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  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/22—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated the carbon skeleton being further substituted by oxygen atoms
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  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
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  • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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  • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/51—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
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  • C07C233/81—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
  • C07C233/82—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/83—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom of an acyclic saturated carbon skeleton
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  • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
  • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
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Definitions

• This invention relates to a novel class of peptide ketoesters, peptide ketoacids, and ketoamides useful for selectively inhibiting serine proteases, selectively inhibiting cysteine proteases, generally inhibiting all serine proteases, and generally inhibiting all cysteine proteases.
• Serine proteases and cysteine proteases are involved in numerous disease states and inhibitors for these enzymes can be used therapeutically for the treatment of diseases involving serine proteases pr " cysteine proteases.
• thafpeptide ⁇ -ketoesters, peptide ⁇ -ketoacids, and ⁇ -ketoamides can be constructed to inhibit selectively individual serine or cysteine proteases or groups of serine or cysteine proteases.
• peptide ketoesters, ketoacids, and ketoamides which contain hydrophobic aromatic amino acid residues in the Pj site are potent inhibitors of chymases and chymotrypsin-like enzymes.
• Ketoesters, acids, and amides containing small hydrophobic amino acid residues at the Pj position are good inhibitors of elastases. Inhibitors of elastases and chymases are useful as anti-inflammatory agents.
• peptide ketoesters, amides, and acids which contain canonic amino acid residues such as Arg and Lys in the Pj site are potent inhibitors of trypsin and blood coagulation enzymes. These inhibitors are thus useful as anticoagulants. Cysteine proteases such as papain, cathepsin B, and calpain I and LT are also inhibited by ketoesters. Ketoesters, acids, and amides with aromatic amino acid residues in the Pj site would be good inhibitors for cathepsin B and papain. Thus, they would have utility as anticancer agents.
• Ketoesters, ketoacids, and ketoamides with either aromatic amino acid residues or small hydrophobic alkyl amino acid residues at Pj are good inhibitors of calpain I and II. These inhibitors are useful as neuroprotectants and can be used as therapeutics for the treatment of neurodegeneration.
• Amino acid residues and blocking groups are designated using standard abbreviations [see J. Biol. Chem. 260, 14-42 (1985) for nomenclature rules; incorporated herein by reference].
• An amino acid residue (AA) in a peptide or inhibitor structure refers to the part structure -NH-CHR1-CO-, where Rj is the side chain of the amino acid residue AA.
• a peptide ⁇ -ketoester residue would be designated -AA-CO-OR which represents the part structure -NH- CHRi-CO-CO-OR.
• the ethyl ketoester derived from benzoyl alanine would be designated Bz-AIa-CO-OEt which represents CgHsCO-NH-CHMe-CO-CO-OEt.
• peptide ketoacid residues and peptide ketoamide residues would be designated -AA-CO-OH and -AA-CO-NH-R respectively.
• the ethyl keto amide derived from Z-Leu-Phe-OH would be designated Z-Leu-Phe-CO-NH-Et which represents C6H5CH2 ⁇ CO-NH-CH(CH2CHMe2 - CO-NH-CH(CH2 p h)-CO-CO-NH-EL 3.
• Serine Proteases play critical roles in several physiological processes such as digestion, blood coagulation, complement activation, fibrinolysis, viral infection, fertilization, and reproduction. Serine proteases are not only a physiological necessity, but also a potential hazard if they are not controlled. Uncontrolled proteolysis by elastases may cause pancreatitis, emphysema, rheumatoid arthritis, bronchial inflammation and adult respiratory distress syndrome.
• ttyptase trypsin-like cellular enzyme
• HTV-1 human immunodeficiency virus type 1
• Hattori et aL FEBS Letters 248, pp.48-52 (1989)
• Plasmin is involved in tumor invasiveness, tissue remodeling, blistering, and clot dissociation.
• proteases should be potent anticoagulants, anti-infiammtory agents, anti-tumor agents and anti-viral agents useful in the treatment of protease-reiated diseases [Powers and Harper, Proteinase Inhibitors, pp 55- 152, Barrett and Salvesen, eds., Elsevier, (1986); incorporated herein by reference].
• protease-reiated diseases Powers and Harper, Proteinase Inhibitors, pp 55- 152, Barrett and Salvesen, eds., Elsevier, (1986); incorporated herein by reference.
• In vitro proteolysis by chymotrypsin, trypsin or the elastase family is a serious problem in the production, purification, isolation, transport or storage of peptides and proteins.
• Elastase inhibitors are anti-inflammatory agents which can be used to treat elastase- associated inflammation including rheumatoid arthritis and emphysema.
• ⁇ l-protease inhibitor ⁇ l-PI
• this protein inhibitor is not widely used clinically due to the high dosage needed for treatment and the difficulty of producing large quantities. Therefore small molecular weight elastase inhibitors are needed for therapy.
• elastase inhibitors have utility for the treatment of emphysema and inflammation (see: l-carpapenem-3-carboxylic esters as anti-inflammatoiy agents, U.S. Patent 4,493,839; N-carboxyl-thienamycin esters and analogs thereof as anti-inflammatory agents, U.S. Patent 4,495,197; incorporated herein by reference).
• Anticoagulants and antithrombotic drugs are used in a variety of thrombotic disorders.
• the 1990 Physician's Desk Reference lists several anticoagulant drugs (heparin, protamine sulfate and warfarin), a few antiplatelet drugs (aspirin) and several thrombolytic agents.
• Heparin and warfarin are commonly used clinically for prevention and treatment of venous thrombosis and pulmonary embolism. Heparin inhibits the blood coagulation activity by accelerating the binding of natural plasma protease inhibitor antithrombin HI with coagulation factors, and warfarin acts as a vitamin K antagonist and inhibits the synthesis of coaguation factors.
• Inhibitors for the trypsin-like enzymes involved in blood coagulation are useful anticoagulants in vivo [see for example: H-D-Phe-Pro-Arg-CH2C1, Hanson and Harker, Proc. Nad. Acad. Sci. 85, 3184-3188 (1988); 7-Amino-4-chloro-3-(3- isothiureidopropoxy)isocoumarin (ACTTIC), Oweida, Ku, Lumsden, Kam, and Powers, Thrombos. Res.58, 191-197 (1990); incorporated herein by reference].
• Cysteine Proteases Cysteine proteases such as calpain use a cysteine residue in their catalytic mechanism in contrast to serine proteases which utilize a serine residue. Cysteine proteases include papain, cathepsin B, calpains, and several viral enzymes. Neural tissues, including brain, are known to possess a large variety of proteases, including at least two calcium stimulated proteases termed calpains. Calpains are present in many tissues in addition to the brain. Calpain I is activated by micromolar concentrations of calcium while calpain II is activated by millimolar concentrations.
• calpain II is the predominant form, but calpain I is found at synaptic endings and is thought to be the form involved in long term potentiation, synaptic plasticity, and cell death.
• Other Ca ⁇ + activated cysteine proteases may exist, and the term “calpain” is used to refer to all Ca ⁇ activated cysteine proteases, including calpain I and calpain II.
• the terms "calpain I” and “calpain IT' are used herein to refer to the micromolar and millimolar activated calpains, respectively, as described above. While calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack.
• 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. Acad. Sci. USA 81, 3572-3576 (1984); incorporated herein by reference].
• calpains and/or accumulation of breakdown products of cytoskeletal elements has been observed in neural tissues of mammals exposed to a wide variety of neurodegenerative diseases and conditions. For example, these phenomena have been observed following ischemia in gerbils and rats, following stroke in humans, following administration of the toxins kainate, trimethyltin or colchicine in rats, and in human Alzheimer's disease.
• Several inhibitors of calpain have been described including peptide aldehydes such as Ac-Leu-Leu-Nle-H and leupeptin (Ac-Leu-Leu- Arg-H), as well as epoxysuccinates such as E- 64.
• 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 have multiple therapeutic uses.
• Ketoesters A few amino acid and peptide ketoesters and ketoacids have been previously reported. Cornforth and Comforth [/. Chem. Soc, 93-96 (1953); incorporated herein by reference] report the synthesis of the ketoacids PhCH2CO-Gly-CO-OH and Ac-Gly- CO-OH upon hydrolysis of heterocyclic molecules. Charles et al. [/. Chem. Soc. PerkinI, 1139-1146 (1980); incorporated herein by reference] use ketoesters for the synthesis of bicyclic heterocycles.
• Ketoamides A single peptide ketoamide is reported in the literature by Hu and Abeles [Arch. Biochem. Biophys. 281, 271-274 (1990)].
• peptide and amino acid ⁇ -ketoester, ⁇ -ketoacid, and ⁇ - ketoamide derivatives are a novel group of inhibitors for serine proteases and cysteine proteases.
• Inhibitors are compounds that reduce or eliminate the catalytic activity of the enzyme.
• peptide and amino acid ⁇ -ketoester, ⁇ -ketoacid, and ⁇ - ketoa ide derivatives which have an amino acid sequence similar to that of good substrates for a particular protease, are good inhibitors for that protease.
• Trypsin and trypsin-like enzymes normally cleave peptide bonds in proteins and peptides where the amino acid residue on the carbonyl side of the split bond (Pi residue) is Lys or Arg.
• Peptide and amino acid ⁇ -ketoester, ⁇ -ketoacid, and ⁇ - ketoamide derivatives which have Lys or Arg at Pj are thus good inhibitors for these enzymes.
• Elastase and elastase-like enzymes cleave peptide bonds where the Pi amino acid is Ala, Val, Ser, Leu and other similar amino acids. Inhibitors with these residues at P- are good elastase inhibitors.
• Chymotrypsin and chymotrypsin-like enzymes hydrolyze peptide bonds where Pi amino acid is Trp, Tyr, Phe, Met, Leu or other amino acid residues which contain aromatic or large alkyl side chains. Inhibitors with these residues at P j are good chymotrypsin and chymase inhibitors. All of the above enzymes have extensive secondary specificity and recognize amino acid residues removed from the Pi residue.
• the new protease inhibitors are useful for controlling tissue damage and various inflammatory conditions mediated by proteases such as blistering.
• the inhibitors for blood coagulation enzymes are useful anticoagulants and could be used to treat thrombosis.
• the peptide and amino acid ⁇ -ketoester, ⁇ -ketoacid, and ⁇ -ketoamide derivatives are also useful in vitro for inhibiting trypsin, elastase, chymotrypsin and other serine proteases of similar specificity, and for inhibiting serine proteases in general.
• the inhibitors can be used to identify new proteolytic enzymes encountered in research. They can also be used in research and industrially to prevent undesired proteolysis that occurs during the production, isolation, purification, transport and storage of valuable peptides and proteins. Such proteolysis often destroys or alters the activity and or function of the peptides and proteins.
• Uses would include the addition of the inhibitors to antibodies, enzymes, plasma proteins, tissue extracts or other proteins and peptides which are widely sold for use in clinical analyses, biomedical research, and for many other reasons. For some uses a specific inhibitor would be desirable, while in other cases, an inhibitor with general specificity would be preferred.
• the peptide and amino acid ⁇ -ketoester, ⁇ -ketoacid, and ⁇ -ketoamide derivatives are also novel and potent inhibitors of cysteine proteases including calpains, cathepsin B, and papain.
• the calpain inhibitors are useful for treatment of various neurodegenerative diseases and conditions, including ischemia, stroke, and Alzheimer's disease.
• Peptide ⁇ -ketoesters, peptide ⁇ -ketoacids, and peptide ⁇ -ketoamides are transition state analog inhibitors for serine proteases and cysteine proteases.
• Peptide ketoesters containing hydrophobic amino acid residues in the P site have been found to be excellent inhibitors of several serine proteases including human leukocyte elastase, porcine pancreatic elastase, human leukocyte cathepsin G, and bovine chymotrypsin.
• Peptide ketoesters containing amino acid residue with canonic side chain in the P- site have been found to be excellent inhibitors of several se ⁇ ne proteases including bovine trypsin, bovine thrombin, human plasma kallikrein, porcine pancreatic kallikrein, human factor XIa and human plasmin.
• 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.
• These structures may be used in vivo to treat diseases such as emphysema, adult respiratory distress syndrome, rheumatoid arthritis and pancreatitis which result from uncontrolled proteolysis by elastase, chymotrypsin, trypsin and related serine proteases.
• These inhibitors may be used in vitro to prevent proteolysis which occurs in the process of production, isolation, purification, storage or transport of peptides and proteins.
• These inhibitors may be useful as therapeutic agents for treatment of neurodegeneration. viral infections, muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption.
• novel class of peptide ⁇ -ketoamides have the following structural formula: M j -AA-NH-CHR2-CO-CO-NR3R4 or a pharmaceutically acceptable salt, wherein M represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-,
• X is selected from the group consisting of Ci-io alkyl, Ci-io fluoroalkyl, Ci-io alkyl substituted with J, Ci-io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K.
• phenyl disubstimted with K phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstimted with K, naphthyl trisubstituted with K, Ci-io alkyl with an attached phenyl group, Ci-io alkyl with two attached phenyl groups, - 10 alkyl with an attached phenyl group substituted with K, Ci-io alkyl with two attached phenyl groups substituted with K, C l . ⁇ o alkyl with an attached phenoxy group, and C ⁇ _ ⁇ o alkyl with an attached phenoxy group substituted with K on the phenoxy group;
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, Ci-io alkylamine, C2-12 dialkylamine, -iO alkyl-O-CO-, Ci-io alkyl-O-CO- NH-, and C i. ⁇ o alkyl-S-; K is selected from the group consisting of halogen, C ⁇ _io alkyl, C ⁇ _ ⁇ o perfluoroalkyl,
• 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, vaiine, leucine, isoieucine, 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
• R3 and R4 are selected independently from the group consisting of H. C ⁇ _20 alkyl. C . 20 cyclized alkyl, C 1.20 alkyl with a phenyl group attached to the C ⁇ _20 alkyl, C ⁇ _20 cyclized alkyl with an attached phenyl group, C ⁇ _20 alkyl with an attached phenyl group substituted with K, C 1.20 alkyl with an attached phenyl group disubstimted with K, C 1.20 alkyl with an attached phenyl group trisubstituted with K, C ⁇ _20 cyclized alkyl with an attached phenyl group substituted with K, C ⁇ io alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C ⁇ IQ alkyl with a piperidi ⁇ e ring attached through nitrogen to the alkyl, C ⁇ _ ⁇ o alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C
• novel class of peptide ⁇ -ketoamides also have the following structural formula: M1-AA2-AA1-CO-NR3R4 or a pharmaceutically acceptable salt, wherein L represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-;
• X is selected from the group consisting of Ci-io alkyl, Ci-io fluoroalkyl, Ci-io alkyl substituted with J, Ci-io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstimted with K, naphthyl trisubstituted witii K, Ci-10 alkyl with an attached phenyl group, Ci-io alkyl with two attached phenyl groups, Ci- 10 alkyl with an attached phenyl group substituted with K, C ⁇ io alkyl with two attached phenyl groups substituted with K, Ci-io alkyl with an attached phenoxy group, and Ci-io alkyl with an attached phen
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ . 10 alkoxy, Ci-io alkylamine, C2-12 dialkylamine, Ci-io alkyl-O-CO-. Ci-io alkyl-O-CO- NH-, andC ⁇ _! ⁇ alkyl-S-;
• K is selected from the group consisting of halogen, C io alkyl, C o perfluoroalkyl, C o alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ iQ alkylamino, C2-12 dialkylamino, C - C 0 acyl, and C . Q alkoxy-CO-, and C ⁇ .
• 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, tiyptophan, serine, threonine, cysteine, tyrosine, asparagine, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine.
• norvaline alpha- aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S- ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH 2 -C ⁇ (CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH 2 -CH(CH2- cyclohexyD-COOH, NH2-CH(CH2- c y cIo P ent 1 )-COOH, NH2-CH(CH
• AA2 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, tiyptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, giutamine, aspartic acid, glutamic acid, Iysine.
• arginine histidine, phenyiglycine, beta-alanine, norleucine, norvaline.
• alpha-aminobutyric acid alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S- ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2 -COOH, alpha-aminoheptanoic acid, NH2-CH(CH 2 -l-napthyl)-COOH, NH 2 -CH(CH2-2-napthyl)-COOH, NH 2 -CH(CH2- cyclohexyl)-CC>OH, NH2-CH( ⁇ i2- c y lo P ent y
• novel class of peptide ⁇ -ketoamides also have the following structural formula: Mi -AA- AA-AA-CO-NR3R4 or a pharmaceutically acceptable salt, wherein Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-- X-NH-CO-, X 2 N-CO-,
• X is selected from the group consisting of -io alkyl, Ci-io fluoroalkyl, Ci-io alkyl substituted with J, -io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substituted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, Ci-io alkyl with an attached phenyl group, -io alkyl with two attached phenyl groups, Ci- 10 alkyl with an attached phenyl group substituted with K, Ci-io alkyl with two attached phenyl groups substituted with K, Ci-io alkyl with an attached phenoxy group, and -io alkyl with an attached phenoxy group substitute
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cj. 10 alkoxy, Ci-io alkylamine, C2-12 dialkylamine, Ci-io alkyl-O-CO-, -io alkyl-O-CO- NH-, and Ci.10 alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ .IQ alkyl, C ⁇ io perfluoroalkyl, C io alkoxy, NO2, CN, OH, CO2H, amino.
• 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsUon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S- ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2
• novel class of peptide ⁇ -ketoamides also have the following structural formula: M 1 -AA-AA-AA-AA-CO-NR3R4 or a pharmaceutically acceptable salt, wherein M X represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-,
• X is selected from the group consisting of C ⁇ _ ⁇ o alkyl, Ci-io fluoroalkyl, C ⁇ io alkyl substimted with J, Ci-io fluoroalkyl substimted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, Ci-io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C ⁇ jQ alkyl-S-;
• T K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl, C ⁇ io alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ io alkylamino, C2-12 dialkylamino, Ci- C10 acyl, and C ⁇ io alkoxy-CO-, and C ⁇ io 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, tiyptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piper
• R3 and R4 are selected independently from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 cyclized alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, C ⁇ 20 cyclized alkyl with an attached phenyl group, C ⁇ o alkyl with an attached phenyl group substimted with K, C ⁇ 20 alkyl with an attached phenyl group disubstimted with K, C ⁇ 20 alkyl with an attached phenyl group trisubstimted with K, C ⁇ 20 cyclized alkyl with an attached phenyl group substituted with K, C ⁇ io al yl ititl a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C ⁇ io alIc y 1 with a p-peridine ring attached through nitrogen to the alkyl, C ⁇ io alkyl with a pyrrolidine ring attached through nitrogen to the alkyl,
• novel class of peptide ⁇ -ketoamides also have the following structural formula: M1-AA-CO-NR3R4 or a pharmaceutically acceptable salt, wherein
• Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-:
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J. C ⁇ io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K. phenyl disubstimted with K, phenyl trisubstituted with K, naphthyl.
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ o alkyl-O-CO- NH-, and C ⁇ 10 alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl, C ⁇ io alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ io alkylamino, C2-12 dialkylamino, Ci- C 0 acyl, and C ⁇ io alkoxy-CO-, and C ⁇ o alkyl-S-;
• a A 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, glycme, serine, threonine, cysteine, tyrosine, asparagine, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carb
• R3 and R4 are selected independently from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 cyclized alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, C ⁇ 20 cyclized alkyl with an attached phenyl group, C ⁇ 20 alkyl with an attached phenyl group substimted with K, C ⁇ 20 a ⁇ yl w*-- 1 an attached phenyl group disubstimted with K, C ⁇ 20 alkyl with an attached phenyl group trisubstimted with K, C ⁇ 20 cyclized alkyl with an attached phenyl group substimted with K, C ⁇ io alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C ⁇ o alty* with a piperidine ring attached through nitrogen to the alkyl, C ⁇ io alkyl with a pyrrolidine ring attached through nitrogen to the alky
• novel class of peptide ⁇ -ketoacids have the following structural formula: M1-AA-NH-CHR2-CO-CO-OH or a pharmaceutically acceptable salt, wherein
• Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-xNH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-;
• B TIT X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C ⁇ xo alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl, C ⁇ io alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ o alkylamino, C2.12 dialkylamino, Ci- C10 acyl, and C ⁇ o alkoxy-CO-, and C ⁇ io 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine,
• R2 represents C ⁇ g branched and unbranched alkyl, C ⁇ g branched and unbranched cyclized alkyl, or C ⁇ g branched and unbranched fluoroalkyl;
• novel class of peptide ⁇ -ketoacids also have the following structural formula: M1-AA2-AA1-CO-OH or a pharmaceutically acceptable salt, wherein
• Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-:
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substimted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naph
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C io lkyl-S-
• K is selected from the group consisting of halogen, C io aIk y 1 - C io perfluoroalkyl, C io alkoxy, NO2, CN, OH, CO2H, amino, C io alkylamino, C2-12 malkylamino, C ⁇ - C10 acyl, and C io alkoxy-CO-, and C io alkyl-S-;
• AAi 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, tiyptophan, serine, threonine, cysteine, tyrosine, asparagine, giutamine, aspartic acid, glutamic acid, Iysine, arginine
• AA2 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S- ethylcysteine, S-benzylcystei ⁇ e, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH 2 -2
• novel class of peptide ⁇ -ketoacids also have the following structural formula: Mi-AA-AA-AA-CO-OH or a pharmaceutically acceptable salt, wherein
• Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-;
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ o fluoroalkyl, C ⁇ io alkyl substituted with J, C ⁇ io fluoroalkyl substimted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthy
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ 10 alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C io alkyl-S--,
• K is selected from the group consisting of halogen, C o alkyl, C io perfluoroalkyl, C ⁇ o alkoxy, NO2, CN, OH, CO2H, amino, C io alkylamino, C2-12 dialkylamino, C - C10 acyl, and C ⁇ io alkoxy-CO-, and C ⁇ o 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxy
• novel class of peptide ⁇ -ketoacids also have the following structural formula: Mi -AA-AA-AA-AA-CO-OH or a pharmaceutically acceptable salt, wherein
• Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, Yi-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-:
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substituted with J, C ⁇ io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io al yl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇ io alkyl
• Yl is selected from the group consisting of C2-10 alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substimted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io lkyl w*--- two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ 10 alkyl with two attached phenyl groups substimted with K; J is selected from the group consisting of halogen, CO
• K is selected from the group consisting of halogen, C ⁇ io alkyl, C io perfluoroalkyl, C ⁇ o alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ io alkylamino, C2-12 dialkylamino, Ci- C 0 acyl, and C io alkoxy-CO-, and CUJQ alkyl-S-;
• AA is a side chain blocked or unbiocsed amino acid with the L configuration, D configuration, or no chirality at the ⁇ -carbo:; selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-
• novel class of peptide ⁇ -ketoacids also have the following structural formula: Mi-AA-CO-OH or a pharmaceutically acceptable salt, wherein
• M ⁇ represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, Y2-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-;
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substituted with J, C ⁇ io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇ io alkyl with
• Y2 is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substimted with J, 1-admantyl, 9-fluorenyl, phenyl substituted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K;
• J is selected from the group consisting of halogen, COOH, OH, CN, NC»2, NH2, C 10 alkoxy, C io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C io alkyl-O-CO- NH-, and C io alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl, C ⁇ io alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ io alkylamino, C2-12 dialkylamino, Ci- C10 acyl, and C ⁇ o alkoxy-CO-, and C ⁇ io 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxy
• novel class of peptide ⁇ -ketoesters have the following structural formula: M1-AA2-AA1-CO-O-R1 ) or a pharmaceutically acceptable salt, wherein
• Mi represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-;
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl 5 substimted with J, C ⁇ io fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstituted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 m ' alkyla ine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C ⁇ io alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ io al y 1 - C ⁇ io perfluoroalkyl, C io alkoxy, NO2, CN, OH, CO2H, amino, C io alkylamino, C2-12 dialkylamino, C - C10 acyl, and C io alkoxy-CO-, and C io alkyl-S-;
• AAi 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha- aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, - homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid
• AA2 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, giutamine, aspartic acid, glutamic acid, Iysine, 5 arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methyl
• Rl is selected from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, and C ⁇ 20 alkyl with an attached phenyl group substimted 5 with K.
• novel class of peptide ⁇ -ketoesters also have the following structural formula: M!-AA-NH-CHR2-CO-CO-O-R or a pharmaceutically acceptable salt, wherein Mi represents H, NH2-CO-.
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substituted with J, C ⁇ io fluoroalkyl substimted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- N ⁇ -, and C ⁇ io alkyl-S-; K is selected from the group consisting of halogen, C ⁇ io alkyl. C io perfluoroalkyl,
• 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.
• R is selected from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, and C ⁇ 20 alkyl with an attached phenyl group substimted with K.
• the novel class of peptide ⁇ -ketoesters also have the following structural formula:
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io lk l substimted with J, C ⁇ io fluoroalkyl substimted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ 10 alkyl with an attached phenyl group
• T is selected from the group consisting of C io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substimted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C2-10 alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K;
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C ⁇ io alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl, C ⁇ io alkoxy, NO2, CN, OH, CO2H, amino, C ⁇ io alkylamino, C2-12 dialkylamino, Ci- C10 acyl, and C ⁇ io alkoxy-CO-, and C io 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine,
• R is selected from the group consisting of H, C2-20 alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, and C ⁇ 20 alkyl with an attached phenyl group substimted with K.
• the novel class of peptide ⁇ -ketoesters also have the following structural formula: M3-AA-AA-NH-CHR2-CO-CO-O-R or a pharmaceutically acceptable salt, wherein
• M 3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X- O-CS-;
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substimted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C ⁇ io dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C ⁇ o alkyl-S-; K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl,
• 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxy
• R is selected from the group consisting of H, C ⁇ 20 alkyl, C 1.20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, and C ⁇ 20 alkyl with an attached phenyl group substimted with K.
• novel class of peptide ⁇ -ketoesters also have the following structural formula: M3-AA4-AA-AA-AA-CO-O-R or a pharmaceutically acceptable salt, wherein
• M 3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X- O-CS-;
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substimted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C io alkyl-S-; K is selected from the group consisting of halogen, C ⁇ io alkyl, C ⁇ io perfluoroalkyl,
• 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, giutamine.
• aspartic acid glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine.
• AA4 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2 -carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylser
• R is selected from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, and C ⁇ 20 alkyl with an attached phenyl group substimted with K.
• novel class of peptide ⁇ -ketoesters also have the following structural formula: Mi-AA-CO-O-R or a pharmaceutically acceptable salt, wherein
• M! represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X 2 N-CO-, X-NH-CS-, X 2 N-CS-, X-NH-SO2-, X 2 N-SO2-, Y-CO-, X-CS-, X-SO2-, X-O-CO-, or X- O-CS-;
• X is selected from the group consisting of C ⁇ io alkyl, C ⁇ io fluoroalkyl, C ⁇ io alkyl substimted with J, C ⁇ io fluoroalkyl substituted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substimted with K, phenyl disubstimted with K, phenyl trisubstimted with K, naphthyl, naphthyl substimted with K, naphthyl disubstimted with K, naphthyl trisubstimted with K, C ⁇ io alkyl with an attached phenyl group, C ⁇ io alkyl with two attached phenyl groups, C ⁇ 10 alkyl with an attached phenyl group substimted with K, and C ⁇ io alkyl with two attached phenyl groups substimted with K, C ⁇ io alkyl with an attached phenoxy group, and C ⁇
• J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C ⁇ 10 alkoxy, C ⁇ io alkylamine, C2-12 dialkylamine, C ⁇ io alkyl-O-CO-, C ⁇ io alkyl-O-CO- NH-, and C ⁇ 10 alkyl-S-;
• K is selected from the group consisting of halogen, C ⁇ o alkyl, C ⁇ io perfluoroalkyl, C ⁇ o alkoxy, NO2, CN, OH, CO2H, amino, C io alkylamino, C2-12 dialkylamino, Ci- C10 acyl, and C io alkoxy-CO-, and C io 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, giutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenyiglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxy
• R is selected from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, and C ⁇ 20 alkyl with an attached phenyl group substimted with K.
• 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) omitting the Blue-Sepharose step.
• Calpain II from rabbit muscle and cathepsin B were purchased from Sigma.
• Papain was purchased from Calbiochem.
• Assay of Inhibitory Potency Peptide ⁇ -ketoamides were assayed as reversible enzyme inhibitors.
• Various concentrations of inhibitors in Me2SO 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 Kj values were determined by either Lineweaver-Burk plots or Dixon plots.
• a 0.1 M Hepes, 0.5 M NaCl, pH 7.5 buffer was utilized for human leukocyte elastase
• HLE porcine pancreatic elastase
• PPE porcine pancreatic elastase
• cathepsin G cathepsin G.
• a 0.1 Hepes, 0.01 M CaC-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 KH2PO4, 12 mM Na2HPO4, 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 CaCl2, 10 mM mercatoethanol, pH 7.2 buffer was utilized for calpain I and calpain ⁇ .
• 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 0979); incorporated herein by reference].
• Human leukocyte cathepsin G and chymotrypsin A ⁇ were assayed with Suc- Val-Pro-Phe-NA [Tanaka et al., Biochemistry 24, 2040-2047 0985); incorporated herein by reference].
• Papain was assayed with Bz-Arg- AMC or Bz-Arg-NA [Kanaoka et al., Chem. Pharm. Bull.25, 3126-3128 (1977); incorporated herein by reference].
• the AMC (7-am ⁇ c ⁇ 4-methylcoumarin) release was followed fluorimetrically (excitation at 380 nm, and emmision at 460 nm).
• Cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and Kirschke, Methods Enzymol.
• Platelet membrane permeability assay Calpain-mediated breakdown of spectrin was measured by quantitative densitometry of the calpain-specific 150/155 kDa spectrin fragment doublet [see Siman et al., Proc. Na . Acad. Sci. USA 81, 3572-3576 (1984)]. Platelets were isolated by a modification of the method of Ferrell and Martin [7. Biol. Chem. 264, 20723- 20729 (1989)]. Blood (15-20 ml) was drawn from male Sprague-Dawley rats into 1/lOth volume of 100 mM EDTA-citrate, and ⁇ ntrifuged 10 minutes at 2000 rpm in a clinical centrifuge at room temperature.
• the plasma was resuspended in 15 ml of buffer 1 (136 mM NaCl, 2.7 mM KC1, 0.42 mM Na ⁇ PO 12 mM NaHCO3, 2 mM MgCb, 2 mg ml BSA (Sigma), 5.6 mM glucose, 22 mM Na3citrate pH 6.5) and platelets were isolated at 2200 rpm at room temperature for 10 minutes. Platelets were washed once in 15 ml buffer 1, then resuspended to 10?
• buffer 1 136 mM NaCl, 2.7 mM KC1, 0.42 mM Na ⁇ PO 12 mM NaHCO3, 2 mM MgCb, 2 mg ml BSA (Sigma), 5.6 mM glucose, 22 mM Na3citrate pH 6.5
• platelets were reisolated at 14,000 rpm for 10 sec in a Beckman microfuge, dissolved in SDS- PAGE sample buffer, and heated to 90 °C for 3 minutes.
• Z-Ala-Ala-Abu-CO-OBzl is a potent inhibitor of elastases, and replacement of the Z group (PhCH2OCO-) by PI1CH2CH2CO-, PI1CH2CH2SO2-,
• PI1CH2NHCO-, and PI1CH2NHCS- would result in good inhibitor structures.
• Changing the R group of Z-Ala-Ala-Abu-CO-OR from ethyl to benzyl or/.-trifluoromethylbenzyl results in equally potent inhibitors of HLE.
• replacement of ethyl by benzyl group in Z-Ala- Ala-CO-OEt makes a better elastase inhibitor.
• Amino acid and peptide ketoesters with Phe in the P site are good inhibitors of chymotrypin and cathepsin G.
• MeO-Suc-Val-Pro-Phe-CO- OR is a potent inhibitor of chymotrypsin and cathepsin G, and replacement of methoxysuccinyl group by Z, benzoyl, PhCH2CH2SO2-, PhC ⁇ NHCO-, or PhC ⁇ NHCS- would result in good inhibitors for chymotrypsin and cathepsin G.
• Table II shows the inhibition constants (Ki) for trypsin, plasmin, and several blood coagulation enzymes. Amino acid and peptide ketoesters with Arg or Lys in the P site are good inhibitors of trypsin, although they inhibit blood coagulation enzymes less potently.
• Bz- Arg-CO-OEt is a better thrombin inhibitor than Bz-Lys-CO-OEt, and tripeptides such as D-Phe- Pro-Arg-CO-OEt and Boc-D-Phe-Pro-Arg-CO-OEt are expected to be potent thrombin inhibitors because the interactions between the enzyme and inhibitor increase.
• H-Gly-Lys-CO-OEt inhibits thrombin better than Bz-Lys-CO-OEt, but this dipeptide ketoester is a less potent inhibitor for human plasma kallikrein. Therefore variation of the blocking group and amino acid sequence in the peptide ketoesters would result in the more specific inhibitors toward individual coagulation enzymes.
• Tables III and IV shows the inhibition constants (K ) for papain, cathepsin B, calpain I, and calpain ⁇ .
• Dipeptide ketoesters with Abu, Phe, or Nle in the P site and Leu in the P2 site are potent inhibitors of calpain I and calpain II.
• Z-Leu- Abu-CO-OEt is a better inhibitor of calpain than Z-Ala-Ala-Abu-CO-OEt by 500-1250 fold.
• Replacement of the Z group (PhCH2 ⁇ CO-) by similar groups such as PhCH2CH2CO-, PhCH2CH2SO2-, PhOr ⁇ NHCO- , and PhCH2NHCS- would also result in good inhibitor structures.
• R group Extending the R group to include longer alkyl groups or alkyl groups substimted with phenyl groups would increase the membrane permeability of this inhibitor.
• Dipeptide and tripeptide ketoesters with small aliphatic amino acid residue or Phe in the Pi site are also good inhibitors for papain and cathepsin B.
• MeO-Suc-Val-Pro-Phe-CO-OMe are potent inhibitors of cathepsin B, and replacement of the Z (PhCH2OCO-) or MeO-Suc- group by PhO ⁇ O ⁇ CO-, PhCH 2 CH 2 SO2-, PhQ ⁇ NHCO-, and PhCH2NHCS- would also result in good inhibitor structures.
• Z-Ala-Ala-Abu-CO-OBzl inhibits papain ca. 30 fold less potently than Z-Ala-Ala-Abu-CO-OEt , thus changing the benzyl group to a smaller alkyl group such as methyl, or propyl would make better papain inhibitors.
• Table IV shows the inhibition constants (Kj) for cathepsin B, calpain I, and calpain II with peptide ketoamides.
• Dipeptide ⁇ -ketoamides with Abu and Phe in the Pi site and Leu in the P2 site are potent inhibitors of calpain I and calpain H Z-Leu- Abu-CONH-Et is a better inhibitor of calpain I than Z-Leu-Phe-CONH-Et by 14 fold.
• Replacement of the Z group (PhCH2OCO-) by similar groups such as PhO ⁇ O bCO-, PI1CH2CH2SO2-, PhO ⁇ NHCO- , and PhCH2NHCS- would also result in good inhibitor structures.
• the best inhibitor of calpain II is Z-Leu-Abu-CONH-(CH2)2-Ph- Changing the R3 and R4 groups significantly improves the inhibitory potency toward calpain H
• the best dipeptide inhibitors are those which have long alkyl side chains (e.g. Z-Leu-Abu-CONH-(CH2)7CH3), alkyl side chains with phenyl substituted on the alkyl group (e.g. Z-Leu-Abu-CONH-(CH2)2-Ph), or alkyl groups with a morpholine ring substituted on the alkyl group [e.g.
• Peptide ⁇ -ketoamides and peptide ketoamides were substantially more stable in both plasma and liver than the corresponding peptide ⁇ -ketoesters (Table IV).
• the peptide ⁇ - ketoamides and ketoacids were also much more effective in the platelet assay. Extending the R3 group to an alkyl group or an alkyl group substimted with a phenyl group increased the membrane permeability of the inhibitors as indicated by increased potency in the platelet assay.
• Inhibition Mechanism A crystal structure of one ⁇ -ketoester bound into the active site of porcine pancreatic elastase has been completed and a schematic drawing of the interactions observed is shown below.
• the active site Ser- 195 oxygen of the enzyme has added to the carbonyl group of the ketoester to form a tetrahedral intermediate which is stabilized by interactions with the oxyanion hole.
• This structure resembles the tetrahedral intermediate involved in peptide bond hydrolysis and proves that ⁇ -ketoesters are transition-state analogs.
• His-57 is hydrogen bonded to the carbonyl group of the ester functional group, the peptide backbone on a section of PPE's 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 Pj amino acid residue is located in the Si pocket of the enzyme.
• ketoamides Interactions with ketoamides would be similar except for that there would be the possibility of forming an additional hydrogen bond with the NH group of the ketoamide functional group if R3 or R4 was H. If R3 and/or R4 arc longer subsmments, then they would make favorable interactions with the S' subsites of the enzyme. In the case of ketoacids, there would be no R group to interact with the S' subsites and thes ketoamides.
• cysteine proteases share 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 keto acids, keto esters, or ketoamides to form an adduct very similar to the structure depicted 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 which would increase the binding affinity and specificity of the inhibitors.
• the peptide and amino acid ⁇ -ketoester, ⁇ -ketoacid, and ⁇ -ketoamide derivatives bind to the enzymes using many of the interactions that are found in complexes of a particular individual enzyme with its substrates.
• R group of the inhibitor can be tailored to interact with the S subsites of the enzyme.
• R branched alkyl groups
• the M group can be tailored to interact with the S subsites of the enzyme.
• Elastase is an enzyme which hydrolyzes most effectively tetra- and tripeptides having ? ⁇ residues with small alkyl side chains such as Ala and Val.
• Suc-Phe-Leu-Phe-NA is an excellent substrate for chymotrypsin, cathepsin G, and mast cell chymases.
• the corresponding ⁇ -ketoester is an excellent inhibitor for these chymotrypsin-like enzymes.
• cysteine protease calpain a good inhibitor sequence is Ac-Leu-Leu-Nle-H.
• ketoesters related in structure such as Z-Leu- Abu-CO-OEt and Z-Leu-Nle-CO-OEt arc potent inhibitors for calpain.
• ketoamides related in structure such as Z-Leu-Abu-CO-NR3R4 and Z- Leu-Phe-CO-NR3R4 are potent inhibitors for calpain.
• the following structures are predicted to be potent inhibitors for the listed enzymes.
• the inhibitor sequences were obtained from peptide substrate and or inhibitor sequences in the protease literature.
• Q-R is -NR3R4 and R3 & R4 are selected independently from the group consisting of H, C ⁇ 20 alkyl, C ⁇ 20 cyclized alkyl, C ⁇ 20 alkyl with a phenyl group attached to the C ⁇ 20 alkyl, C 20 cyclized alkyl with an attached phenyl group, C ⁇ 20 alkyl with an attached phenyl group substimted with K, C 20 alkyl with an attached phenyl group disubstimted with K, C ⁇ 20 alkyl with an attached phenyl group trisubstimted with K, C ⁇ 20 cyclized alkyl with an attached phenyl group substimted with K, C ⁇ IQ alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C io alkyl with a piperidine ring attached through nitrogen to the alkyl, C io al y with a pyrrolidine ring attached through nitrogen to
• novel compounds of this invention are effective in the prevention of unnecessary proteolysis caused by chymotrypsin-like, elastases, and trypsin-like enzymes in the process of purification, transport and storage of peptides and proteins as shown in Tables I, ⁇ , IE, and IV by effective inhibition of chymotrypsin, elastase, trypsin, and other serine & cysteine proteases.
• Effective inhibitors of the proteolytic function of human leukocyte elastase and human cathepsin G would have anti-inflammatory activity and can be used to treat and control emphysema, adult respiratory distress syndrome and rheumatoid arthritis.
• Effective inhibitors of the proteolytic function of chymotrypsin and pancreatic elastase are effective for therapeutic use in treatment of pancreatitis.
• ⁇ -ketoesters have anticoagulant activity as shown in Table ⁇ by effective inhibition of the proteolytic function of blood coagulation enzymes in Hepes buffer.
• Other peptide ⁇ -ketoesters have anti-tumor activity as shown in Table H by the effective inhibition of the proteolytic function of human plasma plasmin.
• Peptide ⁇ -ketoesters can be used to control protein turnover, muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption as shown in Tables m and TV by effective inhibition of lysosomal cathepsin B in buffer. Peptide ⁇ -ketoesters can also be used as neuroprotectants or for the treatment of ischemia, stroke or Alzheimer's disease as shown in Tables HI and IV by effective inhibiton of calpain I and calpain ⁇ .
• Pulmonary emphysema is a disease characterized by progressive loss of lung elasticity due to the destruction of lung elastin and alveoli.
• the destructive changes of lung parentchyma associated with pulmonary emphysema are caused by uncontrolled proteolysis in lung tissues [Janoff, Chest 83, 54-58 (1983); incorporated herein by reference].
• a number of proteases have been shown to induce emphysema in animals [Marco et al., Am. Rev. Respir. Dis. 104, 595-598 (1971); Kaplan, /. Lab. Clin. Med.
• Leukocyte elastase and other mediators of inflammation also appear to play a role in diseases such as mucocutaneous lymph node syndrome [Reiger et al., Eur. J. Pediatr. 140, 92-97 (1983); incorporated herein by reference] and adult respiratory distress syndrome [Stockley, Clinical Science 64, 119-126 (1983); Lee et al.,N. Eng. J. Med. 304, 192-196 (1981); Rinaldo, ibid 301, 900-909 (1982); incorporated herein by reference].
• novel inhibitors described here should be useful for the treatment of emphysema and inflammation.
• Elastase inhibitors have been used orally, by injection, or by instillation in the lungs in animal studies (Powers, Am. Rev. Respir. Dis., 127, s54-s58 (1983); Powers and Bengali, Am. Rev. Respir. Dis. 134, 1097-1100 (1985); these two articles are incorporated herein by reference).
• the inhibitors described above can be used by any of these routes.
• the peptide ⁇ -ketoesters, ⁇ -ketoamides, and ⁇ - ketoacids may be administered orally, topically or parenterally.
• parenteral includes subcutaneous injection, intravenous, intramuscular, intrastemal injection or infusion
• the dosage depends primarily on the specific formulation and on the object of the therapy or prophylaxis.
• the amount of the individual doses as well as the administration is best determined by individually assessing the particular case.
• compositions containing the active ingredient may be in a form suitable for oral use, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs.
• Dosage levels of the order to 0.2 mg to 140 mg per kilogram of body weight per day are useful in the treatment of above-indicated conditions (10 mg to 7 gms per patient per day).
• the amount of active ingredient that may be combined with carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• the therapeutic amount of the peptide ⁇ -ketoesters, ⁇ -ketoamides, and ⁇ - ketoacids or their pharmaceutically acceptable salts will normally be in the dosage range from 0.2 to 140 mg/kg of body weight Administration is made by intravenous, intramuscular or subscutaneous injection.
• pharmaceutical compositions for parenteral administration will contain in a single dosage form about 10 mg to 7 gms of the compounds per dose.
• these pharmaceutical compositions will usually contain a buffer, e.g. a phosphate buffer which keeps the pH in the range from 3.5 to 7 and also sodium chloride, mannitol or sorbitol for adjusting the isotonic pressure.
• a composition for topical application can be formulated as an aqueous solution, lotion, jelly or an oily solution or suspention.
• a composition in the form of an aqueous solution is obtained by dissolving the compounds of this invention in aqueous buffer solution of pH 4 to 6.5 and if desired, adding a polymeric binder.
• An oily formulation for topical application is obtained by suspending the compounds of this invention in an oil, optionally with the addition of a swelling agent such as aluminium stearate and or a surfactant.
• ketoester inhibitors 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.
• SUBSTITUTE SHEET The precursor peptide can be prepared using standard peptide chemistry which is well described in publications such as The Peptides. Analysis, Synthesis, Biology, Vol. 1-9, published in 1979-1987 by Academic Press and Houben-Weyl Methoden der Organischen Chemie, Vol. 15, Parts 1 and 2, Synthese von Peptiden, published by Georg Thieme Verlag, Stuttgart in 1974 (both references incorporated herein by reference).
• the Mi group can be introduced using a number of different reaction schemes. First it could be introduced directly on an amino acid as shown in the following scheme (top), or the Mi group could be introduced by reaction with an amino acid ester, followed by removal of the ester group to give the same product (bottom).
• Reaction with a substimted alkyl or ary 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-SO2-CI would introduce the X-SO2- group.
• reaction with MeO-CO-CH2CH2-CO-Cl would give the X-CO- group where X is a C2 alkyl substimted with a Ci alkyl-OCO- group.
• Reaction with an a substimted alkyl or aryl suifonyl chloride would introduce an X-SO2- group.
• reaction with dansyl chloride would give the X-SO2- derivative where X was a naphthyl group mono substimted with a dimethylamino group.
• Reaction with a substimted alkyl or aryl chloroformate would introduce a X-O-CO- group.
• Reaction with a substimted alkyl or aryl chlorothiofor ate 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 Mi-AA-AA-OH, M -AA- AA-AA-OH, or Mi -AA-AA-AA-OH which could be used in the Dakin-West reaction.
• the substimted peptides Mi-AA-AA-OH, Mi-AA-AA-AA-OH, or M-AA-AA- AA-AA-OH could also be prepared directly from H-AA- AA-OH, H-AA-AA- AA-OH, or H- AA-AA-AA- 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 to give Mi-AA-AA- OR', Mi-AA-AA-AA-OR * , or M -AA-AA-AA-AA-OR', followed by the removal of the blocking group R'.
• the R i 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 Mi-AA-AA-OH with ethyl oxalyl chloride Cl-CO-CO-O-Et gives the keto ester M -AA-AA-CO-O-EL
• reaction of Mi-AA-AA-AA-OH with ⁇ -CO-CO-O-Bzl would give the ketoester M AA-AA-AA- AA-CO-O-Bzl.
• R groups can be introduced into the ketoester strucmre by reaction with various alkyl or arylalkyl oxalyl chlorides (Cl-CO-CO-O-R).
• the 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-0-CO-CO-Cl and n-Bu-O-CO-CO-Cl are prepared by reaction of respectively benzyl alcohol and butanol with oxalyl chloride in yields of 50% and 80% [Warren, C. B., and Malee, E. J., J. Chromatography 64, 219-222 (1972); incorporated herein by reference].
• Ketoamides M1-AA-CO-NR3R4, M-AA-AA-CO-M ⁇ R M-AA-AA-AA-CO-l ⁇ R M-AA-AA-AA-CO-NR3R4 were prepared indirectly from the 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-NR3R4. The illustrated procedure should also work with other protecting groups.
• the corresponding ketoacid could be used as a precursor. Blocking the ketone carbonyl group of the ketoacid and then coupling with an amine H-NR3R4 using standard peptide coupling reagents would yield an intermediate which could then be deblocked to form the ketoamide.
• Amino acid methyl ester hydrochlorides were prepared according to M. Brenner et al.[He/v. 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 was synthesized via Schotten-Baumann reaction [M. Bergmann, L.
• 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-Acylami Acid Methyl Esters (General Procedure).
• N-Acyldipeptide methyl esters were synthesized via the HOBt-DCC method in a DMF solution [K ⁇ nig and Geiger. Chem. Ber. 103, 788 (1970)]. 10
• 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 [K ⁇ nig and Geiger, Chem. Ber. 103, 788 (1970)].
• 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
• Z-Leu-DL-Nva-enol ester the precursor of Z-Leu-DL-Nva-COOEt was synthesized by the same procedure as described in Example 1 and purified by column chromatography, oil, one spot on tic.
• Z-Leu-DL-Phe-enol ester the precursor of Z-Leu-DL-Phe-COOEt was synthesized by the same procedure as described in Example 1 and purified by column chromatography, oil, one spot on tic.
• NMR (CDCI3) d: 0.86 (t, 3H); 0.99 (t, 3H); 1.24 (t, 3H); 1.40 (t, 3H); 1.52 (m, 2H); 1.83 (m, 2H); 4.23 (m, 4H); 4.39 (q, 2H); 5.10 (t, 2H); 5.18 (s, IH); 7.26 (m, 5H); 7.34 (m, 5H); 8.89 (s, IH).
• EXAMPLE 19 EXAMPLE 19
• Z-Leu-DL-Abu-enol ester the precursor of Z-Leu-DL-Abu-COOEt was synthesized by the same procedure as described in Example 1 and purified by column chromatography, oil, one spot on tic.
• EXAMPLE 20 Ala-DL-Lys-COOEt-HCl.
• N-carbobenzyloxyalanyl-Ne- carbobenzyloxylysine (1.88 g, 3.9 mmol)
• 4-dimethylaminopyridine 21 mg, 0.17 mmol
• pyridine 1.0 mL, 12.4 mmol
• THF 7 mL
• ethyl oxalyl chloride 0.9 mL, 8.0 mmol
• the mixture was extracted with ethyl acetate (150 ml) and after separation of the organic layer, the water layer was saturated with solid (NH4)2SO4 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 MgSO4. After evaporation of the solvent the crude enol ester (8.36 g, 98%) was flash-chromatographed on silica gel and the product was
• reaction mixture was stirred at room temperature for 4-5 hours, the ethanol was then evaporated in vacuo (rotavaporator) and the residue treated with 200 ml ethyl ether (or 200 ml ethyl acetate in the case of the tripeptide).
• the ether (ethyl acetate) solution was washed with 2 x 75 ml H,0, 2 x
• EXAMPLE 41 Synthesis ofn-Butyl Oxalyl Chloride. This was prepared by a literature procedure [Warren and Malee, J. Chromat. 64, 219-222 (1972)]. N-Butanol (0.1 mol. 7.41 g) was added dropwise to oxalyl chloride (0.5 mol. 63.5 g) at -10 °C. After the addition was
• EXAMPLE 45 Z-Leu-Phe-COOH.
• Dipeptide Ketoacids General Procedure.
• the color of the reaction mixture turned dark yellow and a small amount of solid was deposited.
• the reaction was ran at room temperature and progress of the hydrolysis was checked on TLC. After 24 h. no more substrate was detected
• the protected ⁇ -ketoester (0.98 g, 1.8 mmole) was dissolved in ethanol (5 ml), cooled to 0-5 °C in a ice bath, and ethylamine was bubbled through the solution until 2.43 g (54 mmole) had been added.
• the reaction mixture was allowed to warm to room temperature slowly, and stirred ovemight.
• the product was a pale yellow solid.
• Example 50
• Example 51 Z-Leu-Phe-CONH-Bzl. This compound was synthesized from the protected ⁇ - ketoester and benzylamine in 40 % yield by the procedure described in Example 47. 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 HC1 (3 x 25 ml), water (1 x 20 ml), saturated sodium chloride (2 x 20 ml), and dried over magnesium sulfate. The solution was evaporated leaving a yellow solid. Chromatography on a silica gel column with CHCI3/CH3OH 30: 1 v/v) afforded a yellow solid.
• Example 54 Z-Leu-Abu-CONH-nPr. This compound was synthesized from the corresponding protected ⁇ -ketoester and propylamine in 47 % yield by the procedure described in Example 47. Single spot on TLC, Rf - 0.28 (CHCI3/CH3OH 50:1); mp 134-135 °C. Anal, calcd. for C22H33N3O5: 419.50; C, 62.98; H, 7.93; N, 10.02. Found: C, 62.84; H, 7.97; N, 9.94. NMR (CDCI3) ok. MS (FAB) m/e « 420 (M+1).
• Example 59 Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)2O. This compound was synthesized from protected ⁇ -ketoester and 4(3-aminopropyl)morpholine in 33 % yield by the procedure described in Example 47. After reacting ovemight, ethyl acetate (80 ml) was added. The mixmre 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 64 Z-Leu-Abu-CONH-(CH2)i7CH3. This compound was synthesized from the corresponding protected ⁇ -ketoester and octadecylamine in 12 % yield by the procedure described in Example 51. The product was a pale yellow
• Example 65 Z-Leu- bu-CONH-CH2-C4H4 .
• This compound was synthesized from the corresponding protected ⁇ -ketoester and 4-(aminomethyl)pyridine in 45 % yield by the procedure described in Example 59.
• NMR (CDCI3) ok.
• MS (FAB) m/e 469 (M+1).
• inhibition constants were measured in 0.05 M Tris-HCl, pH 7.5 buffer , containing 2 mM EDTA, 5 mM cysteine (freshly prepared), 1 % M ⁇ 2SO, and at 25 °C. N ⁇ -Benzoyl-Arg-AMC was used as a substrate.
• Inhibition constants were measured in 88 mM KH 2 PO4, 12 mM Na 2 HPO4, pH 6.0 buffer , containing 1.33 mM EDTA, 2.7 mM cysteine (freshly prepared), and at 25 °C.
• Z-Arg-Arg-AFC was used as a substrate.
• Inhibition constants were measured in 20 mM Hepes, pH 7.2 buffer , containing 10 mM CaCl ,

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