-VD-fmk is 500 and suppressed PARP cracking (attention does not have the 85KDa band) during 250nM concentration fully.Even still kept its most of activity (Fig. 8 A) that suppresses when the concentration of Z-VD-fmk is low to moderate 50nM.By contrast, although Z-VAD-fmk is effective inhibitor (data not shown) of PARP cracking when 5 μ M concentration, its efficient much smaller when 500nM (Fig. 8 B).These test demonstration, and Z-VD-fmk is as at least 10 times of the efficiency ratio Z-VAD-fmk height of PARP cracking inhibitor in the intact cell, in this intact cell programmed cell death model, and the IC of Z-VD-fmk ₅₀Value is less than 50nM.

Embodiment 25

The effect of the cell death that Z-VD-fmk induces TNF-α

Tumor necrosis factor-alpha (TNF-α) can cause programmed cell death by starting the caspase cascade reaction in a large amount of cell types, its apoptosis inducing activity can be suppressed by peptidyl caspase inhibitor.But, good anti-programmed cell death effect be produced, the inhibitor (50 μ M or higher) of high concentration must be wanted.Here determine the anti-programmed cell death usefulness of Z-VD-fmk with cell-line-Hela cell commonly used in the TNF-α cell death research.

In processing preceding 24 hours, the density of Hela cell with 50000 cells/well is inoculated in 48 orifice plates.Then their were cultivated 2 hours with the Z-VD-fmk of variable concentrations is pre-, and with TNF-α (25ng/mL) and cycloheximide (CHX; 30 μ g/mL) stimulate.This culture was cultivated 24 hours again, removed dead cell with the PBS washed twice.Every part of culture uses calcein AM (calcein AM is a kind of preceding fluorescent dye, and it is taken in and be converted into fluorescence-causing substance by living cells) to cultivate again 45 minutes, measures the density of survivaling cell thus.The gained data are with " % relative comparison " value representation (control value is with cycloheximide but does not have the value that the cell of TNF-α incubation obtains).

Fig. 9 has shown the result of the Z-VD-fmk in 0 to the 500nM test concentrations scope.Z-VD-fmk has produced the good cell protective effect during near 100nM in concentration.By contrast, Z-VAD-fmk has lost its most of cytoprotection performance (data not shown) when being lower than 1 μ M.The tetrapeptide inhibitor, such as Z-DEVD-fmk and Ac-DEVD-CHO, efficient very low (data not shown) when being lower than 50 μ M.Therefore, Z-VD-fmk not only suppresses PARP cracking (referring to embodiment 24) in sub-micro molar concentration level, and suppresses cell death in sub-micro molar concentration level, and it is more much effective than known tripeptides and tetrapeptide inhibitor.

Embodiment 26

Z-VD-fmk is to the effect of dna ladder shapeization

In the later stage of programmed cell death, along with cytoplasm fragment decoherence (by bubble) and nuclear decomposition, cell begins to collapse fully.One of evidence of nuclear decomposition is the fragment (being called " dna ladder shapeization ") that genomic DNA is cracked into the nucleosome size.The dna ladder shapeization is considered to irreversible as other later stage programmed cell death incidents, determines that therefore whether anti-programmed cell death medicine can prevent it is very important.

The Jurkat cell that the ability of Z-VD-fmk blocking dna scalariformization utilizes anti-Fas to handle is tested.With the Jurkat cell with 5 * 10 ⁶The density bed board of cell and is cultivated with the Z-VD-fmk of variable concentrations in the 60mm culture dish in advance.They stimulated 4 hours with the anti-Fas of 100ng/mL then, with they results, made it agglomerating and used the PBS washed twice.Genomic DNA utilizes the method for (1996) such as Eldadah to separate.Briefly, with cytolysis in 2mL 7M guanidine hydrochloride and prepare DNA purifying resin (Promega) in a small amount with 1mL Wizard and mix.This resin/DNA compound buffer solution washed twice, DNA TE wash-out.With this DNA sample electrophoresis on 1% agarose/TBE gel of 1-2 μ g, this gel ethidium bromide staining.

Figure 10 has shown the result of dna ladder shape test, and wherein cell is cultivated in advance with Z-VD-fmk or pharmaceutical carrier (DMSO).The vehicle treated cell that stimulates with anti-Fas has shown the characteristic scalariform pattern of DNA, and it expands to about 300bp downwards.Suppressed scalariform formation when by contrast, the dosage of Z-VD-fmk is low to moderate 50nM.

This result shows, Z-VD-fmk can be under the sub-micro molar concentration of the concentration that is equivalent to block cell death and PARP cracking the dangerous later stage programmed cell death (dna ladder shapeization) of blocking-up.Can reach a conclusion based on the data of describing in this experiment and embodiment 24 and 25: Z-VAD-fmk is efficient, the agent of sub-micro mole apoptosis inhibiting of the intact cell model of programmed cell death.

Embodiment 27

Cbz-Val-Asp-CH ₂F is in the Mouse Liver programmed cell death model

Anti-programmed cell death activity

Use the 3-4 female mice in age in week in this research.The anti-mouse Fas of purifying hamster monoclone antibody (clone Jo2 by the anti-mouse Fas of intravenous injection 2-6 μ g antigen, Pharmingen), this antibody makes the liver sex change (Rodriguez etc., 1996) of these mouse with the physiological saline dilution of 80 μ l phosphate-buffered.With the terminal point of lethality as the sex change of assessment liver.Cbz-Val-Asp-CH ₂F is used for drip-feed with the preparation of Tris buffer solution, and through the dosage intravenous administration of tail vein with 1-10mg/kg.After 10 minutes, to animal injection Fas antibody.In the time of 30 minutes, 1,3 and 24 hour, lethality is counted.The control-animal group of only accepting Fas antibody is all arranged for every kind of compound.Those animals of accepting maximum dose level are observed their acute behaviors reflection (for example, calmness, motion activity, gait change, convulsions, straub tail, tremble etc.), then their are closed that cage spends the night and second day inspection toxicity/lethality.

In these experiments, Cbz-Val-Asp-CH ₂F is the wonderful potent inhibitor of the anti-Fas lethality rate of inducing.The single 1mg/kg dosage of intravenous injection just is no more than the injury of having protected mouse to avoid anti-Fas fully in 1 hour behind antibody administration, dosage is low to moderate 0.25mg/kg and has still produced intimate 100% protective effect.By contrast, in the vehicle Control group, all mouse when this time point all in the dust.Cbz-Val-Asp-CH ₂F has also shown the substantive protective effect (28% survival) that is no more than 24 hours.Independently studies show that, weaken relevant with the expectation of the inducing action of liver enzyme SPGT and SGOT the protection of lethality rate.

These results prove, Cbz-Val-Asp-CH ₂F is highly active after to the administration of Mouse Liver programmed cell death model system in vivo.

Embodiment 28

Cbz-Val-Asp-CH ₂F is in the ischemic rat model of focus

Neuroprotection

(i) preparatory operation: the male Fischer-344 rat of the heavy 200-240g of use (Harlan Sprague Dawley, CA).These animals are at first used 3% halothane anesthesia in 30% oxygen and 70% AIR MIXTURES.Fluothane concentration is reduced to 1.5% narcosis that is used for keeping the whole surgery process.Preparatory operation comprises: (a) ductus venosus is implanted: expose left femoral vein, the Teleflex conduit that insertion is filled with carrier is used for administration subsequently up to inferior caval vein.(b) arterial duct is implanted: at the femoral artery cannulate, be used for when ischaemic process, beginning administration and artery when pouring into again, monitoring blood pressure and other physiological conditions comprise pO ₂, pCO ₂, pH, glucose, hematocrit.It is external that artery and ductus venosus all pass through the back of animal, so that can move freely.(c) (Data Sciences International MI) implants the abdominal cavity, with 22 hours body temperature of remote monitoring animal with the PhysioTel converyer.

(ii) physiologic parameters: in surgical procedure, utilize YSI can use temperature probe (YSI Co., Ltd again, Yellow Spring, OH) make core temperature keep 37.5 ℃, described probe and YSI temperature control equipment (73A type, YSI Co., Ltd, Ohio) and electric pad (756 types, Sunbean-Oster Co., Ltd, Hattiesburg MS) connects.After the ischaemic, the PhysioTel converyer is activated, per 5 minutes record core temperatures.In the whole surgery, in medicine (medicine group) drip-feed process, began the back 1,2,3 and 4 hour, the monitoring system blood pressure with ischaemic.At arterial occlusion with when pouring into again, measure other physiological conditions, comprise pO ₂, pCO ₂, pH, glucose and hematocrit.

(iii) instantaneous focus ischaemic model: after the preparatory operation, carry out the neck otch, to expose two CCA along the veutro center line.The right CCA permanent ligation of 4-0 silk ligature, and left CCA clamps with no wound aneurysm clip.Make the otch of a 1cm across perpendicular to line between the side canthus of right eye and the external auditory meatus and with it.Temporalis below cutting also retracts, and makes it directly to observe down in the help of disecting microscope (SZ-STB1 type, Olympus, Japan), and arteria cerebri media (MCA) exposes by a 2mm boring, and this hole pierces 2-3mm to fusion place of zygomatic arch and temporo squamosum.Boring is carried out under the condition with the continuous normal saline flushing.Cutting is also drawn back endocranium to expose the MCA in the rhinoschisis.Because of MCA flows through rhinoschisis, so press from both sides (No. 1) temporary transient closed MCA with the graceful aneurysms of Coud.Interruption with disecting microscope check blood flow.Use the surgical clamp closure of incisions, stop anesthesia, (a few minutes are interior) turned back to them in the cage of oneself after animal revived.Stand the anesthesia once more after MCA is closed back 2.5 hours of instantaneous ischemic rat.After confirming the MCA closure, the clip on MCA and the left CCA is removed, visual observation confirms restoration of blood flow among the MCA.With the otch sealing, rat turns back in the cage of oneself.Allowing needs the animals survived of short-term recovery 24 hours.All animals deep anaesthesia before execution.Brain is taken out, do the section of 2mm coronal-plane and place TTC.It is pale that blocking tissue seems, can differentiate with adjacent living tissue.The image machining software Blind Test of cortex and infracortical infarct size, infarct volume calculates by each measured value with known thickness.

(iv) analyse around score: all physiologic parameters, thermograph and cortex infarct volume to each the animal group in experimental group and the control group carry out the statistics contrast.Statistical analysis utilizes Sigmastat software, and (Jandel Scientific Software, SanRafael CA) carries out.Paired data does not carry out the T check, and ANOVA is used for multiple ratio.The p value is considered to significant less than 0.05.With SigmaPlotv2.01 software (JandelScientific) curve plotting figure.

In two apoplexy research experiments, with the instantaneous focus ischaemic of rat (Fischer-344) model testing Cbz-Val-Asp-CH ₂Neuroprotective performance in the body of F.Ischaemic took place back 10 minutes, venoclysis 20mg/kg Cbz-Val-Asp-CH ₂F, then continuous intravenous infusion 5mg/kg/h.In experiment 1, continuous drip 6 hours.In experiment 2, instil and extend to 12 hours.

In two researchs, all find Cbz-Val-Asp-CH ₂F has significantly reduced the cortex infraction: in the experiment 1 is 46% (p＜0.05), is 57% (p＜0.05) (Figure 11 A and 11B) in the experiment 2.After the administration, blood pressure, flood gas or temperature all do not have to change.These results prove, Cbz-Val-Asp-CH ₂F is well tolerable behind urgent intravenous administration, and it is the strong neuroprotective agent of the rat model of instantaneous focus cerebral ischaemia.

The present invention has been carried out comprehensive description now, those of ordinary skills can understand that the present invention can not influence extensively finishing in disease, preparation and other parameters of equal value of the scope of the invention or its any embodiment.All patents mentioned herein and publication all are incorporated herein by reference in full.

Claims

1, the dipeptides of formula II:

Wherein: R ₁Be the terminal protecting group of N-, be selected from tertbutyloxycarbonyl (Boc), acetyl group (Ac) and benzyloxycarbonyl group (Cbz); R ₃Be C _1-6Alkyl or H; AA is the amino acid residue that is selected from Val, Ile and Leu.

2, dipeptides as claimed in claim 1, wherein R ₃Be methyl or hydrogen.

3, dipeptides as claimed in claim 2, it is Cbz-Val-Asp-CH ₂F.

4, dipeptides as claimed in claim 2, it is Cbz-Leu-Asp-CH ₂F.

5, dipeptides as claimed in claim 2, it is Cbz-Ile-Asp-CH ₂F.

6, dipeptides as claimed in claim 2, it is Ac-Val-Asp-CH ₂F.

7, dipeptides as claimed in claim 2, it is Ac-Leu-Asp-CH ₂F.

8, dipeptides as claimed in claim 2, it is Ac-Ile-Asp-CH ₂F.

9, dipeptides as claimed in claim 2, it is Boc-Val-Asp-CH ₂F.

10, dipeptides as claimed in claim 2, it is Boc-Leu-Asp-CH ₂F.

11, dipeptides as claimed in claim 2, it is Boc-Ile-Asp-CH ₂F.

12, dipeptides as claimed in claim 2, it is Cbz-Val-Asp (OMe)-CH ₂F.

13, dipeptides as claimed in claim 12, it is Cbz-Leu-Asp (OMe)-CH ₂F.

14, dipeptides as claimed in claim 12, it is Cbz-Ile-Asp (OMe)-CH ₂F.

15, a kind of pharmaceutical composition, it comprises the dipeptides and the pharmaceutically acceptable carrier of claim 1.

16, a kind of method that suppresses the death of cell or tissue inner cell, it comprises makes described cell or tissue contact at the described compound of external claim 1 with effective dose.

17, the described compound of claim 1 can be used for treating or improves purposes in the medicine of the maincenter of animal and the cell death in peripheral nervous system, retinal neurons, cardiac muscle or the immune system cell in preparation.

18, purposes as claimed in claim 17, wherein said cell death are in maincenter or peripheral nervous system, and owing to one of underlying cause causes:

(a) be selected from the focus ischaemic that causes because of apoplexy and the ischemic and the exitotoxicity disease of the general ischemic that stops to cause because of heartbeat;

(b) traumatic damage;

(c) virus infections;

(d) radiation-induced nerve cell death; Or

(e) be selected from the neurodegenerative disease of alzheimer's disease, Parkinson's disease, prion disease, multiple sclerosis, amyotrophic lateral sclerosis and spinobulbar atrophy.

19, purposes as claimed in claim 17, wherein said cell death are in maincenter or peripheral nervous system, and are because the expansion of the trinucleotide duplicate block of specific gene causes.

20, purposes as claimed in claim 17, wherein said cell death is owing to Huntington causes.

21, purposes as claimed in claim 17, wherein said cell death is in cardiac muscular tissue, and is because the virus infections of miocardial infarction, congestive heart failure, cardiomyopathy or heart causes.

22, purposes as claimed in claim 17, wherein said cell death is in retinal neurons, and owing to intraocular pressure rising, AMD or retinitis pigmentosa cause.

23, purposes as claimed in claim 17, wherein said cell death is in immune system, and is to cause owing to being selected from acquired immunodeficiency syndrome, severe combined immunodeficiency syndrome and radiation-induced immunosuppressant immune deficiency disorder.

24, purposes as claimed in claim 17, wherein said cell death are owing to the autoimmune disease that is selected from lupus erythematosus, rheumatoid arthritis and type i diabetes causes.

25, purposes as claimed in claim 17, wherein said cell death is owing to type i diabetes causes.

26, the described compound of claim 1 can be used for treating or preventing MCKD of animal or the purposes in anaemia/erythropoietic medicine in preparation.

27, a kind of method of protecting mammiferous organ or tissue to avoid supplying with because of normal blood the cell death that forfeiture causes, this method are included in and external described organ or tissue are contacted with claim 1 compound of effective dose.

28, method as claimed in claim 27, wherein said organ or tissue is present in the storage medium before in being transplanted to mammalian body.

29, method as claimed in claim 27, wherein said contact comprise described compound are injected in the organ or tissue, perhaps described organ or tissue are bathed in the storage medium that contains described compound.

30, the described compound of claim 1 can be used for reducing or prevents donor organ or be organized in purposes in the medicine of being transplanted to the cell death that back among the host causes because of the effect of host immune cell in preparation.

31, a kind of reduce or prevention inseminatio externalis process in used mammalian sperm or the method for egg cell death, be included in and external described sperm or egg cell contacted with claim 1 compound of effective dose.

32, a kind of method that prolongs the valid expiration date of mammal or yeast cells system comprises described cell-line is contacted with claim 1 compound.

33, method as claimed in claim 32, wherein said contact are included in and contain described compound in the cell culture medium.

34, the described compound of claim 1 can be used for treating or improves purposes in the too early grizzled medicine of mammalian hair loss or hair in preparation.

35, purposes as claimed in claim 34, wherein treatment is trichomadesis, and described trichomadesis is owing to male pattern baldness, radiation, chemotherapy or emotional stress cause.

36, the described compound of claim 1 can be used for topical therapeutic or improves purposes in the medicine of the skin injury that mammal causes because of contact high levels of radiation, heat or chemical substance in preparation.

37, purposes as claimed in claim 36, wherein said compound is used as the part of ointment.

38, purposes as claimed in claim 36, wherein said skin injury be owing to serious solar radiation excessively causes, and wherein said treatment has reduced skin and blisters and peel.