Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acids and salts thereof
Reexamination Certificate
1996-12-27
2001-05-22
Shippen, Michael L. (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acids and salts thereof
C544S168000, C548S334100, C548S496000, C548S535000, C560S024000, C560S032000, C560S038000, C560S040000, C560S041000, C560S045000, C560S125000, C560S159000, C560S169000, C562S449000, C562S561000, C564S153000, C564S157000, C564S159000
Reexamination Certificate
active
06235929
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a novel class of peptide 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 or cysteine proteases. We have discovered that peptide &agr;-ketoamides can be constructed to inhibit selectively individual serine or cysteine proteases or groups of serine or cysteine proteases. We have found that peptide ketoamides which contain hydrophobic aromatic amino acid residues in the P
1
site are potent inhibitors of chymases and chymotrypsin-like enzymes. Ketoamides containing small hydrophobic amino acid residues at the P
1
position are good inhibitors of elastases. Inhibitors of elastases and chymases are useful as anti-inflammatory agents. We show that peptide ketoamides which contain cationic amino acid residues such as Arg and Lys in the P
1
site will be 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 II are also inhibited by ketoamides. Ketoamides with aromatic amino acid residues in the P
1
site are good inhibitors for cathepsin B and papain. Thus, they would have utility as anticancer agents. Ketoamides with either aromatic amino acid residues or small hydrophobic alkyl amino acid residues at P
1
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 and stroke.
2. Nomenclature
In discussing the interactions of peptides with serine and cysteine proteases, we have utilized the nomenclature of Schechter and Berger [
Biochem. Biophys. Res. Commun.
27, 157-162 (1967); incorporated herein by reference]. The individual amino acid residues of a substrate or inhibitor are designated P
1
, P
2
, etc. and the corresponding subsites of the enzyme are designated S
1
, S
2
, etc. The scissile bond of the substrate is S
1
-S
1
′. The primary substrate recognition site of serine proteases is S
1
. The most important recognition subsites of cysteine proteases are S
1
and S
2
.
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—CHR
1
—CO—, where R
1
is the side chain of the amino acid residue AA. A peptide &agr;-ketoester residue would be designated —AA—CO—OR which represents the part structure —NH—CHR
1
—CO—CO—OR. Thus, 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 ketoamide residues would be designated —AA—CO—NH—R. Thus, the ethyl keto amide derived from Z—Leu—Phe—OH would be designated Z—Leu—Phe—CO—NH—Et which represents C
6
H
5
CH
2
OCO—NH—CH(CH
2
CHMe
2
)—CO—NH—CH(CH
2
Ph)—CO—CO—NH—Et.
3. Description of the Related Art
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. In the brain, 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
2+
activated cysteine proteases may exist, and the term “calpain” is used to refer to all Ca
2+
activated cysteine proteases, including calpain I and calpain II. The terms “calpain I” and “calpain II” 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. In 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. Thus, 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]. Activation of 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. These compounds are not especially useful at inhibiting calpain in neural tissue in vivo because they are poorly membrane permeant and, accordingly, are not likely to cross the blood brain barrier very well. Also, many of these inhibitors have poor specificity and will inhibit a wide variety of proteases in addition to calpain. Other classes of compounds which inhibit cysteine proteases include peptide diazomethyl ketone (Rich, D. H., in
Protease Inhibitors,
Barrett A. J., and Salversen, G., Eds., Elsevier, New York, 1986, pp 153-178; incorporated herein by reference). Peptide diazomethyl ketones are potentially carcinogenic and are thought to be poorly membrane permeant and to have low specificity. Thus, no effective therapy has yet been developed for most neurodegenerative diseases and conditions. Millions of individuals suffer from neurodegenerative diseases and thus, there is a need for therapies effective in treating and preventing these diseases and conditions.
Cathepsin B is involved in muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption. In addition, a number of viral processing enzymes, which are essential for viral infection, are cysteine proteases, inhibitors of cysteine proteases would have multiple therapeutic uses.
Serine Proteases
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 artritis, bronchial inflammation and adult respiratory distress syndrome. It has been suggested that a new trypsin-like cellular enzyme (tryptase) is involved in the infection of human immunodeficiency virus type 1 [HIV-1; Hattori et al.,
FEBS Letters
248, pp. 48-52 (1989)], which is a causative agent of acquired immunodeficiency syndrome (AIDS). Plasmin is involved in twnor invasiveness, tissue remodeling, blistering, and clot dissociation. Accordingly, specific and selective inhibitors of these proteases should be potent anticoagulants, anti-infla
Deveau Todd
Georgia Tech Research Corporation
Schneider Ryan A.
Shippen Michael L.
Troutman Sanders LLP
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