EP0564552A1 - Utilisation d'inhibiteurs de la calpaine dans l'inhibition et le traitement de la neurodegenerescence - Google Patents

Utilisation d'inhibiteurs de la calpaine dans l'inhibition et le traitement de la neurodegenerescence

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Publication number
EP0564552A1
EP0564552A1 EP92902904A EP92902904A EP0564552A1 EP 0564552 A1 EP0564552 A1 EP 0564552A1 EP 92902904 A EP92902904 A EP 92902904A EP 92902904 A EP92902904 A EP 92902904A EP 0564552 A1 EP0564552 A1 EP 0564552A1
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EP
European Patent Office
Prior art keywords
leu
ala
abu
cooet
peptide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP92902904A
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German (de)
English (en)
Inventor
Raymond T. Bartus
David D. Eveleth, Jr.
Gary S. Lynch
James C. Powers
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Georgia Tech Research Institute
Georgia Tech Research Corp
Cortex Pharmaceuticals Inc
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Georgia Tech Research Institute
Georgia Tech Research Corp
Cortex Pharmaceuticals Inc
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Publication of EP0564552A1 publication Critical patent/EP0564552A1/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates generally to the field of neuroprotectants and more specifically to the use of inhibitors of calcium-stimulated proteases, such as calpain, as therapeutics for neurodegeneration.
  • Neural tissues including brain, are known to possess a large variety of proteases, including at least two calcium-stimulated proteases, termed calpain I and calpain II, which are activated by micromolar and millimolar Ca 2+ concentrations, respectively. Calpains are a family of calcium activated thiol proteases that are present in many tissues. Calpain II is the predominant form, but calpain I is found at synapses and is thought to be the form involved in long term potentiation and synaptic plasticity.
  • Thiol proteases are distinguished from serine proteases, metalloproteases and other proteases by their mechanism of action and by the amino acid residue (cysteine) that participates in substrate attack. Although several thiol proteases are produced by plants, these proteases are not common in mammals, with cathepsin B (a lysosomal enzyme), other cathepsins and the calpains being among the few representatives of this family that have been described in mammals. Calpain I and calpain II are the best described of these, but several other members of the calpain family have been reported.
  • Ca 2 + -activated thiol proteases may exist, such as those reported by Yoshihara et al. in J. Biol. Chem. 265:5809-5815 (1990).
  • the term "Calpain” is used hereinafter to refer to any Ca 2+ -activated thiol proteases including the Yoshihara enzyme and calpains I and II.
  • cytoskeletal proteins While Calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack. In at least some cases, the products of the proteolytic digestion of these proteins by Calpain are distinctive and persistent over time. Since cytoskeletal proteins are major components of certain types of cells, this provides a simple method of detecting Calpain activity in cells and tissues. Specifically, the accumulation of the breakdown products ("BDP's") of spectrin, a cytoskeletal protein, has been associated with the activation of Calpain.
  • BDP's breakdown products
  • Calpain inhibitors of Calpain include peptide aldehydes such as leupeptin (Ac-Leu-Leu-Arg-H), as well as epoxysuccinates such as E-64. These compounds are not useful in inhibiting Calpain in Central Nervous System ("CNS") tissue in vivo because they are poorly membrane permeant and, accordingly, do not cross the blood brain barrier very well. Also, many of these inhibitors are poorly specific and will inhibit a wide variety of proteases in addition to Calpain. These commercially available compounds are based upon peptide structures that are believed to interact with the substrate binding site of Calpain. Active groups associated with the Calpain inhibitors then either block or attack the catalytic moiety of Calpain in order to inhibit the enzyme.
  • CNS Central Nervous System
  • leupeptin can facilitate nerve repair in primates.
  • Loxastatin also known as EST, Ep-460 or E-64d
  • E-64d while not having significant protease inhibitory activity itself, is believed to be converted to more potent forms, such as to E-64c, inside a mammalian body.
  • Intracellular calcium is likely to produce a large number of consequences, including the activation of a large number of enzymes, including proteases, such as Calpain, lipases and kinases. An increase in intracellular calcium is also thought to induce changes in gene expression.
  • Ischemia, head trauma and stroke have all been associated with the release of glutamate in amounts large enough to lead to excitotoxicity, the toxicity resulting from the actions of certain amino acids on neurons of the CNS.
  • the excess glutamate and other factors such as free radical damage of membranes or energy depletion, cause an increase in intracellular Ca 2+ .
  • an excess of intracellular Ca 2+ leads to several effects believed to be associated with neuronal cell damage, including destruction of cell structures through activation of phospholipase and Calpain, as well as free radical production resulting from activation of phospholipase and xanthine oxidase.
  • Many other factors have been associated with neurotoxicity. For example, reductions in action potentials and changes in a wide variety of chemical markers are known to be associated with neurons exposed to ischemic conditions.
  • the present invention provide the use of a Calpain inhibitor compound or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicame t for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
  • the neurodegeneration is occurring due to excitotoxicity, HTV-induced neuropathy, ischemia following denervation or injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease, Huntington's Disease, or Parkinson's Disease.
  • the medicament can comprise a pharmaceutically acceptable carrier and is for parenteral administration, such as for transdermal administration, subcutaneous injection, intravenous, intramuscular or intrastemal injection, intrathecal injection directly into the CNS or infusion.
  • the medicament can also be in a form suitable for oral use.
  • the medicament is for substantially preventing neurodegeneration in a patient undergoing surgery during and subsequent to the surgery, such as for a patient undergoing neurosurgery, cardiovascular surgery or a surgery using general anesthesia.
  • the Calpain inhibitor compound preferably enters tissue of the CNS of the mammal, such as through use of a membrane-permeant Calpin inhibitor.
  • the present invention also provides an in vitro method of selecting Calpain Inhibitors for use as Calpain Inhibitor protectants in the in vivo treatment or inhibition of degeneration.
  • This method includes identifying compounds having Calpain inhibitory activity in vitro, and identifying those compounds with Calpain inhibitory activity that are membrane permeant through an in vitro assay for membrane permeance.
  • the in vitro assay for membrane permeance can include providing a plurality of tissue portions from a mammal; treating at least one, but not all, of the tissue portions with a Calpain Inhibitor; subjecting the tissue portions to an event that can cause degeneration in untreated tissue; measuring the amount of degeneration that occurs in the tissue portions; and comparing the amount of degeneration that occurs in the treated tissue portions with the amount of degeneration occurring in the untreated tissue portions.
  • the amount of degeneration in the treated tissue portions being less than the amount of degeneration in the untreated tissue portions indicates that the Calpain Inhibitor is membrane-permeant.
  • the tissue portions in some embodiments are brain slices, platelets or cells in culture.
  • the measuring step in some embodiments involves analyzing the tissue portions for the presence of the BDP's of a cytoskeletal component such as spectrin, MAP2, actin binding protein or tau.
  • the measuring step can also include measuring the electrical activity of the tissue portions.
  • the in vitro assay for membrane permeance can be performed by measuring the ability of the
  • Calpain Inhibitor to penetrate platelet membranes and inhibit endogenous Calpain of the platelets.
  • the present invention also provides the use of a Substituted Heterocyclic Compound or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
  • the medicament is preferably for inhibiting or treating neurodegeneration of the CNS.
  • Heterocyclic Compound preferably is a member of the Class I Substituted Isocoumarins, the Class II Substituted Isocoumarins or the Class I II Heterocyclic Compounds. More preferably, the Substituted Heterocyclic Compound is 3-chloroisocoumarin; a 3,4- dichloroisocoumarin; a 3-alkoxy-7-amino-4-chloroisocoumarin; or a 7-substituted 3-alkoxy-4- chloroisocoumarin.
  • Another aspect of the present invention provides the use of a Peptide Keto-Compound having Calpain inhibitory activity or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
  • the medicament is preferably for inhibiting or treating neurodegeneration of the CNS.
  • the Peptide Keto-Compound preferably is a peptide ⁇ -ketoester, a peptide ⁇ -ketoacid or a peptide ⁇ -ketoamide.
  • the Peptide Keto-Compound is a member of one of the following subclasses: Dipeptide ⁇ -Ketoesters (Subclass A), Dipeptide ⁇ -Ketoesters (Subclass B), Tripeptide ⁇ -Ketoesters (Subclass A), Tripeptide ⁇ -Ketoesters (Subclass B), Tetrapeptide ⁇ -Ketoesters, Amino Acid Peptide ⁇ -Ketoesters, Dipeptide ⁇ -Ketoacids (Subclass A), Dipeptide ⁇ -Ketoacids (Subclass B), Tripeptide ⁇ -Ketoacids, Tetrapeptide ⁇ -Ketoacids and Amino Acid Peptide ⁇ -Ketoacids, Dipeptide ⁇ -Ketoamides (Subclass A), Dipeptide ⁇ -Ketoamides (Subclass B), Tripeptide ⁇ -Ketoamides, Tetrapeptide ⁇ -Ketoamides or
  • Still another aspect of the present invention provides the use of a Halo-Ketone Peptide having Calpain inhibitory activity or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
  • the medicament is preferably for inhibiting or treating neurodegeneration of the CNS.
  • the Halo-Ketone Peptide can be an amino-halo ketone peptide or a diazo-ketone peptide.
  • the uses of the present invention of Substituted Heterocyclic Compounds, Peptide Keto-Compounds or Halo-Ketone Peptides can be used in connection with neurodegeneration associated with excitotoxicity, HIV-induced neuropathy, ischemia, subarachnoid hemorrhage, stroke, brain seizure, major heart attack, multiple infarction dementia, Alzheimer's Disease, Huntington's Disease or Parkinson's Disease.
  • the medicament of these uses can include a pharmaceutically acceptable carrier, and be for parenteral administration, such as transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or an infusion technique.
  • the medicament can also be in a form suitable for oral use.
  • These uses can also be in connection with neurodegeneration is occurring from ischemia-inducing events, stroke, head injury, major heart attack, brain seizure, near drowning, carbon monoxide poisoning, surgery-related brain damage or another event known to cause neurodegeneration.
  • Another aspect of the present invention provides a method of minimizing proteolysis in an in vitro sample containing peptides or proteins during or following the processing, production, preparation, isolation, purification, storage or transport of the samples, comprising the addition to the sample of a Substituted Heterocyclic Compound or a Peptide
  • Keto-Compound that is a member of one of the following subclasses: Dipeptide ⁇ -Ketoesters (Subclass A), Dipeptide ⁇ -Ketoesters (Subclass B), Tripeptide ⁇ -Ketoesters (Subclass A), Tripeptide ⁇ -Ketoesters (Subclass B), Tetrapeptide ⁇ -Ketoesters, Amino Acid Peptide ⁇ -Ketoesters, Dipeptide ⁇ -Ketoacids (Subdass A), Dipeptide ⁇ -Ketoacids (Subdass B), Tripeptide ⁇ -Ketoacids, Tetrapeptide ⁇ -Ketoacids, Amino Acid Peptide ⁇ -Ketoacids, Dipeptide ⁇ -Ketoamides (Subclass A), Dipeptide ⁇ -Ketoamides (Subclass B), Tripeptide ⁇ -Ketoamides, Tetrapeptide ⁇ -Ketoamides or Amino Acid ⁇
  • the present invention also provides a method of minimizing degradation resulting from Calpain activity in a tissue sample during or following preparation of the sample, comprising the addition to the sample of a Substituted Heterocyclic Compound, Peptide Keto-Compound or a Halo- Ketone Peptide.
  • the sample can be a whole organ and the addition of compound comprises perfusion of the organ with the compound dissolved in fluid.
  • the tissue sample is used in an assay for neurodegeneration wherein the assay comprises a test for the products of Calpain activity in the tissue samples.
  • the addition of compound in either of these methods can include the addition of a Peptide ⁇ -Ketoacid to the sample.
  • this Peptide ⁇ -Ketoacid comprises a compound that is a member of one of the following subdasses: Dipeptide ⁇ -Ketoacids (Subclass A), Dipeptide ⁇ -Ketoacids (Subclass B), Tripeptide ⁇ -Ketoacids, Tetrapeptide ⁇ -Ketoacids or Amino Acid Peptide ⁇ -Ketoacids.
  • compositions for the treatment or inhibition of neurodegeneration include a pharmacologically effective neuroprotective amount of a Substituted Heterocyclic Compound, Peptide Keto-Compound or Halo-Ketone Peptide, or pharmaceutically acceptable salts or derivatives thereof in a pharmaceutically acceptable formulation containing a carrier material.
  • a Peptide Keto-Compound is included in the composition wherein said Peptide Keto-Compound comprises a compound from one of the following subdasses: Dipeptide ⁇ -Ketoesters (Subclass A), Dipeptide ⁇ -Ketoesters (Subdass B), Tripeptide ⁇ -Ketoesters (Subclass A), Tripeptide ⁇ -Ketoesters (Subdass B), Tetrapeptide ⁇ -Ketoesters, or Amino Acid Peptide ⁇ -Ketoesters.
  • Dipeptide ⁇ -Ketoesters Subclass A
  • Dipeptide ⁇ -Ketoesters Subdass B
  • Tripeptide ⁇ -Ketoesters Subclass A
  • Tripeptide ⁇ -Ketoesters Subdass B
  • Tetrapeptide ⁇ -Ketoesters or Amino Acid Peptide ⁇ -Ketoesters.
  • Keto-Compound is preferably one of the following compounds: Bz-DL-Ala-COOEt, Bz-DL-Ala-COOCH2-C 6 H 4 -CF 3 (para), Bz-DL-Lys-COOEt, PhCO-Abu-COOEt, (CH 3 ) 2 CH(CH 2 ) 2 CO-Abu-COOEt, CH 3 CH 2 CH) 2 CHCO-Abu-COOEt or Ph(CH2) 6 CO-Abu-COOEt.
  • Another preferred Peptide Keto-Compound is one of the following compounds: Z-Ala-DL-AIa-COOEt, Z-Ala-DL-Ala-COOBzl,
  • a preferred Peptide Keto-Compound could also be MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe or Z-Ala-Ala-Ala-DL-Ala-COOEt. Still other preferred Peptide Keto-Compounds would be a compound from one of the following subclasses: Dipeptide ⁇ -Ketoacids (Subdass A), Dipeptide ⁇ -Ketoacids (Subclass B), Tripeptide ⁇ -Ketoadds, Tetrapeptide ⁇ -Ketoadds or the Amino Acid Peptide ⁇ -Ketoacids.
  • preferred Peptide Keto-Compounds could be one of the following compounds: Bz-DL-Lys-COOH, Bz-DL-Ala-COOH, Z-Leu-Phe-COOH or Z-Leu-Abu-COOH.
  • a compound from one of the following subdasses: Dipeptide ⁇ -Ketoamides (Subclass A), Dipeptide ⁇ -Ketoamides (Subdass B), Tripeptide ⁇ -Ketoamides, Tetrapeptide ⁇ -Ketoamides or Amino Add ⁇ -Ketoamides, could also be used in the preferred compositions.
  • Another preferred Peptide Keto-Compound would be one of the following compounds: Z-Leu-Phe-CONH-Et, Z-Leu-Phe-CONH-nPr, Z-Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-Bzl, Z-Leu-Phe-CONH- (CH 2 ) 2 Ph, Z-Leu-Abu-CONH-Et, Z-Leu-Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, Z-Leu-Abu-CONH-Bzl, Z-Leu-Abu-CONH-(CH 2 ) 2 Ph, Z-Leu-Abu-CONH-(CH 2 ) 3 - N(CH 2 CH 2 ) 2 O, Z-Leu-Abu-CONH-(CH 2 ) 7 CH 3 , Z-Le
  • composition can also include a Substituted Heterocyclic Compound such as one of the Class I Substituted Isocoumarins, Class II Substituted Isocoumarins or Class III Heterocyclic Compounds. Preferred
  • Substituted Heterocyclic Compounds are 3-chloroisocoumarin, a 3,4-dichloroisocoumarin, a 3-alkoxy-7-amino-4-chloroisocoumarin, a 7-substituted 3-alkoxy-4-chloroisocoumarin; CiTPrOIC, NH 2 -CiTPrOIC, PhCH 2 NHCONH-CiTPrOIC, CH 3 CONH-CiTPrOIC, L-Phe-NH-CiTPrOIC, PhCH 2 NHCONH-CiTEtOIC, PhCH 2 CONH-CiTEtOIC, or D-Phe-NH-CiTEtOIC.
  • the composition is preferably in dosage form comprising from 70 ⁇ g to 7 g of active ingredient in each dose, and the carrier material includes a liquid, wherein the composition is in dosage form and wherein each dose comprises from 0.5 ml to 1 liter of said carrier material.
  • the compositions can additionally include at least one of the following: DMSO or other organic solvent, a lipid carrier, a detergent, a surfactant, or an emulsifying agent. These compositions can be suitable for parenteral administration or in a form suitable for topical application.
  • the compositions can be in a variety of forms, such as an aqueous solution, a lotion, a jelly, an oily solution, or an oily suspension.
  • the present invention also provides the use of a Substituted Heterocyclic Compound as a medicament, the use of a Halo-Ketone Peptide as a medicament, and the use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subdasses: Dipeptide ⁇ -Ketoesters (Subdass A), Dipeptide ⁇ -Ketoesters (Subclass B), Tripeptide ⁇ -Ketoesters (Subdass A), Tripeptide ⁇ -Ketoesters (Subdass B), Tetrapeptide ⁇ -Ketoesters, or Amino Acid Peptide ⁇ -Ketoesters.
  • Preferred Peptide Keto-Compounds in this use include any one of the Peptide Keto- Compounds described above in connection with the pharmaceutical compositions.
  • Figure 1 shows the percentage of inhibition of glutamate-induced cell death through the addition of glutamate and various Calpain Inhibitors relative to control where no glutamate was added.
  • Figure 2 graphically depicts the effects of Z-Leu-Phe-CONH-Et (CX269) and Z-Leu-Abu-CONH-Et (CX275) on the size of infarction produced upon MCA occlusion in male rats.
  • Figure 3 shows the effects of CX216 (Z-Leu-Phe-CO2Et, a Peptide Keto- Compound), and CI1 (Ac-Leu-Leu-Me-H) relative to control slices on survival of hippocampal slices exposed to 10 minutes exposure of anoxic atmosphere where both of these compounds were added at their optimal inhibitory concentration at both 1 hour and 2 hour incubation times.
  • Figure 4 shows the evoked potential amplitude for control, CI1 treated and CX218 treated hippocampal slices over a time course during which the slices are exposed to anoxic atmosphere.
  • Figure 5 shows the percent recovery of EPSP from severe hypoxia over the course of one hour incubation for Z-Leu-Phe-CONH-Et (CX269) and Z-Leu-Phe-CO2Et (CX216).
  • Figure 6 shows a comparison of the effect of the presence of CI1 or CX216 on survival of hippocampal slices expressed as the duration of anoxia (in minutes) before fiber volley disappearance.
  • Figure 7 shows the effects of CI1 compared with control on the behavioral and convulsive effects of kainic acid.
  • Figure 8 shows the amount of spectrin BDP's in rat brains exposed to kainate for control and CI1 treated rats.
  • Calpain activation is an event central to many cases of brain atrophy and degeneration and that inhibition of Calpain alone is sufficient to inhibit or prevent cell deterioration and loss.
  • inhibition of Calpain provides protection from neurotoxicity associated with many neurodegenerative conditions and diseases.
  • one aspect of the present invention is directed to inhibition and treatment of the neurodegeneration and other diseases associated with this digestion through the inhibition of Calpain activity.
  • part of this aspect of the present invention is to prevent the neurodegeneration and other pathology caused by this digestion through the in vivo administration of Calpain inhibitors.
  • diseases and conditions which can be treated using this aspect of the present invention include neurodegeneration following excitotoxicity, HTV-induced neuropathy, ischemia, denervation followingischemia or injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD), Parkinson's Disease, Huntington's Disease, surgery-related brain damage and other neuropathological conditions.
  • AD Alzheimer's Disease
  • Parkinson's Disease Huntington's Disease
  • Calpain activation is localized to the brain areas most vulnerable to the particular pathogenic manipulation.
  • Calpain activation precedes overt evidence of neurodegeneration.
  • Calpain activation is spatially and temporally linked to impending or ongoing cell death in the brain.
  • the activation of Calpains is an early event in the death of cells including neural cells. This is in contrast to other known proteases which are activated at later stages of cell death.
  • inhibition of Calpain activity provides intervention at an early stage of cell death, prior to significant deterioration of cellular machinery.
  • Calpains Another aspect of the involvement of Calpains in neurodegeneration is the involvement of these proteins in regenerating systems. It is known that developing or regenerating axons are somehow inhibited from further development in a stabilization process called the "stop pathway.” This stabilization can occur when axons have reached their targets; however, in some systems stabilization can also occur at inappropriate places.
  • This stop pathway operates at least in part by the activation of intracellular Calpain and that inhibition of Calpain can interfere with stabilization (Luizzi, 1990).
  • Calpain inhibitors when used in accordance with the present invention, can advantageously aid regeneration and recovery of neural tissue after injury, in addition to inhibiting neurodegeneration.
  • Another aspect of the present invention is our discovery that at least three classes of compounds, the substituted isocoumarins, the peptide keto-compounds and the Halo- Ketone Peptides have Calpain inhibitory activity.
  • these three classes of compounds exhibit additional properties that render them especially useful as therapeutically effective compounds in the treatment of neurodegenerative conditions and diseases.
  • One particular class of compounds exhibiting Calpain inhibitory activity when used in accordance with the present invention, are the substituted heterocyclic compounds. These compounds include the substituted isocoumarins.
  • the substituted heterocyclic compounds are known to be excellent inhibitors of serine proteases. As discussed here in below, we have now discovered that these compounds are also inhibitors of calpain I and calpain II, and also of other Calpains. Additionally, as also discussed below, we have found that, unlike most known inhibitors of Calpains, these substituted heterocyclic compounds are not effective as inhibitors of papain or cathepsin B. Thus, we believe that the substituted heterocyclic compounds provide a relatively specific means of inhibiting Calpains while not affecting other thiol proteases.
  • the Class I Substituted Isocoumarins are known to be excellent inhibitors of several serine proteases, including bovine thrombin, human thrombin, human factor Xa, human factor XIa, human factor XIIa, bovine trypsin, human plasma plasmin, human tissue plasminogen activator, human lung tryptase, rat skin tryptase, human leukocyte elastase, porcine pancreatic elastase, bovine chymotrypsin and human leukocyte cathepsin G.
  • the Class I Substituted Isocoumarins inhibit the serine proteases by reaction with the active site serine to form an acyl enzyme, which in some cases may further react with another active site nudeophile to form an additional covalent bond.
  • the Class I Substituted Isocoumarins also react with Calpain. We believe that the mechanism of action of Calpain inhibition is similar to that of the inhibition of serine proteases since the reaction mechanism of Calpains is similar to that of the serine proteases.
  • the Class I Substituted Isocoumarins having Calpain inhibitory activity have the following structural formula:
  • Z is selected from the group consisting of C1-6 alkoxy with an amino group attached to the alkoxy group, C1-6 alkoxy with an isothiureido group attached to the alkoxy group, C1-6 alkoxy with a guanidino group attached to the alkoxy group, C1-6 alkoxy with an amidino group attached to the alkoxy group, C1-6 alkyl with an amino group attached to the alkyl group, C1-6 alkyl with an isothiureido group attached to the alkyl group, C1-6 alkyl with an guanidino group attached to the alkyl group, C1-6 alkyl with an amidino group attached to the alkyl group,
  • AA represents alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine or sarcosine,
  • M represents NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X-NH-CS, X- NH-SO2, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-,
  • X represents C1-6 alkyl, C1-6 fluoroalkyl, C1-6 alkyl substituted with K, C1-6 fluoroalkyl substituted with K, phenyl, phenyl substituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, naphthyl, naphthyl substituted with J, naphthyl disubstituted with J, naphthyl trisubstituted with J, C1-6 alkyl with an attached phenyl group, C1-6 alkyl with two attached phenyl groups, C1-6 alkyl with an attached phenyl group substituted with J, or C1-6 alkyl with two attached phenyl groups substituted with J,
  • J represents halogen, COOH, OH, CN, NO2, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamine, C1-6 dialkylamine, or C1-6 alkyl-O-CO-,
  • K represents halogen, COOH, OH, CN, NO2, NH2, C1-6 alkylamine, C1-6 dialkylamine, or C1-6 alkyl-O-CO-,
  • Y is selected from the group consisting of H, halogen, trifiuoromethyl, methyl, OH and methoxy.
  • the compounds of Formula (I) can also contain one or more substituents at position B as shown in the following structure:
  • electronegative substituents such as NO2, CN, Cl, COOR, and COOH will increase the reactivity of the isocoumarin
  • electropositive substituents such as NH2, OH, alkoxy, thioalkyl, alkyl, alkylamino, and dialkylamino will increase its stability.
  • Neutral substituents could also increase the stability of acyl enzyme and improve the effectiveness of the inhibitors.
  • Isocoumarins with basic substituents are also known to be effective inhibitors of serine proteases. See Powers et al, U.S. Patent No. 4,845,242, the disclosure of which is hereby incorporated by reference.
  • This class of compounds, referred to herein as the "ClassII Substituted Isocoumarins," along with the other substituted heterocyclic compounds, is believed to be effective in the use of the present invention.
  • the Class II Substituted Isocoumarins have the following structural formula:
  • Z is selected from the group consisting of H, halogen, C 1 -6 alkyl, C 1 -6 alkyl with an attached phenyl, C 1 -6 fluorinated alkyl, C 1 -6 alkyl with an attached hydroxyl, C 1-6 alkyl with an attached C 1-6 alkoxy, C 1-6 alkoxy, C 1-6 fluorinated alkoxy, C 1-6 alkoxy with an attached phenyl, benzyloxy, 4-fluorobenzyloxy, -OCH 2 C 6 H 4 R' (2-substituent), -OCH 2 C 6 H 4 R' (3- substituent), -OCH 2 C 6 H 4 R' (4-substituent), -OCH 2 C 6 H 3 R 2 ' (2,3-substituents), -OCH 2 C 6 H 3 R 2 ' (2,4-substituents), -OCH 2 C 6 H 3 R 2 ' (2,5-substituents), O
  • R' is selected from the group consisting of H, halogen, trifluoromethyl, NO 2 , cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino.
  • Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH, and methoxy.
  • ClassII Substituted Isocoumarins are represented by structure (II) where,
  • Z is selected from the group consisting of C 1 -6 alkoxy with an attached isothiureido, C 1 -6 alkoxy with an attached guanidino, C 1 -6 alkoxy with an attached amidino, C 1 -6 alkyl with an attached amino, C 1 -6 alkyl with an attached isothiureido, C 1 -6 alkyl with an attached guanidino, C 1 -6 alkyl with an attached amidino,
  • R is selected from the group consisting of H, OH, NH 2 , NO 2 halogen, C 1 -6 alkoxy,C 1 -6 fluorinated alkoxy, C 1 -6 alkoxy with an attached amidino , M-AA-NH-, M-AA-O-, wherein AA represents alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, beta-alanine, norleucine, norvaline, alpha-aminobutyric and epsilon-aminocaponic acid, citrulline, hydroxyproline, ornithine and sarcosine,
  • M represents H, lower alkanoyl having 1 to 6 carbons, carboxyalkanoyl, hydroxyalkanoyl, amin-alkanoyl, benzene sulfonyl, tosyl, benzoyl, and lower alkyl sulfonyl having 1 to 6 carbons,
  • Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH and methoxy.
  • Class II Substituted Isocoumarins are represented by structure (II) where
  • Z is selected from the group consisting of C 1 -6 alkoxy with an attached amino, C 1 -6 alkoxy with an attached isothiureido, C 1 -6 alkoxy with an attached guanidino, C 1 -6 alkoxy with an attached amidino, C 1 -6 alkyl with an attached amino, C 1 -6 alkyl with an attached guanidino, C 1 -6 alkyl with an attached amidino,
  • Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH and methoxy.
  • Z is selected from the group consisting of CO, SO, SO 2 , CCl and CF,
  • Y is selected from the group consisting of O, S and NH,
  • X is selected from the group consisting of N and CH, and
  • R is selected from the group consisting of C 1 -6 alkyl (such as methyl, ethyl and propyl), C 1 -4 alkyl containing a phenyl (such as benzyl), and C 1 -6 fluoroalkyl (such as trifluoromethyl, pentafluoroethyl, and heptafluoropropyl).
  • the Z group must be electrophilic since it interacts with the active site serine OH group of the serine protease.
  • the R group must be uncharged and hydrophobic. One or more of the carbons in the R group could be replaced by O, S, NH and other such atomic groups as long as the R group maintains its hydrophobic character.
  • Heterocyclic Compound shall be used to refer to any particular species of these compounds.
  • Boc-D-Phe (0.33 g, 1.2 mmole) reacted with 1,3-dicyclohexylcarbodiimide (0.13 g, 0.6 mmole) in 10 ml THF at 0°C for 1 hour to form the symmetric anhydride, and then
  • 7-Boc-alanylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin 7-benzoylamino- Ala-4-chloro-3(2-isothiureidoethoxy) isocoumarin
  • 7-Boc-va lylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared by the same procedure.
  • 7- substituted-4-chloro-3-(2-bromoethoxy) isocoumarin can be synthesized by reacting 7-amino- 4-chloro-3-(2-bromoethoxy) isocoumarin with appropriate acid chloride or sulfonyl chloride in the presence of Et 3 N.
  • 7-Ethoxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin 7-benzyloxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin
  • 7-phenoxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy)isocoumarin can be prepared from the reaction of 7-substituted-4-chloro-3-(2-bromoethoxy) isocoumarin with thiourea.
  • Peptide ⁇ -ketoesters, peptide ⁇ -ketoacids, and peptide ⁇ -ketoamides are transition state analog inhibitors for serine proteases and cysteine proteases. While these subdasses of compounds are chemically distinguishable, for simplicity, all of these compounds will be referred to collectively herein as the "Peptide Keto-Compounds".
  • the interactions of peptides with serine and cysteine proteases are designated herein using the nomendature of Schechter, I., and Berger, A., 1967, Biochem. Biophys. Res Commun. 27: 157-162 (incorporated herein by reference).
  • the individual amino acid residues of a substrate or inhibitor are designated P1, P2, etc. and the corresponding subsites of the enzyme are designated S1, S2, etc.
  • the scissile bond of the substrate is
  • Amino acid residues and blocking groups are designated using standard abbreviations [see J. Biol. Chem. 260, 14-42 (1985) for nomendature rules; incorporated herein by reference].
  • An amino acid residue (AA) in a peptide or inhibitor structure refers to the part structure -NH-CHR1-CO-, where R1 is the side chain of the amino acid AA.
  • a peptide ⁇ -ketoester residue would be designated -AA-CO-OR which represents the part structure -NH-CHR1-CO-CO-OR.
  • the ethyl ketoester derived from benzoyl alanine would be designated Bz-Ala-CO-OEt which represents C6H5CO-NH-CHMe-CO-CO-OEt.
  • peptide ketoacid residues residues would be designated -AA-CO-OH.
  • peptide ketoamide residues are designated -AA-CO-NH-R.
  • the ethyl keto amide derived from Z-Leu-Phe-OH would be designated Z-Leu-Phe-CO-NH-Et which represents C 6 H 5 CH 2 OCO-NH-CH(CH 2 CHMe 2 )-CO-NH-CH(CH 2 Ph)-CO-CO-NH-Et.
  • Peptide ⁇ -ketoesters containing amino acid residues with hydrophobic side chain at the P1 site have also been found to be excellent inhibitors of several cysteine proteases including papain, cathepsin B and calpain.
  • Calpains can be inhibited by peptide inhibitors having several different active groups Structure-activity relationships with the commercially available in vitro inhibitors of Calpain such as peptide aldehydes, have revealed that Calpains strongly prefer Leu or Val in the P2 position. These enzymes are inhibited by inhibitors having a wide variety of amino acids in the P1 position, but are generally more effectively inhibited by inhibitors having amino acids with nonpolar or hydrophobic side chains in the P1 position.
  • Another particular class of compounds exhibiting Calpain inhibitory activity when used in accordance with the present invention, are the Peptide Keto-Compounds. These are compounds of the general structure:
  • M represents NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X-NH-CS-, X-NH-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-, H, acetyl, carbobenzoxy, succinyl, methyloxysuccinyl, butyloxycarbonyl;
  • X is selected from the group consisting of C1-6 alkyl, C1-6 fluoroalkyl, C1-6 alkyl substituted with J, C1-6 fluoroalkyl substituted with J, 1-admantyl, 9-ftuorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-6 alkyl with an attached phenyl group, C1-6 alkyl with two attached phenyl groups, C1-6 alkyl with an attached phenyl group substituted with K, and C1-6 alkyl with two attached phenyl groups substituted with K, and C1-6 alkyl with two attached phenyl groups substituted with
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C1-6 alkoxy, C1-6 alkylamine, C1-6 dialkylamine, C1-6 alkyl-O-CO-, C1-6 alkyl-O-CO-NH, and C1-6 alkyl-S-;
  • K is selected from the group consisting of halogen, C1-6 alkyl, C1-6 perfluoroalkyl, C1-6 alkoxy, NO2, CN, OH, CO2H, amino, C1-6 alkylamino, C2-12 dialkylarnino, C1-C6 acyl, and C1-6 alkoxy-CO-, and C1-6 alkyl-S-;
  • aa represents a blocked or unblocked amino acid of the L or D configuration, preferably selected from the group consisting of: alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine (nle), norvaline (nva), alpha-aminobutyric acid (abu), epsilonaminocaproic acid, citrulline, hydroxyproline, homoarginine, ornithine or sarcosine; n is a number from 1 to 20;
  • Q is O or NH
  • R represents H, C1-6 alkyl, C1-6 fluoroalkyl, C1-6 chloroalkyl, benzyl, C1-6 alkyl substituted with phenyl, C1-6 alkyl with an attached phenyl group substituted with K.
  • the Peptide Keto-Compounds can be divided into the Peptide Ketoesters, Peptide Ketoacids and Peptide Ketoamides.
  • Each of the compounds can also be rougeified based on the number of amino acids contained within the compound, such as an amino acid peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide and so on.
  • Dipeptide ⁇ -Ketoesters are compounds of the formula:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CSX 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-10 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-10 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA 1 is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methyl
  • AA 2 is a side chain blocked or unblocked amino acid with the L configuration, configuration, or no chirality at the ⁇ -carbon selected from the group consisting of leucine isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alphaaminobutyric add, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic add), O-methylserine, O-
  • R 1 is selected from the group consisting of H, C 1-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K.
  • Dipeptide ⁇ -Ketoesters are compounds of the structure:
  • Mi represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leudne, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carb
  • R 2 represents C 1-8 branched and unbranched alkyl, C 1-8 branched and unbranched cyclized alkyl, or C 1-8 branched and unbranched fluoroalkyl;
  • R is selected from the group consisting of H, C 1-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K
  • Tripeptide ⁇ -Ketoesters (Subdass A) are compounds of the structure:
  • M 3 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-,X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, T-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • T is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 2-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-12 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • Kis selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • R is selected from the group consisting of H, C 2-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K
  • Tripeptide ⁇ -Ketoesters (Subdass B) are compounds of the structure:
  • M 3 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, T-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • T is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 2-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1 - 10 alkoxy, C 1-10 alkylamine, C 1-10 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citruiline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carb
  • R 2 represents C 1-8 branched and unbranched alkyl, C 1-8 branched and unbranche cyclized alkyl, or C 1-8 branched and unbranched fluoroalkyl;
  • R is selected from the group consisting of H, C 1-20 alkyl, C 1-20 alkyl with a pheny group attached to the C 1-8 alkyl, and C 1-20 alkyl with an attached phenyl group substituted withK
  • Tetrapeptide ⁇ -Ketoesters are compounds of the strudure:
  • M 3 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, T-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • T is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 2-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C lm 10 alkoxy, C 1-10 alkylamine, C 2-12 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid glutamic acid, lysine, arginine, histidine, phenylglycine,
  • AA 4 is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of leucine, isoleucine, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyri acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O
  • R is selected from the group consisting of H, C 1-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K
  • the Amino Acid Peptide ⁇ -Ketoesters are compounds of the structure:
  • Mi represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, Y-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • Y is selected from the group consisting of C 6-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-12 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • Kis selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic add, pipecolinic acid (2-piperidine carboxylic acid
  • R is selected from the group consisting of H, C 1-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K
  • Peptide Ketoester compounds are representative of the Peptide Ket Compounds found to be useful as Calpain inhibitors within the context of the prese invention:
  • Peptide Ketoacid Compounds We have found certain subdasses of Peptide Ketoacid Compounds to be particularly useful when used in accordance with the present invention. These are subclasses are the Dipeptide ⁇ -Ketoacids (Subdass A), the Dipeptide ⁇ -Ketoacids (Subclass B), the Tripeptide ⁇ -Ketoadds, the Tetrapeptide ⁇ -Ketoacids and the Amino Acid peptide ⁇ -Ketoacids. All of these are considered to be within the class of Peptide Keto-Compounds.
  • Dipeptide ⁇ -Ketoacids (Subclass A) are compounds of the structure:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K naphthyl trisubstituted with K C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • R 2 represents C 1-8 branched and unbranched alkyl, C 1-8 branched and unbranched cyclized alkyl, or C 1-8 branched and unbranched fluoroalkyl.
  • Dipeptide ⁇ -Ketoacids (Subclass B) are compounds of the structure:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA 1 is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic add, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methyl
  • AA 2 is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic
  • Tripeptide ⁇ -Ketoacids are compounds of the structure:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-X 2 N-CS-, X-NH-SO 2 -, X2N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alky substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, pheny substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attache phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on the
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylarnine, C 2-12 dialkylamine, C 1-10 alktyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1- C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric add, epsilon-aminocaproic add, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic add (2-piperidine carboxylic add
  • Tetrapeptide ⁇ -Ketoadds are compounds of the structure:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, Y r CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disub
  • Y 1 is selected from the group consisting of C 2-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-12 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic add, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • the Amino Acid Peptide ⁇ -Ketoacids are compounds of the structure: M 1 -AA-CO-OH
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS- X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, Y 2 -CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attache phenyl groups substituted with K, C 1-10 alkyl with an attached phenoxy group, and C 1-10 alkyl with an attached phenoxy group substituted with K on
  • Y 2 is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K naphthyl disubstituted with K, naphthyl trisubstituted with K C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attache phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1 - 10 alkoxy, C 1-10 alkylamine, C 2-12 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • Peptide Ketoacid compounds are representative of the Peptide Keto- Compounds found to be useful as Calpain inhibitors within the context of the present invention:
  • the peptide ⁇ -ketoamides are transition state analogue inhibitors for cysteine proteases, such as Calpain.
  • cysteine proteases such as Calpain.
  • Peptide ⁇ -ketoamides containing amino acid residues with hydrophobic side chains at the P 1 site are excellent inhibitors of several cysteine proteases including calpain I and calpain II.
  • Dipeptide ⁇ -Ketoamides (Subdass A) have the following structural formula:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substitute
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ⁇ -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • R 2 is selected from the group consisting of C 1-8 branched and unbranched alkyl, C 1-8 branched and unbranched cyclized alkyl, and C 1-8 branched and unbranched fluoroalkyl;
  • R 3 and R 4 are selected independently from the group consisting of H, C 1-20 alkyl, C 1-20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, C 1-20 alkyl with an attached phenyl group substituted with K, C 1-20 alkyl with an attached phenyl group disubstituted with K, C 1-20 alkyl with an attached phenyl group trisubstituted with K, C 1-20 cyclized alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with a morpholine [-N(CH 2 CH 2 )O] ring attached through nitrogen to the alkyl, C 1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C 1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C 1-20 alkyl with an
  • Dipeptide ⁇ -Ketoamides (Subclass B) have the following strudural formula:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K C 1-10 alkyl with two attached phenyl groups substituted with K C 1-10 alkyl with an attached phenoxy group, and C 1
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-12 diallylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA 1 is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ⁇ -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic add, pipecolinic acid (2-piperidine carboxylic acid), O-methyl
  • AA 2 is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ⁇ -aminobutyric add, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic add, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic
  • R 3 and R 4 are selected independently from the group consisting of H, C 1-20 alkyl, C 1-20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, C 1-20 alkyl with an attached phenyl group substituted with K, C 1-20 alkyl with an attached phenyl group disubstituted with K, C 1-20 alkyl with an attached phenyl group trisubstituted with K, C 1-20 cyclized alkyl with an attached phenyl group substituted with K C 1-10 alkyl with a morpholine [-N(CH 2 CH 2 )O] ring attached through nitrogen to the alkyl, C 1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C 1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C 1-20 alkyl with an OH
  • Tripeptide ⁇ -Ketoamides have the following structural formula:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substitute
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ⁇ -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • R 3 and R 4 are selected independently from the group consisting of H, C 1-20 alkyl, C 1-20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, C 1-20 alkyl with an attached phenyl group substituted with K, C 1-20 alkyl with an attached phenyl group disubstituted with K, C 1-20 alkyl with an attached phenyl group trisubstituted with K, C 1-20 cyclized alkyl with an attached phenyl group substituted with K C 1-10 alkyl with a morpholine [-N(CH 2 CH 2 )O] ring attached through nitrogen to the alkyl, C 1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C 1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C 1-20 alkyl with an OH
  • Tetrapeptide ⁇ -Ketoamides have the following structural formula:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO 2 -, X 2 N-SO 2 -, X-CO-, X-CS-, X-SO 2 -, X-O-CO-, or X-O-CS-;
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyi, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substitute
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-12 clialkylarnine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 allyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 2-12 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ⁇ -aminobutyric acid, epsilon-aminocaproic acid, dtrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic add, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxy
  • R 3 and R 4 are selected independently from the group consisting of H, C 1-20 alkyl, C 1-20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, C 1-20 alkyl with an attached phenyl group substituted with K C 1-20 alkyl with an attached phenyl group.
  • the Amino Acid ⁇ -Ketoamides have the following structural formula:
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-,
  • X is selected from the group consisting of C 1-10 alkyl, C 1-10 fluoroalkyl, C 1-10 alkyl substituted with J, C 1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C 1-10 alkyl with an attached phenyl group, C 1-10 alkyl with two attached phenyl groups, C 1-10 alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substituted with K, C 1-10 alkyl with two attached phenyl groups substitute
  • J is selected from the group consisting of halogen, COOH, OH, CN, NO 2 , NH 2 , C 1- 10 alkoxy, C 1-10 alkylamine, C 2-12 dialkylamine, C 1-10 alkyl-O-CO-, C 1-10 alkyl-O-CO-NH-, and C 1-10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl, C 1-10 alkoxy, NO 2 , CN, OH, CO 2 H, amino, C 1-10 alkylamino, C 1-10 dialkylamino, C 1 -C 10 acyl, and C 1-10 alkoxy-CO-, and C 1-10 alkyl-S-;
  • AA is a side chain blocked or unblocked amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic add, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ⁇ -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
  • R 3 and R 4 are selected independently from the group consisting of H, C 1-20 alkyl, C 1-20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, C 1-20 alkyl with an attached phenyl group substituted with K, C 1-20 alkyl with an attached phenyl group disubstituted with K, C 1J2 ⁇ alkyl with an attached phenyl group trisubstituted with K, C 1-20 cydized alkyl with an attached phenyl group substituted with K, C 1-10 alkyl with a mo ⁇ holine [-N( CH 2 CH 2 )O] ring attached through nitrogen to the alkyl, C 1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C 1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C 1-20 alky
  • This compound is Z-Phe-NHCH 2 CO-CO-NH-Et (Z-Phe-Gly-CO-NH-Et).
  • the compound is reported in Hu and Abeles [Arch. Biochem. Biophys.. 281, 271-274 (1990)] to be an inhibitor of papain and cathepsin B.
  • Keto-Compounds found to be useful as Calpain inhibitors within the context of the present invention are Keto-Compounds found to be useful as Calpain inhibitors within the context of the present invention:
  • His-57 is hydrogen bonded to the carbonyl group of the ester functional group, the peptide backbone on a section of the polypeptide backbone hydrogen bonds to the inhibitor to form a ß-sheet, and the benzyl ester is directed toward the S' subsites.
  • the side chain of the P1 amino acid residue is located in the SI pocket of the enzyme. Interactions with ketoamides would be similar except that there is the possibility of forming an additional hydrogen bond with the NH group of the ketoamide functional group.
  • ketoacids there would be no R group to interact with the S' subsites. Therefore, these inhibitors would be expected to be slightly less potent than the ketoesters and ketoamides.
  • certain ketoacid compounds have been found to have surprisingly high activity when used in the context of the present invention.
  • Z-Leu-Phe-COOH and Z-Leu-Abu-COOH have been found to be extremely potent inhibitors of Calpains.
  • cysteine proteases shares several features in common with serine proteases including an active site histidine residue.
  • cysteine proteases In place of the Ser-195, cysteine proteases have an active site cysteine residue which would add to the ketonic carbonyl group of the peptide ketoadds, ketoesters, or ketoamides to form an adduct very similar to the structure described above except with a cysteine residue replacing the serine-195 residue. Additional interactions would occur between the extended substrate binding site of the cysteine protease and the inhibitor that would increase the binding affinity and specificity of the inhibitors.
  • the Peptide Keto-Compounds bind to the proteases inhibited thereby using many of the interactions that are found in complexes of a particular individual enzyme with its substrates.
  • a known inhibitor sequence is the peptide aldehyde, Ac-Leu-Leu-Nle-H (also known as Calpain Inhibitor 1 and hereinafter designated as "CI1").
  • This inhibitor in addition to a rdated peptide aldehyde inhibitor Ac-Leu-Leu-Nme-H (also known as Calpain Inhibitor II) are commerdally available from Calbiochem of La Jolla,
  • peptide ⁇ -ketoesters with aromatic amino acid residues in PI are good inhibitors of the thiol proteases, cathepsin B, papain and Calpain. Additionally, we have discovered that peptide ⁇ -ketoester and peptide ⁇ -ketoamides with either aromatic amino acid residues or small hydrophobic alkyl amino acid residues at PI are good inhibitors of Calpain.
  • Papain was assayed with Bz-Arg-AMC or Bz-Arg-NA [Kanaoka et al., Chem. Pharm. Bull. 25, 3126-3128 (1977)], and the AMC (7-amino-4-methylcoumarin) rdease was followed fluorimetrically (excitation at 380 nm, and emission at 460 nm).
  • Cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and Kirschke, Methods Enzymol.
  • Table PKC1 shows the inhibition constants (K i ) for papain, cathepsin B, calpain I, and calpain II.
  • the peptide ⁇ -ketoesters are prepared by a two step Dakin-West procedure. This procedure can be utilized with either amino acid derivatives, dipeptide derivatives, tripeptide derivatives, or tetrapeptide derivatives as shown in the following scheme:
  • the precursor peptide ((AA) n ) can be prepared using standard peptide chemistry procedures, including those that are well described in publications such as The Peptides,
  • the M group can be introduced using a number of different reaction schemes. For example, it could be introduced directly on an amino acid as shown in the following scheme:
  • the M group can be introduced by reaction with an amino acid ester, followed by removal of the ester group to give the same product, as shown in the following scheme:
  • Reaction with NH 2 S 4 O 2 Cl would introduce the NH 2 SO 2 -group.
  • Reaction with a substituted alkyl or aryl isocyanate would introduce the X-NH-CO-group where X is a substituted alkyl or aryl group.
  • Reaction with a substituted alkyl or aryl isothiocyanate would introduce the X-NH-CS- group where X is a substituted alkyl or aryl group.
  • Reaction with X-SO 2 -CI would introduce the X-SO 2 - group.
  • reaction with MeO-CO-CH 2 CH 2 -CO-CI would give the Y-CO- group when Y is a C2 alkyl substituted with a C1 alkyl-OCO- group.
  • Readion with an a substituted alkyl or aryl sulfonyl chloride would introduce an X-SO2- group.
  • reaction with dansyl chloride would give the X-SO2- derivative where X was a napthyl group monosubstituted with a dimethylamino group.
  • Reaction with a substituted alkyl or aryl chloroformate would introduce a X-O-CO- group.
  • Reaction with a substituted alkyl or aryl chlorothioformate would introduce a X-O-CS-.
  • There are many alternate reaction schemes which could be used to introduce all of the above M groups to give either M-AA-OH or
  • M-AA-OR' The M-AA-OH derivatives could then be used directly in the Dakin-West reaction or could be converted into the dipeptides, tripeptides, and tetrapeptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH which could be used in the Dakin-West reaction.
  • the substituted peptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH could also be prepared directly from H-AA-AA-OH,
  • H-AA-AA-AA-OH or H-AA-AA-AA-OH using the reactions described above for introduction of the M group.
  • the M group could be introduced by reaction with carboxyl blocked peptides M-AA-AA-OR', M-AA-AA-AA-OR', or M-AA-AA-AA-AA-OR' followed by the removal of the blocking group R '.
  • the R group in the ketoester structures is introduced during the Dakin-West reaction by reaction with an oxalyl chloride Cl-CO-CO-O-R.
  • reaction of M-AA-AA-OH with ethyl oxalyl chloride Cl-CO-CO-O-Et gives the keto ester M-AA-AA-CO-O-Et.
  • Reaction of M-AA-AA-AA-OH with Cl-CO-CO-O-Bzl would give the ketoester M-AA-AA-AA-AA-CO-O-Bzl.
  • R groups can be introduced into the ketoester strudure by reaction with various alkyl or arylalkyl oxalyl chlorides (Cl-CO-CO-O-R).
  • oxalyl chlorides are easily prepared by reaction of an alkyl or arylalkyl alcohol with oxalyl chloride Cl-CO-CO-Cl,
  • Cl-CO-CO-Cl oxalyl chloride
  • Bzl-O-CO-CO-Cl and n-Bu-O-CO-CO-Cl are prepared by reaction of benzyl alcohol and butanol respectively, with oxalyl chloride in yields of 50% and 80% [Warren, C. B., and Malee, E. J., J. Chromatography 64, 219-222
  • Ketoacids M-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-AA-CO-OH, M-AA-AA-CO-OH are generally prepared from the corresponding ketoesters M-AA-CO-OR, M-AA-AA-CO-OR, M-AA-AA-AA-CO-OR, M-AA-AA-AA-CO-OR by alkaline hydrolysis.
  • R Bzl
  • R t-butyl
  • a ketoacid could be used as a precursor to produce a corresponding ketoamide.
  • Blocking the ketone carbonyl group of the ketoacid and then coupling with an amine H-NR 3 R 4 using standard peptide coupling reagents would yield an intermediate which could then be deblocked to form the ketoamide.
  • N-Acylamino acids was synthesized via Schotten-Baumann reaction [M. Bergman
  • N-Acylamino acids with 4-methylpentanoic, 2-(1- propyl)pentanoic and 7-phenylheptanoic group was synthesized in a two step synthesis.
  • the N-acylamino acid methyl ester was obtained first and then was hydrolysed to the free N-acylamino acid.
  • N-Acylamino Acid Methyl Esters (General Procedure). To a chilled (10 oC) slurry of the appropriate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene was added slowly (temp. 10-15 oC) 40 mmol triethylamine or N- methylmo ⁇ holine and then the reaction mixture was stirred for 30 minutes at this temperature. Then 18 mmol of appropriate acid chloride (temp. 10-15 °C) was added slowly to the reaction mixture and the reaction mixture was stirred overnight at room temperature.
  • the precipiatated hydrochloride was filtered, washed on a funnel with 2 ⁇ 20 ml benzene, and the collected filtrate was washed successively with 2 ⁇ 50 ml 1 M HCl, 2 ⁇ 50 ml 5% NaHCO 3 , 1 ⁇ 100 ml H 2 O, 2 ⁇ 50 ml satd. NaCl and dried over MgSO 4 . After evaporation of the solvent in vacuo (rotavaporator), the residue was checked for purity (TLC) and used for the next step (hydrolysis).
  • the collected organic layer was washed with 2 ⁇ 50 ml H 2 O, decolorized with carbon, and dried over MgSO 4 . After evaporation of the solvent in vacuo (rotavaporator), the residue was checked for purity (TLC) and in the case of contamination was crystallized from a appropriate solvent.
  • N-Acyldipeptide methyl esters were synthesized via the HOBt-DCC method in DMF solution [K ⁇ nig and Geiger, Chem. Ber. 103, 788 (1970)].
  • N-Acyldipeptides were obtained by hydrolysis of the appropriate methyl esters via a general hydrolysis procedure.
  • 1 equivalent of the methyl ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH because of form a sulfonamide sodium salt. Yield (%) mp (oC) TLC (R j , duent)
  • N-Acytripeptide methyl esters were synthesized via HOBt- DCC method in DMF solution [König and Geiger, Chem. Ber. 103, 788 (1970)].
  • N-Acyltripeptide were obtained through hydrolysis of the appropriate methyl esters via general hydrolysis procedure.
  • 1 -.quivalent of methyl ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH to form the sulfonamide sodium salt.
  • MeO-Suc-Ala-DL-Ala-CO 2 Me This compound was prepared from MeO-Suc-Ala-OH in 22% yield by the same procedure as described in Example PKC1, except that sodium methoxide in methanol was used for enol ester hydrolysis, single spot on tlc, R f 2
  • Bz-DL-Ala-COOH The hydrolysis procedure of Tsushima et al. [J. Org. Chem. 49, 1163-1169 (1984)] was used. Bz-DL-Ala-CO 2 Et (540 mg, 2.2 mmol) was added to a solution of 650 mg of sodium bicarbonate in an aqueous 50% 2-propanol solution (7.5 mL of H 2 O and 2-propanol) and stirred at 40 °C under nitrogen. After adding ethyl acetate and a saline solution to the reaction mixture, the aqueous layer was separated and acidified with 2N HCl and extracted with ethyl acetate.
  • Z-Leu-DL-Nva-enol ester the precursor of Z-Leu-DL-Nva-COOEt was synthesized by the same procedure as described in Example FKC1 and purified by column chromatography, oil, one spot on tlc.
  • Z-Leu-DL-Phe-enol ester the precursor of Z-Leu-DL-Phe-COOEt was synthesized by the same procedure as described in Example PKC1 and purified by column chromatography, oil, one spot on tic.
  • the precursor, Bz-DL-Lys(Z)- COOEt was prepared from Bz-Lys(Z)-OH in 100% yield by the procedure described in
  • H-Phe-DL-Lys-COOEt ⁇ 2HCl.
  • the mixture was extracted with ethyl acetate (150 ml) and after separation of the organic layer, the water layer was saturated with solid (NH 4 ) 2 SO 4 and re-extracted 2-times with 25 ml ethyl acetate.
  • the combined organic phases were washed 2-times with 75 ml water, 2-times with 50 ml of satd. NaCl, decolorized with carbon and dried over MgSO 4 .
  • the crude enol ester (8,36 g, 98%) was flash-chromatographed on silica gel and the product was eluted with a AcOEt.
  • Z-Leu-Phe-CO 2 Bzl This compound was prepared from Z-Leu- Phe-OH and benzyl oxalyl chloride in 17% yield by the procedure described in the synthesis of Z-Leu-Phe-CO 2 Et, except that benzyl oxalyl chloride was used in place of ethyl oxalyl chloride and sodium benzyloxide in benzyl alcohol was used for enol ester hydrolysis.
  • the ⁇ -carbonyl group of Z-Leu-Phe-COOEt was proteded by following procedure.
  • a solution of Z-Leu-Phe-COOEt (1 g, 2.13 mmole) in 5 ml of CH 2 Cl 2 was added 1,2-ethanedithiol (0.214 ml, 2.55 mmole), followed by 0.5 ml of boron trifluoride etherate.
  • the solution was stirred overnight at room temperature. Water (20 ml) and ethyl ether (20 ml) were added.
  • Halomethyl ketone peptides are irreversible inhibitors for serine proteases and cysteine proteases. This class of compounds includes peptides having a variety of halomethyl groups at their C-terminus. These halomethyl groups include -CH 2 X, -CHX 2 and CX 3 , where X represents any halogen. A number of analogous compounds have been synthesized, including the amino-halo ketones and the diazo-ketone peptides.
  • haloketones The reactivity of haloketones has generally been found to be in the order I > Br > Cl > F. However, increasing the reactivity of the haloketone in this way can lead to accderation of competing side effects. Thus, it is preferable to increase the reactivity of the halomethyl ketone peptides by altering the peptide structure.
  • Halo-Ketone Peptides are available commercially. For example, Leu-CH 2 Cl, Phe-CH 2 Cl, Z-lys-CH 2 Cl, TosyI-Lys CH 2 Cl (TLCK), Tosyl-PheCH 2 Cl (TPCK), Z-Gly-Leu-Phe-CH 2 Cl, Z-Phe-Ala- CH 2 Cl, z-Phe-Phe-CH 2 Cl, D-Phe-Pro-Arg-CH 2 Cl, MeoSuc-Phe-GIy-Gly-Ala-CH 2 Cl, MeoSuc-A_a-A_a-Pro-Ala-CH 2 Cl, MeoSuc-Ala- Ala-Pro-Val-CH 2 Cl, Ala-Ala-Pro-Val-CH 2 Cl, Ala-Ala-Phe-CH 2 Cl, Suc-Ala-Ala-Pro-Phe-CH 2 Cl and D-Val-Leu-Lys- CH 2 Cl are all available from suppliers such
  • diazomethane with the appropriate add activated by means of dicyclohexylcarbodiimide (DCCI), by the mixed anhydride method.
  • DCCI dicyclohexylcarbodiimide
  • Unblocked amino acid chloromethyl ketones can be prepared by reaction of benzyloxycarbonyl blocked derivatives with HBr or HOAc, trifluoroacetic acid, or by hydrogenation.
  • Synthesis of peptide chloromethyl ketones can be accomplished simply by coupling an appropriate peptide or amino acid with an unblocked amino acid chloromethyl ketone. A few dipeptides can be converted directly to the chloromethyl ketone using the mixed anhydride and CH 2 N 2 followed by HCL
  • Halo-Ketone Peptides A number of examples of the preparation of Halo-Ketone Peptides have been reported in the literature, including a comprehensive review of over 100 amino acid derivatives and approximatley 60 peptide derivatives listed in J.C. Powers, in "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins," Vol. 4, Dekker, New York (1977), the disdosure of which is hereby incorporated by reference. Those of skill in the art will recognize how to locate a multitude of examples of the production of the HaloKetone Peptides. Accordingly, no additional examples are provided herein.
  • Calpain Inhibitor may be used in vitro for a variety of purposes to inhibit unwanted Calpain activity.
  • the Calpain Inhibitors may be used in vitro to prevent proteolysis that occurs in the process of production, isolation, purification, storage or transport of peptides and proteins.
  • Calpain Inhibitors described herein can also be used in vitro to prevent further degradation of tissue samples from c-ccujring after preparation of the samples. This in vitro prevention of degradation can be especially useful in the preparation of assays for neurodegeneration wherein the assay comprises a test for the products of
  • Calpain adivity in the tissues such as assays for breakdown products (BDP's) of cytoskeletal components such as spectrin, MAP2, actin binding protein and tau.
  • BDP's breakdown products
  • cytoskeletal components such as spectrin, MAP2, actin binding protein and tau.
  • the Calpain Inhibitors of this invention are also useful in a variety of other experimental procedures where proteolysis due to Calpains is a significant problem. For example, indusion of the Calpain Inhibitors in radioimmunoassay experiments can result in higher sensitivity. The use of the Calpain Inhibitors in plasma fractionation procedures can result in higher yields of valuable plasma proteins and can make purification of the proteins easier. The Calpain Inhibitors disdosed here can be used in doning experiments utilizing recombinant or transf ected bacterial or eukaryotic cell cultures in order to increase yield of purified recombinant product.
  • the Calpain Inhibitors are dissolved in an organic acid, such as dimethylsulfoxide (DMSO) or ethanol and are added to an aqueous solution containing the protease which is to be inhibited, such that the final concentration of organic solvent is 25% or less.
  • organic acid such as dimethylsulfoxide (DMSO) or ethanol
  • DMSO dimethylsulfoxide
  • the Calpain Inhibitors may also be added as solids or in suspension.
  • Calpain Inhibitors are useful in vivo to treat pathologies in which excess proteolysis by Calpains is involved.
  • pathologies are believed to include neuropathologies such as neurodegeneration resulting from excitotoxicity, HIV-induced neuropathy, ischemia, denervation, injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD), Huntington's Disease, surgery-related brain damage, Parkinson's Disease, and other pathological conditions.
  • Calpain Inhibitors that are useful in the practice of the present invention for treatment or inhibition of neurodegenerative conditions and diseases, it is important to identify those inhibitors posessing significant Calpain inhibitory activity. It is also important to identify those Calpain Inhibitors having a high degree of specificity for inhibition of Calpain, in order to avoid interference with other biological processes when the Calpain Inhibitor is introduced into a mammal requiring treatment for neurodegeneration. Because all thiol proteases are believed to exert their effect through a similar mechanism of action, our primary concern was to identify those Calpain Inhibitors having substantial inhibitory activity against Calpain, but relatively weak or no activity against other thiol proteases.
  • Calpain Inhibitors in order to identify such Calpain Inhibitors, we tested a variety of Calpain Inhibitors for their ability to inhibit calpains I and II, and compared this data with the ability of the same Calpain Inhibitors to inhibit Cathepsin B, another thiol protease. Those Calpain Inhibitors with high in vitro inhibitory activity against Calpain and a rdativdy lower activity against Cathepsin B are believed to be most useful for in vivo therapy. Examples 1A through 1C show the results of these studies for a variety of Calpain Inhibitors.
  • the isocoumarins are irreversible inhibitors of Calpain. We obtained IC 50 values for a variety of these Calpain Inhibitors as a kinetic analysis of these compounds.
  • Purified Calpains can be assayed using the fluorogenic substrate succinyl-leucine ⁇ tyrosine-methylan ⁇ nocoumarin (available commercially) or by measuring the release of acid-soluble peptides from casein because we have found that the isocoumarins inhibit casein proteolysis by Calpain.
  • Calpains I and II were purified by the method of (Yoshimura, et al. 1983).
  • Calpain II may alternatively be purchased from Sigma Chemical Co. as "Calcium Activated Neutral Protease.”
  • purified Calpain was incubated with 14 C-methylated casein in the presence of various Heterocyclic Compounds and the amount of acid-soluble radioactivity released by the action of Calpain was measured.
  • the IC 50 values were determined as the concentration of Heterocyclic Compound compound at which 50% of the Calpain activity was inhibited.
  • Table 1A shows IC 50 values for various Isocoumarin Compounds.
  • Compounds have significant Calpain inhibitory activity at low concentrations.
  • the Peptide Keto-Compounds are reversible inhibitors of Calpains and other thiol proteases.
  • the K- values for the inhibition of calpain I, calpain II and Cathepsin B were determined for several Peptide Keto-Compounds, Inhibition of calpain I from human erythrocytes and calpain II from rabbit musde were assayed using
  • Table 1B(i) shows the results of the studies of Example 1B(i).
  • the Ki value for the inhibition of Calpains and cathepsin B by several Peptide Keto-Compounds are shown in ⁇ M (micromolar).
  • the values for leupeptin, which is commercially available from Calbiochem of La Jolla, California, are shown for comparison.
  • Table 1B(ii) shows the inhibition constants (K I ) for cathepsin B, calpain I, and calpain II with peptide ketoamides.
  • Dipeptide Ketoamides with Abu and Phe in the P 1 site and Leu in the P 2 site are potent inhibitors of calpain I and calpain II.
  • Z-Leu-Abu-CONH-Et is a better inhibitor of calpain I than Z-Leu-Phe-CONH-Et by 14 fold.
  • Replacement of the Z group (PhCH 2 OCO-) by similar groups such as PhCH 2 CH 2 CO-,
  • PhCH 2 CH 2 SO 2 -, PhCH 2 NHCO-, and PhCH 2 NHCS- would also result in good inhibitor structures.
  • the best inhibitor of calpain II is Z-Leu-Abu-CONH-(CH 2 ) 2 -Ph. Changing the R 3 and R 4 groups significantly improves the inhibitory potency toward calpain II.
  • the best Dipeptide Ketoamide inhibitors are those which have long alkyl side chains (e.g. Z-Leu-Abu-CONH-(CH 2 ) 7 CH 3 ), alkyl side chains with phenyl substituted on the alkyl group (e.g.
  • PhCH 2 CH 2 SO 2 -, PhCH 2 NHCO-, and PhCH 2 NHCS- would also result in good inhibitor structures.
  • the Halo-Ketone Peptides like the substituted isocoumarins, are irreversible inhibitors of Calpain.
  • K app /[I] values for various members of this ensemble of compounds against Calpains I and II.
  • these values against the additional thiol proteases Papain and Cathepsin B for at least one Halo-Ketone Peptide.
  • [P ⁇ ] represents the concentration of product formed at a time approaching infinity
  • A is the K app in the presence of substrate (S)
  • K is the Michaelis constant
  • [Y] is the concentration of the inhibitor. Since [S] and [Y] are known and V and K can be determined, K app can be readily determined.
  • the kinetic constants of other irreversible Calpain Inhibitors include the following with K app /[I] in parentheses: E-64 (7500), E64-d (23000) and Z-leu-leu-tyr-CHN 2 (230000).
  • E-64 is commercially available from Sigma Chemical Co., and is shown here to be a poor inhibitor of Calpain.
  • Z-leu-leu-tyr-CHN 2 is a diazomethyl peptide compound, here shown to possess significant Calpain inhibitory activity.
  • Calpain in crude brain extracts was measured by examining the Ca 2+ -stimulated proteolysis of the endogenous substrate spectrin.
  • Various Calpain Inhibitors were added to the supernatant in a DMSO vehicle and a calcium salt (final effective concentration about 1.2mM) added to start the reaction.
  • Proteolysis of spectrin was evaluated by western blot as described by Seubert, et al. (Brain Research,
  • a known quantity of a spectrin-containing sample treated with Calpain is separated by SDS-PAGE and immunoblotted with anti-spectrin antibody.
  • the amount of spectrin immunoreactivity found corresponding to the characteristic BDP's is indicative of the amount of spectrin activity present in the sample.
  • the proteins in the gels are transferred to nitrocellulose and the spectrin and BDP's detected using a rabbit polydonal anti-spectrin antibody and established immunodetection methods.
  • the amount of spectrin and BDP's in each sample can be quantitated by densitrometric scanning of the developed nitrocellulose.
  • Substituted Heterocyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides, in addition to leupeptin and CI1, provide inhibition in brain homogenates.
  • leupeptin is poorly membrane permeant. Therefore, leupeptin is not expected to cross the blood-brain barrier ("BBB") very well. Accordingly, in order to provide the brain with suffident leupeptin to adequately inhibit Calpain activation, we used brain infusion techniques. Through the use of these techniques we were able to subjed brain tissues to intimate contact with leupeptin for sustained periods of time.
  • Example 3A is provided to show the in vivo protection from neurodegeneration found during one such study.
  • a small cannula was implanted in the right lateral ventride of adult gerbils, and secured to the skull with dental cement.
  • An Alzet micro-osmotic pump was attached to the cannula for intracerebroventricular perfusion.
  • the pump was filled with either saline alone (control) or leupeptin (20 mg/ml in saline).
  • transient ischemia was induced by bilaterally damping the carotid arteries for a period of ten minutes. Core temperatures were taken during and following ischemia, with no differences noted between control and leupeptin treated animals.
  • Example 3A cannot be explained by changes in thermoregulation, since core temperatures did not differ between the groups. Accordingly, we believe that the Calpain inhibitory activity of leupeptin is responsible for the observed differences in neuronal cell loss. In order to further quantitate the differences, and verify that leupeptin produced a Calpain inhibitory effect within the observed regions of the brain, we performed a related series of experiments. In this series of experiments, spectrin BDP's were measured in the leupeptin treated and control animals. As discussed above, these BDP's are indicative of the amount of Calpain activity occurring within the tissue. Example 3B is provided to demonstrate the results of these experiments.
  • TPCK The CA1 region of the hippocampus was then dissected.
  • the samples from both control and leupeptin treated animals were then prepared for SDS-PAGE and immunoblotting with labeled anti-spectrin antibody, as described above in connection with in vitro uses of the Calpain Inhibitors.
  • the control animals exhibited a marked increase in the levels of BDP's rdative to the gerbils not subjeded to ischemia. These BDP's co-migrated with BDP's observed after in vitro proteolysis of spectrin with Calpain.
  • the brain tissue from the leupeptin treated gerbils exhibited approximately 25% of the BDP's observed in the control ischemia treated gerbils.
  • results indicate that the observed proteolysis of spectrin was an effect of ischemia, and not secondary to the reoxygenation. Accordingly, the results indicate that inhibition of Calpain activity in vivo produces a neuroprotective effect.
  • leupeptin can inhibit neurodegeneration in vivo
  • leupeptin is not the therapeutic drug of choice because of the need to infuse the drug directly into the brain for an extended period of time to exert its neuroprotective effect. This is due to the rdatively poor ability of this compound to cross the BBB. Accordingly, it is believed that a more therapeutically practical way to inhibit neurodegeneration would be to use more membrane permeant Inhibitor of Calpain.
  • Platelets were isolated by a modification of the method of Ferrell and Martin, 1 Biol. Chem.. 264:20723-20729 (1989), the disdosure of which is hereby incorporated by reference. Blood (15-20 ml) was drawn from male Sprague-Dawley rats into 100mM EDTA-citrate containing 10 units heparin, and centrifuged 30 minutes at 1600 rpm at room temperature.
  • the plasma was resuspended in 15ml buffer 1 (136mM NaCl, 2.7mM KCl, 0.42mM NaH 2 PO 4 , 12mM NaHCO 3 , 2mM MgCl 2 , 2 mg/ml BSA (Sigma), 5.6mM glucose, 22mM Na 3 Citrate pH 65) and platelets were isolated at 2200 rpm at room temperature of 25 minutes. Platelets were resuspended to 10 7 cells/ml in buffer 2 (136mM NaCl, 2.7mM KCl, 0.42 NaH 2 PO 4 , 12mM NaHCO 3 , 2mM MgO, 1 mg/ml
  • Example 2 Among those compounds found to exhibit Calpain inhibitory activity in the homogenate system of Example 2, we found at least three compounds which failed to exhibit Calpain inhibitory activity in the platelet system of Example 4. These compounds are leupeptin, MeO-Suc-Val-Pro-D,L-Phe-COOMe and Bz-D,L-Phe-COOEt. Leupeptin is known to be poorly membrane permeant, thus corifirming that the platelet assay will exclude known poorly membrane permeant compounds.
  • the two Peptide Ketocompounds found not to provide Calpain inhibitory activity within platdets are also believed to be poorly membrane permeant, and would not be expected to cross the BBB.

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  • Cardiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne l'utilisation de composés inhibiteurs de la calpaïne et également des compositions pharmaceutiques contenant lesdits composés inhibiteurs de la calpaïne. On peut utiliser lesdits composés dans le traitement d'une pathologie neurodégénérative chez l'homme. L'invention concerne également d'autres utilisations et des compositions pharmaceutiques contenant les composés inhibiteurs de la calpaïne, tels que des céto-amides peptidiques, des céto-acides peptidiques, et des céto-esters peptidiques.
EP92902904A 1990-12-28 1991-12-27 Utilisation d'inhibiteurs de la calpaine dans l'inhibition et le traitement de la neurodegenerescence Withdrawn EP0564552A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US635952 1984-07-30
US63595290A 1990-12-28 1990-12-28

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EP0564552A1 true EP0564552A1 (fr) 1993-10-13

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EP92902904A Withdrawn EP0564552A1 (fr) 1990-12-28 1991-12-27 Utilisation d'inhibiteurs de la calpaine dans l'inhibition et le traitement de la neurodegenerescence

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Country Link
EP (1) EP0564552A1 (fr)
JP (1) JPH06504061A (fr)
AU (2) AU667463B2 (fr)
CA (1) CA2098609A1 (fr)
WO (1) WO1992011850A2 (fr)

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Also Published As

Publication number Publication date
WO1992011850A2 (fr) 1992-07-23
AU5590596A (en) 1996-08-22
AU9152791A (en) 1992-08-17
WO1992011850A3 (fr) 1992-09-03
JPH06504061A (ja) 1994-05-12
AU667463B2 (en) 1996-03-28
CA2098609A1 (fr) 1992-06-29

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