EP0650368A1 - Verwendung von calpain inhibitoren in der inhibition und behandlung von medizinischen zuständen die mit einer erhöhten calpainaktivität assoziiert sind - Google Patents

Verwendung von calpain inhibitoren in der inhibition und behandlung von medizinischen zuständen die mit einer erhöhten calpainaktivität assoziiert sind

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Publication number
EP0650368A1
EP0650368A1 EP93915482A EP93915482A EP0650368A1 EP 0650368 A1 EP0650368 A1 EP 0650368A1 EP 93915482 A EP93915482 A EP 93915482A EP 93915482 A EP93915482 A EP 93915482A EP 0650368 A1 EP0650368 A1 EP 0650368A1
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EP
European Patent Office
Prior art keywords
leu
mammal
group
alkyl
administering
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EP93915482A
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English (en)
French (fr)
Inventor
David D. Eveleth, Jr.
Gary Lynch
James C. Powers
Raymond T. Bartus
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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 EP0650368A1 publication Critical patent/EP0650368A1/de
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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
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates generally to medical treatments involving the inhibition of calcium-activated proteases, such as Calpain. More specifically, the present invention relates to the treatment of neurodegenerative conditions, coronary disease, circulatory pathology, cataract formation, and other medical conditions associated with calcium-activated protease activity using inhibitors of these proteases.
  • 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 and use a cysteine residue in their catalytic mechanism.
  • 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, synaptic plasticity and cell death.
  • 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 /. 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 Although 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.
  • calpain activation can be measured indirectly by assaying the proteolysis of the cytoskeletal protein spectrin, which produces a large, distinctive and biologically persistent breakdown product when attacked by calpain (Siman, Baudry, and Lynch, Proc. Natl.
  • Calpains In neural tissues, activation of Calpains, as evidenced by accumulation of these BDP's, has been observed in many neurodegenerative conditions. For example, these phenomena have been observed after denervation resulting from focal electrolytic lesions, in genetic abnormalities, after excitotoxicity, following ischemia in gerbils and rats, following administration of the toxins kainate and colchicine in rats, an in human Alzheimer's disease. Calpains have also been shown to degrade the lens proteins alpha-crystallin, vimentin, and actin in vitro, and have been implicated in the degradation of cardiac muscle proteins and other tissues.
  • Calpain Commercially available in vitro inhibitors of Calpain include peptide aldehydes such as leupeptin (Ac-Leu-Leu-Arg-H) and Ac-Leu-Leu-Nle-H, as well as epoxysuccinates such as E-64. These compounds are not useful in inhibiting Calpain in
  • CNS Central Nervous System
  • Cathepsin B is involved in muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption.
  • a number of viral processing enzymes, which are essential for viral infection* are cysteine proteases.
  • Inhibitors of cysteine proteases would thus have multiple therapeutic uses. 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.
  • 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, upases 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.
  • Calpain action results in the irreversible cleavage of cellular proteins and alterations in their function, and this degradative function fits in well with a possible role in cell death. Further, leupeptin, a calpain inhibitor, has been shown to reduce ischemic damage in gerbils and to reduce hypoxic damage in rat hippocampal slices.
  • calpains are ubiquitously distributed in mammalian cells but apparently do not contribute to normal protein catabolism or general protein turnover, they appear to serve a regulatory role in such cells. However, the mechanisms of such regulation have not been well studied. While some calpain inhibitors have been shown to inhibit cellular proliferation and thus cell cycling, the specific point in the reproductive cycle at which such inhibition occurs is not yet known. An understanding of the regulation of cell cycling is relevant to the development of treatments for cancer, because cancer cells grow without regulation of such cell cycling. Chemotherapy treatments for cancer sometime take the form of administering chemicals which will kill cells that are passing through the cell cycle and actively dividing while sparing those cells which are not dividing.
  • drugs which interfere with the replication of the DNA of cells during the "S" (synthesis) phase of the cell cycle are administered to a patient.
  • This treatment will only be effective in killing cells in the S phase.
  • a drug must be present in a patient's body for long enough so that all of the cancer cells in the patient progress through the S phase. Since chemotherapeutic agents kill non-cancerous cells which are dividing as well as cancerous cells, the timing and duration of chemotherapeutic drug administration is critical to successful therapy.
  • proteases such as calpain have also been linked to the regulation of smooth muscle contraction.
  • the mechanism by which contractility and the maintenance of the tonically contracted state is regulated in smooth muscle is not well understood.
  • Many agents which act to decrease contractility of smooth muscle have little or no efficacy at inhibiting the establishment of the tonic state or reversing the tonic contractile state once established.
  • the tonic contraction of smooth muscle is a normal process. In some cases, however, such tonic contraction can lead to serious pathological conditions. For example, contraction of the bronchial smooth muscle leads to shortness of breath and other symptoms of asthma. Contraction of the coronary arteries can lead to angina, partial coronary hypoxia and subsequent loss of coronary function. Contraction of the smooth muscle in cerebral arteries can lead to cerebral vasospasm and hypoxia of the brain tissue, a serious condition that can leave patients mentally disabled and permanently brain damaged.
  • One aspect of the present invention is a method of synchronizing the reproductive cycle of actively dividing cells.
  • the Calpain Inhibitor which is pharmacologically effective to block the progression of cells from G 1 phase into S phase is administered to the cells.
  • the Calpain Inhibitor can be one of the Peptide Keto-Compounds, the Halo-Ketone Peptides, or the Substituted Heterocyclic Compounds.
  • the cells to be treated in this method are located in vivo in a mammal, so that the administering step of the method comprises administering a Calpain Inhibitor to cells in a mammal.
  • the administering step can comprise administering a Calpain Inhibitor to cells in vitro.
  • the administering step of this method comprises administering a Peptide Keto-Compound.
  • Calpain Inhibitors can be administered in this method either intravenously, intramuscularly, intraperitoneally, topically, orally, or by direct application to cells.
  • the present invention comprises a method of blocking the progression of the cell cycle from G j phase to S phase in actively dividing cells in a mammal.
  • a mammal is administered an amount of a Calpain Inhibitor which is pharmacologically effective to block the progression of the cell cycles of actively dividing cells in the mammal from G j phase into S phase.
  • the Calpain Inhibitor can be one of the Peptide Keto-Compounds, the Halo-Ketone Peptides, or the Substituted Heterocyclic Compounds.
  • the Calpain Inhibitor is a Peptide Keto-Compound.
  • Calpain Inhibitors can be administered according to this method either intravenously, intramuscularly, intraperitoneally, topically, orally, or by direct application to living cells.
  • the Calpai Inhibitor is administered by direct application, where such direct application can comprise either applying a gel to an area of living cells, driving microspheres loaded with the Calpain Inhibitor into tissue comprising the living cells, or injecting a solution containing the Calpain Inhibitor directly into tissue comprising such living cells.
  • the present invention comprises a method of enhancing the efficacy of chemotherapy in the treatment of cancer in a human patient.
  • This method comprises administering to the cancerous cells of the patient an amount of a Calpain Inhibitor which is pharmacologically effective to block the progression of the cell cycles of such cancerous cells from G j phase to S phase, and thereafter administering to the cells a chemotherapeutic agent.
  • the Calpain Inhibitor in this method is selected from the group consisting of Peptide Keto-Compounds, Halo-
  • the Calpain Inhibitor is a Peptide Keto-Compound.
  • the Calpain Inhibitor in this method can be administered intravenously, intramuscularly, intraperitoneally, topically, orally, or by direct application to the cancerous cells.
  • the chemotherapeutic agent can be administered beginning 24-48 hours after the administration of the Calpain Inhibitor, at which time the cell cycles of the patient's cancerous cells which were treatable with the Calpain Inhibitor will be synchronized.
  • a further aspect of the present invention includes a method of determining the effectiveness of a chemotherapeutic agent, comprising growing cancerous cells in vitro, administering to such cancerous cells an amount of a Calpain Inhibitor which is effective to block the progression of the cells from G 1 phase into S phase, administering to the cells the chemotherapeutic agent in an amount sufficient to kill the cells, and thereafter determining the amount of cell death that occurs.
  • the amount of cell death that occurs in this method is indicative of the effectiveness the chemotherapeutic agent tested.
  • Another aspect of the present invention is a method of increasing the efficiency of cell transformation and thus increasing the efficiency of integration of foreign DNA into living cells.
  • This method comprises administering to a population of cells comprising actively dividing cells an amount of a Calpain Inhibitor which is pharmacologically effective to block the progression of the cell cycles of the cells from G j phase into S phase, discontinuing the administration of the Calpain Inhibitor, and thereafter introducing foreign DNA into the population of cells.
  • the Calpain Inhibitor in this method is selected from the group consisting of Peptide Keto-Compounds, Halo- Ketone Peptides, and Substituted Heterocyclic Compounds.
  • the Calpain Inhibitor is a Peptide Keto-Compound.
  • the administration of the Calpain Inhibitor in this method can continue for the length of one cell cycle in the population of living cells.
  • the target of the Calpain Inhibitor can be a population of cells located in a mammal, which can be administered a Calpain Inhibitor intravenously, intramuscularly, intraperitoneally, topically, orally, or by direct application to the population of cells in the mammal.
  • the Calpain Inhibitor is administered instead to a population of cells in vitro.
  • the present invention provides methods of treating a variety of medical conditions associated with calcium-activated protease activity in a mammal by administering the Calpain inhibitors of the present invention to that mammal.
  • Calpain inhibitors are Peptide Keto-Compounds, Halo-Ketone Peptides, and Substituted Heterocyclic Compounds.
  • Particularly preferred compounds for this use include the Peptide Ketoamides, such as Z-Leu-Abu-CONH-Et, Z-Leu-Phe-CONH-Et and Z-Leu-Phe-CONH(CH 2 ) 2 C 6 H 5 .
  • Administration of the inhibitors can be through any of a variety of routes.
  • These routes include all of the following types of administration: intravenous, intraperitoneal, intramuscular, oral, topical treatment such as through ointments (including ophthalmic ointments), eye drops, contact lenses, catheter, directly onto tissues such as blood vessels or cardiac tissue during surgery, or injection into the pericardial space.
  • Specific medical conditions which can be treated with these Calpain Inhibitors include cardiac muscle tissue damage. After a mammal with cardiac muscle tissue damage has been identified, that mammal can be treated with a Calpain Inhibitor. Mammals at risk for developing cardiac muscle tissue damage can also be treated with the present Calpain Inhibitors. Administering these Inhibitors to such mammals protects them from the cardiac tissue damage experienced by mammals which are not so protected.
  • cataracts are treated by the administration of a Calpain Inhibitor. If a mammal has already developed cataracts, the development of the cataracts can be slowed or arrested through the administration of a Calpain Inhibitor. On the other hand, if a mammal has been identified as being a risk for developing cataracts in the future, the development of cataracts in such a mammal can be prevented or slowed through the administration of a Calpain Inhibitor.
  • a variety of other tissues and conditions can also be treated with the novel Calpain Inhibitors of the present invention.
  • Skeletal and smooth muscle damage for example, can be treated by identifying a mammal with such tissue damage and administering a Calpain Inhibitor to that mammal.
  • Vasospasm a condition of a particular kind of smooth muscle, the vascular tissue, can also be reversed in a mamma identified as having this condition by the administration of Calpain Inhibitors.
  • Erythrocytes damaged by the proteolytic activity of Calpain in hypertensive mammals can also be treated with the Calpain Inhibitors of the present invention.
  • the present invention provides methods of halting or inhibiting the proliferation of smooth muscle cells both in vivo and in vitro by administering a Calpain Inhibitor.
  • Calpain Inhibitors are Peptide Keto-Compounds, Halo- Ketone Peptides, and Substituted Heterocyclic Compounds.
  • Particularly preferred compounds for this use include the Peptide Ketoamides, such as Z-Leu-Phe-CONH-Et
  • Preferred Peptide Keto- Compounds useful in the present invention include (Ph) 2 CHCO-Leu-Phe-CONH-CH 2 - 2-Py; Z-Leu-Nva-CONH-CH 2 -2-Py; Z-Leu-Phe-CONH-CH 2 CH(OH)Ph; (Ph) 2 CHCO- Leu-Abu-CONH-CH 2 CH(OH)Ph; Z-Leu-Phe-CONH 2 ; Z-Leu-Abu-CONH- CH 2 CH(OH)Ph; and Z-Leu-Phe-CONHEt.
  • Direct application of the Calpain Inhibitors can be through various means. Such means include using a gel or ointment containing the inhibitor to coat the surface of the balloon of a balloon catheter or onto another surgical instrument that is inserted into the blood vessel during angioplasty. Alternatively, the gel may be applied directly to an area of vascular tissue which has been treated by angioplasty during the surgical procedure.
  • Another route of administration comprises driving microspheres which have been loaded with a Calpain Inhibitor directly into the mammal's blood vessel. This can be accomplished by applying the microspheres to the surface of the balloon or other surgical instrument used during the angioplasty procedure. The microspheres are driven into the arterial wall, where they lodge and release the Calpain Inhibitor over time.
  • Calpain Inhibitors include the treatment of a mammal to prevent restenosis of a blood vessel following angioplasty. After a mammal which has undergone angioplasty has been identified, that mammal can be treated with a Calpain Inhibitor. Mammals at risk for developing restenosis can also be treated with the present Calpain Inhibitors. Administering these Inhibitors to such mammals protects them from the smooth muscle cell proliferation experienced by mammals which are not so protected.
  • the present invention provides a method of inhibiting tonic smooth muscle contraction in a mammal susceptible to inappropriate contraction in a smooth muscle thereof.
  • the method includes administering to the smooth muscle an amount of a Calpain Inhibitor that is pharmacologically effective to suppress the contraction thereof.
  • the Calpain Inhibitor is one of the Peptide Keto-Compounds, Halo-Ketone Peptides or Substituted Heterocylic Compounds.
  • the inhibitor is administered intravenously, intramuscularly, intraperitoneally, topically, orally, by injection into cerebrospinal fluid, by inhalation, or by direct application to the smooth muscle, such as by applying directly to an area of smooth muscle. Direct application can also be by driving microspheres loaded with the Calpain Inhibitor into the smooth muscle. Relaxation of the smooth muscle is preferably induced.
  • the present invention provides a method of treating coronary vasospasm in a mammal.
  • the method includes administering t the mammal an amount of a Calpain Inhibitor which is pharmacologically effective to stop vasospasm of coronary tissue in the mammal.
  • the Calpain Inhibitor is one of the Peptide Keto-Compounds, Halo-Ketone Peptides or Substituted Heterocylic Compounds.
  • the coronary tissue is surgically exposed and a solution of Calpain Inhibitor is applied directly to the tissue.
  • the coronar tissue comprises a coronary artery.
  • the mammal is suffering from angina and the method comprises a treatment for the angin
  • a method of treating bronchial vasospasm in a mammal includes administering to the mamm an amount of a Calpain Inhibitor which is pharmacologically effective to stop vasospasm of bronchial tissue in the mammal.
  • the Calpain Inhibitor is one of the
  • the bronchial tissue can be surgically exposed and a solution of Calpain Inhibitor applied directly to the tissue.
  • the mammal is suffering from asthma and the method comprises a treatment for the asthma.
  • Yet another aspect of the invention relates to a method of treating cerebral vasospasm in a mammal.
  • This method includes administering to the mammal an amount of a Calpain Inhibitor which is pharmacologically effective to stop vasospasm cerebral tissue in the mammal.
  • the Calpain Inhibitor is one of the Peptide Keto- Compounds, Halo-Ketone Peptides and Substituted Heterocylic Compounds.
  • the cerebral tissue can be surgically exposed and a solution of Calpain Inhibitor applied directly to the tissue.
  • the Calpain Inhibitor can be injected into the mammal's cerebrospinal fluid.
  • One aspect of the present invention provides a method of medical treatment f a medical condition in a mammal.
  • a pharmaceutical composition containing a morpholine Peptide Keto-Compound is administered to the mammal.
  • Th composition is administered in an amount that is pharmacologically effective to treat the condition.
  • the condition is one which is associated with increased proteolytic activity of Calpain.
  • the morpholine Peptide Keto-Compound can be either a C-terminal or N-terminal morpholine Peptide Keto-Compound, such as cardiac muscle tissue damage, cataracts, skeletal muscle damage, vasospasm or restenosis following cardiac angioplasty.
  • Another aspect of the present invention also provides a method of medical treatment for a medical condition in a mammal.
  • a pharmaceutical composition containing a Peptide Ketoamide, Subclass C is administered to the mammal.
  • This composition is administered in an amount that is pharmacologically effective to treat the condition.
  • the condition that can be treated with this method is also one associated with increased proteolytic activity of Calpain, such as cardiac muscle tissue damage, cataracts, skeletal muscle damage, vasospasm or restenosis following cardiac angioplasty.
  • the present Calpain Inhibitors can be used to counteract the harmful effects associated with calpain activity which arise in a number of medical conditions and diseases. Therefore, the treatment of such conditions with the present Calpain Inhibitors is within the scope of the present invention.
  • 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.
  • FIG. 2 shows that Calpain inhibitor reduces cell death following glutamate exposure.
  • PC12 cells were exposed to 7.5mM glutamate with the indicated concentration of inhibitor, as described in the text, for 24 hours. Cell viability was assayed using the Mi 1 assay. Values are expressed as % of naive control ⁇ sem.
  • Figure 3 shows the dependence of the ability of Calpain inhibitors to reduce cel death on glutamate concentration. PC 12 cells were incubated with the indicated concentration of glutamate and no inhibitor (circles), 20uM Z-Leu-Nva-CONH(CH 2 ) 3 morpholine (triangles), or 30uM Z-Leu-Phe-CONHCH 2 CH (squares) for 24 hours an cell viability was assayed by MTT. Values expressed as % of naive control ⁇ sem.
  • FIG. 4 Delayed addition of calpain inhibitor.
  • Glutamate (7.5mM) was adde at 0 time and Z-Leu-Phe-CONHCH 2 CH 3 (squares) or Z-Leu-Nva-CONH(CH 2 ) 3 morpholine (triangles) added at the indicated times to final concentrations of lOOuM each.
  • Cell viability was measured 24 hours after the addition of glutamate by the MT assay. Values expressed as % of naive control ⁇ sem.
  • Figure 5 graphically depicts the effects of Z-Leu-Phe-CONH-Et and Z-Leu- Abu-CONH-Et on the size of infarction produced upon MCA occlusion in male rats.
  • Figure 6 shows the effects of Z-Leu-Abu-C0 2 Et, a Peptide Keto-Compound, and CIl (Ac-Leu-Leu-Nle-H) relative to control slices on survival of hippocampal slice 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 7 shows the evoked potential amplitude for control, CIl treated and Z-
  • Figure 8 shows the percent recovery of EPSP from severe hypoxia over the course of one hour incubation for Z-Leu-Phe-CONH-Et and Z-Leu-Phe-C0 2 Et.
  • Figure 9 shows a comparison of the effect of the presence of CIl or Z-Leu-Ph
  • Figure 10 shows the effects of CIl compared with control on the behavioral an convulsive effects of kainic acid.
  • Figure 11 shows the amount of spectrin BDP's in rat brains exposed to kainate for control and CIl treated rats.
  • Figure 12 graphically depicts the effect of several different Calpain Inhibitors contraction of isolated arteries induced by endothelin (ET-1).
  • Drug A is Z-Leu-Abu- CONHEt
  • Drug B is Z-Leu-Phe-CONHEt
  • Drug C is 1,10-Phenanthroline
  • Drug is TLCK (Tosyl-Lysine-chloromethylketone).
  • Figure 13 graphically depicts the effect of several other Calpain Inhibitors on contraction of isolated arteries induced by endothelin (ET-1).
  • Drug E is Z-Leu-Phe
  • Drug F is Z-Leu-Phe-CONHEt (the same as drug B)
  • Drug G is Z-Leu-Phe- CONH(CH2) 2 Ph
  • Drug H is Ac-Leu-Leu-Nle-H (Calpain Inhibitor I)
  • Drug I is Gly- Gly-Gly
  • Drug J is (Ph) 2 CHCO-Leu-Abu-CONH-CH 2 CH(OH)Ph.
  • Figure 14 shows the effect of Calpain Inhibitors on contraction of isolated arteries induced by phorbol dibutyrate (PDB).
  • Drugs E through J are as in Figure 15.
  • FIG 15 graphically depicts the effect of Calpain Inhibitors on smooth muscle resting tension. Drugs E through J are as in Figure 13.
  • Figure 16 shows the dose-dependent inhibition of oxyhemoglobin-induced constriction by a Calpain Inhibitor, Z-Leu-Phe-CONH(CH 2 ) 3 , of the present invention.
  • Figure 17 shows an example of the time course of artery constriction in an artery constricted by subarachnoid hemorrhage (SAH) and treated with a Calpain Inhibitor, Z-Leu-Phe-CONH(CH 2 ) 3 , of the present invention.
  • SAH subarachnoid hemorrhage
  • Figure 18 shows the summary of data from three animals in which a Calpain Inhibitor, Z-Leu-Phe-CONH(CH 2 ) 3 , of the present invention reversed constrictions caused by SAH.
  • Figure 19 graphically depicts the effects of Z-Leu-Phe-CONHEt and Ph 2 CHCO-Leu-Abu-CONH-CH 2 CH(OH)Ph on the proliferation of cultured bovine smooth muscle cells.
  • Figure 20 shows the continued viability of smooth muscle cells after treatment with a Calpain Inhibitor, despite a complete inhibition of cell proliferation.
  • Figure 21 graphically depicts the blocking of the progression into S phase of bovine aortic smooth muscle cells (BASMC) after treatment with the Calpain Inhibitor Ph 2 -CHCO-Leu-Abu-CONH-CH 2 CH(OH)Ph.
  • “Drug C” is Ph 2 -CHCO- Leu-Abu-CONH-CH 2 CH(OH)Ph ("Drug C" elsewhere may be a different compound).
  • Figure 22 graphically depicts the synchronous progression into S phase of HeLa and AT-2 cells after the Calpain Inhibitor Ph 2 -CHCO-Leu-Abu-CONH-CH 2 CH(OH)Ph was washed out of the medium in which such cells were maintained.
  • Calpain activation is an event central to many cases o 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, HI induced neuropathy, ischemia, denervation following ischemia or injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD),
  • AD Alzheimer's Disease
  • Parkinson's Disease Huntington's Disease, surgery-related brain damage and other neuropathological conditions.
  • 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.
  • Calpain activation is an important mechanism of cell damage and death in many pathological conditions, including neuropathological conditions.
  • the activation of Calpains is an early event in the death of cells includin neural cells.
  • 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.
  • Calpain has also been implicated in the pathogenesis of a number of other medical conditions.
  • the inhibition of Calpain is capable of slowing the progress of these diseases and of preventing certain conditions altogether.
  • the formation of cataracts, for exarasle has been linked to Calpain activity in mammalian lenses.
  • increased Calpain activity has been documented just before the onset of detectable cataract formation.
  • Calpain activity has also been observed to decrease after a cataract has formed in a lens, leading to the inference that calpain activity is involved in the formation of cataracts.
  • spectrin breakdown products there are increased levels of spectrin breakdown products found in in vitro models of cataract formation. The presence of such spectrin breakdown products is known to be reflective of increased Calpain activity.
  • Calpain activity has also been implicated in producing myocardial infarctions. Calpain activity is regulated by intracellular calcium concentrations, and increased intracellular calcium in myocardial tissues has been observed when the myocardium is cut off from its supply of oxygen due to ischemia. Cell damage and ultimately cell death results from such ischemia. The increased proteolytic activity of Calpain due to increased levels of intracellular calcium during ischemia is therefore a contributor to o direct cause of cell death during cardiac ischemia. Cardiac tissue damage can thus be prevented or minimized with the present Calpain Inhibitors.
  • Calpain is also believed to be an important regulator of cell growth.
  • Calpain Inhibitors have been found, for example, to inhibit smooth muscle cell proliferation. Such proliferation is in fact necessary to repair injured smooth muscle tissue. Following therapeutic angioplasty, however, smooth muscle cell proliferation may result in restenosis of the opened blood vessel. Calpain Inhibitors may thus be used to prevent the smooth muscle cell proliferation which results in the restenosis of blood vessels.
  • Calpain Inhibitors can be treated with Calpain Inhibitors as well.
  • Calpa has been shown to degrade the constituents of skeletal and smooth muscle cells, and has been implicated in causing vasospasm.
  • Increased Calpain activity has also been shown in the blood cells of hypertensive patients, and has been shown to be five times as active in degrading proteins in such cells as in the cells of non-hypertensive patients Calpain Inhibitors therefore can reduce or eliminate the harmful effects of Calpain activity in these tissues.
  • Calpain Inhibitors inhibit tonic smooth muscle contraction. These compounds are useful in the treatment of animals or humans for the purpose of preventing or reducing the smooth muscle contraction associated with vasospasm and bronchospasm.
  • the present invention includes the use of a variety of Calpain Inhibitors and methods for using these inhibitors to treat disease conditions. Specifically, Substituted
  • Heterocyclic Compounds, Peptide Keto-Compounds, and Halo-Ketone Peptides have been found to be effective in treating the foregoing conditions as well as other disease Unless otherwise stated, the Calpain Inhibitors of the present invention refers to the novel Substituted Heterocyclic Compounds, Peptide Keto-Compounds, and Halo- Ketone Peptides described herein.
  • Calpain Inhibitors have also been found to play a role in the regulation of the reproductive cycle of the cell. These compounds can be used in the treatment of cancer in animals or humans along with other chemotherapeutic agents in order to enhance the effectiveness of such agents. By synchronizing the growth of rapidly dividing cells, these compounds can increase the effectiveness of chemotherapeutics that act at a specific stage in the cell cycle, such as at DNA replication. By synchronizing the cell cycles of cells, Calpain Inhibitors are also useful in increasing the efficiency of cell transformation. Transformation results from the incorporation of foreign DNA into a cell. Such incorporation is increased when cells are synthesizing DNA. Thus, by synchronizing cells to the DNA synthetic portion of the cell cycle, the cells will be more efficiently transformed by foreign DNA introduced into the cells.
  • 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 hereinbelow, 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 Xlla, bovine trypsin, human plasm plasmin, human tissue plasminogen activator, human lung tryptase, rat skin tryptase, human leukocyte elastase, porcine pancreatic elastase, bovine chymotrypsin and huma leukocyte cathepsin G.
  • bovine thrombin human thrombin
  • human factor Xa human factor XIa
  • human factor Xlla bovine trypsin
  • human plasm plasmin human tissue plasminogen activator
  • human lung tryptase rat skin tryptase
  • human leukocyte elastase human leukocyte elastase
  • the Class I Substituted Isocoumarins inhibit the serine proteases by reaction with the active site serine to form an acyl enzyme, which in som cases may further react with another active site nucleophile 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 th following structural formula:
  • M-AA-AA-O- wherein 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, wherein M represents NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X-NH-CS,
  • Y is selected from the group consisting of H, halogen, trifluoromethyl, 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 N0 2 , CN, Cl, COOR, and COOH will increase the reactivity of the isocoumarin
  • electropositive substituents such as NH 2 , 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 "Class II 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 _ 6 alkyl, C- ⁇ g alkyl wit an attached phenyl, C ⁇ fluorinated alkyl, C j _ 6 alkyl with an attached hydroxyl, C j .
  • R' is selected from the group consisting of H, halogen, trifluoromethyl, N0 2 , cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino.
  • 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 ⁇ g alkoxy with an attached isothiureido, C w alkoxy with an attached guanidino, C ⁇ alkoxy with an attached amidino, C ⁇ alkyl with an attached amino, C _ 6 alkyl with an attached isothiureido,
  • C j . alkyl with an attached guanidino C ⁇ g alkyl with an attached amidino
  • R is selected from the group consisting of H, OH, NH 2 , N0 2 halogen, C j .g alkoxy, C l 6 fluorinated alkoxy, C ⁇ g alkyl, C-_ 6 alkyl with an attached amino, M-AA- NH-, M-AA-O-, wherein AA represents alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine-- ⁇ ferine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, beta-alanine, norleucine, norvaline, alpha-aminobutyric and epsilon-aminocaponic acid, cit
  • 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 ⁇ 6 alkoxy with an attached amino, C j _ alkoxy with an attached isothiureido, C- ⁇ g alkoxy with an attached guanidino, C ⁇ g alkoxy with an attached amidino, C j _ 6 alkyl with an attached amino, C j _ 6 alkyl with an attached guanidino, C j .g 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, S0 2 , CC1 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
  • R is selected from the group consisting of C ⁇ g alkyl (such as methyl, ethyl and propyl), C ⁇ alkyl containing a phenyl (such as benzyl), and C ⁇ 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.
  • 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-amino-4-chloro-3(2-bromoethoxy) isocoumarin (0.2g, 0.6 mmole) was added.
  • 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.
  • 7-Ethoxycarbonylamino-4-chloro-3-(2-bromoethoxy) isocoumarin, 7- benzyloxycarbonylamino-4-chloro-3-(2-bromoethoxy) isocoumarin and 7- phenoxycarbonylamino-4-chloro-3-(2-bromoethoxy) isocoumarin can be synthesized by reacting 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin with the corresponding chloroformate.
  • Peptide ⁇ -ketoesters, peptide ⁇ -ketoacids, and peptide ⁇ -ketoamides are transition state analog inhibitors for serine proteases and cysteine proteases. While these subclasses 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 nomenclature of Schechter and Berger, Biochem. Biophys. Res.
  • the individual amino acid residues of a substrate or inhibitor are designated PI, P2, etc. and the corresponding subsites of the enzyme are designated SI, S2, etc.
  • the scissile bond of the substrate is Pl-Pl'.
  • the primary recognition site of serine proteases is SI.
  • the most important recognition subsites of cysteine proteases are SI and S2.
  • There are additional recognition sites at the prime subsites such as SI' and S2'.
  • Amino acid residues and blocking groups are designated using standard abbreviations using nomenclature rules presented in /. Biol. Chem., 260:14-42 (1985), inco ⁇ orated herein by reference.
  • amino acid residue (AA) in a peptide or inhibitor structure refers to the part structure -NH-CHR j -CO-, where R j 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-CHR r CO-CO-OR.
  • the ethyl ketoester derived from benzoyl alanine would be designated Bz-Ala-CO-OEt which represents C 6 H 5 CO-NH-CHMe-CO-CO-OEt.
  • peptide ketoacid residues would be designated -AA-CO-OH.
  • peptide ketoamide residues are designated -AA-CO-NH-R.
  • 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 PI 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 PI position, but are generally more effectively inhibited by inhibitors having amino acids with nonpolar or hydrophobic side chains in the PI position. Thus, we have discovered that 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 NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X-NH-CS-, X-NH-SO r , X-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X-O-CS-, H, acetyl, carbobenzoxy, succinyl, methyloxysuccinyl, butyloxycarbonyl;
  • X is selected from the group consisting of C w alkyl, C ⁇ fluoroalkyl, C w alkyl substituted with J, C ⁇ 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, naph
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C ⁇ alkoxy, C j .g alkylamine, C ⁇ _ 6 dialkylamine, C j _g alkyl-O-CO-,
  • K is selected from the group consisting of halogen, C j . 6 alkyl, Cl-6 perfluoroalkyl, C ⁇ g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C j . alkylamino, C 2 .
  • 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), epsilon-aminocaproic acid, citrulline, hydroxyproline, homoarginine, ornithine o sarcosine; n
  • the Peptide Keto-Compounds can be divided into the Peptide Ketoesters Peptide Ketoacids and Peptide Ketoamides.
  • Each of the compounds can also be classified based on the number of amino acids contained within the compound, such a an amino acid peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide and so on.
  • Dipeptide ⁇ -Ketoesters are compounds of the formula: M 1 -AA 2 -AA 1 -CO-0-R 1 or a pharmaceutically acceptable salt, wherein
  • M- represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO , X 2 N-S0 2 -, X-CO-, X-CS-, X-SO r , X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C ⁇ j g alkyl, 1 0 fluoroalkyl, C ⁇ . alkyl substituted with J, C j .-g 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, 1 0 alkyl with an attached phenyl group, C ⁇ alkyl with two attached phenyl groups, C ⁇ g alkyl with an attached phenyl group substituted with K, and C 1 0 alkyl with two attached phenyl groups substituted with K, ⁇ g alkyl with an attached phenyl groups substituted with K, ⁇ g alkyl
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C j ⁇ Q alkoxy, C ⁇ alkylamine, C 2 . 12 dialkylamine, C ⁇ g alkyl-O-CO-, C ⁇ g alkyl-O-CO- NH-, and C w0 alkyl-S-;
  • K is selected from the group consisting of halogen, C ⁇ g alkyl, C ⁇ perfluoroalkyl, C- ⁇ g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 _ 12 dialkylamino, C 1 -C 10 acyl, and C ⁇ g alkoxy-CO-, and C ⁇ alkyl-S-;
  • AA j 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-pi ⁇ eridine carboxylic acid
  • CH(CH 2 CHEt 2 )-COOH alpha-aminoheptanoic acid, NH 2 -CH(CH 2 -l-napthyl)-COO NH 2 -CH(CH 2 -2-napthyl)-COOH, NH 2 -CH(CH 2 -cyclohexyl)-COOH, NH 2 -CH(CH 2 - cyclopentyl)-COOH, NH 2 -CH(CH 2 -cyclobutyl)-COOH, NH 2 -CH(CH 2 -cyclopropyl)- COOH, trifluoroleucine, and hexafluoroleucine; 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 leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspart 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-methylserin O-ethylserine, S-methylcysteine, S-e
  • R x is selected from the group consisting of H, C ⁇ alkyl, C ⁇ g alkyl with a phenyl group attached to the C ⁇ g alkyl, and C- ⁇ g alkyl with an attached phenyl grou substituted with K.
  • Dipeptide ⁇ -Ketoesters are compounds of the structure: M r AA-NH-CHR 2 -CO-CO-0-R or a pharmaceutically acceptable salt, wherein
  • M ⁇ represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SQ 2 -, X 2 N-SO r , X-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X
  • X is selected from the group consisting of C- ⁇ g alkyl, C ⁇ fluoroalkyl, C j _ 10 alkyl substituted with J, l l0 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- ⁇ g alkyl with an attached phenyl group, C ⁇ g alkyl with two attached phenyl groups, C j .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 ,
  • K is selected from the group consisting of halogen, C ⁇ alkyl, C ⁇ perfluoroalkyl, C 1 0 alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 . 12 dialkylamino, C j -C j acyl, and C ⁇ alkoxy-CO-, and C j . 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
  • R 2 represents C ⁇ g branched and unbranched alkyl, C 8 branched and unbranched cyclized alkyl, or C ⁇ g branched and unbranched fluoroalkyl; R is selected from the group consisting of H, C ⁇ g alkyl, C ⁇ alkyl with a phenyl group attached to the C j _ 20 alkyl, and C ⁇ g alkyl with an attached phenyl grou substituted with K.
  • Tripeptide ⁇ -Ketoesters are compounds of the structure: M 3 -AA-AA-AA-CO-0-R or a pharmaceutically acceptable salt, wherein
  • M 3 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO r , X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO r , X 2 N-S0 2 -, X-CO-, X-CS-, X-SO , T-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C j _ 10 alkyl, C ⁇ g fluoroalkyl, C 1 ⁇ 0 alkyl substituted with J, C ⁇ g 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 di
  • T is selected from the group consisting of C 1 ⁇ 0 alkyl, C 0 fluoroalkyl, C j . 10 alkyl substituted with J, C ⁇ g 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 .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH C j ⁇ g alkoxy, C ⁇ g alkylamine, C ⁇ dialkylamine, C ⁇ J Q alkyl-O-CO-, C ⁇ g alkyl-O-C NH-, and C wo alkyl-S-; ,
  • K is selected from the group consisting of halogen, C ⁇ g alkyl, C 1 _ 10 perfluoroalkyl, C ⁇ Q alkoxy, N0 2 , CN, OH, C0 2 H, amino, C- ⁇ g alkylamino, C 2 .
  • 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,
  • R is selected from the group consisting of H, C 2 . 20 alkyl, C wo alkyl with a phenyl group attached to the C j _ 20 alkyl, and C ⁇ g alkyl with an attached phenyl group substituted with K.
  • Tripeptide ⁇ -Ketoesters are compounds of the structure: M 3 -AA-AA-NH-CHR 2 -CO-CO-0-R or a pharmaceutically acceptable salt, wherein M 3 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO r , X-NH-CO-, X 2 N-CO-,
  • X is selected from the group consisting of C ⁇ g alkyl, C j . 10 fluoroalkyl, C- ⁇ g alkyl substituted with J, C ⁇ g 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 j . j g alkyl with an attached phenyl group, C j .
  • T is selected from the group consisting of C 1 ⁇ 0 alkyl, x _ w fluoroalkyl, C ] _ 10 alkyl substituted with J, C j .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C ⁇ g alkoxy, C ⁇ alkylamine, C- ⁇ g dialkylamine, C ⁇ g alkyl-O-CO-, C ⁇ g alkyl-O-CO- NH-, and C wo alkyl-S-;
  • K is selected from the group consisting of halogen, C j . j g alkyl, C ⁇ jg perfluoroalkyl, C l l0 alkoxy, N0 2 , CN, OH, C0 2 H, amino, C u0 alkylamino, C 2 . 12 dialkylamino, C j -C jg acyl, and C ⁇ g 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, beta- alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid
  • R 2 represents C ⁇ g branched and unbranched alkyl, C j . branched and unbranched cyclized alkyl, or C ⁇ g branched and unbranched fluoroalkyl;
  • R is selected from the group consisting of H, C ] _ 20 alkyl, C ⁇ g alkyl with a phenyl group attached to the C ⁇ g alkyl, and C ⁇ alkyl with an attached phenyl group substituted with K.
  • the Tetrapeptide ⁇ -Ketoesters are compounds of the structure: M 3 -AA 4 -AA-AA-AA-CO-0-R or a pharmaceutically acceptable salt, wherein
  • M 3 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO r , X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-S0 2 -, X 2 N-SO , X-CO-, X-CS-, X-SO r , T-O-CO-, or X-
  • X is selected from the group consisting of 1 0 alkyl, C j . j g fluoroalkyl, C ⁇ g alkyl substituted with J, C ⁇ g 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 ⁇ j g alkyl with an attached phenyl group, i l0 alkyl with two attached phenyl groups, C ⁇ g alkyl with an attached phenyl group substituted with K, and C j . jg alkyl with two attached phenyl groups substituted with K, 1 0 alkyl with an attached phenoxy group, and C
  • T is selected from the group consisting of C ⁇ J Q alkyl, C j . j fluoroalkyl, C ⁇ alkyl substituted with J, C j _ 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 j . 10 alkyl with two attached phenyl groups, C l l0 alkyl with an attached phenyl group substituted with K, and CJ.J Q alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C 1 0 alkoxy, C ⁇ g alkylamine, C 2 . 12 dialkylamine, C 1 ⁇ 0 alkyl-O-CO-, C ⁇ g alkyl-O-CO- NH-, and C u0 alkyl-S-;
  • K is selected from the group consisting of halogen, ⁇ g alkyl, C ⁇ perfluoroalkyl, C- ⁇ g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 12 dialkylamino, C ⁇ C j g acyl, and C j . 10 alkoxy-CO-, and C ⁇ j 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 carboxy
  • CH(CH 2 CHEt2)-COOH alpha-aminoheptanoic acid
  • NH 2 -CH(CH 2 -l-napthyl)-COOH NH 2 -CH(CH 2 -2-napthyl)-COOH
  • NH 2 -CH(CH 2 -cyclohexyl)-COOH NH 2 -CH(CH 2 - cyclopentyl)-COOH
  • NH 2 -CH(CH 2 -cyclobutyl)-COOH NH 2 -CH(CH 2 -cyclopropyl)- COOH, trifluoroleucine, and hexafluoroleucine
  • 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 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-aminobutyric 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-ethylserine, S-methylcysteine, S-e
  • R is selected from the group consisting of H, C ⁇ o alkyl, C ⁇ g alkyl with a phenyl group attached to the C 1 _ 20 alkyl, and C ⁇ g alkyl with an attached phenyl group substituted with K.
  • the Amino Acid Peptide ⁇ -Ketoesters are compounds of the structure:
  • M represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-S0 2 -, X 2 N-SO r , Y-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X-
  • X is selected from the group consisting of C-_ 1Q alkyl, C ⁇ fluoroalkyl, C 1 . 10 alkyl substituted with J, C ⁇ g 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 ⁇ g alkyl with an attached phenyl group, C ⁇ alkyl with two attached phenyl groups, C j _ 10 alkyl with an attached phenyl group substituted with K, and CJ.J Q alkyl with two attached phenyl groups substituted with K, C ⁇ g alkyl with an attached phenoxy group, and C ⁇ g alkyl
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C j . jg alkoxy, C ⁇ alkylamine, C 2 . 12 dialkylamine, l ⁇ 0 alkyl-O-CO-, C _ 10 alkyl-O-CO- NH-, and C ⁇ g alkyl-S-;
  • K is selected from the group consisting of halogen, 1 ⁇ 0 alkyl, C ⁇ g perfluoroalkyl, C j . j g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C j . 10 alkylamino, C 2 _ 12 dialkylamino, C j -C j g acyl, and C ⁇ j alkoxy-CO-, and C ⁇ j g 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 carboxy
  • R is selected from the group consisting of H, C ⁇ g alkyl, C ⁇ g alkyl with a phenyl group attached to the C ⁇ g alkyl, and C 1 2 o al yl with an attached phenyl group substituted with K.
  • ketoesters for th synthesis of bicyclic heterocycles. They report the synthesis of n-Bu-CO-Ala-CO-OEt, Pr-CO-Ala-CO-OEt, cyclopentyl-CO-Ala-CO-OEt, Pr-CO-Phg-CO-OEt, and Bz-Ala-CO-OEt. Hori et al.
  • Peptide Ketoester compounds are representative of the Peptide Keto-Compounds found to be useful as Calpain inhibitors within the context of the present invention:
  • Acid peptide ⁇ -Ketoacids All of these are considered to be within the class of Peptid Keto-Compounds.
  • Dipeptide ⁇ -Ketoacids are compounds of the structure: M r AA-NH-CHR 2 -CO-CO-OH or a pharmaceutically acceptable salt, wherein
  • M t represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO r , X 2 N-SO r , X-CO-, X-CS-, X-SO , X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C j . j g alkyl, C wo fluoroalkyl, C ⁇ alkyl substituted with J, C ⁇ 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 ⁇ g alkyl with an attached phenyl group, C j .
  • J is selected from the group consisting of halogen, COOH, OH, CN ⁇ 0 2 , NH 2> C j . j g alkoxy, C j . j alkylamine, C 2 . 1 dialkylamine, C 0 alkyl-O-CO-, ⁇ _ 10 alkyl-O-CO- NH-, and C w0 alkyl-S-;
  • K is selected from the group consisting of halogen, C ⁇ alkyl, C j . 10 perfluoroalkyl, C ⁇ alkoxy, N0 2 , CN, OH, C0 2 H, amino, C 0 alkylamino, C 2 . 12 dialkylamino, ⁇ -C ⁇ acyl, and C ⁇ g alkoxy-CO-, and C j . 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
  • R 2 represents C 1 . g branched and unbranched alkyl, C ⁇ g branched and unbranched cyclized alkyl, or C ⁇ g branched and unbranched fluoroalkyl.
  • the Dipeptide ⁇ -Ketoacids are compounds of the structure: M 1 -AA 2 -AA ⁇ -CO-OH or a pharmaceutically acceptable salt, wherein M x represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-,
  • X-NH-CS- X 2 N-CS-, X-NH-SO , X 2 N-S0 2 -, X-CO-, X-CS-, X-SO , X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C ⁇ j alkyl, C j .
  • j g fluoroalkyl C ⁇ g alkyl substituted with J, C- ⁇ 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 j . j g alkyl with an attached phenyl group, C 1 .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 ,
  • K is selected from the group consisting of halogen, C j . j g alkyl, C ⁇ g perfluoroalkyl, C l lQ alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 . 12 dialkylamino, ⁇ C ⁇ acyl, and C ⁇ g alkoxy-CO-, and C _ 10 alkyl-S-;
  • AA j 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
  • 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 carboxy
  • Tripeptide ⁇ -Ketoacids are compounds of the structure:
  • M j represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-S0 2 -, X 2 N-SO r , X-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C j . 10 alkyl, C ⁇ .g fluoroalkyl, C ⁇ g 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 ⁇ g alkyl with an attached phenyl group, C 0 alkyl with two attached phenyl groups, C ⁇ g alkyl with an attached phenyl group substituted with K, and C ⁇ g alkyl with two attached phenyl groups substituted with K, C ⁇ g alkyl with an attached phenoxy group, and C ⁇ J Q alkyl with an attached
  • K is selected from the group consisting of halogen, C ⁇ g alkyl, C- ⁇ g perfluoroalkyl, C ⁇ g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C 1 . 10 alkylamino, C 2 . 12 dialkylamino, C r C 10 acyl, and C j _ 10 alkoxy-CO-, and C 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,
  • Tetrapeptide ⁇ -Ketoacids are compounds of the structure: M r AA-AA-AA-CO-OH or a pharmaceutically acceptable salt, wherein M ⁇ represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO r , X-NH-CO-, X 2 N-CO-,
  • X is selected from the group consisting of C 0 alkyl, C j _ 10 fluoroalkyl, C l i0 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, ⁇ g alkyl with an attached phenyl group, C j .
  • Y 1 is selected from the group consisting of C 2 . 10 alkyl, C ⁇ j g fluoroalkyl, C ⁇ jg 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 ⁇ g alkyl with an attached phenyl group, C ⁇ j g alkyl with two attached phenyl groups, C ⁇ g alkyl with an attached phenyl group substituted with K, and C j . j g alkyl with two attached phenyl groups substituted with K;
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C j . j g alkoxy, C ⁇ g alkylamine, C 2 -_2 dialkylamine, C 1 . 10 alkyl-O-CO-, C ⁇ g alkyl-O-CO- NH-, and C w0 alkyl-S-;
  • K is selected from the group consisting of halogen, 1 ⁇ 0 alkyl, C ⁇ Q perfluoroalkyl, C ⁇ g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 . 12 dialkylamino, C j -C ⁇ acyl, and 1 0 alkoxy-CO-, and C ⁇ j g 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,
  • the Amino Acid Peptide ⁇ -Ketoacids are compounds of the structure: M r AA-CO-OH or a pharmaceutically acceptable salt, wherein M ⁇ represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -SO r , X-NH-CO-, X 2 N-CO-,
  • X is selected from the group consisting of ⁇ _ 10 alkyl, C ⁇ J Q fluoroalkyl, C j . j 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, i 0 alkyl with an attached phenyl group, C ⁇ g alkyl with two attached phenyl groups, C- ⁇ alkyl with an attached phenyl group substituted with K, and C j .
  • Y 2 is selected from the group consisting of C j . j alkyl, C j . j g fluoroalkyl, C ⁇ Q alkyl substituted with J, C j .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C j . j g alkoxy, C j _ 10 alkylamine, C 2 . 12 dialkylamine, l0 alkyl-O-CO-, C 1 0 alkyl-O-CO- NH-, and C l , 10 alkyl-S-;
  • K is selected from the group consisting of halogen, C 0 alkyl, C j . 10 perfluoroalkyl, C j . j g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C 1 0 alkylamino, C 2 . 12 dialkylamino, j -C 1Q acyl, and C ⁇ g alkoxy-CO-, and C ⁇ g 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, 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
  • 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 j site are excellent inhibitors of several cysteine proteases including calpain I and calpain II.
  • Dipeptide ⁇ -Ketoamides Dipeptide ⁇ -Ketoamides
  • Dipeptide ⁇ -Ketoamides Dipeptide ⁇ -Ketoamides
  • Dipeptide ⁇ -Ketoamides Subclass B
  • Dipeptide ⁇ -Ketoamides Subclass C, Types 1 through 6
  • Tripeptide ⁇ -Ketoamides Tetrapeptide ⁇ -Ketoamides
  • Amino Acid ⁇ -Ketoamides All of these subclasses are considered herein to be within the class of Peptide Keto-Compounds.
  • Dipeptide ⁇ -Ketoamides (Subclass A) have the following structural formula: M r AA-NH-CHR 2 -CO-CO-NR 3 R 4 or a pharmaceutically acceptable salt, wherein
  • M j represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO , X 2 N-S0 2 -, X-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C 1 .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , CJ.JO alkoxy, C 1 0 alkylamine, C 2 . 12 dialkylamine, C ⁇ g alkyl-O-CO-, C j _ 10 alkyl-O-CO- NH-, and C uo alkyl-S-;
  • K is selected from the group consisting of halogen, C ⁇ alkyl, 1 0 perfluoroalkyl, C 1 0 alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 . 12 dialkylamino, C j -C j acyl, C 1 0 alkoxy-CO-, and C ⁇ g 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, beta- alanine, norleucine, norvaline, ⁇ -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid
  • R 2 is selected from the group consisting of C ⁇ g branched and unbranched alkyl C j . branched and unbranched cyclized alkyl, and C- ⁇ g branched and unbranched fluoroalkyl;
  • R 3 and R 4 are selected independently from the group consisting of H, C ⁇ alkyl, C 1 2Q cyclized alkyl, C ⁇ g alkyl with a phenyl group attached to the C 1 . 20 alkyl, C- l -20 cyclized alkyl with an attached phenyl group, C j . 20 alkyl with an attached phenyl group substituted with K, C 1 .
  • the Dipeptide ⁇ -Ketoamides (Subclass B) have the following structural formula:
  • M j represents H, NH 2 -CO-, NH 2 -CS-, NR 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-S0 2 -, X 2 N-SO r , X-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C ⁇ j g alkyl, C w0 fluoroalkyl, 1 0 alkyl substituted with J, C ⁇ g 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 ⁇ g alkyl with an attached phenyl group, C j .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 ,
  • K is selected from the group consisting of halogen, C j . j g alkyl, C ⁇ _ 10 perfluoroalkyl, C 1 . 10 alkoxy, N0 2 , CN, OH, C0 2 H, amino, C M0 alkylamino, C 2 _ 12 dialkylamino, C j -C jg acyl, and C lA0 alkoxy-CO-, and C j . jg alkyl-S-;
  • AA j 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 acid, pipecolinic acid (2-piperidine carboxylic acid), O
  • 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 acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxy
  • R 3 and R are selected independently from the group consisting of H, C ⁇ g alkyl, C ⁇ cyclized alkyl, C ⁇ g alkyl with a phenyl group attached to the C ⁇ g alkyl, C j .20 cyclized alkyl with an attached phenyl group, C
  • Dipeptide ⁇ -Ketoamides (Subclass C, Type 1) have the following structural formula:
  • M j is selected from the group consisting of C l4 alkyl monosubstituted with phenyl, C 1-4 alkyl disubstituted with phenyl, C ⁇ alkyl monosubstituted with 1-naphthyl, C 1-4 alkyl monosubstituted with 2-naphthyl, C 1- alkoxy monosubstituted with phenyl, C M alkoxy disubstituted with phenyl, ArCH 2 0-, rO-, ArCH 2 NH-, and ArNH-; wherein Ar is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, 1-naphthyl, 1-naphthyl monosubstituted with J, 2-nap
  • J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH, C0 2 Me, C0 2 Et, CF 3 , C 1-4 alkoxy, C M alkylamine, C 2 .
  • AA 2 is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid,
  • AA j is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 CHE
  • R j is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl,
  • R 2 represents C ⁇ alkyl substituted with phenyl, phenyl and phenyl substituted with J.
  • Dipeptide ⁇ -Ketoamides (Subclass C, Type 2) have the following structural formula:
  • M ⁇ is selected from the group consisting of C w alkyl monosubstituted with phenyl, C ⁇ alkyl disubstituted with phenyl, C w alkyl monosubstituted with 1-naphthyl,
  • J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH, C0 2 Me, C0 2 Et, CF 3 , C 1 ⁇ ⁇ alkoxy, C 1-4 alkylamine, C 2 . 8 dialkylamine, C 1 _ 4 perfluoroalkyl, and -N(CH 2 CH 2 ) 2 0;
  • AA 2 is an amino acid with the L configuration, D c" duration, or DL configuration at the a-carbon selected from the group con g of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 -cyclohexyl)-COOH, NH 2 -CH(CH 2 -cyclopentyl)-COOH, NH 2 -CH(CH 2 -cyclo
  • AA j is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 CHE
  • R 3 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with J, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J,
  • Dipeptide ⁇ -Ketoamides (Subclass C, Type 3) have the following structural formula:
  • M 3 -(CH 2 ) q -CO-AA 2 -AA 1 -CO-NH-CH 2 CH(OH)-R 1 or a pharmaceutically acceptable salt, wherein M 3 is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl,
  • 3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, 1-tetrahydroquinolinyl, 1-isoquinolinyl, 2-tetrahydroisoquinolinyl, and -N(CH 2 CH 2 ) 2 0; q 0-2;
  • AA 2 is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 -cyclohexyl)-COOH, NH 2 -CH(CH 2 -cyclopentyl)-COOH,
  • AA ⁇ is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 CHE
  • R j is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl,
  • R 2 represents C l4 alkyl substituted with phenyl, phenyl and phenyl substituted with J.
  • J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH, C0 2 Me, C0 2 Et, CF 3 , C w alkoxy, C 1- alkylamine, C 2 . 8 dialkylamine, C 1 _ 4 perfluoroalkyl, and N(CH 2 CH 2 ) 2 0;
  • Dipeptide ⁇ -Ketoamides (Subclass C, Type 4) have the following structural formula:
  • 3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, 1-tetrahydroquinolinyl, 1-isoquinolinyl, 2-tetrahydroisoquinolinyl, and -N(CH 2 CH 2 ) 2 0; q 0-2;
  • AA 2 is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2- c H( CH 2 HEt 2)" OOH ' alpha-aminoheptanoic acid, NH 2 -CH(CH 2 -cyclohexyl)-COOH, NH 2 -CH(CH 2 -cyclopentyl)-COOH,
  • AA j is an amino acid with the L configuration, D configuration, or DL configuration at the ⁇ -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine
  • R 3 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with J, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J,
  • H 2 ) 4 CONH(CH 2 ) 2 J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH, C0 2 Me, C0 2 Et, CF 3 , C ⁇ alkoxy, C w alkylamine, C 2 . 8 dialkylamine, C M perfluoroalkyl, and N(CH 2 CH 2 ) 2 0;
  • Dipeptide ⁇ -Ketoamides (Subclass C, Type 5) have the following structural formula:
  • M 4 -(CH 2 ) q -0-CO-AA 2 -AA 1 -CO-NH-CH 2 CH(OH)-R 1 or a pharmaceutically acceptable salt, wherein M 4 is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl,
  • AA 2 is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 -cyclohexyl)-COOH, NH 2 -CH(CH 2 -cyclopentyl)-COOH,
  • AA j ⁇ is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2
  • R j is selected from the group consisting of phenyl, phenyl monosubstituted wit J, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl,
  • R 2 represents C 1 _ 4 alkyl substituted with phenyl, phenyl and phenyl substituted with J.
  • J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH C0 2 Me, C0 2 Et, CF 3 , C lJ ⁇ alkoxy, C 1 _ 4 alkylamine, C 2 . 8 dialkylamine, C 1-4 perfluoroalkyl, and N(CH 2 CH 2 ) 2 0;
  • Dipeptide ⁇ -Ketoamides (Subclass C, Type 6) have the following structural formula:
  • AA 2 is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-aminoheptanoic acid, NH 2 -CH(CH 2 -cyclohexyl)-COOH, NH 2 -CH(CH 2 -cyclopentyl)-COOH,
  • AA j is an amino acid with the L configuration, D configuration, or DL configuration at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine, arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine
  • R 3 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with J, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J,
  • Tripeptide ⁇ -Ketoamides have the following structural formula: M r AA-AA-AA-CO-NR 3 R 4 or a pharmaceutically acceptable salt, wherein
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-S0 2 -, X 2 N-SO , X-CO-, X-CS-, X-S0 2 -, X-O-CO-, or X-
  • X is selected from the group consisting of C ⁇ g alkyl, C 1 . 10 fluoroalkyl, C ⁇ g alkyl substituted with J, C l l0 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 ⁇ g alkyl with an attached phenyl group, C ⁇ g alkyl with two attached phenyl groups, C ⁇ g alkyl with an attached phenyl group substituted with K, C j . jg alkyl with two attached phenyl groups substituted with K, C ⁇ g alkyl with an attached phenyl group substituted with K, C j
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C ⁇ o alkoxy, 10 alkylamine, C 2 . 12 dialkylamine, C- ⁇ g alkyl-O-CO-, C ⁇ g alkyl-O-CO- NH-, and C ⁇ g alkyl-S-;
  • K is selected from the group consisting of halogen, C j . j g alkyl, C ⁇ j g perfluoroalkyl, C 1 0 alkoxy, N0 2 , CN, OH, C0 2 H, amino, C 1 o alkylamino, C 2 . 12 dialkylamino, C j -C j acyl, and C j . j alkoxy-CO-, and C ⁇ g 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
  • R 3 and R 4 are selected independently from the group consisting of H, C ⁇ alkyl, C j . ⁇ cyclized alkyl, C ⁇ g alkyl with a phenyl group attached to the C ⁇ alkyl, C- i -2 0 cyclized alkyl with an attached phenyl group, C j _ 20 alkyl with an attached phenyl group substituted with K, C ⁇ alkyl with an attached phenyl group disubstituted with K, C j .2 0 alkyl with an attached phenyl group trisubstituted with K, C- ⁇ g cyclized alkyl with an attached phenyl group substituted with K, C- ⁇ g alkyl with a mo ⁇ holine [-N(CH 2 CH 2 )0] ring attached through nitrogen to the alkyl, C j _ 10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C j ⁇ g alkyl with a pyr
  • M 1 represents H, NH 2 -CO-, NH 2 -CS-, NH 2 -S0 2 -, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO r , X 2 N-SO r , X-CO-, X-CS-, X-SO r , X-O-CO-, or X- O-CS-;
  • X is selected from the group consisting of C . j g alkyl, C M0 fluoroalkyl, C ⁇ Q alkyl substituted with J, C ⁇ g 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 ⁇ g alkyl with an attached phenyl group, C 1 .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 ,
  • K is selected from the group consisting of halogen, C ⁇ g alkyl, C j . 10 perfluoroalkyl, C j . j alkoxy, N0 2 , CN, OH, C0 2 H, amino, C ⁇ g alkylamino, C 2 . 12 dialkylamino, C j -C j g acyl, and C ⁇ alkoxy-CO-, and C ⁇ 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,
  • R 3 and R 4 are selected independently from the group consisting of H, C ⁇ g alkyl, CJ.- Q cyclized alkyl, C ⁇ g alkyl with a phenyl group attached to the C 1 2 ⁇ alkyl, ⁇ 1-2 0 cyclized alkyl with an attached phenyl group, C ⁇ o alkyl with an attached phenyl group substituted with K, C ⁇ g alkyl with an attached phenyl group disubstituted with
  • the Amino Acid ⁇ -Ketoamides have the following structural formula: M r AA-CO-NR 3 R 4 or a pharmaceutically acceptable salt, wherein M.
  • X is selected from the group consisting of C j . 10 alkyl, C ⁇ g fluoroalkyl, C ⁇ g alkyl substituted with J, C ⁇ 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 j _ 10 alkyl with an attached phenyl group, C j .
  • J is selected from the group consisting of halogen, COOH, OH, CN, N0 2 , NH 2 , C 1 0 alkoxy, C- ⁇ g alkylamine, C 2 . 12 dialkylamine, C 0 alkyl-O-CO-, C l 0 alkyl-O-CO- NH-, and C 1AQ alkyl-S-;
  • K is selected from the group consisting of halogen, C 1 _ 10 alkyl, C w perfluoroalkyl, CJ.J Q alkoxy, N0 2 , CN, OH, C0 2 H, amino, C 1A0 alkylamino, C 2 . 12 dialkylamino, C j -C j g acyl, and C ⁇ j g alkoxy-CO-, and C j . 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,
  • R 3 and R are selected independently from the group consisting of H, C 1 . 20 alkyl, C 1 2Q cyclized alkyl, C l 20 alkyl with a phenyl group attached to the C ⁇ g alkyl, C--1-2 0 cyclized alkyl with an attached phenyl group, C ⁇ g alkyl with an attached phenyl group substituted with K, C ⁇ Q alkyl with an attached phenyl group disubstituted with K, C j ⁇ alkyl with an attached phenyl group trisubstituted with K, C j .
  • Peptide Ketoamide compounds are representative of the Peptide Keto-Compounds found to be useful as Calpain inhibitors within the context of the present invention: Z-Leu-Phe-CONH-Et
  • His-57 is hydrogen bonded to the carbonyl group of the ester functional group, the peptide backbone on a section of the PPE polypeptide backbone hydrogen bonds to the inhibitor to form a ⁇ -sheet, and the benzyl ester is directed toward the S' subsites.
  • the side chain of the PI 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. If R is a longer substituent, then it would make favorable interactions with the S' subsites of the enzyme.
  • 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 su ⁇ risingly 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.
  • the active site of 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 ketoacids, 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.
  • This design strategy will also work when other classes of peptide inhibitors are used in place of the peptide substrate to gain information on the appropriate sequence to place in the Peptide Keto-Compound inhibitor.
  • Additional interactions with the enzyme can be obtained by tailoring the R group of the inhibitor to imitate the amino acid residues which are preferred by an individual protease at the SI' and S2' subsites.
  • the Ml group can be tailored to interact with the S subsites of the enzyme. This design strategy will also work when other classes of peptide inhibitors are used in place of the peptide substrate to gain information on the appropriate sequence to place in the ketoamide inhibitor.
  • a cysteine protease a known inhibitor sequence is the peptide aldehyde, Ac-Leu-Leu-Nle-H (also known as Calpain Inhibitor 1 and hereinafter designated as "CH").
  • This inhibitor in addition to a related peptide aldehyde inhibitor Ac-Leu-Leu-Nme-H (also known as Calpain Inhibitor II) are commercially available from Calbiochem of La Jolla, California.
  • peptide ⁇ -ketoesters with aromatic amino acid residues in PI are good inhibitors of the thiol proteases, cathepsin B, papain and Calpain.
  • 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.
  • Calpain I from human erythrocytes and calpain II from rabbit were assayed using Suc-Leu-Tyr-AMC (Sasaki et al., /. Biol. Chem. 259:12489-12494 (1984), hereby inco ⁇ orated by reference), and the AMC (7-amino-4- methylcoumarin) release was followed fluorimetrically (excitation at 380 nm, and emission at 460 nm). Enzymatic hydrolysis rates were measured at various substrate and inhibitor concentrations, and K j values were determined by Dixon plot.
  • Table PKC1 shows the inhibition constants (K ) for papain, cathepsin B, calpain I, and calpain II.
  • the inhibition constants for papain shown in Table PKC1 were measured in 0.05 M Tris-HCl, pH 7.5 buffer, containing 2mM EDTA, 5mM cysteine (freshly prepared), 1% DMSO, at 25° C, using N e -Benzoyl- Arg-AMC as a substrate, except that those values of inhibition constants for papain marked with an "e" in Table PKC1 were measured in 50 mM Tris-HCl, pH 7.5 buffer, containing 20 mM EDTA, 5 mM cysteine, 9% DMSO, at 25° C, using N ⁇ -Benzoyl-Arg-NA as a substrate. TABLE PKC1
  • Keto-Compound inhibitors we believe that Peptide Keto-Compounds based on these and similar structures will exhibit Calpain inhibitory activity.
  • HEPES, heparin, and A23187 were obtained from Calbiochem.
  • Suc-Leu-Tyr-AMC and chromogenic substrates were obtained from Sigma.
  • Calpain I was purified from human erythrocytes according to the method of Kitahara (Kitahara, et al, J. Biochem. 95:1759-1766 (1984)) omitting the Blue-Sepharose step.
  • Calpain II from rabbit muscle and cathepsin B were purchased from Sigma. Papain was purchased from Calbiochem.
  • Peptide ⁇ -ketoamides were assayed as reversible enzyme inhibitors.
  • Various concentrations of inhibitors in Me 2 SO were added to the assay mixture which contained buffer and substrate. The reaction was started by the addition of the enzyme and the hydrolysis rates were followed spectrophotometrically or fluorimetrically.
  • the AMC (7-amino-4-methylcoumarin) release was followed fluorimetrically (excitation at 380 nm, and emmision at 460 nm).
  • Enzymatic hydrolysis rates were measured at various substrate and inhibitor concentrations, and K j values were determined by either Lineweaver-Burk plots or Dixon plots.
  • HLE human leukocyte elastase
  • PPE porcine pancreatic elastase
  • chymotrypsin cathepsin G
  • 0.1 Hepes, 0.01 M CaCl 2 , pH 7.5 buffer was utilized for trypsin, plasmin, and coagulation enzymes.
  • a 50 mM Tris.HCl, 2 mM EDTA, 5 mM cysteine, pH 7.5 was used as a buffer for papain.
  • a 88 mM KH 2 P0 4 , 12 mM Na 2 HP0 4 , 1.33 mM EDTA, 2.7 mM cysteine, pH 6.0 solution was used as a buffer for cathepsin B.
  • a 20 mM Hepes, 10 mM CaCl 2 , 10 mM mercatoethanol, pH 7.2 buffer was utilized for calpain I and calpain II.
  • HLE and PPE were assayed with MeO-Suc-Ala-Ala-Pro-Val-NA and Suc-Ala-Ala-NA, respectively (Nakajima et al, J. Biol Chem. 254:4027-4032 (1979); inco ⁇ orated herein by reference).
  • Human leukocyte cathepsin G and chymotrypsin Aa were assayed with Suc-Val-Pro-Phe-NA (Tanaka et al, Biochemistry 24:2040-2047
  • Dipeptide ⁇ -ketoamides with Abu, Phe, and Nva in the PI site and Leu in the P2 site are potent inhibitors of these cysteine proteases.
  • the presence of a hydrogen bond donor in the SI' subsite of the cysteine proteases which may be interacting with the N-H on the ketoamide functional group is indicated since disubstituted amides were much less effective inhibitors.
  • Derivatives of Z-Leu-AA-CONHR where the R group contained a hydroxy or alkoxy group, such as (CH 2 ) 5 OH and CH 2 CH(OC 2 H 5 ) 2 are very good inhibitors of the calpains.
  • the prescence of an aromatic group in PI' position of the peptide ketoamide inhibitor resulted in improved inhibitory potency for calpains which indicates the prescence of hydrophobic residues in the S' subsites of both calpains.
  • the derivatives Z-Leu-AA-CO-NOH ⁇ CH ⁇ R where R was phenyl, phenyl substituted with hydroxy or alkoxy groups and naphthyl, are also very good inhibitors of calpains and cathepsin B.
  • Z-Leu-Abu-CONH(CH 2 ) n R where the R group contained a heterocylic group which has both a hydrophobic moiety with an electronnegative atom, are among the best inhibitors for calpains and cathepsin B.
  • Z-Leu-Nva-CONHCH 2 -2-pyridyl is the best inhibitor of calpain I.
  • Z-Leu-Abu-CONHCH 2 -2-pyridyl is the best inhibitor of calpain II respectively in this series, but its isomers, Z-Leu-Abu-CONH-CH 2 -3-pyridyl and Z-Leu-Abu-CONH-CH 2 -4-pyridyl, are substantially poorer inhibitors.
  • AA a-aminobutyric acid (CH 2 ) 2 OH 0.8 0.078
  • AA phenylalanine
  • Table PKC3 shows the inhibition constants (K j ) of Z-Leu-AA- CONH-CH 2 CH(OH)R.
  • the hydrophobic moiety substituted with CH 2 CH-X (X electronegative atoms such as O, N) resulted in good inhibitor structures.
  • Z-Leu-Abu-CONH-CH 2 CH(OH)C 6 F 5 is the best inhibitor for calpain I, and
  • Ph 2 CH (CH 2 ) 3 -4-mo ⁇ holinyl 0.76 0.0743.8 Preparation of peptide a-ketoesters.
  • 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: O
  • 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. Analysis. Synthesis. Biology. 1-9 (1979-1987), published by Academic
  • 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 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-S0 2 -C1 would introduce the X-S0 2 - group.
  • reaction with MeO-CO-CH 2 CH 2 -CO-Cl would give the Y-CO- group when Y is a C 2 alkyl substituted with a Cl alkyl-OCO- group.
  • Reaction with an a substituted alkyl or aryl sulfonyl chloride would introduce an X-S0 2 - group.
  • reaction with dansyl chloride would give the X-S0 2 - 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-.
  • 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 be used in the Dakin-West reaction.
  • the substituted peptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-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 oxaiyl 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-CO-O-Bzl.
  • R groups can be introduced into the ketoester structure 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 C1-CO-CO-C1.
  • oxalyl chloride C1-CO-CO-C1 For example, 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 and Malee, /. Chromat,
  • Ketoacids M-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-AA-CO-OH, M-AA-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-AA-CO-OR by alkaline hydrolysis.
  • R Bzl
  • R acid cleavage
  • the alternate methods would be used when the M group was labile to alkaline hydrolysis.
  • the various peptide ketoamide subclasses including M-AA-NH-CHR 2 -CO-CO- NR 3 R 4 (Dipeptide Ketoamides, Subclass A), M-AA-AA-CO-NR 3 R 4 (Dipeptide Ketoamides, Subclass B), M 1 CO-AA 2 -AA 1 -CO-NH-CH 2 CH(OH)-R 1 and five others presented above (Dipeptide ⁇ -Ketoamides, Subclass C, Types 1 through 6), M-AA-AA- AA-CO-NR 3 R 4 (Tripeptide Ketoamides), M-AA-AA-AA-AA-CO-NR 3 R 4 (Tetrapeptide Ketoamides) and M 1 -AA-CO-NR 3 R 4 (Amino Acid Ketoamides), were prepared indirectly from the corresponding ketoesters. The ketone carbonyl group was first protected as shown in the following scheme and then the ketoamide was prepared by reaction with an amine H-NR 3 R 4 . The illustrated procedure should also
  • 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.
  • Ketoamides M j CO-AA-AA-CONHR were prepared indirectly from the ketoesters.
  • the ketone carbonyl group is first protected as shown in the following scheme and then the ketoamide is prepared by reaction with an amine RNH 2 .
  • the product is easily isolated from the reaction mixture when using this procedure. This procedure will also work with other ketone protecting groups.
  • the corresponding ketoacid can be used as a precursor to the a-ketoamide via coupling with an amine RNH 2 using standard peptide coupling reagents would result in formation of the peptide a-
  • Amino acid methyl ester hydrochlorides were prepared according to M. Brenner et al., Helv. Chem. Acta 33:568 (1950); 36:1109 (1953) in a scale over 10 mmol or according to Rachele, /. Org. Chem. 28:2898 (1963) in a scale of 0.1-1.0 mmol.
  • N-Acylamino acids with 4-methylpentanoic, 2-(l- 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 °C) slurry of the appropriate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene was added slowly (temp. 10-15 °C) 40 mmol triethylamine or N- methylmo ⁇ holine and then the reaction mixture was stirred for 30 minutes at this temperature.
  • the collected organic layer was washed with 2 x 50 ml H 2 0, decolorized with carbon, and dried over MgS0 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 an appropriate solvent.
  • N-Acyldipeptide methyl esters were synthesized via the HOBt-DCC method in a DMF solution as in 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.
  • N-Acytripeptide methyl esters were synthesized via HOBt- DCC method in DMF solution as in K ⁇ nig and Geiger, supra.
  • N-Acyltripeptide were obtained through hydrolysis of the appropriate methyl esters via general hydrolysis procedure.
  • 1 equivalent of methyl ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH to form the sulfonamide sodium salt.
  • M is selected from the group consisting of C 1-4 alkyl monosubstituted with phenyl,
  • AT- is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, 1-naphthyl, 1-naphthyl monosubstituted with J, 2-naphthyl, and 2-naphthyl monosubstituted with J; J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH,
  • AA j , AA 2 and AA 3 are side chain blocked or unblocked a-amino acids with the L configuration, D configuration, or DL configuration at the a-carbon selected independently from the group consisting of alanine, valine, leucine, isoleucine, histidine, proline, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, arginine, lysine, tryptophan, glycine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-
  • R is selected from the group consisting of H, C ⁇ g alkyl, C ⁇ o cyclized alkyl, C ⁇ alkyl with a phenyl group attached to the C j _ 2 g alkyl, C j . 2 cyclized alkyl with an attached phenyl group, C ⁇ alkyl with an attached phenyl group substituted with K, C 1 . 2g alkyl with an attached phenyl group disubstituted with K, C ⁇ g alkyl with an attached phenyl group trisubstituted with K, C ⁇ g cyclized alkyl with an attached phenyl group substituted with K, C j .
  • K is selected from the group consisting of halogen, C ⁇ g alkyl, C ⁇ perfluoroalkyl,
  • Ar 2 is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl, C 6 H 4 (3-OR 2 ), C 6 H 4 (4-OR 2 ), C 6 H 3 (3,4-(OR 2 ) 2 , C 6 H 2 (2,4,6-(OR 2 ) 3 , 1-naphthyl, 1-naphthyl monosubstituted with J, 1-naphthyl disubstituted with J, 2-naphthyl, 2-naphthyl monosubstituted with J, 2-naphthyl disubstituted with J, 2-pyridyl, 2-quinolinyl, and 1-isoquinolinyl;
  • R 2 represents C 1-4 alkyl substituted with phenyl, phenyl and phenyl substituted with J.
  • Heterocycle 2 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with J, 2-tetrahydrofuryl, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J, 2-pyrazinyl, 2-quinolinyl, 2-quinr ,1 -nyl monosubstituted with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted wit'r consider, 1-tetrahydroquinolinyl, 2-tetrahydroisoquinolinyl, 3-indolyl, 2-pyridyl-N-oxide, 3-pyridyl-N-oxide, 4-pyridyl-N-oxide, 2-(N-methyl-2-pyrrolyl), 1-imid
  • R 6 is selected from the group consisting of C 6 alkyls and C ⁇ g alkyls monosubstituted with phenyl, by treatment with a blocking reagent in the presence of a Lewis acid in an organic solvent at 0-100 °C for 1-48 hours, wherein the preferred blocking reagent is 1,2-ethanedithiol; the preferred Lewis acids are selected from the group consisting of BF3.Et 2 0, 4-toluene sulfonic acid, A1C1 3 and ZnCl 2 ; the preferred organic solvents are selected from the group consisting of CH 2 C1 2 , CHC1 3 , Et 2 0 and THF;
  • M is selected from the group consisting of C 1-4 alkyl monosubstituted with phenyl,
  • Ar j is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, 1-naphthyl, 1-naphthyl monosubstituted with J, 2-naphthyl, and 2-naphthyl monosubstituted with J; J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH,
  • AA j , AA 2 and AA 3 are side chain blocked or unblocked a-amino acids with the L configuration, D configuration, or DL configuration at the a-carbon selected independently from the group consisting of alanine, valine, leucine, isoleucine, histidine, proline, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, arginine, lysine, tryptophan, glycine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-
  • R is selected from the group consisting of H, C._ 20 alkyl, C j ⁇ cyclized alkyl, C ⁇ alkyl with a phenyl group attached to the C ⁇ g alkyl, C j _ 20 cyclized alkyl with an attached phenyl group, C ⁇ Q alkyl with an attached phenyl group substituted with K, C ⁇ alkyl with an attached phenyl group disubstituted with K, C 20 alkyl with an attached phenyl group trisubstituted with K, C ⁇ cyclized alkyl with an attached phenyl group substituted with K, C j .
  • alkyl with an OH group attached to the alkyl -CH 2 CH 2 OCH 2 CH 2 OH, C w0 with an attached 4-pyridyl group, C ⁇ g with an attached 3-pyridyl group, C ⁇ g with an attached 2-pyridyl group, C ⁇ g with an attached cyclohexyl group, -NH-CH 2 CH 2 -(4-hydroxyphenyl), -NH-CH 2 CH 2 -(3-indolyl), CH 2 CH(OH)-Ar 2 and (CH 2 ) n -Heterocycle 2 ;
  • K is selected from the group consisting of halogen, C j . 10 alkyl, C ⁇ g perfluoroalkyl, C 1 - i o alkoxy, N0 2 , CN, OH, C0 2 H, amino, C j . j g alkylamino, C 2 . ]2 dialkylamino, C ⁇ j g acyl, and C j . jg alkoxy-CO-, and C j . j g alkyl-S-;
  • Ar 2 is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl, C 6 H 4 (3-OR 2 ), C 6 H 4 (4-OR 2 ), C 6 H 3 (3,4-(OR 2 ) 2 , C 6 H 2 (2,4,6-(OR 2 ) 3 , 1-naphthyl, 1-naphthyl monosubstituted with J, 1-naphthyl disubstituted with J, 2-naphthyl, 2-naphthyl monosubstituted with J, 2-naphthyl disubstituted with J, 2-pyridyl, 2-quinolinyl, and 1-isoquinolinyl;
  • R 2 represents C w alkyl substituted with phenyl, phenyl and phenyl substituted with J.
  • Heterocycle 2 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with J, 2-tetrahydrofuryl, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J, 2-pyrazinyl, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J, 1-tetrahydroquinolinyl, 2-tetrahydroisoquinolinyl, 3-indolyl, 2-pyridyl-N-oxide, 3-pyridyl-N-oxide, 4-pyri
  • R 6 is selected from the group consisting of C ⁇ alkyls and C j _ 6 alkyls monosubstituted with phenyl; by treating the peptidyl ⁇ -ketoester with a hydrolysis reagent in an appropriate solvent at
  • the preferred hydrolysis reagents are selected from the group consisting of NaOH, KOH, EtONa and EtOK; the preferred solvent are selected from the group consisting of water, MeOH, EtOH, THF and DMF;
  • M is selected from the group consisting of C 4 alkyl monosubstituted with phenyl, ⁇ alkyl disubstituted with phenyl, C j _ 4 alkyl monosubstituted with 1-naphthyl, C ⁇ alkyl monosubstituted with 2-naphthyl, C ⁇ 4 alkoxy monosubstituted with phenyl, C w alkoxy disubstituted with phenyl, and
  • a ⁇ - is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, 1-naphthyl, 1-naphthyl monosubstituted with J, 2-naphthyl, and 2-naphthyl monosubstituted with J;
  • J is selected from the group consisting of halogen, OH, CN, N0 2 , NH 2 , COOH, C0 2 Me, C0 2 Et, CF 3 , C 1 ⁇ ⁇ alkoxy, C M alkylamine, C 2 _ 8 dialkylamine, C M perfluoroalkyl, and -N(CH 2 CH 2 ) 2 0;
  • Heterocycle j ⁇ is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, 2-tetrahydroquinolinyl,
  • AA ⁇ AA 2 and AA 3 are side chain blocked or unblocked a-amino acids with the L configuration, D configuration, or DL configuration at the a-carbon selected independently from the group consisting of alanine, valine, leucine, isoleucine, histidine, proline, methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine, norleucine, norvaline, arginine, lysine, tryptophan, glycine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH 2 -CH(CH 2 CHEt 2 )-COOH, alpha-amino
  • R is selected from the group consisting of H, C j .
  • K is selected from the group consisting of halogen, C j . j g alkyl, C 1 ⁇ 0 perfluoroalkyl, C j . j g alkoxy, N0 2 , CN, OH, C0 2 H, amino, C i ⁇ 0 alkylamino, C 2 . 12 dialkylamino, C w0 acyl, and C 1 Q alkoxy-CO-, and C j . j g alkyl-S-;
  • Ar 2 is selected from the group consisting of phenyl, phenyl monosubstituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl,
  • Heterocycle 2 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with J, 2-tetrahydrofuryl, 2-pyridyl, 2-pyridyl monosubstituted with J,
  • 3-pyridyl 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J, 2- ⁇ yrazinyl, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl,
  • Rg is selected from the group consisting of C ⁇ g alkyls and C ⁇ alkyl monosubstituted with phenyl; with a primary amine RNH 2 in an organic solvent at 0-100 °C for 1-72 hours to give th desired peptidyl ⁇ -ketoamide, wherein the preferred organic solvents are selected from the group consisting of CH 2 C1 2 EtOH, DMF and THF.
  • PKC1-PKC65 are given to illustrate the synthesis of Peptide Keto-Compounds:
  • EXAMPLE PKC1 Z-AIa-DL-AIa-COOEt This compound was synthesized by a modified Dakin-West procedure as in Charles et al, /. Chem. Soc. Perkin 1:1139-1146 (1980). To a stirred solution of Z-Ala-Ala-OH (880 mg, 3 mmole), 4-dimethylaminopyridine (15 mg, 0.31 mmole), and pyridine (0.8 mL, 10 mmole) in tetrahydrofuran (3 mL) was added ethyl oxalyl chloride (0.7 mL, 6 mmole) at a rate sufficient to initiate refluxing. The mixture was gently refluxed for 3.5 h. The mixture was treated with water (3 mL) and stirred vigorously at room temperature for 30 min. The mixture was extracted with ethyl acetate.
  • Bz-DL-Ala-COOH The hydrolysis procedure of Tsushima et al., /. Org. Chem., 49:1163-1169 (1984) was used.
  • Bz-DL-Ala-C0 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 0 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 PKC1 and purified by column chromatography, oil, one spot on tic.
  • Z-Leu-DL-Abu-enol ester the precursor of Z-Leu-DL-Abu-COOEt was synthesized by the same procedure as described in Example PKC1 and purified by column chromatography, oil, one spot on tic.
  • 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 S0 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 MgS0 4 .
  • the crude enol ester (8,36 g, 98%) was flash-chromatographed on silica gel and the product was eluted with a AcOEt.
  • R f 0.71, K; 0.54, C.
  • EXAMPLE PKC38 2-NapS0 2 -Leu-Abu-COOEt This was prepared by the preceding general procedure.
  • Z-Leu-Phe-C0 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-C0 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.
  • EXAMPLE PKC52 Z-Leu-Phe-CONH-BzI This compound was synthesized from the protected ⁇ - ketoester and benzylamine in 40 % yield by the procedure described in Example PKC48. After reacting overnight, ethyl acetate (60 ml) was added. The mixture was filtered to remove a white precipitate. The solution was washed with cooled 1 N HCl
  • EXAMPLE PKC60 Z-Leu-Abu-C0NH-(CH 2 ) 3 -N(CH 2 CH 2 ) 2 0.
  • This compound was synthesized from protected ⁇ -ketoester and 4(3-aminopropyl)mo ⁇ holine in 33 % yield by the procedure described in Example PKC48. After reacting overnight, ethyl acetate (80 ml) was added. The mixture was filtered to remove a white precipitate. The solution was washed with water (3 x 20 ml), saturated sodium chloride (2 x 20 ml), and dried over magnesium sulfate. The solution was evaporated leaving a yellow oil.
  • EXAMPLE PKC67 Z-Leu-Abu-CONH-(CH 2 ) 5 OH. This compound was synthesized from 1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 5-amino-l-pentanol. To a solution of protected a-ketoester (1 mmol) in ethanol (3 mL) was added 5-amino-l-pentanol (3 mmol) and stirred overnight at r.t. To the mixture was added AcOEt (25 mL) and white precipitate was filtered.
  • EXAMPLE PKC68 Z-Leu-Abu-CONH-(CH 2 ) 2 OH This is an alternative synthesis for the compound designated in Example PKC 62.
  • This compound was synthesized from 1,3-dithiolane derivative of Z-Leu-Abu-COOEt and ethanolamine by the procedure described in Example PKC67, and purified by column chromatography using solvent CHCl 3 /CH 3 OH 10:1 (40% yield).
  • White solid, single spot on TLC, R f 0.42 (CHCl 3 /CH 3 OH 10:1), mp 151-154 C.
  • 1H NMR (CDC1 3 ) ok, MS (FAB) m/e 422 (M+ l).
  • EXAMPLE PKC69 Z-Leu-Abu-CONH-(CH 2 ) 2 0(CH 2 ) 2 OH.
  • This is an alternative synthesis for the compound designated in Example PKC 63.
  • This compound was synthesized from 1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-(2-aminoethoxy)ethanol by the procedure described in Example PKC67, and purified by column chromatography using solvent CHCl 3 /CH 3 OH 10:1 (34% yield).
  • White solid, single spot on TLC, R f 0.42 (CHCl 3 /CH 3 OH 10:1), mp 103-105 C.
  • EXAMPLE PKC74 Z-Leu-Abu-CONH-(CH 2 ) 2 C 6 H 4 (2-OCH 3 ).
  • EXAMPLE PKC104 Z-Leu-Abu-CONH-CH 2 -2-Pyridyl. This compound was synthesized from 1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-aminomethylpyridine. After reacting overnight at r.t., to the mixture was added AcOEt (25 mL) and white precipitate was filtered. The filtrate was washed with water (3 x 10 mL), saturated NaCl (2 x 10 mL) and dried over MgS0 .
  • EXAMPLE PKC114 Z-Leu-Abu-CONH-CH 2 -2-QuinolinyI. This compound was prepared from 1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-aminomethylquinoline by the procedure described in Example PKC104, and purified by column chromatography using solvent AcOEt/hexane 2:1 (16% yield). YeUow solid, single spot on TLC, R f
  • EXAMPLE PKC120 Z-Leu-Abu-CONH-(CH 2 ) 2 NH-Biotinyl This compound was prepared from Z-Leu-Abu-COOH and biotmylethylenediamine hydrochloride. Biotin (1 g, 4.1 mmol was dissolved in 20 mL of DMF at 70 _C and cooled to 40 _C, CDI (0.97 g, 6 mmole in 3 mL of DMF was then added and white precipitate were appeared. After stirring at r.t. for two hours, ethylenediamine (1.34 mL, 20 mmole) in 10 mL of DMF was added and stirred for another 3 hours.
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