CA2098702A1

CA2098702A1 - Peptide ketoamides, ketoacids, and ketoesters

Info

Publication number: CA2098702A1
Application number: CA002098702A
Authority: CA
Inventors: James C. Powers
Original assignee: Individual
Current assignee: Georgia Tech Research Corp
Priority date: 1990-12-28
Filing date: 1991-12-27
Publication date: 1992-06-29
Also published as: AU654834B2; WO1992012140A1; AU9155391A; JPH06504547A; EP0564561A1; EP0564561A4

Abstract

Peptides ketoamides, ketoacids, and ketoesters, their use in inhibiting serine proteases and cysteine proteases.

Description

20,987~2 PEPTIDE KETOA;~IDES, KETOACIDS. AND KETOESTERS

BACKGROUND OF THE INVENTION
1. Field of the Invention sThis invention relates to a novel class of peptide ketoesUrs. peptide ketoæids, and ketoamides useful for selectively inhibiting serine proteases. selectively inhibiting eysteine proteases, generally inhibiting all serine proteases. and generally inhibiting all eysteine 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 10 serine proteases ,or cysteine proteases. We have discovered that~eptide a-ketoesters, peptide a-ketoacids, and a-ketoamides can be constructed to inhibit selectively individual serine or cysteine proteases or groups of serine or cysteine proteases. We have found that peptide ketoesters. ketoacids, and ketoamides whieh contain hydrophobic aromatic amino aeid residues in the P I site are potent inhibitors of chymases and chymotrypsin-like enzymes. Ketoesters.
15 acids, and amides containing small hydrophobie amino aeid residues at the Pl position are good inhibitors of elastases. Inhibitors of elastases and ehymases are useful as anti-inflammatory agents. We have found that peptide ketoesters, amides, and acids which contain cationie amino acid residues such as Arg and Lys in the Pl site are potent inhibitors of trypsin and blood coagulation enzymes. These inhibitors are thus useful as anticoagulants. Cysteine proteases 'O such as papain, cathepsin B, and ealpain I and II are also inhibited by ketoesters. Kctocstc~s, acids, and amides with aromatie arnino aeid residues in the Pl site would be good inhibitors for cathepsin B and papain. Thus, they would have utility as antieaneer agents. Ketoesters, ketoaeids, and ketoamides with either aromatie amino æid residues or small hydrophobie alkyl amino aeid residues at Pl are good inhibitors of ealpain I and II. These inhibitors are useful as 'S neuroproteetants and can be used as therapeutics for the treatment of neurodegeneration.

2. Nomenelature In diseussing the interaetions of peptides with serine and eysteine proteases, we have utilizcd the nomenelature of Schechter and Berger [Biochem. Biophys. Res. Commun. 27, 157-162 (196~); incorporated herein by reference~. The individual amino aeidresidues of a 30 substrate or inhibitor are designated Pl, P2, etc. and the corresponding subsites of the enzyme are designated Sl, S~, etc. The scissile bond of the substra~e is Sl-SI'. The pnmary substra~e reeognition si~e of serine proteases is S 1 The most irnportant recognition subsiles of cysteine proteases are S I and S~.
Amino acid residues and bloeking groups are designated using standard abbreviations ~3 ~se- 1. Biol. Chem. 260, 14-42 ( 1985) for nomenclature rules; incorporated herein by referenee]. An arnino aeid tesidue (AA) in a peptide or inhibitor strueture refers to the part structure -NH-CHR I -CO-. where R I is the side chain of the amino aeid residue AA. A pepude -ketoester residue would be designated -AA-CO-OR which represents the part structure -~
C~l-CO-CO-OR, Thus. the ethyl ketoester derived from benzoyl alanine would be SUBSTITU~E SHEEl WO 92/12140 PCr/US91/Og801 2~

designated Bz-Ala-CO-OEt whch represents COHsCO-~-CHMe-CO-CO-OEt. Likewise, peptide ketoacid rcsidues and pepude ketoamide residues would be designated -AA-CO OH and -AA-CO-NH-R respcctively. Thus. the ethyl keto arnide derived from Z-Lcu-Phe-OH would be designated Z-Leu-Phe-CO-~-Et which represents coHscH2oco-NH-cH(cH~cHMe2) CO-NH-CH(CH2Ph)-CO-CO~ Et.

3. Description of the Related Art Serine Proreoses. Serine proteases play criuical roles in several physiological processes such as digestion, blood coagulation, complement activation, fibrinolysis, viral infedon, fertilizaion, 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 panc~atitis, emphyserna, rheumatoid arthrttis, 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 irnmunodeficiency virus type I ~V- I; Hattori et aL, FEBS
Lerrers 248, pp. 48-52 (1989)], which is a causadve agent of acquired immunodeficieney syndrome (AlDS). Plasrnin is involved in tumor invasiveness, tissue remodeLng, blistering, and clot dissociation. Accordingly, specific and seleetive inhibitors of these proteases should be potent anticoagulants, anti-infl=tory agents, anti-tumor agents and anui-viral agents useful in the treatment of protease-relatet tiseases ~Powers and Harper, Protein~lse Inhibitors, pp S5-152, Barrett ant Salvesen, eds., Elsevier, (1986); ineorporated herein by referenee]. In vitro ~O proteolysis by ehymotrypsin, trypsin or the elastase farnily is a serious problem in the production, purifieation, isolauion. transport or storage of peptides and proteins.
Elastase inhibitors are anti-inflammatory agents which can be uset to treat elastæ-associated inflasnmadon including rheumatoid arthritis and emphysema. Although the naturally occumng protease inhibitor, I-protease inhibitor (al-PI) has been used to ~cat patients with ~5 emphysema, this protein inhibitor is not widely uset clinically due to the high dosage needed for trearment and the tifficulty of produeing large quantities. Therefore small molecular weight elasttse inhibitors are neetet for therapy. Other low molecular weight elastase inhibitors have utility for the tn a~nent of emphysema and inflammation (see: I-carpapenem-3-carboxylie esters as anti-inflamntatory agents, U.S. Patent 4,493,839; N-carboxyl-thienamycin esters and analogs thereof as anti-inflarmnalory agents, U.S. Patent 4.495.197: incorporatet herein by reference).
Anticoagulants and antithrombotic drugs are used in a variety of thrombouic disorders.
The 1990 Physician's Desk Reference lists several anticoagulant drugs (heparin. protamine sulfate and warfarin), a few anaplatelet drugs (aspirin) and several thrombolytic agents.
Heparin and warfarin are commonly used clinically for p~:venrion and treaument of venous thrombosis and pulmonary embolism. Heparin inhibits the blood coagulation activity by accelerating the binding of namral plasma protease inhibitor antithrombin m with coagulation faetors, and warfarin ~cts as a vit~nin K antagonist and inhibits the synthesis of coaguation factors. None of the anticoagulant drugs, an~ithrombotic drugs, fibrinolytic agents and SUBSTITUTE SHEET

WO92/12140 2a~8702 PCr/US9~

anoplatelet drugs are highly effecive in all clinical situa~ions and many induce side reacions [VonKaulla, Burger'sMedicinalCfiemisrrv,Parrll, pp 1081-1132.Wolff,ed..(1979);
incorporated herein by ref=ce]. Coagulation disorders such as disseminated intravascular coagulaion, bleeding complications of medical and surgical procedures and bleeding S complicaions of systemic illness are sill difficult to manage [Ingram, Brozo~ric and Slater, Bleeting D~sorters, pp 1-413, Blackwell Scientific Publicaions, (1982); incorporated heTcin by reference]. In the treatment of paients with coagulaion problerns, anticoagulant or antithromboic agents of diverse mechanisms are urgently sought in o~er to provide better medical care. Inhibitors for the trypsin-like enzymes involved in blood coagulation are useful 10 anoicoagulants in vivo [see for example: H-D-Phe-Pr~Arg CH2Cl, Hanson and Harlcer, Proc.
Narl. Acad. Sci. 85, 3184-3188 (1988); 7-Amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin ~AClllC), Oweida~ Ku, Lumsden, Kam. and Powers, T))rombos. Res. 58, 191-197 (1990); incorporated herein by reference].
Cysreine Proteases. Cysteine proteases such as calpain use a cysteine residue in their 15 catalytic mechanism in contras~ to serine proteases which utilize a serine tesidue. 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 simulated proteases tetmed calpains. Calpains are present in many tissues in addition to the brain. Calpain I is aetivated by micromolar eoncentrations of caleium while calpain II is 20 activated by millimolar eoneentrations. In the brain, calpain n is thc predominant fonn, but calpain I is found at synaptic endings and is thought to be the form involved in long term potentiation, synapic plasticity, and eell death. Other Ca2+ aeivated eysteine proteases may exist, and the term "calpain" is used to refer to all Ca2+ activated cysteine proteases, including calpain I ant calpain 11. The terms "calpain 1" and "calpain II ' are used herein to refer to the '5 micromolar and millimolar aetivated calpains. respectively, as described above. While calpains degrade a wide variety of protein substrates. cytoskeletal proteins seem to be partieulariy suscepible to attack. In some cases, the products of the prouolytic digestion of these proteins by ealpain are distincive and persistent over ime. Since cytoskeletal prouins are mapr components of cerlain types of cells. this provides a simple method of detecting calpain acivity 30 in cells and tissues. Thus, ealpain acivation can be measured indirectly by assaying the proteolysis of the cytoskeietal protein spectrin. which produces a large, disinctive 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~.
Acivation of calpains and/or aecumulation of breakdown products of cytoskeletal elements has 35 been observed in neural tissues of mammals exposed to a wide varjety of neurodegenerative diseases and condiions. For example. these phenomena have been observed following isehetnia in gerbils and rats, following stroke in humans. following administration of the toxins kainate. rrimethyltin or colchicine in rats. and in human Alzheimer s disease.

~UBSTITlITE SHEET

WO 92/12140 PCI'/US91/0g~01 2~9~

Several inhibitors of calpain have beeD descnbed including peptide aldehydes such as Ac-Leu-Leu-Nle-H and leupeptin (Ac-Leu-Leu-Arg-H), as well as epoxysueeinates such as E-64. These compounds are not especially useful at inhibiting calpain in neu~l tissue in vn~o because they are poorly membrane permeant and. aceordingly, are not likely to cross the blood brain barner very well. Also, many of these inhibitors have poor speeifieity and will inhibit a wide variety of proteases in addition to calpain. In addition, other elasses of compounds whieh inhibit cysteine proteases include peptide diazonuthyl ketone (Rieh, D. H, in Proteasc Inhibitors, Barrett A. J., and Salversen, G., Eds., Elsevier, New York, 1986, pp 153-178:
incorporated herein by reference). Peptide diazomethyl ke~ones are potentially carcinogenie and are thought to be poorly membrane permeant and to have low speeifieity. Thus, no effective therapy has yet been developed for most neurodegenerative diseascs and eonditions. Millions of individuals suffer from neurodegenerative discases and thus. there is a need for therapies effective in treating and preventing these diseases and eonditions.
Cathepsin B is involved in museular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption. In addition, a number of viral proeessing enzymes, whieh are essential for viral infeeion. are cysteine proteases. Inhibitors of cysteine proteases would have multiple therapeutic uses.
Ketoesters. A few amino aeid and peptide ketoesters ant ketoaeids have been previously reported. Cornforth and Cornforth [J. Chcm. Soc., 93-96 (19S3); ineorporated ''O herein by refereneel report the synthesis of the ketoaeids PhCH~CO-Gly-CO-OH and Ae-Gly-CO-OH upon hydrolysis of heterocyclic molecules. Charles et al. [J. Chcm. Soc. Perldn 1, 1139-1146 (1980); incorporated herein by referenee~ use ketoesters for the synthesis of bicyelic heterocyeles. They report the synthesis of n-BuCO-Ala-CO-OEL PrCO-Ala-CO-OEL
cyclopentylCO-Ala-CO-OEt. PrCO-PhGly-CO-OEt. and Bz-Ala-CO-OEL Hori et al.
~5 [Pepndes: Srrucrure and Funcr~on-Proceedings of rhe Ninrh Arnencan P~pride Syrnposium (Deber, Hruby, and Kopple, Eds.) Pierce Chemieal Co., pp 819-822 (198S); ineorporated heretn by referenee] ~port Bz-Ala-CO~Et. Bz-Ala-CO-OH. Z-Ala-Ala-Abu-CO-OEL Z-Ala-Ala-Abu-CO-OBzl. and Z-Ala-Ala-Ala-Ala-CO-OEt (Abu = 2-aminobutanoie acid or a-aminobutyrie acid) and report that these compounds inhibit elastase. Trainer [Trends Pharm.
Sci. 8,303-30~ (1987); incorporated herein by reference] comments on one of thiscompounds. Bur~hart. J.. Peet. .~T. P., and Bey, P. [Terrahedron Lerr. 29. 3433-3436 (1988);
incorporated herein bv reterence] report the synthoeis of Z-Val-Phe-C~OMe and Bz-Phe-CO-OMe.
.Uehdi et al. [Biochem. Biophys. Res. Comm. 166. 595-600 (1990); incorporated 35 herein by reference] report the inhibition of human neucrophil elastase and cathepsin G by peptide a-ketoesters Angelastro et al.. [J. Med. Chem. 33. 13-16 (1990): ineorporated herein by referencel repor~ some a-ketoesters which are inhibi~ors of calpain and chymot~ypsin. Hu and Abeles ~Arch. Biochem. Biophvs. 281, 271-274 (1990): incorporated herein by ref=ce]
repon some pcptityl a-ketoamides and a-ketoaeids which are inhibitors of cathepsin B and SUBSTlTUTE SHEET

2 0 9 ~ ~ 0 2 papain. Peet et al. ~J. Med. Chem. 33, 394-407 (1990); incorporated herein by refesencel report some peptidyl a-ketoesters which are inhibitors of porcine panaeatic clastase. human neutrophil elastase, and ra~ & human neutsophil cathepsin G.
f~etoarnudes. A single peptide ketoamide is reported in the literature by Hu and Abeles [Arch. Biochem. Biopkvs. 281, 271-274 (1990)]. This compound Z-Phe-NHCH2CO-CO-NH-Et or Z-Phe-Gly-CO-NH-Et is reported to be an inhibitor of papain (Kl = 1.5 ~LM) and cathepsin B (KI = 4 ~lM).

SUMMARY OF T~ ~NVENTION
We have discovered that peptide and amino acid a-ketoester, -ketoacid, and a-ketoamide derivatives are a novel group of inhibitots for serine pTOteaSes and cysteine proteases. Inhibitors are compounds that reduce or eliminate the catalytic activity of the enzyme. We have discovered that peptide and amino acid a-ketoester, a-ketoacid, and -ketoamide derivatives, which have an amino acid sequence similar to that of good substrates for a particular protease, are good inhibitors for that ptotease. Thus, we are able to predict the structure of new inhibitors for other serine and cystehe proteases based on knowledge of their substrate specificities.
We have tiscovered some peptide and amino acit a-ketoester, a-ketoacid, and -ketoamide derivadves which are specific inhibitors for trypsin, elastase, chymotrypsin, ~0 granzymes, and other serine proteases, and some of the derivadves which ase genesal inhibitoss for groups of serine proteases. l;ypsin and trypsin-like enzymes norsslally cleave pepdde bonds in proteins and peptides where the amino acid residue on the carbonyl side of the split bond (Pl residue) is Lys or Arg. Peptide and amino acid a-ketoester. a-ke~oacid, and a-ketoamide derivatives which have Lys or Arg at Pl are thus good inhibitors for these enzymes.
'5 Elastase and elastase-like enzymes cleave peptide bonds where the Pl amino acid is Ala, Val, Ser, Leu and other similar amino acids. Inhibitors with these sesidues at Pl are good elastase inhibitors. Chymotrypsin and chymotrypsin-like enzyrnes hydrolyze pepdde bonds where P
amino acid is Trp, Tyr, Phe, Met. Leu or other amino acid residues which contain aromatic or large allcyl side chains. Inhibitors with these residues at Pl are good chymotrypsin and chymase inhibitoss. All of the above enzymes have e1~tensive secondary specificity and recognize amino acid residues semoved from the Pl residue.
The new psotease inhibi~ors, especially the elastase inhibitors. trypsin inhibitoss, and chymase inhibitors are useful for controlling issue damage and various inflammatory condidons mediated by proteases such as blistering. The inhibitors for blood coagulation enzymes are useful andcoagulants and could be used to treat thsombosis.
The pepdde and amino acid a-ketoester. a-ketoacid. and a-ketoamide derivadves are also useful in vi-ro for inhibidng Irypsin, elastase. chymotrypsin and other sesine proteases of si nilar specificity, and for inhibiting serine proteases in general. The inhibitors can be used to identify new pToteolydc enzymes encountered in research. l~ey can also be used in research SUBSTI, UTE SHEET

WO 92/12140 Pcl/US91/O~X

~ 6-and industrially to preven~ undesired proteolysis that occurs during the production. isolat~, purificauon. transport and storage of valuable peptides and proteins. Such proteolysis often destroys or alters the activity and/or function of the peptides and proteins. I~ses would include the addition of the inhibitors to antibodies, enzymes, plasma proteins, tissue e%tracs or other 5 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.
rhe peptide and arnino acid -ketoester, a-ketoacid, and a-ketoarnide derivattves are also novel and potent inhibitors of cysteine proteases including calpains, cathepsin B, and 10 papain. The calpain inhibitors are useful for treatment of various neurodegenerative diseases and conditions, including ischemia, stroke, and Alzheirner's disease.

DEI A~LED DESCRI~l'ION OF THE lNVENTION
Pepide a-ketoesters, pepide a-ketoacids, and peptide a-ketoamides are transition state 15 analog inhibitors for serine proteases and cysteine proteases. Pepide ketoesters containing hydrophobic arnino acid residues in the Pl site have been found to be excellent inhibitors of several serine proteases including hurnan leukocyte elastase, porcine pancreatic elastase, human leukocyte cathepsin G, and bovine chymotrypsin. Peptide ketoesters containing amino acid residue with cationic side chain h the Pl site have been found to be excellent hhibitors of 20 several serme proteases hcluding bovine t~ypsin, bovhe thrombin, human plasma kallLlcrein, porcine pancreatic kallikrein, human factor XIa and human plasmin. Peptide ketoesters containing amino acid residues with hydrophobic side chain at the Pl site have also been found to be excellent inhibitors of several cysteine proteases including papain. cathepsin B and calpaun. These st uctures may be used in vivo to treat diseases such as emphyserna. adult '5 respiratory distress syndrome, rheumatoid arthritis and pancreatitis which result from uncontrolled proteolysis by elastase. chymotrypsin. tTypsin and related serine proteases. These inhibitors may be used in vitro to prevent proteolvsis which occurs in the process of production. isolation, puri~lcation. storage or tTansport of peptides and pro~eins. These inhibisors may be useful as therapeuoc agents for tTeatment of neurodegeneration. viral 30 infections, muscular dystrophy, myocardial tissue damage, tumor metastasis. and bone resorption.
The novel class of peptide tt-ketoamides have the following slructural formula:
~ I -AA-NH-CHR~-C~CO-NR3R4 or a phannaceutically acceptable salt. wherein i5 ~I represents H, NH2-CO-. NH2-CS-. NH2-SO~-, X-NH-CO-. X~N-CO-.
X-NH-CS-, X~N-CS-. X-NH-S02-, X~N-SO2-, X-CO-. X-CS-, X-S02-, X-O-CO-. or X-O-CS-;
X is select~d from the gTOUp consisting of Cl 10 alkyl, Cl lo fluoroalkyl, Cl lo alkyl substituted with J. C l 10 fluoroalkyl substituted with J, l-admantyl, 9-fluorenyl, phenyl.

SUBSTITUTE SHEET

20!~7(3~

phenyl substituted with K. phenyl disubstituted with K, phenyl trisubsutuud with K. naphthyl, naphthyl substituted with K. naphthyl disubstitutcd with K. naphthyl trisubstituted with K, C l lo alkyl with an attached phenvl group, C l l o alkyl with two attached phenyl groups, C
lo alkyl with an attached phenyl group substi~uted with K. Cl.lo alkyl with two attached 5 phenyl groups substituted with K. Cl lo aL~cyl with an attached phenoxy group, and Cl lo 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
all~oxy, Cl lo alkylamine, C2-l2 dialkylamine, Cl 10 alkyl-O-CO-, Cl lo alkyl-O-CO-~H-, and C 1-10 alkyl-S-;
K is selected from the group consisting of halogen, C 1-10 alkyl, C 1-10 perfluoroal~yl, Cl lo allcoxy, N02, CN, OH, C02H. amino, Cl lo aLI~ylamino, C2 12 diallcylamino, Cl-Clo acyl, and Cl lo alkoxy-CO-. and Cl lo allcyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configurauon, D
configuration, or no chiraliy at the -carbon seleeted from the group consisting of alanine, IS valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanhe, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid.
glutamic acid, Iysine. arginine. hisudine, phenylglyeine. beta-alanine, norleucine, norvaline.
alpha-aminobuyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarghhe, sareosine, indolhe 2-earboxylie acid, 2-azetidineearboxylic acid, pipeeolinic '0 acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methyleystehe, S-ethylcysteine. S-benzyleysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoie acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH. NH2-CH(CH2-cyelohexyl)-COOH. NH2-CH(CH2-eyelopentyl)-COOH. NH2-CH(CH2-cyelobutyl)-COOH, NH2-CH(CH~-eyelopropyl)-COOH. trifluoroleuche, and hexafluoroleucine:
~5 R2 is selected from the group consisthg of C 1-8 branched and unbranched allcyl, C 1-8 branched and unbranched cyclizcd alkyl, and C1 8 branched and unbranched fluoroalkyl;
R3 and R4 are selected independently from the group consisting of H. C 1-20 alkyl, C I
20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the Cl -20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, Cl 20 alkvl with an atuched phenvl group substituted with K. C1 20 alkyl with an attached phenyl group disubstituted with K. C1 20 aLlcyl with an anaehed phenyl group trisubstituted with K. C 1-20 cyclized alkyl with an artaehed phenyl group substituted with K. Cl lo alkyl with a morpholine ~-N(CH2CH2)0] ring attached through nitrogen to the alkyl. C I l o allcyl with a plperidine ring attached through nitrogen to the aLt~yl, Cl lo aL~vl with a pyrrolidine ring attached through nitrogen to the aL~cyl, Cl 20 iS aL~cyl with an OH group attached to the alkyl. -CH~CH~OCH~CH~OH. Cl lo with an attached ~pyridyl group, C 1-10 with an a~tached 3-pyridyl group, C I 10 with an a~tached 2-pyridyl group, C l lo with an attached cyclohexyl group. -.~H-CH~CH~-(4-hvdroxyphenyl), and -~H-CH~CH2-(3-indolyl).
The novel class of peptide -ketoamides also have the following structural formula:

SVBSTITUTE SH~ET

WO 92/12140 PCT/USg1/09801 ~,~9~

.U~ CO-NR3E~4 or a pharmaceuucally acceptable salt. wherein .~11 represents H. ~iH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-. X~N-CO-, X-NH-CS-, X2N-CS-, X~ S02-, X2N-S02-, X-CO-, X-CS-. X-SO~-, X-O-CO-, or X-S O-CS-;
X is selected from the group consisting of C l lo al~yl, C l l o fluoroallcyl, C l lo alkyl subsntuted with J, Cl lo fluoroal~yl substituted with J, l-admantyl, 9-fluorenyl, phenyl, pheDyl substituted with K. phenyl disubstituted with K, phenyl ttisubstituted with K, naphthyl, naphthyl subsututed with K, naphthyl disubstituted with K, naphthyl trisubstituted with K.
CI 10 alkyl with an attached phenyl group, CI-10 alkyl with two attached phenyl groups, C1-lo alkyl with an attached phenyl group substituted with K, Cl lo al~yl with two attached phenyl groups substituted with K, Cl lo alkyl with an attached phenoxy group, and Cl lo al~yl with an attached phenoxy group substituted with K on the phenoxy gTOUp;
J is selected from the group consisting of halogen, COOH. OH. CN, NO2, NH2, C
10 alkoxy, C I -10 aLlcylamine, C2- 12 dialkylamine, C 1-10 alkyl-O-CO-. C I -10 alkyl-O-CO-NH-, and C 1-10 a~kyl-S-:
K is selected from the group consisting of halogen, C 1-10 alkyl, C l l o perfluoroalkyl, C 1-10 alkoxy, NO ~, CN. OH. CO2H, amino, C 1-10 al~ylamino, C2- 1~ diallcylamino, C 1-Clo ac~i. and Cl lo al~oxy-CO-, and Cl lo alkyl-S-;
AA I is a side chain blocked or unblocked amino acid with the L configuration, Dconfiguration, or no chirality at the c~-carbon selected from the group consisthg of alanine, valine, Ieucine, isoleucine, proline, methionhe, methionine sulfoxide, phenylalanhe, tryptophan. senne, thrconine. cystehe. tyrosine, asparagine, glutamine, aspartic acid, glutamic acid. lysine. arginine, histidine. phenylglycine. beta-alanhe. norleucine. norvaline. alpha-'S aminobutyric acid, epsilon-aminocaproic acid, citrullhe, hydroxyproline. ornithine, homginhe, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid. pipecolinic acid (2-piperidine carboxylic acid), O-methylserine. O-ethylsenne, S-methylcysteine. S-ethylcysteine, S-benzylcysteine. NH2-CH(CH2CHEt2)-COOH. alpha-aminoheptanoic acid, ~2-CH(CH2- 1 -napthyl)-COOH. NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH. NH2-CH(CH2-cyclopentyl)-COOH. NH~-CH(CH~-cyclobutyl~COOH, NH2-CH(CH2-cyclopropyl)-COOH, t~ifluoroleucine, and hexafluoroleucine:
AA2 is a side chain blocked or unblocked amino acid with rhe L configuration, D
configuraùon, or no chi3ality at the ~-carbon selected from the group consisting of alanine, valine. Ieucine. isoleucine, proline. methionine. methionine sulfoxide, phenylalanine.
;5 tryptophan, glycine. serine. threonine. cysteine. tyrosine. asparagine, glutamine, aspartic acid.
glutamic acid, Iysine. arginine. histidine. phenylglycine. beta-alanine. norleucine, norvaline.
alpha-aminobutyric acid. epsilon-arninocaproic acid. citrulline. hydroxyproline. ornithine.
homoarginine. sarcosine. indoline 2-carboxylic acid. 2-azetidinecarboxylic acid, pipecolinic acid (2-pipe3idine carboxylic acid), O-methylsezine. O-ethylserine. S-methylcysteine. S-SUBSTITUTE~ SHET

WO 92/12140 PCllUS91/09801 209~7~2 ethylcvsteine. S-benzylcysteine. NH2-CH(CH2CHEt2)-COOH. alpha-aminohcptanoic aeid.
~H2-CH(CH ~- I-napthyl)-COOH. NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH. NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl~cooH~
NH2-CH(CH~-cyclopropyl)-COOH, trifluoroleucine~ and he~afluoroleueine:
R3 and R4 are seleeted independently from the group eonsisdng of H. Cl.20 allcyl, Cl cyelizcd aL~cyl, C1.20 alkyl with a phenyl group attached to the Cl 20 aL~yl, Cl.20 cyelized aL~cyl with an attaehed phenyl group, C 1-20 alkyl with an attached phenyl group subsdtuted with K, C 1-20 alkyl with an attached phenyl group disubstituted with K, C 1-20 aL~yl ~vith an attaehed phenyl group trisubstituted with K, C 1-20 eyelized allcyl with an attached phenyl group subsdtuted with K, Cl lo allcyl with a morpholine [-N(CH2CH2)0] ting attached through nitrogen to the alkyl, CI lo alkyl with a piperidine ting attaehed through nitrogen to the alkyl, C 1-10 alkyl with a pyrrolidine nng attaehed through nittogen to the alkyl, C 1-20 alkyl with an OH group attaehed to the alkyl, -CH~CH~OCH2CH2OH, C l- 10 with an attached ~pyridyl group, Cl lo with an attached 3-pyridyl group, Cl.lo with an attaehed 2-pyridyl group, Cl.lo with an attaehed cyelohexyl group, -NH-CH2CH2-(~hydroxyphenyl), and -NH-CH2CH~-(3-indolyl).
The novel elass of peptide -ketoamides also have the following struetural formula:
~1 1 -AA-AA-AA-CO-NR3E~4 or a pharmaceuueaUy aeeeptable salt, wherein ~0 Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X~N-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 eonsisting of C l . lo alkyl, C l 1o fluoroalkyl, C l 1o alkyl substituted with J. Cl.lo fluoroalkyl substituted with J. I-admantyl, 9-fluorenyl, phenyl, '5 phenyl substituted with K, phenyl disubstituted with K. phenyl trisubsututed with K. naphthyl, naphthyl substituted with K, naphthyl disubstituted with K. naphthyl trisubstituted with R.
C 1-10 alkyl with an attaehed phenyl group, C I .10 alkyl with two attaehed phenyl groups. C l 10 aIkyl with an attaehed phenyl group subsituted with K. C1~10 alkyl with two attached phenyl groups substituted with K, Cl 1o aIkyl with an attached phenoxy group, and Cl.lo 30 alkyl with an attached phenoxy group subsituted with K on the phenoxy group:
J is seleeted from the group consisting of halogen. COOH, OH. CN. ~102, NH2, C
10 alkoxy, C I -10 alkylamine- C2- 12 dialkylamine. C I . I0 alkvl-aCO-. C I- 10 aLkyl-aCO-NH-. and C 1-10 alkyl-S-:
K is seleeted from the group eonsising of halogen. Cl.lo alkyl, Cl.lo pcrfluoroaLkvl.
3~ C 1-10 alkoxy. NO~. CN. OH. CO~H, amino. C I . I o alkvlamino, C2. 12 dialkylamino. C I -C 10 aeyl, and C I . I o aLkoxy-CO-, and C I 10 aLkvl-S-;
AA is a side ehain bloeked or unbloeked a~runo acid with the L conf~gurauon. D
configuraion. or no chirality a~ the ~-carbon seleeted from the group eonsisting of alanine.
valine, Ieueine. isoleueine. proline. methionine. methionine sulfoxide. phenylalanme.

SUBSTITUTE SHER

WO 92/12140 q PCl/US91/09801 q ~ IJ

tryptophan. glycine, serine. threonine. cysteine. tyrosine. asparagine. glutamine. aspa~oc aci~
glutamic acid, Iysine, arginine. histidine, phenvlglycine, beta-alanine, norleucine, norvaline.
alpha-aminobutyric acid. epsilon-an~inocaproic acid. citrulline. hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid. 2-azetidinecarboxylic acid. pipecolinic 5 acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine. NH2-CH(CH2CHEt~)-COOH. alpha-aminoheptanoic acid, NH~-CH(CH2-1-napthyl)-COOH. NH2-CH(CH2-2-napthyl)-COOH. NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH. trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, Cl 20 alltyl, Cl 20 cyclized alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, C 1-20 cyclized alkyl with an attached phenyl group, C 1-20 alkyl with an attached phenyl gr~up substituud with K. C 1-20 alkyl with an attached phenyl group disubstituted with K, C 1-20 alkyl with an attached phenyl group trisubstituted with K, C1 20 cyclized alkyl with an attached phenyl 15 group substiruted with K, Cl lo alkyl with a morpholine [-N(CH~CH2)O~ ring attached through nitrogen to the alkyl, C l lo alkyl with a piperidine ring attached through nitrogen to the alkyl, Cl lo alkyl with a pyrrolidine ring attached through nitrogen to the allcyl, C1 20 alkyl with an OH group attached to the alkyl, -CH2CH20CH2CH20H, C l lo with an attached ~pyridyl group, CI lo with an attached 3-pyridyl group, CI lo with an attached 2-pyridyl 20 group, Cl lo with an attached cyclohexyl group, -NH-CH2CH2-(4-hydro~yphenyl), and -NH-CH~CH2-(3-indolyl).
The novel class of peptide a-ketoamides also have the following structural formula:
Ml-AA-AA-AA-AA-CO-NR3R4 or a phannaceudcally acceptable salt. whescin ''5 .~11 represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X~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 consisdng of C1.1o alkyl, Cl 10 fluoroalkyl, Cl lo alkyl substituted with J, Cl lo fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, 30 phenyl subsdtuted with K. phenyl disubstituted with K, phenyl trisubsdtuted with K, naphthyl, naphthyl substituted with K. naphthyl disubstituted with K, naphthyl trisubsuruted with K, C l. lo alkyl with an attached phenyl group, C l 10 alkyl with two attached phenyl groups, C
10 alkyl with an attached phenvl group substituud with K, C l lo aLIcyl with two attached phenyl groups substituted with K. Cl lo alkyl with an attached phenoxy group, and Cl lo 35 aLtcyl with an attached phenoxy group substituud with K on the phenoxv group:J is selected from the group consisung of halogen. COOH. OH. C~. .NO2, NH2, C I
allcoxy, C I lo aL~cylanune. C2- 12 dialkylamine, C I l 0 aL~cyl-O-CO-~ C I l o alkyl-O-CO-~JH-, and Cl lo alkyl-S-:

SJIB~.~ S~

WO 92/12140 2 ~ 9 2 7 ~ 2 PCI /US91/09801 K is selected from the group consising of halogen, C 1-10 alkyl, C 1-10 perfluoroalkyl.
Cl lo aLkoxy, N02, CN, OH. C02H, amino, Cl.lo aLkylamino. C -12 dialhylamino, Cl-Clo acyl, and Cl.lo all~oxy-CO-. and Cl.lo aLkyl-S-:
AA is a side chain blocked or unbloeked amino aeid with the L configura~ion, D
configuration, or no chirality at she a-earbon seleeted from the group consisting of alanine, valine, leueine, isoleucine. proline, methionine, methionine sulfoxide, phenylalanine, tryp~ophan, glyeine, serine, threonine. cysteine, tyrosine, asparagine, glutamine. aspanie aeid.
glutamic aeid, Iysine. arginine, histidine, phenylglyeine, beta-alanine. norleueine, norvaline, alpha-aminobutync aeid, epsilon-aminoeaproie aeid, citrulline, hydroxyproline, ornithine, 10 homoarginine, sarcosine, indoline 2-carboxylie aeid, 2-azetidinecarboxylie aeid, pipeeolis~ie acid (2-piperidine earboxylic acid), O-methylserine, O-ethylserine, S-methyleysteine, S-ethyleysteine, S-benzyleysteine. NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoie aeid, NH~-CH(CH2- 1 -napthyl)-COOH. NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH~-cyclohexyl)-COOH, NH2-CH(CH2-cyelopentyl)-COOH, NH2-CH(CH2-cyelobutyl)-COOH, 15 NH~-CH(CH ~-cyelopropyl)-COOH. trifluoroleueine, and hexafluoroleueine:
R3 and R4 are seleeted independently from the group eonsisting of H, Cl.20 allcyl, Cl 20 cyclized aLkyl, C1 20 aLkyl with a phenyl group attached to the Cl 20 alkyl, C1 20 eyelized aLkyl with an attached phenyl group, C 1-20 aLkyl with an attached phenyl group substitutcd with K, C 1 20 alkyl with an attaehed phenyl group disubstituted with K, C l 20 aLkyl with an 20 attaehed phenyl group trisubstituted with K, C 1-20 eyelized allcyl with an attaehet phenyl group subsituted with K, CI.10 alkyl with a morpholine [-N(CH2CH~)O1 ring attached through nitrogen to the aLkyl, Cl lo alkyl with a piperidine ring attaehed through nir~ogen to the alkyl, C 1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C 1-20 alkyl with an OH group attaehed to the alkyl, -CH2CH20CH2CH20H. Cl.lo with an attaehed '5 1-pyridyl group, Cl.lo with an attaehed 3-pyridyl group, Cl.lo with an attaehed 2-pyridyl group, Cl lo with an attaehed eyelohexyl group, -NH-CH2CH2-(4 hydro~yphenyl), and -NH-CH2CH2-(3-indolyl).
The novel elass of pepide a-ketoamides also have the following structural fonnula:
Ml-AA-CO-NR3R4 30 or a pharmaceutieaLly aceeptable salt. wherein ~vll represents H, NH2-CO-. NH2-CS-. NH2-SO2-, X-NH-CO-. X~N-CO-, X-NH-CS-, X~N-CS-. X-NH-SO~-, ,Y~N-SO2-, X-CO-, X-CS-. X-SO~-, X-O-CO-, or X-O-CS-:
X is seleeted from the group eonsisting of Cl.lo aLkyl, Cl.lo fluoroaLkyl, Cl lo allcyl 35 subsututed with J. Cl lo fluoroalkyl subsututed with J, l-admantyl. 9-fluorenyl. phenyl, phenyl subsututed with K. phenyl disubstituted with K, phenyl trisubstituted with K. naphthyl.
naphthyl subsuituted with K. naphthyl disubstituted with K. naphthyl trisubstituted with K.
C 1-10 alkyl with an ar~aehed phenyl group, C I .10 aLkyl with two at~aehed phenyl groups, C 1-10 alkyl with an attaehed phenyl group substituted with K, C 1-10 aLkyl with two a~tached S'J~3STITUTE SHEET

WO 92/12140 ~ PCI/US91/09B01 q ~93~

phenyl eroups subsrituted with K. Cl lo ,aLcyl with an attached phenoxy group. and Cl lo alkyl with an aetached phenoxy group substituted with K on the phenoxy group;
J is seleeted from the group consisting of halogen. COOH, OH, CN, ~1O2, .~H2, C 1-10 alkoxy, Cl lo alkylamine, C2 12 dialkyl~mine. Cl 10 alkyl-~CO-. Cl lo alkyl-O-CO-~-. and C I lo alkyl-S-;
K is selected from the group consising of halogen, C l lo alkyl, C l l o perfluoroallcyl.
C I . Io alkoxy, NO2, CN. OH. CO2H. amino. C l lo alkylam~no. C2 12 dialkylamino, C 1-C lo acyl, and C l lo alkoxy-CO-. and C l lo allcyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
10 configuraion. or no chi~lity at the a-carbon selected from the gxup eonsising of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine. threonine, cysteine. tyrosine. asparagine, glutamine, aspartic aeid.
glutarnic acid, Iysine. arginine. histidine, phenylglycine. beta-alanine, norleucine, no~valine.
alpha-aminobutytic aeid. epsilon-aminaproic acid, citrulline, hydroxyproline, omithine, lS homoarginine, sareosine, indoline 2-carboxylic aeid. 2-azeidineearboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine. O-ethylsenne, S-methylcysteine, S-ethylcysteine, S-benzyleysteine, NH2-CH(CH2C~IEt2)-COOH, alpha-aminoheptanoie aeid.
NH2-CH(CH2- 1 -napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-eyelohexyl)-COOH, NH2-CH(CH2-eyelopentyl)-COOH, NH2-CH(CH2-eyelobutyl)-COOH, 'O ~H2-C~(CH2-eyelopropyl)-COOH, trifluoroleueine. and hexafluoroleueine;
R3 and R4 are seleeted independently from the group eonsisting of H. Cl -20 alkyl, C l 20 eyelized allcyl, C 1-20 alkyl with a phenyl g~up attached to the C 1-20 alkyl, C 1-20 eyelized allcyl with an attaehed phenyl group, Cl 20 alkyl with an attaehed phenyl group substituted with K. C 1-20 alkyl with an attaehed phenyl group disubstituted with K, Cl 20 alkyl with an attaehed phenyl group trisubstituted with K, C 1-20 eyelized alkyl with an attaehed phenyl group substituted with K, Cl lo alkyl with a morpholine [-N(CH~CH~7)O~ ring attaehed thrwgh nitrogen to the aL~cyl, C l lo alkyl with a piperidine ring attaehed through nitrogen to the alkyl, Cl lo alkyl with a pyrrolidine ring attaehed through nitrogen to the alkyl, Cl 20 alkyl with an OH group attaehed to the alkyl, -CH~CH~OCH2CH2OH. C 1-10 with an attached 30 1 pyridyl group, CI lo with an attacned 3-pyndyl group, CI lo with an attached 2-pyndyl group, C l lo with an ar~aehed cycloh ~xyl group. -NH-CH~CH~-(~hvdroxyphenyl). and -~'H-CH~CH2-(3-indolyl).
The novel class of pepude c~-ketoaeids have the following structural formula:
.\11-AA-NH-CHR2-CO-CO-OH
3~ or a phannaeeuucally acceptable salt. wherein Ml represen~s H. ~'H2-CO-, NH2-CS-, .~ 2-SO~-. X-NH-CO-. X2N-CO-, X-NH-CS-. X2N-CS-. X-NH-SO~-, X2N-SO2-. X-CO-. X-CS-, X-S02-, X-O-CO-, or X-O-CS-;

SUP~STITUTE SHEE~

WO 92/12140 PCr/US91/0g801 20~70~

X is selected from the group consisting of C I I o alkyl. C l - ~o fluoroal~cyl~ C l l o aLkyl substiNted with J, Cl lo fluoroallcyl substi~uted with J. l-adsslantyl, 9-fluorenyl, phenyl, phenyl substiZuted with K, phenyl disubstitu~ed with K. phenyl trisubstiNted with K, naphthyl, naphthyl substituted with K, naphthyl disubstiNted with K, naphthyl trisubstiNted with K, 5 C I 10 alkyl with an attached phenyl group, C I 10 allcyl with two attached phenyl groups. C l l o al~yl with an attached phenyl grwp substiNZed with K. C 1-10 allcyl with two attaehed phenyl groups substiNted with K. CI 10 alk~/l With an attached phenoxy group, arld Cl lo 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
lo allcoxy, CI-10 allcylamine. C2 12 diallcylamine, CI lo allcyl-O-CO-, CI.lo allcyl-O-CO-NH-, and C I 10 allcyl-S-;
K is selected from the group consisting of halogen, C I l o allcyl, C 1 1o pcsnuoroa Cl lo allcoxy, N02, CN. OH. C02H. amino. Cl lo allcylamino, C2 12 diallcylamino, Cl-Clo acyl, and Cl lo allcoxy-CO-, and C1 1o a~cyl-S-;
AA is a side chain blociced or unbloeked asnino acid with the L configuration, Dconfiguration, or no chirality at the c~-carbon seleeted from the gsoup eonsisting of alanine, valine, leucine, isoleucine, proline, snethionine. methionine sulfoxide, phenylalanine, tryptophan, glycine. serine. threonine, cysteine, tyrosine, asparagine, gluiamine, aspartie aeid, gluurnie aeid, Iysine, arginine, histidine, phenylgiyeine, beu-aianine, norleueine, nonraline, alpha-arl~inobutyZie aeid, epsilon-arninoeaproie aeid, eitrulline, hydroxyproline, o~ithine, homoarginine, sareosine, indoline 2-earboxylic acid. 2-azetidinecarboxylie aeid, pipeeolinie aeid (2-pipesidine carboxylie aeid), O-methylserine, O-ethylserine, S-methyleysteine, S-ethyleysteine, S-benzyleysteine. NH2-CH(CH~CHEt2)-COOH, alpha-aminoheptanoie aeid, NH2-CH(CH2- I-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyelohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-eyclobutyl)-COOH, NH2-CH(CH2-cyelopsopyl)-COOH. tsifluoroleucine, and he~afluosoleueine;
R2 represents C 1-8 branched and unbranched alkyl, C 1-8 branched and unbranehedcyelized alkyl, or Cl 8 branched and unbranched fluoroalkyl;
The novel class of peptide -ketoacids also haYe the following structural forrnula:
Ml-AA2-AAI-CO-OH
or a pharmaeeutically aeceptabk salt, wherein Ml represents H. NH2-CO-. NH2-CS-. NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-. X-CS-. X-SO2-, X-O-CO-, or X-O-CS-;
;5 X is seleeled from the group consisung of Cl 10 aL~cyl, Cl lo fluoroalkyl, Cl lo al~yl substituted with J. Cl lo fluoroallcyl subsutu~ed with J. I-admantyl, 9-fluoxnyl, phenyl, phenyl subsututed with K. phenyl disubstituted with K. phenyl trisubstitu~ed with K, naphthyl.
naphthyl subsuNted with K. naphthyl disubs~tuted with K. naphthyl trisubstiNted with K, C 1-10 allcyl with an attaehed phenyl group, C I - l 0 alkyl with two attaehed phenyl groups, C I

SUE~STITUTE SHEET

WO 92tl2140 ' PCrtUS91/09801 ~9Q~ J 14 10 alkyl with an a~ached phcnyl gT'OUp substituted with K. and Cl lo allcyl with two attached phenyl groups substitutcd with K. Cl lo alkyl with an attached phenoxy group, and Cl-10 alkyl with an attachcd phenoxy gT~Up substituted with K on the phenoxy group;
J is selected from the group consisting of halogcn, COOH. OH. CN, No2"~H2~ Cl S 10 alkoxy, Cl lo al~ylaminc, C2 12 dialkylaminc, Cl 10 alkyl-O-CO-, Cl lo alkyl-O-CO-NH-, and Cl lo alkyl-S-;
K is selcctcd from the group consisting of halogcn, C l- l0 alkyl, C I lo perfluoroallcyl, Cl lo alkoxy, N02, CN, OH, C02H, amino, Cl lo alkylamino. C~ 12 dialkylamino, Cl-Clo acyl, and Cl lo alkoxy-CO-, and Cl 1o alkyl-S-;
AAl is a sidc chain blockcd or unblockcd amino acid with thc L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteinc, tyrosine. asparagine, glutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline. alpha-aminobutyric acid. epsilon-aminocapToic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2- 1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-~H(CH ~-cyclopropyl)-COOH, trifluoroleucine, and hcxafluoT~leucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chi~ality at the -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine. methionine sulfoxide, phenylalanine, '5 tryptophan, glycine, serine, threonine. cysteine. tyT~sine, asparagine, glutaminc. aspartic acid.
glutamic acid, Iysine, arginine, histidine. phenylglycine, beta-alanine, norleucine. noT~aline.
alpha-aminobutyric acid, epsilon-aminocaproic acid. citrulline. hydT~xyproline, ornishine, homoarginine, sarcosine, indoline 2-carboxylic acid. 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylsenne. O-ethylserine. S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH~-CH(CH2CHEt~)-COOH, alpha-arninoheptanoic acid.
~'H2-CH(CH2- 1-napthyl)-COOH. NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH ~-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl)-COOH.
NH~-CH(CH~-cyclopropyl)-COOH, trifluoroleucine. and hexafluoT~leucine;
The novel class of pepude c~-ketoacids also have the following strucluTal formula:
~ I -AA-AA-AA-CO-OH
or a phaTmaceuucally acceptable salt. wherein ~I represents H. NH2-CO-. .NH2-cs-~ NH2-S02-, X-NH-CO-. ,Y~N-CO-, X-NH-CS-, X2N-CS-, X-NH-so2-~ X2N-so2-l X-CO-, X-CS-, X-SO2-, X-O-CO-. or X-O-CS-;

S-,'BSTITUTE ~;KEEt wo 92/12140 2 0 9 ~ 7 0 2 Pcr/usg1/Qg8o1 ,5 X is selected from the group consisting of C I 10 alkyl. C I 10 fluoroalkyl, C I -10 aL~cyl substituted with J, C I l o fluoroalkyl substituted with J, 1 -admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubsituted with K. phenyl trisubstituted with K. naphthyl.
naphthyl subsituted with K, naphthyl disubstituted with K. naphthyl tnsubsututed with K, Cl lo alkyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl lo alkyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached phenyl groups substituted with K. Cl 10 alkyl with an attached phenoxy group, and Cl lo aL~cyl with an attached phenoxy gTOUp substituted with K on the phenoxy group;
J is selected from the group consisuing of halogen, COOH, OH, C~, NO~, ~H2, C
10 alkoxy, Cl lo alkylamine, C2 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl lo alkyl-O-CO-NH-, and C 1-10 alkyl-S- , K is selected from the group eonsisting of halogen, Cl lo alkyl, Cl lo perfluoroall~l, Cl 1o alkoxy, NO~, CN, OH, CO~H, amino. Cl lo alkylamino, C2 12 diaLkylamino, Cl-C l o acyl, and C I 10 alkoxy-CO-. and C I 10 alkyl-S-;
AA is a side chain blocked or unbloeked amino acid with the L configuration, D
configuration, or no chirality at the a-earbon seleeted from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine. cysteine, tyrosine. asparagine, gluta~rune, aspartic acid, glutamie aeid, Iysine, arginine, histidine, phenylglyeine, beta-alanine, norleueine, norvaline, 'O alpha-aminobutyric acid. epsilon-aminoeaproie aeid. citrulline, hydroxyproline, omithine, homoarginine, sareosine. indoline 2-carboxylie acid, 2-azetidinecarboxylic aeid, pipeeolinie aeid (2-piperidine earboxylic acid), O-methylserine, O-ethylserine, S-methyleysteine, S-ethylcysuine, S-benzyleysuine, NH2-CH(CH2CHEt2)-COOH, alpha-arninoheptanoie aeid, ~H2-CH(CH2- 1 -napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH. NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl)-COOH
NH2-CH(CH2-cyclopropyl)-COOH. trifluoroleucine, and hexafluoroleueine:
The novel class of peptide c~-ketoaeids also have the following structural formula:
Ml-AA-AA-AA-AA-CO-OH
or a phaTmaeeuically accep~able salt. wherein Ml represents H, NH2-CO-. NH2-CS-, NH2-so2-~ X-NH-CO-, X2N-CO-, X-~H-CS-, X~N-CS-. X-NH-S02-, X2N-S02-, Yl-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-:
X is selected from the group consising of C1-10 alkyl, CI 10 fluoroalkyl, C1 lo alkvl subsituted with J, Cl lo fluoroalkyl subsituted with J. I-admanryl, 9-fluorenyl, phenyl.
phenyl subsi~uted with K. phenyl disubstituted with K, phenyl tnsubstituted with K, naphthvl naphthyl subsututed with K. naphthyl disubstituted with K. naphthyl trisubsituted with K.
Cl lo aL~cyl with an attaehed phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl lo al~yl with an a~tached phenyl group substituted wi~h K. and Cl lo alkyl with two attached SUBSTITUTE SHEET

wo 92/]2140 q.,~9 PCI/US91/09801 phenyl groups substituted with K. Cl lo alkyl with an attached phenoxy group, and Cl )o aL~I with an attached phenoxy group substituted with K on the phenoxy group;
Yl is selected from the group consisting of C2 10 a~kyl, Cl lo fluoroalkyl, Cl lo aL~cyl substituted with J, C I lo fluoroalkyl substituted with J. I -admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl lo alkyl with an attæhed phenyl group, Cl lo alkyl with ~wo attached phenyl groups, Cl 10 al~yl with an atr~ched phenyl group substituted with K, and Cl lo aLlcyl with two attached phenyl groups substituted with K:
J is selected from the gTOUp consisting of halogen, COOH, OH, CN, NO2, NH2, C
10 alkoxy, Cl 10 alkylamine, C2-l2 dialkylamine, Cl lo alkyl-O-CO-, Cl 10 aL~cyl-O-CO-NH-, and Cl 1o a~kyl-S-;
K is selected from the group consisting of halogen, CI lo alkyl, CI 10 perfluoroalkyl, Cl lo alkoxy, N02, CN. OH. CO~H, amino, Cl lo aLIcylamino. C2 12 dialkylamino, Cl-C l o acyl, and C l l o alkoxy-CO-, and C 1 -10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality a~ the -carbon selected from the group consis~ing of alanine, valine. Ieucine, isoleucine, proline. methionine. methionine sulfoxide, phenylalanine, tryptophan, glycine, serine. threonine, cysteine, tyrosine, asparagine, glutamine, aspar~c acid, 20 glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, nonraline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-pipe~idine carboxylic acid), ~methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine. NH2-CH(CH2CHEt2)-COOH. alpha-aminoheptanoic acid, ~H~-CH(CH2-1-napthyl)-COOH. NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH?-cyclobutyl~COOH, ~iH_-CH(CH2-cyclop~pyl)-COOH, trifluoroleucine, and hexafluoroleucine;
The novel class of peptide c~-ketoacids also have the following stTuctural formula:
Ml-AA-CO-OH
or a pharmaceutically acceptable salt. wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X~N-CO-, X-.~H-CS-. X?N-CS-. X-NH-S02-, X?N-S02-, Y~-CO-, X-CS-. X-SO?-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of CI Io alkyl, CI-10 fluoroalkyl, CI-10 alkyl subsututed with J, C l lo fluoroalkyl substituted with J. I-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K. phenyl disubstituted with K, phenyl trisubsuluted with K, naphthyl.
naphthyl substituted with K. naphthyl disubstituted with K, naphthyl trisubsuituted with K, Cl lo aLlcyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, and C I -10 aL~cyl wi~h two attached SUBSTITUTE SHEET

wO 92/~2140 2 0 9 3 7 0 2 PCI/US91/09801 phenyl groups substituted with K. C l 10 alkyl with an attached phenoxy group, and C I lo aLtcyl with an attached phenoxy group substituted with K on the phenoxy group;
Y2 is selected from the group consisting of C 1-10 alkyl, C I -10 fluoroalkyl, Cl 10 aL~yl substituted with J, Cl lo fluoroalkyl substituted with J. l-adTnantyl~ 9-fluorenyl, phenyl substituted with K. phenyl disubstituted with K. phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl lo alkyl with an attached phenyl group, Cl lo alkyl with two attached phenyl groups, Cl lo alkyl with an attached phenyl group substituted with K, and Cl lo alkyl with two attached phenyl gtoups substituted with K:
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C
lo alkoxy, Cl lo aL~ylamine, C2 12 dialkylamine, Cl lo alkyl-O-CO-, Cl lo alkyl-O-C~
NH-, and Cl lo alkyl-S-:
K is selected from the group consisting of halogen, C l- 10 alkvl, C 1-10 perfluoroallcyl, Cl lo alkoxy, N02, CN, OH, C02H, amino, Cl lo aLlcylamino, C2 12 dialkylamino, Cl-Cloacyl,andCl loalkoxy-CO-,andCl loalkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, 20 glutamic acid, Iysine, arginine, hisddine, phenylglycine, beu-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azeddinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid.
.~H2-CH~CH2- 1 -napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH.
~H2-CH(CH2-cyclopropyl)-COOH, ttifluoroleucine, and hexafluoroleucine;
The novel class of peptide a-ketoesters have the following structural formula:
Ml-AA2-AAI-CO-O-RI
or a pha~naceuucally acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-. NH2-S02-, 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 consisung of Cl lo alkyl, Cl lo fluoroalkyl, Cl lo aL~cyl subsututed with J, Cl lo fluoroalkyl substituted with J, l-admantyl~ 9-fluorenyl, phenyl, phenyl subsututed with K. phenyl disubstituted with K. phenyl trisubstituted with K. naphthyl, naphthyl subsututed with K. naphthyl disubstituted with K. naphthyl trisubstituted with K, C l lo alkyl with an attached phenyl gTwp, C l lo allcyl with two attached phenyl groups, C l 10 allcyl with an attached phenyl group substituted with K. and Cl lo alkyl with two attached SUBSTITUTE Sl`l~T

phenyl groups substituted with K. Cl 10 alkyl with an attached phenoxy group, and Cl ~o alkyl with an attached phenoxy group substituted with K on the phenoxy group:
J is selccted from the group consisting of halogen. COOH. OH, CN, ~'O2, NH2, C
10 alkoxy, Cl 10 alkylamine, C2-l2 dialkylamine, Cl 10 alkyl-O-CO-, Cl lo all~yl-O-CO-l~TH-, and Cl 1o aL~cyl-S-;
K is selected from the group consisting of halogen, C I 10 alkyl, C I 10 pcrfluoroa~yl, C l lo alkoxy, N02, CN, OH. C02H, amino, C l 1o aLtcylarnino, C2 12 dialkylamino, C l-Clo acyl, and Cl lo alkoxy-CO-, and Cl lo al~yl-S-;
AAl is a side chain blocked or unbloclced amino acid with the L configuration, Dconfiguration, or no chirality at the -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalasline, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutarnine, aspartic acid, glutamic acid, lysine, arginine. histidine, phenylglycine. beta-alanine. norleucine, nonaline, alpha-aminobutyric acid. epsilon-aminocaproic acid, citrulline. hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcys~eine, S-ethylcysteine, S-benzylcysteine. NH~-CH(CH2CHEt2)-COOH. alpha-aminoheptanoic acid, NH2-CH(CH2- I-nap~hyl)-COOH, ~I2-CH(CH~-2-napthyl)-COOH, NH~-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, 20 NH2-CH(CH ~-cyclopropyl)-COOH. trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine. cysteine. tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, Iysine.
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-pipesidine carboxylic acid), O-methylsesine. O-ethylserine. S-methylcysteine, S-ethylcysteine. S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH. alpha-aminoheptanoic acid, NH2-CH(CH2- 1 -napthyl)-COOH.
l~H2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohcxyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutvl)-COOH, NH~-CH(CH~-cyclopropyl)-COOH.
trifluoroleucine. and hexafluoroleucine:
Rl is selected from the group consisting of H. C 1-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C l 20 aLIcyl, and C 1 20 al~yl with an attached phenyl group substituted with K.
The novel class of peptide a-ketoesters also have the following structural forrnula:
M I -AA-NH-CHR~-co-co-o-R
or a pharmaceuucally acceptable salt. wherein SUB~TITUT~ S~!E~T

wO 92/12~40 2 ~ ~ 8 7 ~ 2 PCr/US91/Og801 Ml represents H, NH2-CO-. .NH2-CS-~ ~JH2-SO2-, X-1~1H-CO-. X~N-CO-, X-l~-CS-, X2N-CS-, X-NH-S02-, X~N-so2-~ X-CO-, X-CS-. X-so2-~ X-O-CO-. or %-O-CS-;
X is selected from the group consisting of Cl lo alkyl, Cl lo fluoroalkyl, Cl l~ alkyl substituted with J, Cl lo fluoroalkyl substituted with 1, I-adman~yl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with K, Cl lo al~yl with an attached phenyl group, Cl lo allcyl with two attached phenyl groups, Cl 10 aLI~l with an attached phenyl gTOUp substituted with K, and Cl 1o aLtcyl with two attached 10 phenyl groups substituted with K, Cl lo alkyl with an attached phenoxy group, and Cl 1o 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, N02, NH2, Cl 10 alkoxy, Cl 10 alkylamine. C2 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl lo alkyl-~CO-.~-, and Cl lo alkyl-S-:
K is selected from thc group consisting of halogen, C1-10 aLkyl, Cl-10 perfluoroallcyl, C I -10 alkoxy, NO2, CN, OH, CO~H, arnino, C I -10 alkylamino, C2 12 dialkylamino, C1-C lo acyl, and C I I o alkoxy-CO-, and C l lo alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuraion, D
configuration, or no chirality at the -carbon selected from the group consising of alanine, '0 valine. Ieucine, isoleucine, proline. methionine, methionine sulfoxide, phenylalanine, trvptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glu~nic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azctidinecarboxylic acid, pipecolinic '5 acid (2-piperidine carboxylic acid), O-methylserine, O-ethylsenne, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid.
-H ~-CH(CH2- 1-napthyl)-COOH, NH~-CH(CH2-2-napthyl)-COOH, NH~-CH(CH ~-c-clohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH.
~H2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents C1 8 branched and unbranched alkyl, Cl 8 branched and unbranched cyclized alkyl, or Cl 8 branched and unbranched fluoroaLkyl;
R is selected from the group consisting of H, C 1-20 aLkyl, C 1-20 alkyl with a phenyl group attached to the Cl 20 alkyl. and Cl 20 alkvl with an attached phenvl group substituted with K.
The novel class of peptide a-ke~oestcrs also have the following structural fotmula:

or a pharmaceuucaLly acceptable salt. wherein SUBSTITUTE SH~ET

WO 92/12140 c PCr/US91/09801 ~lf~
C~ C~ 20-M3 represents H. NH2-CO-, NH2-CS-. NH2-SO2-, X-~iH-CO-, X ~N-CO-, X-NH-CS-, X~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 I 10 alkyl, C l lo fluoroalkyl, C l- lo allcvl substituted with J, Cl lo fluoroalkyl substituted with J, I-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstitutcd with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubsituted with K, Cl lo alkyl with an ar~ached phenyl group, Cl 10 alkyl with rwo attached phenyl groups, Cl lo alkyl with an ar~ached phenyl group substiruted with K, and Cl lo allcyl with two attached phenyl groups subsiruted with K, Cl lo allcyl with an attached phenoxy gTOUp, and Cl lo alkyl with an attached phenoxy group substiruted with K on the phenoxy group;
T is selected from the group consisting of Cl lo aLlcyl, Cl lo fluoroallcyl, Cl lo allcyl substituted with J, Cl lo fluoroalkyl substituted with J, I-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K. phenyl trisubsituted with K. naphthyl.
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C2 10 alkyl with an attached phenyl group, Cl lo alkyl with two attached phenyl groups, Cl lo alkyl with an attached phenyl group substituted with K, and C l lo aLt~yl with two attached phenyl groups subsituted with K;
J is selectcd from the group consisuing of halogen, COOH, OH, CN, NO2, NH2, C
lo alkoxy, CI-10 alkylamine, C2 12 dialkylamine, C1-10 alkyl-O-CO-, C1 lo alkyl-aCa NH-, and C l lo aL1cyl-S-;
K is selectcd from the group consising of halogen, C 1 lo alkyl, C I lo pesfluoroalkyl, C l lo aL~coxy, N02, CN, OH, C02H, amino, C l lo alkylamino, C2 12 dialkylamino, C l-Clo acyl, and Cl lo aL~coxy-CO-, and Cl lo alkyl-S-;
'5 AA is a side chain blocked or unblocked amino acid with the L configurauon, D
configurarion, or no chirality at the c~-carbon selected from the group consisting of alanine, valine. Ieucine. isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, t~yptophan, glycine, serine. threonine, cysteine, tyrosine, asparagine, glutamine, asparic acid.
glutamic acid, Iysine, argim-. e, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-an~nobutyric acid, epsilon-aminocaproic acid, cirrulline, hydroxyproline, ornithine, homoarginine. sarcosine, indoline 2-carboxylic acid. 2-azetidinecarboxylic acid. pipccolinic acid (2-piperidine carboxylic acid), amethylserine. O-ethylserine, S-methylcysteine. S-ethylcysuine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH. alpha-aminoheptanoic acid, NH2-CH(CH2- 1-napthyl)-COOH, NH ~-CH(CH2-2-napthyl)-COOH, NH2-CH(CH ~-i~ cyclohexyl)-COOH. NH2-CH(CH~-cyclopentyl)-COOH. NH~-CH(CH2-cyclobutyl)-COOHNH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine. and hexafluoroleucine;
R is selected from the group consisung of H. C2 20 alkyl, C 1-20 alkyl with a phenyl group at~ached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K.

SUBSTITUTE SHE~

WO 92/12140 2 0 3 ~ 7 0 2 PCI/US91/09801 The novel class of pepide -ketoesters also have the following structural formula:

or a pharmaceutically acceptable salt, whercin M3 represents H. NH2-co-~lNH2-cs-~NH2-so2-~x-~H-co-~ X2N-CO-, X-NH-CS-, X2N-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 Cl lo alkyl, Cl lo fluoroaL~yl, Cl lo alkyl substituted with J, C l lo fluoroalkyl substituted with J, I-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubsituted with K, naphthyl, 10 naphthyl subsututed with K, naphthyl disubsituted with K. naphthyl tnsubsituted with K, C l 1o alkyl with an attached phenyl group, C l l o alkyl with r.~o attached phenyl groups, C l 10 alkyl with an attached phenyl group substituted with K, and Cl lo aLkyl with two attached phenyl groups substituted with K, Cl lo aL~cyl with an a~tached phenoxy group, and Cl lo aL~yl with an attached phenoxy group subsituted with K on the phenoxy group;
T is selected from the group consisting of Cl lo alkyl, Cl lo fluoroalkyl, Cl lo allcyl substituted with J, Cl lo fluoroalkyl substituted with J, I-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K. phenyl disubstituted with K, phenyl trisubsituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C2 10 alkyl with an attached phenyl group, Cl lo alkyl with two attached phenyl groups, Cl ' 10 alkyl with an attached phenyl group substituted with K, and C 1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen. COOH, OH, CN, NO2, NH2, Cl 10 aLlcoxv, Cl lo aL~ylamine. Cl lo dialkylamine, Cl lo alkyl-O-CO-, Cl lo alkyl-aCO-NH-, and C I l0 allcyl-S-:
~5 K is selected from the group consisting of halogen. C I 1o alkyl, C I l o perfluoroaLlcyl, Cl io aLI~oxy, N02, C~, OH, CO~H, amino, Cl lo alkylamino, C2 12 dialkylamino, Cl-C lo acyl, and C l- 10 aLkoxy-CO-, and C l- 10 alkyl-S-;
AA is a side chain blocked or unbloclced amino acid with the L configuration, D
configuration, or no chi~litv at the c~-carbon selected from the group consisting of alanine, 30 valine, Ieu~ine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, t~yptophan, glycine. serine, threonine, cysteine, tyrosine. asparagine, glutamine. aspartic acid.
glutamic acid, Iysine, arginine, histidine, phenylglycine. beta-alanine, norleucine, nor~aline, alpha-ammobutyric acid. epsilon-aminocaproic acid. citrulline. hydroxyproline, omithine.
homoarginine, sarcosine. indoline 2-carboxylic acid. 2-azetidinecarboxylic acid. pipecolinic 3~ acid (2-piperidine carboxylic acid), O-methylserine. O-ethylsenne, S-methylcys~eine, S-ethylcysteme, S-benzylcysteine. lNH~-CH(CH2CHEt~)-COOH, alpha-aminoheptanoic acid.
~H2-CH(CH~- I -napthyl)-COOH. NH~-CH(CH~-2-napthyl)-COOH, NH~-CH(CH~-cyclohexyl)-COOH. ~iH~-CH(CH~-cyclopentyl)-COOH. NH~-CH(CH2-cyclobutyl)-COOH, ,~rH2 CH(CH2-cyclopropyl)-COOH. trifluoroleucine. and hexafluoroleucine:

SUBSTITUTE SHEET

wO 92/12140 PCr/US91/09801 2~ n, c~
R2 rcpresents C 1-8 branched and unbranched alkyl, C 1-8 branched and unbranchedcyclized alkyl, or C 1-8 branched and unbranched fluoroalkyl;
R is selected from the group consisting of H, C 1-20 aLkyl, C 1-20 aL~cyl wilh a phenyl group attached ~o the C 1-20 alkyl, and C 1-20 alkyl with an auached phenyl group substituted 5 with K.
The novel class of peptide a-ketoesters also have the following structural formula:
M3-AA4-AA-AA-AA-CO~R
or a pharrnaceutically aeeeptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-so2-~ X-NH-CO-, X~N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X-O-CS-;
X is selected from thc g~oup consisting of Cl lo aL~cyl, Cl lo fluoroalkyl, Cl lo alkyl substituted with J, Cl lo fluoroalkyl substituted with J, l-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubsti~uted with K. naphthyl trisubstituted with K, Cl lo aIkyl with an at~aehcd phenyl group, Cl lo alkyl with two attaehed phenyl groups, Cl 10 alkyl with an attaehed phenyl group substituted with K, and Cl lo alkyl with two attaehed phenyl groups substitutcd with K. C I -10 alkyl with an attached phenoxy group, and C 1-10 aLkyl with an attaehcd phenoxy group substitutcd with K on the phenoxy g~oup;
T is sclceted from thc group consisting of Cl.lo alkyl, Cl lo fluoroalkyl, Cl lo allcyl substituted with J, Cl lo fluoroalkyl substitutcd with J, l-admantyl, 9-fluorenyl, phcnyl, phenyl substituted with K, phcnyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstitutcd with K, naphthyl trisubstituted with K, C2 10 alkyl with an attaehcd phenyl group, CI-10 alkyl with two attachcd phenyl groups, C1-10 allcyl with an attaehed phenyl group substituted with K, and Cl lo alkyl with two attaehed phcnyl groups substitutcd with K;
J is seleeted from thc group consisting of halogcn, COOH, OH. C~, NO2, NH2, C
10 alkoxy, Cl-10 alkylamine, C2-12 dialkylamine, Cl 10 all~yl-O-CO-, Cl lo alkyl-O-C~
NH-, and Cl lo alkyl-S-;
K is selcctcd from the group consisting of halogcn, Cl lo alkyl, Cl lo perfluoroalkyl, Cl lo alkoxy, N02, C~, OH, C02H. amino. Cl lo aLkylamino. C2 12 dialkylamino. Cl-Cloacyl,andCI Ioalkoxy-CO-,andCl loallcyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configurauon. D
configuration. or no chirality at the a-carbon selected from the group consisting of alanine, valinc, leucine. isoleucine, proline. methionine. methionine sulfoxide. phenylalanine.
ttyptophan, glycine, serinc, thrconine. cysteine. tyrosine. asparagine, glutamine. aspartic acid.
glutamic acid, Iysinc, arginine, histidine, phenylglycine. beta-alanine, norleucine. norvalinc, alpha-aminobutyric acid, cpsilon-aminocaproic acid. cirrulline. hydroxyproline. ornithine.
ho noargininc, sarcosinc, indolinc 2-carboxylic acid. 2-azctidinccarboxylic acid. pipecolinic SUBSTITUTE SHEET

WO 92/12140 2 0 ~ 8 7 0 ~ Pcr/usgl/ng801 acid (2-piperidine earboxylic acid), O-metbylserine. O-ethylserine. S-metbyleysteine, S-ethylcysteine, S-benzylcysteine. .~H~-CH(CH~CHEt~)-COOH. alpha-aminoheptanoic acid.
~'H~-CH(CH ~- I-napthyl)-COOH, NH 7-CH(CH2-2-napthyl)-COOH, NH~-CH(CH~-cyclohexyl)-COOH, NH2-CH(CH~-cyclopentyl)-COOH. NH2-CH(CH2-eyelobutyl)-COOH, S .~ -CH(CH2-cyclopropyl)-COOH. trifluoroleucine, and hexafluoroleucine;;
AA4 is a side chain blocked or unblocked amino acid witb the L configuration, D
configuration. or no chirality at the cl-carbon selected from the group consisting of Ieucine, isoleucine, methionine, methionine sulfoxide, phenylalanine, tTyptopban, glycine, scnne, threonine, cysteine, tyrosine, asparagine. glutamine, aspartic acid, glutamic aeid, Iysine.
10 arginine, histidine, phenylglycine. beta-alanine. norleucine, norvaline, alpha-amirlobutyne aeid.
epsilon-aminocaproie acid, citruLline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2 carboxylic acid, 2-azetidinecarboxylic æid, pipeeolinic acid (2-piperidine earboxylic aeid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, ~H~-CH(CH ~CHEt2)-COOH, alpha-aminoheptanoie æit, NH~-CH(CH~- l-napthyl)-COOH, 15 NH~-CH(CH~-2-napthyl)-COOH, ~H~-CH(CH~-eyelohexyl)-COOH, NH2-CH(CH~-cyclopentyl)-COOH. NH2-CH(CH2-cyelobutyl)-COOH, NH2-CH(CH2-eyelopropyl)-COOH, trifluoroleucine. and hexafluoroleueine;
R is seleeted from the group eonsisting of H, C 1-20 alkyl, C 1-20 aLkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 aLkyl with an attaehed phenyl group subsdtuted ~0 with K.
The novel class of peptide ~-ketoesters also have the following struetural formula:
Ml-AA-CaO-R
or a pharmaceutically aeeeptable salt, wherein Ml represents H, NH2-CO-, NH2-cs-~ NH2-SO2-~ X-NH-CO-. X2N-CO-, ~5 X-.~H-CS-, X~N-CS-, X-NH-SO~-, X2N-SO2-, Y-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-:
X is selected from the group eonsisting of CI lo aLlcyl, C1-10 fluoroaLkyl, C1-10 aLkyl substituted with J, Cl lo fluoroaLkyl substituted with J. l-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K. phenyl disubstituted with K. phenyl trisubstituted with K. naphthyl, 30 naphthyl substituted with K. naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl lo aLkyl with an attaehed phenyl group, Cl lo aLkvl with two attaehed phenyl groups, Cl 10 alkyl with an attaehed phenyl group subsoitu~ed with K. and C 1-10 alkyl with two attaehed phenyl groups substi~uted with K. Cl lo alkyl with an attaehed phenoxy group, and Cl lo alkyl with an attaehed phenoxy group substituted with K on the phenoxy group;
3~ Y is seleeted from the group consisoing of C6 10 alkyl, CI 10 fluoroallcyl. CI 10 alh i subsotuted with 1, C 1-10 fluoroalkyl substituted with J. l-admantyl, 9-fluorenyl, phenyl substi~uted with K. phenyl disubstituted with K, phenvl trisubstituted with K, naphthyl.
napnthyl substituted with K. naphthyl disubstituted with K. naphthyl trisubstituted with K. C I
10 alkyl with an attached phenyl group. Cl lo alkyl with two ar~aehed phenyl g~oups. Cl lo SUBSTITUTE SltEET

WO 92/12140 ,~ 0~, PCr/US91/098~1 yl with an attached phenyl gsoup substituted with K. and C l l 0 alkyl with two attached phenyl groups substituted with K:
J is selected from the group consisting of halogen. COOH. OH. C~J. N02, NH2, C
l o alkoxy, C l l o aLIcylamine. C2- l 2 dialkylamine. C l 1 o alkyl-C~CO-, C l lo alkyl-O-CO-NH-, and C 1- l0 alkyl-S-;
K is selected from the gsoup consisting of halogen, C l- lo alkyl, C 1-10 perfluoroal~yl, Cl lo aL~coxy, N02, CN, OH. C02H, amino, Cl lo alkylamino, C2 12 dialkylamino, Cl-Clo acyl, and Cl lo alkoxy-CO-, and C1 1o aIkyl-S-;
AA is a side chain blocked or unblocked amino acid with thc L configuration, D
10 configuration, or no chirality a~ the -carbon selected from the group consisting of alanine, valine, leucine, isoleucine, p~oline. methionine, methionine sulfoxide, phenylalanine, t~yptophan, glycine, serine. threoninc, cystcine, tyrosine, asparagine, glutaminc, aspartic acid, glutamic acid, Iysine, arginine. histidine. phenylglycine, beta-alaninc. norleucinc, norvaline.
alpha-aminobutysic acid, epsilon-aminocaproic acid. citrulline. hydroxyproline. omithine, 15 homoarginine, sarcosine. indoline 2-carboxylic acid~ 2-azctidinccarboxylic æid, pipccolinic acid (2-piperidinc carboxylic acid), O-methylsesine, O-ethylscsinc. S-mcthylcysteine, S-ethylcystcine. S-bcnzylcystcine. NH~-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, ~H~-CH(CH~-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl~COOH, 20 NH~-CH(CH2-cyclopropyl)-COOH, trifluorolcucine, and hcxafluoroleucine;
R is sclccted from the group consisting of H, C 1-20 alkyl, C 1-20 alkyl with a phenyl group attached to the C 1-20 alkyl, and C 1-20 alkyl with an attached phenyl group substituted with K.
The following compounds are representatives of the invention:
25 Z-Leu-Phe-CONH-E~
Z-Leu-Phe-CONH-nPr Z-Leu-Phe-CONH-nBu Z-Leu-Phe-CONH-iBu Z-Leu-Phe-CONH-Bzl 30 Z-Leu-Phe-CONH-(CH~)2Ph Z-Leu-Abu-CONH-Et Z-Leu-Abu-CONH-nPr Z-Leu-Abu-CONH-nBu Z-Leu-Abu-CONH-iBu 3~ Z-Leu-Abu-CONH-Bzl Z-ku-Abu-CONH-(CH~ )2Ph Z-Leu-Abu-CONH-(CH2)3-1~(CH'CH')2o Z-Leu-Abu-CONH-(CH ~)7CH3 Z-Leu-Abu-CONH-(CH2)20H

SUBSTITUTE SHEET

WO 92/12140 2 0 ~ ~ ~ O ~ Pcr/usg1/09801 Z-Leu-Abu-CONH-(CH2)2O(CH~ )~OH
Z-Leu-Abu-CONH-(CH2) 1 7CH3 ZLeu-Abu-coNH-cH2-c6H3(ocH3)2 Z-Leu-Abu-CONH-CH2-C4H4N
5 Bz-DL-Ala-COOEt Bz-DL-Ala-COOBzl Bz-DL-Ala-COO n-Bu Bz-D~Ala-COOH
Bz-DL-Phe-COOE~
10 Bz-DL-Ala-COOCH2-C6H4-CF3 (para Bz-DL,Arg~OOEt Bz-DL-Lys-COOEt Bz-DL-Lys-COOH
Z-Ala-DL-Ala-COOEt 15 Z-Ala-DL-Ala-COOBzl Z-Ala-DL-Ala-COan-Bu Me~Suc-Ala-DL-Ala-COOMe Z-Leu-Nva-COOEt Z-Leu-Nle-COOEt 20 Z-Leu-Phe-COOEt Z-Leu-Abu-COOEt Z-Phe-DL-Phe-COOEt H-Gly-DL-Lys-COOEt H-Ala-DL-Lys-COOEt ~5 H-Pr~DL-Lys-COOEt H-Phe-DL-Lys-COOEt Z-Ala-Ala-DL-Ala-COOEt Z-Ala-Pr~DL-Ala-COOEt Z-Ala-Ala-DL-Abu-COOEt 30 ZAla-Ala-DL-Abu-COOBzl Z-Ala-Ala-DL-Abu-COOCH2-C6H4-CF3 (para) MeaSuc-Val-Pr~DL-Phc-COOMc H-Leu-Ala-DL-Lys-COOEt Z-Ala-Ala-Ala-DL-Ala-COOEt 35 .M~Suc-Ala-Ala-PT~DL-Abu-COOMe Z-Leu-Phe-COOE~
2-Leu-Nva-COOEt ZLen-Abu-COOEt PhCO-Abu-COOEt Sl '8ST5T~T.~ S~1E~T

wO 92/12140 PCl'/USglt~9801 (CH3)2CH(CH2)2CO-Abu-COOEt CH3cH~cH)2cHco-Abu-cooEt Ph(CH2)6CO-Abu-COOEt Z-Leu4~a-Phe-COOEt 5 Z-Leu-Leu-Abu-COOEt Z-Leu-Leu-Phe-COOEt 2-NapS02-Leu-Abu-COOEt 2-NapS02-Leu-Leu-Abu-COOEt Z-Leu-Met-C02Et I O Z-Leu-NLeu-CO ~E~
Z-Leu-Phc-C02Bu Z-Leu-Abu-C02Bu Z-Leu-Phe-C02Bzl Z-Leu-Abu-CO"Bzl 15 Z-Leu-Phe-COOH
Z-Leu-Abu-COOH

Materials and Methods. HEPES, heparin, and A23187 were obtained from Calbiochem. Suc-Leu-Tyr-AMC and chromogenic substrates were obtained from Sigma.20 Calpain I was purifled from human erythrocyscs according to the method of Kitahara (Kitahara et al., J. Biochem. 95, 1759-1766) omitsing the Blue-Sepharose step. Calpain II from rabbit muscle and cathepsin B were purchased from Sigma. Papain was purchased from Calbiochem.
Ass~y of Inhibifory Potency. Pepside a-kesoamides were assayed as reversiblc enzyme inhibitors. Various concentrations of inhibitors in Me2SO were added to the assay mixture 'S which contained buffer and substrate. The reaction was st~ted by the addition of she enzyme and the hydrolysis rates were followed specsrophotometrically or fluorimetrically.
Calpain I from human erythrocytes and calpain II from rabbit were assayed using Suc-Leu-Tyr-AMC [Sasaki et al.. J. Biol. Chem. 259, 12489-12494 (1984); incorporated herein by reference3, and the AMC (7-amino~methylcotunarin) release was followed fluorimetrically 30 (excitation at 380 nm, and emmision at 460 nm). Calpains were assayed in 25 mM Tris pH =
8.0, 10 mM CaC12. Fluorescence was followed using a Gilson FL- I A fluorometer or a Perkin-Elmer 203 Fluorescence spectrometer. Cathepsin B was assayed in 20 mM sodium acetate pH
= S.2, 0.5 mM dithiothreitol using Bz-Phe-Val-Arg-~nitroanilide as substrate. Alternately, cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and Kirschke. Merhods En~. mol. 80, 35 535-561 (1981); incorporated herein by refercnce], and ~he AFC (7-amino-~
trifluoromethylcoumarin) release was followed fluorimetrically (excitation at 400 nm and emmision at 505 nm). Papain was assayed in 100 mM KPO4, 1 mM EDTA. 2.5 mM cysteine pH = 6.0 using Bz-Arg-AMC or Bz-Arg-NA [Kanaoka et al.. Chem. Phanm. Bull. 25, 3126-3128 (1977): incorporated herein by t~fcrence] as a substrate. The AMC (7-amino~

SVBSTITUTE SHEET

wO 92/12140 2 PCr/US~1/09801 ~ 927~ 7 ~

methvlcourn~rin) release was followed fluorirnetrically (excitation at 380 nrn, and emrnision at 460 nm). Enzymauc hydrolysis rates were measured at various substrate and inhibitor concentrations. and KI values were determined by either Lineweaver-Burk plots or Dixon plots.
A 0.1 M Hepes, O.S M ~aCl. pH 7.5 buffer was utilized for human leukocyte elastase (HLE), porcine panc~eatic elastase (PPE), chymotrypsin and cathepsin G. A 0.1 Hepes, 0.01 \~I CaC12, pH 7.5 buffer was utilized for trypsin, plasmin, and coagulation enzymes. A 50 rnM
Tns HCI,2 mM EDTA,5 rnM cysteine, pH 7.S was used as a buffer for papain. A 88 mM
KH2P04, 12 mM Na2HP04, 1.33 rnM EDTA, 2.7 mM cysteine, pH 6.0 so]ution was used as a buffer for cathepsin B. A 20 rnM Hepes, 10 mM CaC12, 10 rnM mercatoethanol, pH 7.2 buffer was utilized for calpain I and calpain ~.
HLE and ~k were assayed with Me~Suc-Ala-Ala-Pro Val-NA and Suc-Ala-Ala-Ala-~IA, respectively tNakajima et al.. J. Biol. Chem. 2S4, 4027-4032 (1979); incorporated he~cin by reference]. Hurnan leukocyte cathepsin G and chymotrypsin Aa were assayed with Suc-Val-pro-phe-NA [Tanaka et al.. Biochemistry 24, 2040-2047 (1985); incorporated herein by reference]. The hydrolysis of peptide 4-nitroanilides was measured at 410 nm [e410 = 8800 ~l~lcm~l; Erlanger et al., Arch. Biochem. Biophys. 95, pp 271-278 (1961); incorporated herein by reference~. Trypsin, thrombin, human plasma kallikrein. poreine pancreatic kallikrein, human factor Xla, and hurnan plasmin were assayed with Z-Arg-SBzl or Z-Gly-Arg-SBu-i tMeRae et al., Bioehemistry 20, 7196-7206 (1981); ineorporated herein by refereneel. All peptide thioester hydrolysis rates were measured with assay mixtures eontaining

4.4'-dithiodipyridine ~e324 = 19800 M- lem~ 1; Grasetti & Murray, Arch. Biochem. Biophys.
119, pp 4149 (1967); ineorporated herein by reference~. Papain was assayed with Bz-Arg-AMC or Bz-Arg-NA [Kanaoka et al., Chem. Pharm. Bull. 25, 3126-3128 (1977); ineorporated '5 herein by referenee]. The AMC (7-amino~methyleoumarin) release was followed fluorimetrieally (excitation at 380 nm, and emmision at 460 nm). Cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and Kirschke, Methods Enzymol. 80.535-561 (1981);
incorporated herein by refereneel, and the AFC (7-amino 1 ~ifluoromethylcournarin) release was followed fluorimetrically (excitation at 400 nm, and emmision at 505 nm). Calpain I fiom human erythnocytes and ealpain II from rabbit were assayed using Suc-Leu-Tyr-AMC [Sasaki et al.. J. Biol. Chem. 259, 12489-12494 (1984); incorporated herein by reference], and the AMC (7-amino 4 methyleoumarin) release was followed fluorimetrieally (excitation at 380 nm.
and emrnision at 460 nm). Enzymatic hydrolysis rates were measured at various substrate and inhibitor eoncentrations, and KI values were deterrnined by either I ineweaver-Burk plots or ~5 Dixon plots.
Pla~elet mern~rane penneability assav. Calpain-mediated breakdown of spectrin was rneasured by quantitative densitome~y of the calpain-speeific 150/155 kDa spectrin fragment doublet [see Sirnan et al., Proc. Narl. Acad. Sci. USA 81, 3572-3576 (1984)]. Platelets were isolated by a modifieauon of the me~hod of Fe~Tell and Martin ~J. Biol. Chem. 264, 20723-SU~3STITU I SHEET

WO 92/12140 PCr/US91/0~801 3f~ 8-~0729 (1989)1. Blood (15-20 ml) was drawn from male Spraguc-Dawley rats in~o lil0th volume of 100 mM EDTA-citrate, and centrifuged 10 minuus at 2000 rpm in a ciinical centrifuge at room temperature. The plasma was resuspended in 15 ml of buffer l (136 mM
~aCI. ~.7 mM KCI, 0.42 mM NaH2P04, 12 mM NaHC03, 2 mM MgCl 7, 2 myml BSA
(Sigma), 5.6 mM glucose, 22 mM Na3citrate pH 6.5) and platelets werc isolated at 2200 rpm at room temperarure for 10 minutes. Platelets wcre washed once in 15 ml buffer 1, then resuspended to 107 cells/ml in buffer 2 (136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH2P04, 12 mM NaHCO3, 2mM MgCl, 1 mg/ml BSA (Sigma), 5.6 mM glucose, 20 mM HE'PES (Sigma) pH 7.4) and allowed to "rest" for a minimum of 10 minutes at room temperaturc before usc.
Inhibitors were added from stock solutions made fresh in DMSO. 100 ~LI platelets, suspended to 107 cells/ml in buffer 2, were incubated with 1 ~ll of an inhibitor solution for ~
minutes at room temperaD prior to the addition of 2 mM Ca2+ and 1 uM A23187. After 10 minuses total exposure to inhibitor (5 minutes exposure to ionophore) at room temperature, platele~s were reisolated at 14,000 rpm for 10 sec in a Beckman microfuge, dissolved in SDS-I ~ PAGE sample buffer, and heated to 90 C for 3 minutes.
Samples were subjected to SDS-PAGE in 4-12% gTadient mini gels (Novex) and transferred to nitrocellulose (Schleicher and Schuell 0.45 um) by electroblotting. Filters were blocked for 10 rninutes in 0.2S% gelatin, 1% BSA. 0.25% triton Xl00, 0.9% NaCl, 10 mM
Tris-CI pH 7.5, incubated overnight in the same solution containing antibody to rat spec~in, 20 washed 3 x 10 minutes with 10 mM Tris-Cl pH 7.5, 0.5% triton X 100, incubated 4 hours in wash buffer plus ~Ik~line phosphatase conjugated goat anti-rabbit antibody (Biorad), and washed as above. Blots wcre developed using the Biorad AP conjugate substrate kit.
Quantitative densitomctry was used to obtain values for the intact spectrin bands and the 150/155 kDa breakdown product doublet.
Structure-Activirv Relationships. Tables I and IV shows the inhibition constants (~I) for human leukocycte elastase (HLE), porcine pancreatic dastase (PPE), chymotIypsin and cathepsin G. Tripeptide and tetrapeptidc ketoesters with Ala, Abu, or Nva in the Pl site are potent inhibitors of HLE and PPE. Amino acid and dipeptide ketoesters with Ala in the P1 site are less potent than the tripeptides. Z-Ala-Ala-Abu-CO-OBzl is a potent inhibitor of elastases, and replacement of the Z group (PhCH2OCO-) by PhCH2CH~CO-, PhCH~CH~SO2-, PhCH~NHCO-. and PhCH2NHCS- would result in good inhibitor structures. Changing the R
group of Z-Ala-Ala-Abu-CO-OR from ethyl to bcnzyl or p-trifluoromethylbenzyl results in equally potent inhibitors of HLE. However, replacement of ethyl by benzyl group in Z-Ala-Ala-CO-OEt makes a better elastase inhibitor. Amino acid and peptide kesoesters with Phc in 3~ the Pl sitc 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 me~hoxvsuccin- l group by Z. benzoyl, PhCH~CH~SO2-, PhCH~'HCO-, or PhCH~NHCS- would rcsult in good inhibitors for chymotrypsin and cathepsin G.

51)S~ITllTE SHET

WO 92/12140 2 ~ 9 8 7 0 2 PCr/US91/09801 Tablc II shows the inhibition constanls (KI) for trypsin. plasmin, and sevcral blood coagulation enzymes. Amino acid and peptidc kctoes~ers with Arg or Lys in the Pl site are good inhibitors of trypsin. although thcy 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-

5 Pro-Arg-CO-OEt and Boc-D-Phe-Pr~Arg-CO-OEt arc expectcd to bc potcnt thrombin inhibitors because the interactions between thc enzyme and inhibitor increase.
H-Gly-Lys-CO-OEt inhibits thrombin bctter than Bz-Lys-CO-OEt. but this dipeptide ketoestcr is a less potent inhibitor for hurnan plasma kallikrcin. Therefore variation of thc blocking group and arnino acid sequence in the peptidc ketocsurs would result in the more specific inhibitors 10 toward individual coagulation enzymes.
Tablcs III and IV shows the inhibition constants (KI) for papain, cathepsin B, calpain I.
and calpain ~. Dipcptide ketocsters vith Abu, Phe, or Nle in the P 1 site and Leu in the P2 site are potent inhibitors of calpain I and calpain lI. 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 (PhCH20CO-) by similar groups such as PhCH2CH2CO-, PhCH2CH~SO--, PhCH-7NHCO-, and PhCH2NHCS- would also result in good inhibitor structures. Extending the R group to include longer aL~cvl groups or alkyl groups substituted 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 Pl site are also good inhibitors for papain and cathepsin B. Z-Phe-Phe-CO-OEt~ Z-Ala-Ala-Nva-CO-OEt, and MeO-Suc-Val-Pro-Phc-CO-OMe are potent inhibitors of cathepsin B, and rcplacement of the Z
(PhCH20CO-) or McO-Suc- group by PhCH2CH2CO-, PhCH~CH2S02-, PhCH~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-C~OEt, thus changing thc benzvl ~5 group to a smallcr alkyl group such as methyl, or propyl would make better papain inhibitors.
Tablc IV shows the inhibition constants (Kl) for cathepsin B, calpain I, and calpain II
with pcptide ketoamides. Dipeptide a-ketoamides with Abu and Phe in the Pl site and Lcu in the P2 site are potent inhibitors of calpain I and calpain II. Z-Leu-Abu-CONH-Et is a better inhibitor of calpain I than Z-Leu-Phe-CONH-Et by 14 fold. Replacement of the Z group (PhCH20CO-) by sirnilar groups such as PhCH2CH ~CO-, PhCH~CH2S02-, PhCH2NHCO-, and PhCH~NHCS- would also result in good inhibitor strucnlres. The best inhibitor of calpain II is Z-Leu-Abu-CONH-(CH~)2-Ph. Chan~ing thc R3 and R4 groups significantly improves the inhibitory potency loward calpain ~. The best dipeptide inhibitors are those which havc long alkyl side chains (e.g. Z-Leu-Abu-CONH-(CH2)7CH3), alkyl side chains ^a with phenyl substituted on the alkyl group (e.g. Z-Leu-Abu-CONH-(CH2)2-Ph), or alkyl groups with a morpholinc nng substitutcd on the alkyl group ~e.g. Z-Leu-Abu-CONH-(CH2)3-Mpl, Mpl = -N(CH~CH2)2O1 Dipeptide a-kctoamides with a small aliphatic amino acid residuc or a Phc in the Pl site are also good inhibitors for cathcpsin B. The best inhibitor is Z-Leu-Abu-CONH-Et 2nd replacement of the Z (PhCH~OCO-) by PhCH~CH2CO-, SUBS~lTUTE SHEET

W092/12140 7~39~ ~3~'J PCJ/US~1/09801 PhCH2CH2S02-, PhCH2NHCO-. and PhCH2NHCS- would also result in good inhibitor structures.
Peptide a-ke~oamides and peptide ketoamides were substantially more stable in both plasma and liver than the corresponding peptide a-ke~oesters (Table IV). The pepide a-S ketoamides and ketoacids were also much more effective in the platelet assay. Extending theR3 group to an alkyl group or an alkyl group substituted with a phenyl group increased the membrane permeability of the inhibitors as indicated by increased potency in the platelet assay.
Inhb~non Mec)~nism. A crystal structure of one a-ketoester bound into ~he active site of porcine pancreatic elastase has been compleud and a schernatic drawing of the interactions 10 observed is shown below. The active site Ser-195 oxygen of the enzyme has addcd to the carbonyl group of the ketoester to forrn a tetrahedral inunnediau which is stabilized by inuractions with the oxyanion hole. This structure resembles the utrahedral intermediate involved in peptide bond hydrolysis and proves that a-ketoesters are transition-state analogs.
His-57 is hydrogen bonded to the carbonyl gTOUp of the esur functional group, the peptide 15 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' subsitcs. The side chain of the Pl amino acid residue is located in the S1 pocket of the cnzyme. Interactions with ketoamides would be similar except for that there would be the possibility of forrning an additional hydrogen bond with the NH group of the ketoamide functional group if R3 or R4 was H. If R3 and/or R4 are ~0 longer substutuents, 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 these inhibitors would be slightly less potent than the ketoesters and ketoamides.
S~ ~I Ser-195 ~ ~ ~ N ~ _ S' subsites ,0 H ,0~- "
H O XYhaolnen ~ His-57 Val 216 Phe-214 Se 214 ~5 The acrive site of cysteine proteases share several features in cornmon with serine proteases including an acive si~e histidine residue. Irl place of the Ser- 195, cysteine pro eases have arl active site cysteine residue which would add to the ketonic carbonyl group of the pepide keto acids, keto esters, or ke~oarnides to forrn an adduct very similar to the structure depicted a~ove except with a cysteine residue replacing the serine- 195 residue. Additional SUBSTITUTE S~

interacttons 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.
Inhibiror Des~gn and Selec~ion The peptide and amino acid a-kctocstcr~ a-ketoacid, and -kuoamide derivatives, as shown in the above crystal structure, bind to the enzymes a using many of the interactions that are found in complexes of a particular individual enzyme with its substrates. In order to design an inhibitor for a parttcular serine or cysteine protease, it is necessary to: l ) find the amino acid sequences of good peptide substrates for that enzyme, and 2) pla~e those or similar amino acid sequences into a -ketoester, a-ketoacid, or a-ketoamide structure. Additional interacsions with the enyme can be obtained by tailoring the R
10 g~roup of the inhibitor to imitate the amino acid xsidues which are preferred by an individual protease at the S l' and S2' subsites. For e~cample, ketoesters with R = branched allcyl groups would interact effectively with senne and cysteine proteases which prefer Leu, ne, and Val residues at P I ' and/or P2', while ketoesters and amides with R = alkyl substituted with phenyl would interact eKectively with serine and cysteine proteases which prefer Phe, Tyr, Trp I ~ residues at Pl' and/or P2'. Likewise, the Ml group can be tailored to interact with the S
subsites of the enzyme. This design strategy will also worlc when other classes of peptide inhibitors are used in place of the peptide substrate to gain infonnation on the appropriate sequence to place in the ketoester, ketoacid, or ketoamide inhibitor. Thus, we are able to predict the structuse of new inhibitors for other serine and cysteine proteases based on ~0 knowledge of their substrate specificities. Once a good inhibitor structure for a particular enzyme is found, it is then possible to change other characteristics such as solubility or hydrophobicity by adding substituents to the M1 or R, R3, and R4 groups.
Elastase is an enzyme which hydrolyzes most effectively tetra- and tripeptides having P
residues with small alkyl side chains such as Ala and Val. MeO-Suc-Ala-Ala-Ala-Val-NA and ~5 Z-Ala-Ala-Ala-Ala-NA are good substrates (NA = 4-nitroanilide). Thus the corresponding c~-ketoesters Z-Ala-Ala-Ala-DL-Ala-COOEt and MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe are excellent elastase inhibitors. Suc-Phe-Leu-Phe-NA is an excellent substrate for chymotrypsin, cathepsin G, and mast cell chymases. Thus, the corresponding a-ketoester is an excellent inhibitor for these chymotrypsin-like enzyrnes. In the case of the cysteine protease calpain. a v 30 good inhibitor sequence is Ac-Leu-Leu-Nle-H. We have found that ketoesters related in structure such as Z-Leu-Abu-CO-OEt and Z-Leu-Nle-CO-OEt are potent inhibitors for calpain.
We have also found that ketoarnides related in siructure 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 pount inhibitors for the listed enzymes.
3~ The inhibitor sequences were obtained from peptide substrate and/or inhibitor sequences in the protease literature.
Z-Gly-Leu-Phe-CaQ-R for cathepsin G and RMCP Il Me(~Suc-Ala-Ala-Pr~Met-CO-~R for cathepsin G
Boc-Ala-Ala-Asp-CaQ-R for hurnan Iymphocyte granzyme B

SUBSf~TUTE Sl~EE~

wO 92/12140 9 ~ ?~ -32- PCI/US91/09801 Suc-Pro Leu-Phe-CO-Q-R and Boe-Ala-Ala-Phe-CO-Q-R
for RMCP I (RMCP = rat rnast cell proteasc Boc-Gly-Leu-Phe-CO-Q-R. Suc-Phe-Leu-Phe-CO-Q-R
for hurnan and dog skin ehymase Boc-Ala-Ala-Glu-CO-Q-R for S. au~us V-8 protease Z-Gly-Gly-Pro-CO-Q-R for human prolyl endopepadase Ala-Pr~CO-Q-R for DPP IV
Suc-Ala-Ala-Pro-Val-CO-Q-R for PPE
Sue-Lys(Cbz)-Val-Pro-Val-CO-~R. adamantyl-S02-Lys(COCH2CH2C02H)-Ala-Val-CO-Q-R, adarnantyl-CH2CH20CO-Glu(O-t-Bu)-Pro Val-CO-Q-R. and adasnantyl-SO2-Lys(CO-C6H4C02H) Ala-Val-CO-Q-R for hurnan Ieukoeyte (neutrophil) elastase Sue-Ala-Ala-Pr~Leu-CO-Q-R for elastolytie proteinase from "Sehistosoma mansorli"5 Glu-Phe-Lys-CO-Q-R and Dns-Ala-Phe-Lys-CO-Q-R
for plasmin D-Val-Gly-Arg-CO-Q-R and Dns-Glu-Gly-Arg-CO-Q-R
for factor Xa Z-Phe-Arg-CO-Q-R and Z-Tr~Arg-CO-Q-R
for poreine panereaae and human plasma kallilcreins Z-Lys-Arg-C~Q-R for human skin tryptase Z-Gly-Arg-CO-Q-R for human lung tryptase Z-Ile-Ala-Gly-Arg-CO-Q-R for faetors IXa, Xa, XIa. XIIa and bovine plasma kallikre~
Giu-Gly-Arg-CO-Q-R for urokinase Dns-Phe-Pro-Arg-CO-Q-R for plasminogen aetivator Dns-Ile-Pro-Arg-CO-Q-R for ac~vated protein C
Z-Tr~Arg-CO-Q-R for bovine faetor IXa ;0 Z-Gly-Arg-CO-Q-R for bovine faetor Xa and XIa Z-Phe-Arg-CO-Q-R for bo~ine factor XIIa Dns-Glu-Gly-Arg-CO-Q-R for human factor Xa D-Phe-Pro-Arg-CO-Q-R. D-~IePhe-Pro-Arg-CO-Q-R. and Boe-D-Phe-Pro-Arg-CO-Q-R for human thrombin Z-Phe-Gly-Arg-CO-Q-R for trypsin Cl-C6H4CH~OCO-Phe-Gly-CO-O-n-Bu for papain C6H5CH 71~lHCO-Gly-Phe-Gly-CO-O-n-Pr for eathepsin B

SU~3ST~TUT~ SHEET

WO 92/12140 2 0 9 8 7 0 2 PCr/US91/09801 where Q is O for ke~oesters; and R is selected from the group consisting of H. C 1-20 aLkyl, C 1-20 aLkyl with a phenyl group atlached to the C 1-20 alkyl, and C 1-20 aLkyl with an attached phenyl group substituted with K.
where Q-R is OH for ketoacids.
where Q-R is -NR3R4 and R3 & R4 are selecred independently from the group consisting of H, C 1-20 aLkyl, C 1-20 cyclized aLkyl, C 1-20 aLkyl with a phenyl group attached to the C 1-20 aL~yl, C 1-20 cyclized alkyl with an attached phenyl g~oup, C 1-20 aLkyl with an attached phenyl group substituted with K, C 1-20 alkyl with an attaehed phenyl group disubstituted with K, Cl 20 aLkyl with an attaehed phenyl group trisubstituted with K. Cl 20 cyclized aLkyl with an attaehed phenyl group substituted with K, Cl 1o aIkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C 1- 10 aLkyl with a piperidine ring attached through nitrogen to the aIkyl, Cl lo aLkyl with a pyrrolidine ring attached through nitrogen to the aLkyl, Cl 20 aLkyl with an OH group attaehed to the aLkyl, -CH~CH2OCH2CH~OH, Cl lo with an attaehed 4-pyridyl group, Cl lo with an attached 3-pyridyl group, Cl lo with an attached 2-pyridyl group, Cl 1o with an attaehed cyclohexyl group, -.~H-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).
/n virro Uses. To use the above inhibitors in vitro, they are dissolved in an organie solvent such as dimethylsulfoxide or ethanol, and are added to an aqueous solution containing serine and/or cysteine proteases. The final coneentration of îhe organie solvent should be less than 25%. The inhibitors may also be added as solids or in suspension. The serine and cysteine protease inhibitors of this invention would be useful in a variety of experimental proeedures where proteolysis is a signifieant problem. Inelusion of these inhibilors in a radioimmunoassay experiments would result in higher sensitivity. The use of Ihese inhibitors in plasma fractionation procedures would resull in higher yields of valuable plasma proteins and ~5 would make purifieation of the proteins easier. The inhibitors diselosed here could be used in cloning experiments utilizing bacterial cultures, yeasl and human cells IO yield a purified eloned produet in higher yield.
The novel compounds of this invention are effeetive in the prevention of unnecessary proteolysis eaused by chymotTypsin-like, elastases, and trypsin-like enzymes in the process of purification, transport and storage of peptides and proteins as shown in Tables I. II, m. and IV
by effective inhibiion of chymotrypsim elastase. trypsin. and other serine & cysteine proleases.
/n vivo C;ses. Effecive inhibitors of the proteolylic funcuion of human leukocyte elastase and human cathepsin G (Tables I and IV) would have anti-inflalTuna~ory activity and can be used IO treat and eontrol emphysema. adult respiratory distress syndrome and rheumaloid arthritis. Effeeive inhibi~ors of the pro~eolyde funeuon of chymotrypsin and pancreaue elastase (Tables I and rV) are effeetive for therapeutic use in treatment of pancreatitis.
Various ~ etoesters have anucoagulant aeuiviry as shown in Table II by effeetiveinhibition of the proteolytic funeion of blood eoagulaion enzymes in Hcpes buffer. Other SUBSTITUTE SHEFr WO 92/~2140 PC~/US91/098~1 ~ 5~J~1 3~

peptide a-ketoesters have anti-tumor activity as shown in Table rl by the effective inhjbition of the proteoly~c function of human plasma plasmin Peptide a-ketoesters can be used to control protein tusnover, muscular dystrophy, myocasdial tissue damage, tumor metastasis, and bone resorption as shown in Tables m and IV
5 by effective inhibition of Iysosomal cathepsin B in buffer. Peptide a-ketocsters can also be used as neuroprotectants or for the treatment of ischemia, stroke or Alzheimer s disease as shown in Tables m and IV by effective inhibiton of calpain I and calpain II.
Considerable evidence has shown that leukocyte elastase and/or related enzymes play a role in tumor cell metastasis [Salo et al., Int. J. Cancer 30, pp o69-673 (1973); Kao et aL, Biochem. Biophys. Res. Comm. 10S, pp 383-389 (1982); Powers, J. C. in Modification of Proteins, R. E. Feeney and J. R. Whitaker, eds., Adv. Chem. Ser 198, Amer. Chem. Soc., Wash., D. C. pp 347-367 (1982); all incorporated herein by reference], therefore it is suggested that compounds of this invention may have anti-tumor activity.
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 parentehyma associated with pulmonary emphysema are caused by uncontrolled proteolysis in lung tissues [Janoff, Chesr 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, l. ~b. Clin. Mcd. 82, 349-356 (1973); incorporated herein by referencel, particularly human leukocyte elastase ~Janoff, ibid 1 lS,461-478 (1977);
incorporated herein by reference]. Leukocyte elastase and other mediators of inflammation also appear to play a role in diseases such as mucocutaneous Iymph node syndrome [Reiger et al., Eur. J. Pediatr. 140,92-97 (1983); incorpoRted herein by reference] and adult respiratory distress syndrome [Stockley, Clinical Science 64, 119-126 (1983); Lee et al., N. Eng. J. Med.
'5 304, 192-196 (1981); Rinaldo, ibid 301, 900-909 (1982); incorporated herein by referencel.
It is known that in vitro activity of elastase inhibitors correlates with in vivo activity in animal models of emphysema and inflammation [Otterness et al., editors, Advances in Inflammation Research, Vol. 11, Raven Press 1986; incorporated herein by reference].
Prophylactic administration of an inhibitor of elastase significantly diminishes the extent of elastase-induced emphysema [Kleinerman et al., Am. Rev. Resir. Dis. 121, 381-387 (1980);
Lucey et al., Eur. Respir. J. 2, 421-427 (1989); incorporated he~in by referencel. Thus the novel inhibitors described here should be useful for the treatment of emphysema and inflarnmation. Elastase inhibitors have been used orally, by injection, or by instillation in the lungs in anirnal studies (Powers, Am. Rev. Respir. Dis., 127, s54 s58 (1983); Powers and Bengali, Am. Rev. Respir. Dis. 134, 1097- 1100 (1986): these tWO articles are incorporated herein by reference). The inhibitors described above can be used by any of these routes.
Drug Delivery. For therapeutic use, the peptide -ketoesters, -ketoamides. and -ketoacids may be administered orally. topically or parenterally. The term parenteral as used includes subcutaneous injection. intravenous, intramuscular. intrasternal injection or infusion SuBsTlTuTE S~EE~ ' WO 92/12140 2 0 9 ~ 7 0 2 Pcr/us91/09801 techniques. 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 administ~ration is best determined by individually assessing the particular case.
The pharrnaceutical compositions containing the active ingredient may be in a forrn 5 suitable for oral use, for example as tablets, troches, lozenges, aqueous or oily suspcnsions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs. I~osage 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 maurials to produce a single dosage forrn 10 will vary depending upon the host treated and the particular mode of administration.
For injection, the therapeutic amount of the peptide r~-ketoesters, a-ketoamides, and a-ke~oacids or their pharmaceutica~ly accepuble salts will normally be in the dosage range from 0.2 to 140 mg/kg of body weight. Administration is rnade by intravenous, intramuscular or subscutaneous injection. Accordingly, pharmaceutical compositions for parenteral15 administration will contain in a single dosage form about 10 mg to 7 gms of the compounds per dose. In addition to the active ingredient, these pha~rnaceutical 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 ean be forrnulated as an aqueous solution, lotion, ~0 jelly or an oily solution or suspention. A composition in the forrn of an aqueous solution is obuined by dissolving the compounds of this invention in aqueous buffer solution of pH 4 to 65 and if desired, adding a polymeric binder. An oily forrnulation 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.
~5 SYNT~IlC MEl~IODS
The ketoester inhibitors are prepared by a two step Dakin-West procedure. This procedure can be utilized with either arnino acid derivatives, dipeptide derivatives, tripeptide derivatives, or terrapep~de derivatives as shown in the following scheme.
M,--AA--OH EnolEster ~ M,--AA--CO-O-R

M,--M--AA--OH Enol Ester - ~ M.--AA--AA--CO-O-R

M.--M--M--M--OH - ~ Enol Ester ~M,--AA--AA--AA--CO-O-R

M,--M--M--AA--AA--OH ~ Enol Ester--M,--M--AA--AA--M--CO-O-R

SlJBSTlTUTt SHEET

The precursor peptide can be prepared using standard pepide chemistry which is wcll dcscribed in publications such as The Peptides. Analysis, Syn-hesis, Biology, Vol. 1-9, published in 19~9-1987 by Academic Press and Houben-Weyl Methoden der Organischen Chcmic, Vol 15, Parts 1 and 2, Synthese von Peptiden. published by Georg Thicme Verlag, Stuttgart in 1974 S (both references incorporatcd hercin by rcfercncc).
The M I group can be introduced using a number of differcnt reaction schcmcs, First it could bc introduced directly on an anuno acid as shown in the following schemc (top), or the M I group could be introduced by reaction with an amino acid cster, followcd by rcmoval of the es~er group to give the sarne product (bottom).
H--M--OH -- M,--M--OH

H--M--O R' -- M1--M--O R'- ~ M1--M--OH
The tcchniqucs for introduction of thc M 1 group is well docurnented in the The Peptides, Houbcn-Wcycl, and many other tcxtbooks on organic synthcsis. For examplc reaction with cyanate or p-nitrophenyl cyanate would introduce a carbamyl group (Ml =
IS ~ CO-). Reaction with Me2NCOCI would introduce thc Mc2NCO- group. Reaction with p-nitrophenyl thiocarbarnatc would introduce a thio carbamyl group (Ml = NH2CS-). Rcaction with NH2SO2CI would introducc thc NH2SO2- group. Reaction with Mc2NSO2CI would introducc thc Mc2NSO ~- group. Rcaction with a substituted alkyl or aryl isocyanate would introducc thc X-~H-CO- group where X is a substituted alkyl or aryl group. Reaction with a ~0 substituted aL~cyl or aryl isothiocyanatc would introducc thc X-NH-CS- group whcrc X is a substututed alkyl or aryl group. Rcaction with X-S02-CI would introduce thc X-S02- group.
Rcaction with a substitutcd alkyl or aryl acid chloride would introduce an acyl group (M = X-CO-) For cxarnplc. reacuon with MeO-CO-CH2CH~-CO-CI would give ~hc X-CO- group wherc X is a C2 alkyl subsututcd wi~h a C I al~yl-OCO- g~oup. Rcaction with a substituted ~5 aLkyl or aryl thioacid chloridc would introducc a thioacyl group (M = X-CS-). Rcaction with an a substitutcd alkyl or aryl sulfonyl chloridc would introducc an X-S02- group. For cxarnplc reacuon with dansyl chloridc would give the X-SO~- dcrivative where X was a naphthyl group mono substituted with a dirncthylamino group. Reaction with a substitutcd aLlcyl or aryl chloroforrnatc wouid introduce a X-O-CO- group. Reaction with a substituted alkyl or aryl 30 chlorothioformatc would introducc a X-O-CS-. There are many altcrnatc rcaction schcmcs wnich could bc uscd ~o introducc all of the abovc M I groups to give either M I -AA-OH or M I -AA-OR'.
The M I -AA-OH derivativcs could thcrl bc uscd dircctly in the Dakin-West reaction or could bc convcrtcd into thc dipepudcs. tripcptides, and tctrapcptidcs Ml-AA-AA-OH, M1-AA-5 AA-AA-OH, or M I -AA-AA-AA-AA-oH which could ~c bc uscd in thc Dakin-Wcst reaction.
Thc subsututed pcptidcs M1-AA-AA-OH. Ml-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH
could also bc prcparcd dircctly from H-AA-AA-OH, H-AA-AA-AA-OH~ or H-AA-AA-AA-SU~3STlTUTE SHE~T

WO 92/12140 2 0 ~ ~ 7 0 2 PCI/US91/09801 AA-OH using the reactions described above for introduction of the M group. Alternately, ~he M group could be introduced by reaction with carboxyl blocked peptides to give Ml AA AA
OR', M I -AA-AA-AA-OR', or M I -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 Dalcin-West reaction by reaction with an oxalyl chloride Cl-CO-C~O-R. For exarnple, reaction of Ml-AA-AA-OH
with ethyl oxalyl chloride a-CO-CO~Et gives the ke~o ester M l-AA-AA-CaO-EL Re~rtion of Ml-AA-AA-AA-AA-OH with a-CO-CO~Bzl would give the ketoester Ml-AA-AA-AA-AA-CO-o-Bzl~ Clearly a wide variety of R groups can be introduced into the ketoester structure by reaction with vatious alkyl or arylalkyl oxalyl chlorides (a-CO-CO-O-R). The oxalyl chlorides are easily prepared by reaction of an alkyl or arylalkyl alcohol with oxalyl chloride Cl-CO-CO-Cl. For exarnple, Bzl-O-CO-CO-CI and n-Bu-O-CO-CO-Cl are pT~pard by reaction of respectively benzyl alcohol and butanol with oxalyl chloride in yields of 50~O and 80% [Warren, C. B., and Malee, E. J., J. Chromatography 64, 219-222 (1972); incorporated herein by reference~.
Ketoacids Ml-AA-CO-OH, Ml-AA-AA-CO-OH, Ml-AA-AA-AA-CO-OH, Ml-AA-AA-AA-AA-CO-OH, are generally prepaxd from the colTesponding kesoesters M l-AA~:O-OR, Ml-AA-AA-CO~R, Ml-AA-AA-AA-CO-OR, Ml-AA-AA-AA-AA-CO-OR by al~aline hydrolysis. In some cases, it may be necessary to use other methods such as hydrogenolysis of a benzyl group (R = Bzl) or acid cleavage (R = t-butyl) to obtain the ke~oacid. The alurnau methods would be used when the M group was labile to aLkaline hydrolysis.
Ketoamides Ml-AA-CO-NR3R4, M-AA-AA-CO-NR3R4, M-AA-AA-AA-CO-NR3R4, M-AA-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 reacion with an amine H-NR3R4. The illustrated procedure should also work with other protecting groups. In addition, the corrcsponding 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 reagen~s would yield an intermediatç which could then be deblocked to form the ketoamide.

WO 92/12140 PCr/US~l/09801 " Oq, M, -AA2' ~N ~ R4 Rl O
General Synthenc Me-hods. Unless otherwise noted, materials were obtained fi~m commercial suppliers and used withou~ further purifieation. Melting points were taken with a Bùchi capillary apparatus and are uncorreeted. lH Nl~ speetra were determined on a Varian Gemini 30G. Chemieal shifts ar~ expressed in ppm (o) relative ~o internal tetramethylsilane.
Flash column chromatography was performed with Universal Scientific Inc. siliea gel ~63.
Elecrron-irnpact mass speetra (MS) of novel eompounds were determined with a Varian MAT
l l'S speetrometer. The puri~y of all compounds was checked by thin-layer ehroma~ography on Baker Si250F siliea gel plates using the following solvent system: A, CHC13:MeOH = 20: 1 vlv:B,CHC13:.~feOH= l00:1v/v;C,AeOEt:D,CHC13:MeOH= 10:1 v/v;E, n-BuOH:AeOH;py:H2O = 4:1:1:2 v/v: F, CHC13:MeOH = 5:1 v/v: G. AeOEt:MeOH = 10:1 v/v: H, (i-Pr)2O: I, CHC13:MeOH:AeOH = 80:10:5 v/v: J, CHC13:MeOH:AeOH = 95:5:3 v/v;
K. AeOEt:AeOH = 200: I v/v; L, CHC13; M, CHC13:MeOH = 50: I v/v Amino aeid me~hyl ester hydroehlorides wc~ prepared according to M. Brenner et al.[Helv. Chem. Acta 33,568 (1950); 36, 1109 (1953)] in a scale over 10 mmol or according to Raehele [J. Org. Chem. 28, 2898 (1963)] in a seale of 0.1-1.0 mmol.
Yield (%) mp (C) m.p. (literaturc) DL-~va-OCH3 HCI, 100 113- 116 116- 117 L-Ile-OCH3 HCl, 98 90-91 98-100 '0 L-Phe-OCH3 HCI, 98 159-161 158-160 DL-Abu-OCH3 HCI, 100 148-IS0 150-151 L-Leu-OCH3-HC1 100 145.5- 146.5 147 DL-.~i'le-OCH3 ~IC1 93 120-121 1'2-123 1 -Cl-Phe-OCH3 HC1 98 184- 185 (decomp. ) 185- 186 'S ~-Aeylarnino aei~s was synthesized via Schotten-Baumann reaction [M. Bergsnann. L.
Zervas. Chem. Ber. 65, 1192 (1932)] in the case when the acyl group was phenylsulphonyl, '-naph~hylsulphonyl or benzoyl.

SU~3STITUTE SHEET

wo 92/12140 2 0 " ~ 7 0 7 PCl/US9t/09801 Yield (C~c) mp (C) TLC (Rf~ eluent '-~apSO2-L-Leu-OH ~9 115-116 0.58 1 '-~'apSO2-DL-Abu-OH 51 150- 151 0.50 2-NapSO2-L-Phe-OH 57 148- 148.5 0.48 K
PheSO,-DL-Abu-OH 44 142- 143 0.51 K
PhCO-DL-Abu-OH 64 141-142 0.64 K
N-Acylamino acids with ~methylpentanoic, 2-(1-propyl)pentanoic and 7-phenylheptanoic group was synthesized in a two step synthesis. The N-acylarnino acid methyl ester was obtained first and then was hydrolysed to the free N-acylamino acid.
N-Acvlamino Acid Merky~ Esters (General Proce~re). To a chilled (10 C) slurry of the appropnate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene was added slowly (temp. 10-15 C) 40 mmol triethylamine or N-methylmorpholine and then the reac~tion mixture was stirred for 30 minutes a~ this temperature. Then 18 mmol of appropnate acid chloride (temp. 10-15 C) was added slowly to the reaction mixture and the reaction mixture 15 was stirred overnight at room temperature. The precipitated hydrochlonde was filtered, washed on a funnel with 2 x 20 ml benzene, and the collected filtrate was washed successively with 2 x 50 ml I M HCI, 2 x 50 ml 5% NaHC03, I x 100 ml H20, 2 x 50 ml satd. NaCI and dried over MgSO4. After evaporation of the solvent in vacuo (rotavaporator), the residue was check~d for purity (TLC) and used for the next step (hydrolysis).
'0 Yield (%) mp (C) (cH3)2cH(cH2)2co-DL-Abu-ocH3 80 oil (cH3cH2cH2)2cHco-DL-Abu-ocH3 96 117-118 Ph(cH2)6co-DL-Abu-ocH3 72 oil Hydrolysis (General Proced~re) To a solution of 10 mmole of the appropriate ~5 .~-acvlamino acid methyl ester in 100 ml of me~hanol was added in one portion 11.25 ml of I
~I NaOH (11.25 mmol) and the reaction rnixture was stirred three hours at room temperature.
Then the reaction mixture was cooled to 0 C (ice-salt bath) and acidifled to pH = 2 with I M
HCI aq. To this reaction mixture was added 100 ml ethyl acetate, transferred to a separatory funnel and organic layer separated. The water lay was saturated with solid NaCI or (NH ~)2S04 and reextracted with 2 x 50 ml AcOEL The collected organic layer was washed with 2 x 50 ml H2O, decolorized with carbon. and dried over MgSO1. After evaporation of the solvent in vacuo (rotavaporator), the residue was checked for puriry (TLC) and in the case of contaminauon was crystallized from an appropriate solvent.
Yield (%) mp (C) 35 (cH3)2cH(cH~)2co-DL-Abu-oH 92 110.5-112 (CH3CH2CH,,), CHCO-DL-Abu-OH 99 12~127 (n~ctane Ph(CH2)6CO-DL-Abu-OH 89 110-112(n-octane) ~ -Acyldipeptide methyl esters were synthesized via the HOBt-DCC me~hod in a DMF
soluuon [Konig and Geiger. Chem. Ber 103, 788 (1970)]

SUBSTITUTE SHEET

wO 92/t2140 PCr/US91/09801 ? Q ~ Yield (%) mp (C) TLC (Rf, eluent) Z-Leu-DL-NVa-OCH3 80 112- 113 0,37 B
Z-Leu-L-Phe-OCH3 83 86-87 0.85 A
0.39 B
Z-Leu-L-Ile-OCH3 97 oil 0.79 A
0.43 B
Z-Leu-DL-Abu OCH3 99 86-88 0,33 B
0.26 H
Z-Leu-L-Leu-OCH3 80 91 -92 0.79 G
Z-Leu-DL-NLeu-OCH3 97 111 - 111.5 Z-Leu 4 a-Phe-OCH3 65 112-132 0.77 J
(liquid crystal?)0.68 K
2-NapSO,-Leu-DL-Abu-OCH3 99 oil 0,59 A
2-~1apSO,,-Leu-L-Leu-OCH3 90 97-98.5 0,63 A
IS N-Acyldipeptid~s wcre obtaincd by hydrolysis of the appropriate mcthyl estc,rs via a general hydrolysis pr.~cdure. In thc casc of N-sulphonyldipcptide methyl cstcrs, 1 e~uivalcnt of thc mcthyl ester was hydrolyzed with 2.25 cquivalcnt of 1 molar NaOH bccausc of form a sulfonamide sodium salt.
Yicld (%) mp (C) TLC (Rr eluent~
'O Z-Leu-DL-NVa-OH 100 117-118,5 0.11 A
Z-Leu-L-Phe-OH 92 10S-106.5 0.28 C
0.55 G
Z-Leu-L-ILe-OH 79 77-79 0.22 A
0.S2 C
'5 Z-Leu-DL-Abu-OH 99 glass 0.61 G
Z-Leu-L-Leu-OH 97 glass 0.56 I
Z-Leu-DL-NLeu-OH 98 95-96 Z-Leu 4 a-Phe-OH 87 104-114 0.48 K
(liquid crystal?) 2-NapSO2-Leu-~L-Abu-OH 97.4 180-195 (decomp) 0.58 2-~apS02-Leu-L-Leu-OH 94.0 68-70 0.52 1 N-Acytripepode methyl esters were synthesized ~ia HOBt-DCC method in DMF
solution lKonig and Geiger. Chem. Ber. 103. 788 (1970)].
Yield (%) mp (C) TLC ~r eluent) ~5 Z-Leu-Leu-Abu-OCH3 87 140-141.5 0.50 A
Z-Leu-Leu-Phe-OCH3 76 158-159 0.83 J
2-NapSO2-Leu-Leu-Abu-OCH3 97 >200 0.52 A
N-Acyltnpepude were obtained through h,vdrolysis of the appropriate methyl esters via gcneral hyds~lysis procedure. In the case of N-sulphonyltripepode methyl estcr, 1 equivalen~

wO 92/12140 2 0 9 8 7 0 2 PCI/US91/09801 of methyl estcr was hydrolyzed with 2.25 equivalent of 1 molar i~iaOH to form thc sulfonarnidc sodium salL
Yield mp (C) TLC (Rf. eluen Z-Leu-Leu-Abu-OH 97 glass 0.69 1 Z-Leu-Leu-Phc-OH 98 glass 0.44 K
~-~'apSO~-Leu-Leu-Abu-OH 85 193-195 (decomp) 0.53 0.32 J

The following detailed examples are given to illustrate thc invention and are not intenW
10 to limit it in any manner.
EXAI~T F I
Z-Ala-DL-Ala-COOEt. This compound was synthesized by a modified Dalcin-Wcst procedure [Charles et al., J. Chem. Soc. PerJcin 1, 1139-1146, (1980)]. To a stirred solution of Z-Ala-Ala-OH (880 mg, 3 mmole), 4-dimethylaminopyridine (15 mg, 0.31 mmole)~ and pyndine (0.8 rnL. 10 mrnole) in tetrahydrofuran (3 mL) was added ethyl oxalyl chlonde (0.7 mL. 6 rnmole) at a rate sufficient to initiate refluxing. The mixture was gently refluxed for 3.5 h. The rnixture was treated with water (3 mL) and stin~d vigorously at room tempcrature for 30 rnin. The rnixture was extracted with ethyl acetate. The organic extracts wc dried and eva?orated to obtain the residue (1.45 g). The residue was chromatographed on silica gcl and '0 eluted with CH2C12 to give the enol ester product, oil (500 mg,37%); single spot on tlc, Rf2 =
0.67 (CHC13:MeOH = 9: 1); MS, m/e = 451 (M++ 1). To a stirred suspension of the enol ester ~210 mg, 0.47 mmol) in anhydrous cthanol (1 mL) at room temperature was added dropwise a solution of sodium ethoxide in ethanol until a clear ycllow solution resulted. The ethanol was then removed and the residue was treated with ether. The ether solution was washed with 'S water, dried~ and evaporated to give a residue. This residue was chromatographcd on a silica gel and the product was eluted with methylene chloride. The solvent was rcmoved, and the peptide ketocstcr Z-Ala-DL-Ala-C02Et was obtained as an scmi-solid (150 mg,92 %); single spoton tlc, Rfl 0.58 (CHC13:MeOH = 5:1); MS, m/c = 351 (M++l). Anal. Calcd. for CI7H.2O61~2 1/3 H~O: C, 57.29: H, 6.22: N, 7.86. Found: C. 57. 3; H. 6.36; N, 8.17.
EXA~LE 2 Z-Ala-Ala-DL-Ala-C02Et. This compound was prepared from Z-Ala-Ala-Ala-OH
using thc samc procedu~ as descnbed in Example 1. The product was crystallized from ethyl etherin23%yield;singlespotontlc,Rf2=0.31 (CHC13:MeOH=9:1);mp 143-144C;MS.
mJe = 421 (M+). Anal. Calcd. for C~oH27O7N3: C. 56.99; H, 6.46; .~. 9.97. Found: C, 56.96: H. 6.49; ~. 9.92.
EXA~T F 3 - Z-Ala-Ala-DL-Abu-CO2Et. This compound was prepared from Z-Ala-Ala-DL-Abu-OH in 11% yield by the procedure describcd in Example 1: single spot on tlc. Rf- = 0.60 SUBS~lTU~E SHEET

wo 92~12140 ~ PCr/US91/~g801 q~9~ 12-(CHC13:MeOH = 9:1); mp 111-113 C; .~S. rme = 436 (M~-l). Anal Calcd. for C~IH~gO7N3 1/3 H20: C. 57.13; H, 6 75; .~J. 9 51. Found: C, 57.38: H. 6 82: .~, 9 62 Z-Ala-Ala-DL-~va-C02Et. This compound was prepared from Z-Ala-l~lva-OH in 20% yield by the procedure described in Example 1; single spot on tlc, Rfl = 0,64 (CHC13:MeOH=5:1);MS.mle=450(M++l) AnaLcalcd.forc~aH3lo7N3-H~o:
5651: H. 7 11; N, 8.99 Found: C. 56.42; H, 7.08; N, 9.06.
EXA~MPLE 5 Z-Ala-Pro-DL-Ala-CO2Et. This compound was prepared from Z-Ala-Pro-Ala-OH.dicyclohexylalTLine in 19% yield by the procedure described in Example l; singlc spot on tlc, Rf2 = 0.55 (CHa3:MeOH = 9:1); MS, rn/e = 447 (M+). Anal. Calcd. for C22H~gO7N3-1/2 H20: C, 57.88: H, 6.62; N, 9.21. Found: C. 57.65; H, 6.68; N, 9.17.
EXA~LE 6 Z-Ala-Ala-Ala-DL-Ala-CO2Et. The compound was prepared from Z-Ala-Ala-Ala-Ala-OH in 7% yield by the procedure described in Example l; single spot on ~Ic, Rf2 =0.40 (CHC13:MeOH = 9:1); mp. 163-165 C; MS. rn/e = 493 (M++1). Anal. Calcd. for C23H32OgN4-1/2 H2O: C, 55.08; H. 6.63; N. 11.17. Found: C, 54.85; H. 6.53; N, 11.14.

Bz-DL-Phe-CO2Et. This compound was prepared from Bz-Phe-OH in 36% yicld by the procedure described in Example 1, oil, single spot on tlc. Rf2 = 0.61 (CHC13:McOH =
9: 1); MS, m/e = 325 (M+). Anal. Calcd. for ClgH1gO4N.1/3 H2O: C, 68.86; H. 5.98; N, 4.22. Found: C. 69.10; H, 6.09; N, 4.38.
EXAhIPT F 8 MeO-Suc Ala-DL-Ala-CO2Me. This compound was prcpared from MeO-Suc-'5 Ala-Ala-OH in 22% yield by the same procedure as described in Example 1, except that sodium methoxide in mcthanol was used for enol ester hydrolysis, single spot on tlc, Rf2 = o 43 (CHC13:MeOH = 9:1); MS, m/e = 317 (M++l). Anal. Calcd. for C13H20O7N4.1/3 H~O: C, 48.44; H, 6.46; N, 8.69. Found: C. 48.56: H. 6.39; N, 8.69.

MeO-Suc-Ala Ala-Pro-DL-Abu-C02Me. This compound was prepared from eo-suc-Ala-Ala-pro-DL-Abu-oH in 22% yield by the procedure descnbed in Example 8;
foam, single spot on tlc, Rfl = 0.66 (CHC13:MeOH = j: 1). Anal. Calcd. for C~H34OgN4 H~O: C, 51.53; H, 7.02; N, 10.85. Found: C. 51.11; H. 7.03: .~. 10.88. EXAMPLE 10 ~eO-Suc-Val-Pro-DL-Phe-CO~Me. This compound was prepared from MeO-Suc-Val-Pro-Phe-OH in 42% yield by the same procedure as described in Exarnple 8: foam.
single spoton ~Ic, Rf2 0.57 (CHC13:MeOH = 9:1): MS. m/e = 517 (M+). Anal. Calcd. for C26H3sOgN3-2!3 H20: C. 58.96: H. 6.90; 1~', 7.93. Found: C, 58.92: H. 6.96: ;~, 7.89.
EXA.~lPLE 11 SU~STITUTE SHEET

WO 92/12140 2 ~ n ~ 7 ~ 2 PCr/US91/09801 -~3-Bz-DL-Ala.CO2-n-Bu. This compound was prepared from Bz-Ala-OH in 45%
yield by the proredure described in Example 1, except that n-butyl oxalylehloride was used for the Dakin-West reaetion and sodium n-butoxide in n-butanol was used for enol es~:r hydrolysis: colorless oil, single spot on tlc, Rf2 = 0.72 (CHC13:MeOH = 9:1); MS. rn/e = 277 (~+)-Bz-DL-Ala-COtBzl. This compound was prepared from Bz-Ala-OH in 26% yield by the procedure deseribed in Example 1, exeept that benzyl oxalyl chloride was used in piaee of ethyl oxayl ehloride and sodium benzyloxide in benzyl alcohol was used for enol ester hydrolysis; single spot on tlc, Rf2 =0.69 (CHC13:MeOH = 9:1); mp 95-97 C; MS, mie = 312 (M++l). Anal. Caled. for ClgH17O4N 1/2 H2O: C, 67.48; H, S.66; N, 4.37. Found: C, 67.78; H, 5.55; N, 4.66.
EXA~LE 13 Z-Ala-DL-Ala CO~ n-Bu. This eompound was prepared from Z-Ala-Ala-OH in lS 14~o yield by the procedure deseribed in Example 1, excep~ that n-butyl oxalyl ehloride was used in the Dakin-West reaetior. and sodium n-butoxide was used for enol ester hydrolysis; oil, single spoton tlc, Rf2 = 0.45 (CHC13:MeOH = 9:1); MS, srJe = 378 (M+). Anal. CalcL for ClgH26O6N2.1/3 H2O: C. 59.35; H, 7.00; N, 7.29. Found: C, 59.41; H, 7.03; N, 7.10.

Z-Ala-DL-Ala-C02Bzl. This eompound was prepared from Z-Ala-Ala-OH in 36%
yield by the procedure described in Example 1, except that benzyl oxalyl ehloride was used in the Dakin-West reaetion and sodium benzyloxide in benzyl aleohol was used for enol ester hydrolysis; single spot on tlc, Rf2 = 0.55 (CHC13:MeOH = 9:1); MS, rn/e = 413 (M++l), Anal. Caled. fbr C22H24O6N2: C, 64.06; H, 5.87; N, 6.79. Found: C, 63.79; H, 5.95; N, '5 6.72.
EXA~LE 15 Z-Ala-Ala-DL-Abu-CO2Bzl. This compound was prepared from Z-Ala-Ala-Abu-OH in 31 % yield by the proeedure described in Example 1, exeept that benzyl oxalyl ehloride was used in the Dakin-West reaction and sodium benzyloxide in benzyl alcohol was used for enol estcr hydrolysis: single spot on tlc, Rf2 = 0.40 (CHC13:MeOH = 9: 1); mp 12~125 C;
~IS. mle = 498 (M+-l). Anal. Calcd. forC26H3107N3 2/3 H20: C. 61.28; H, 6.39; N,8.24. Found: C. 61.14; H. 6.65; N. 7.94.
EXA~LE 16 Bz-DL-Ala-COOH. The hydrolysis procedure of Tsushima et al. [J. Org. Chem.
~5 49, 1163-1169 (1984)~ was used. Bz-DL-Ala-CO2Et (540 mg, 2.' rnrnol) was added to a solution of 650 mg of sodium bicarbonate in an aqueous 50% 2-propanol solution (7.5 mL of H2O and 2-propanol) and surred at 40 C under nitrogen. After adding ethyl acetate and a saline solution to the reaction mixture. the aqueous layer was separated and aeidified with 2N
HCI and extraeted with ethyl aeetate. The organie layer was dried over magnesium sulfate and SU~STITUTE SHEE~r .~ -44-q ~
the solvent was remo~éd under reduced prcssure. The crude hydrolysis product was chromatog~aphed on silica gel and eluted with methylc~e chloride and methanol lo obtain an oil (ISOmg,31%):singlespotontlc.Rf4=0.68(n-butanol:aceticacid:pyndine:H~0=4:1:1:2).
Anal. Calcd. for C I lHI 104N 3/4 H~O: C, 56.28; H, 5.37; 1~1. 5.97. Found: C. 56.21; H.
5 5.46: j.66.

Z-Leu-DL-Nva-COOEt. This compound was prepared from Z-Leu-Nva-OH in 60 C~c yield by the pt~cedure described in Example 1; oil, one spot on tlc. Rf = 0.49 (CHC13:MeOH = 20:1). NMR (CDC13) d: 0.91 (t, 9H), CH3; I.'S (t, 3H), CH3; 1.38 (q, 2H), OCH2CH3; 1.64 (m, 6H), CH2; 1.85 (m, lH), CH(CH3)2; 4.34 (m, lH) CH2CH(NHCOOCH2Ph?CONH; S.12 (d, 3H) NHCH(CO)CH2 and OCH2Ph; 5.32 (d, lH) NH; 6.71 (d, lH) NH; 7.36 (s, 5H) Ph.
Z-Leu-DL-N-~a-enol ester, the precursor of Z-Leu-DL-Nva-COOEt was synthesized bythe same procedure as described in Example 1 and purified by column chromatography, oil, one spot on tlc. Nh~ (CDC13) d: 0.96 (t, 9H); 1.25 (t, 3H); 1.41 (t, 2H); 1.54 (m, 4H); 1.72 (m, 3H); 2.80 (t, 2H); 4.20 (q, 2H); 4.43 (q, 2H); S.16 (q, 2H); 5.23 (s, lH); 7.37 (m, 5H);
11.33 (s, lH).

Z-Leu-DL-Phe-COOEt. This compound was prcparcd from Z-Lcu-Phc-OH in 30 ~0 % yield by the procedure described in Example l; oil, one spot on tlc, Rf = 0.47 (CHC13:MeOH = 50:1). NMR (CDC13) d: 0.88 (d, 9H), OCH2CH3 and (CH3)2CH; 1.3S (q,2H), OCH2CH3; 1.56 (q, 2H), (CH3)2CHCH2CH: 3.03 (m, lH), (CH3)2CH; 4.32 (m, 2H), NHCH(CO)CH2; 5.08 (s, 4H) CH2Ph; 5.40 (m, IH) NH; 6.61 (d, lH) NH; 7.31 (s,5H) Ph; 7.35.(s, SH) Ph.
Z-Leu-DL-Phe-enol esur. the precursor of Z-Leu-DL-Phe-COOEt was synthesizcd by ~he same procedure as described in Example 1 and purified by column chromalography, oil, one spot on tlc. ~MR (CDC13) 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).

Z~Leu-DL-Abu-COOEt. This compound was prepared from Z-Leu-Abu-OH in 33 % yield by the procedure described in Example l; oil. one spot on tlc, Rf = 0.66(CHC13:~IeOH = 20:1). Nh~ (CDC13) d: 0.96 (t, 9H), OCH2CH3 and (CH3)2CH: 1.'6 (t, 3H), CH2CH2CH3; 1.37 (q, 2H). OCH2CH3; 1.66 (q, 'H), (CH3)~CHCH~CH; 2.00 (m.
lH), CH(CH3)2: 4.12 (q, 2H) CHCH~CH3; 4 34 (m. lH) NHCH(CONH)CH~CH(CH3)2;
5.12 (q, 3H) CH~Ph and CONH(Et)CHCOCOO: S.'9 (t. lH) NH: 6.79 (d. lH) NH; 7.35 (s, ~H) Ph.
Z-Leu-DL-Abu-enol ester. the precursor of Z-Leu-DL-Abu-COOEt was synthesized by the same procedure as described in Example I and purified by column chromatography, oil.

SUBSTITUTE SHEET

wO 92/12140 2 G 9 8 7 O ~ Pcr/usgl/og8ol one spot on tlc. NMR (CDC13) d: 0.98 (t, 6H): 1.12 (t, 3H): 1.24 (t. 3H); 1 41 (t. 3H~: 1 73 (m, 4H): 2.86 (q, 2H); 4.20 (q, 2H): 4 31 (m. IH): 4 42 (q, 'H); S.IS (q, ~H~; S ' l (s, IH):
7 34 (m, SH); 11.'9 (s, IH).
EXA,V~PT F 20 S Ala-DL-Lys-COOEt-HCI. To a solution of N-carbobenzyloxyalanyl-Ne-carbobenzyloxylysine (1.88 g, 3.9 mmol),4-dimethylaminopyridine (21 mg, 0.17 mmol), and pyridine (1 0 mL,12.4 mmol) in THF (7 mL) was added ethyl oxalyl chloride (0.9 mL, 8.0 mmol) at a rate sufficient to start reflux~ng. The mixture was refluxed gently for 3 hr, treated with water (4 mL), and stirred vigorously at room temperature for 30 min. The mixture was extracted with ethyl acetate, the organic extracts were washed with water, dried over ~vIgSO4 and evaporated to give an oily residue (1.56 g). To a solution of the enol ester (1.56 g,2.7 mmol) in anhvdrous ethanol was added dropwise a solution of sodium ethoxide in ethanol at room temperature until the solution turned clear yellow. Ethanol was removed and the residue was dissolved in ethyl aceute. The organic solution was washed with water, dried over ~IgSO4, and evaporated to give a residue. This residue was then purified by column chromatography and the pt~duct was eluted with chloroform-methanol. The solvent was removed and Z-Ala-DL-Lys(Z)-CO~Et was obtained as a hygroscopic powder (328 mg, 16 %), single spot on tlc. Rf2 = 0.53 (CHC13:MeOH = 9:1); MS, m/e = 542 (M++l).
N-Carbobenzoxyalanyl-DL-Necarbobenwxylysine keto cthyl ester, Z-Ala-DL-Lys(Z~
CO2Et (328 mg,0.61 rnmol) was deprotected with liquid HF conuining anisole at 0 C for 30 min. The HF was removed under reduced pressure. The residual oil was dissolved in absolute ethanol. HCVethanol was added to the solution, and ethanol was removed in vacuo. The residue was washed by decantation with ether to give a semi solid (216 mg,100 %): single spot on tlc (n-bu~anol:acetic acid:pyridine:H2O = 4: 1: 1 :').
~5 EXA~LE 21 Bz-DL-l,ys-COOEt HCI. This compound was prepared from Bz-DL-Lys(Z)-COOEt in 62% yield by the pr~cedure described in Exarnple 20; one spot on tlc, Rf4 = 0.57 (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2). The precursor, Bz-DL-Lys(Z)-COOEt was prepared from Bz-Lys(Z)-OH in 1009O yield by the procedure described in Example l; powder, one spot on tlc, Rf2 = 0.75 (CHC13:MeOH = 9: 1); MS. m/e = 440 (M+). Anal. Calcd. for C24H2gO6~2-2/3 H20: C. 63.70: H, 6.53; N, 6.19. Found: C, 63.49: H, 6.5': N. 5.92.

Bz-DL-Arg-COOEt HCI. This compound was prepared from Bz-DL-Arg(Z)-COOEt in 99% yield by the procedure described in Example 20: one spot on tlc, Rf4 = 0.71 (n-butanol:aceuc acid:pyridine:H20 = 4:1:1:2), Sakaguchi reagent positive. Bz-DL-Arg(Z)-COOEt was prepared from Bz-DL-Arg(Z)-OH in 19% yield by the procedure described in Example 20. Rf2 = 0.38 (CHC13:MeOH = 9:1): mp 140-142 C: MS, m/e = 468 (M 1) Anal.
Calcd. for C24H2gO6N4: C, 61.53: H, 6.02: N. 11.96. Found: C. 61.96; H. 6.48~ '. 12.34 SUBSTITUTE SHEET

H-Gly-DL-I,ys-COOEt 2HCI. This compound was prepared from Z-Gly-DL-Lys(Z)-COOEt in 92% yield by the procedurc described in Example 20; Rf = 0.21 (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). Z-Gly-DL-Lys(Z)-COOEt was preparcd from Z-Gly-Lys(Z)-OH in 9% yield by dle procedure described in Example 20, one spot on tlc, Rfl =
0.68 (CHC13:MeOH = 5:1); MS, mie = 528 (M++1).

H-Pro-DL-Lys-COOEt 2HCI. This compound was prepared from Z-Pro-DL-Lys(Z)-COOEt in 100% yield by the procedure described in Example 20; one spot on dc (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). Z-Pro-DL-Lys(Z)-COOEt was prepared from Z-Pro-Lys(Z)-OH in 15% yield by the procedure described in Example 20: Rf2 = 0,73 (CHC13:MeOH = 9:1); MS, m/e 568 (M++l).
EXAMPLE '5 H-Phe DL.Lys-COOEt 2HCI. This compound was prepared from Z-Phe-DL-15 Lys(Z)-COOEt in 39% yield by the procedure describcd in Example 20: one spot on tlc (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2). Z-Phe-DL-Lys(Z)-COOEt was prepared from Z-Phe-Lys(Z)-OH as previously described in 9% yield. Rf2 = 0.68 (CHC13:MeOH = 9:1); MS, n~e = 482 (M+).

'0 H-Leu Ala-DL-Lys-COOEt 2HCI. This compound was prepared from Z-Leu-Ala-DL-Lys(Z)-COOEt in 52% yield by the procedure described in Example 20; one spot on tlc (n-butanol:acetic acid:pyridine:H20 = 4~ 2).
Z-Leu-Ala-DL-Lys(Z)-COOEt was prepared from Z-Leu-Ala-DL-Lys(Z)-OH in 5%
yield by the previously described Dakin West reaction, Rf3 = 0.34 (cHcl3:MeoH = 19:1);
'5 ~S. m/e= 609 (M+-OCH2CH3).

Sirnple Amino Acid. Di- and Tripeptidc Enol Esters (General Procedu~e). A modified Dakin-West procedure was used [Charles et al., J. Chem. Soc. Perkin 1. 1139 (1980)~ and is illustrated with the synthesis of Z-Leu-DL-Phe-EE. To a stirred solution of Z-Leu-Phe-OH
(6.19 g, l5.0 mmol), 4-dimethylaminopytidine (0.183 g; 1.5 mrnol) and pyridine (4.75 g, 4.85 ml, 60 mmol) in ~etrahydrofuran (45 ml) wanned 50 C was added ethyl oxalyl chloride (4.30 g, 3.52 ml, 31.5 mmol) at a rate sufficient to initiate refluxing. The mixture was then heated at a gentle reflux for 4 h. After cooling to room umperature the mixture was treated with water (25 ml) and stirred vigorously at room temperature for 30 min. The mixture was extracted with ethyl acetate ( 150 mi) and after separation of the organic layer, the water layer was saturated with solid (NH4)2S04 and re-exttacted 2-times with 25 ml ethyl acetate. The combined organic phases were washed 2-dmes with 75 ml water, 2-dmes with 50 ml of satd.
NaCI, 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 SUBSrITUTE SHEFr wO 92/12140 2 0 ~ ~ 7 0 2 PCr/US91/09801 -~7-eluted with a AcoEl~ The solvent was evaporated in vacuo (rotavaporator~ and the pure enol ester was obtained as a oil (7.'2 g, 85%); single spot on TLC. Rf = 0.84. A: 0.68. C.
Z-Leu-Nva-EE. This compound was prepared from Z-Leu-.~iva-OH using the gene~l procedure and purified by flash chromatography on silica gel using CHC13:~IeOH = 50:1 vlv ~s eluent. Yield 95%, single spot on TLC, Rf = 0.92, C; 0.28 ,L.
Z-Leu-Abu-EE. This compound was prepared from Z-Leu-Abu-OH in 78% yield the general procedure described above. Purification by flash obr~matography on silica gel. Eluent, CHC13~ IeOH = 50:1 v/v, single spot on TLC, Rf = 0.86, A.
PhCO-Abu-EE. This compound was prepared from PhCO-Abu-OH in 26% yield by 10 the general procedure as described above. Purification by flash chromatography on silica gel.
Eluent CHC13, single spot on TLC, Rf = 0.60, M.
(CH3)2CH(CH2)2CO-Abu-EE. This compound was prepared from (CH3)2CH~CH2)2CO-Abu-OH in 82% yield by the general procedure as described above.
Purification by flash chromatography on silica gel. Eluent AcOEt, single spot on TLC, Rf =
IS 0 7', C.
(CH3CH2CH2)2 CH CO-Abu-EE. This compound was prepared from (CH3CH,,CH~)2CH CO-Abu-OH in 100% yield by the general procedure described above.
Purification by flash chromatography on silica gel. Eluent AcOEt. single spot on TLC, Rf =
0.78, C; 0.81, K.
'O Ph~CH2)6CO-Abu-EE. This compound was prepared from Ph(CH2)6CO-Abu-OH in 86% yield by the general procedure described above. Purification by flash chromatography on silica gel. Eluent AcOE~ Single spot on TLC, Rf =0.74, C.
Z-Leu4-CI-Phe-EE. This compound was prepared ft~m Z-Leu4-CI-Phe-OH in 69%
yield by the general procedure described above. Purification by flash chromatography on silica 'S gel. Eluent AcOEt, single spot on TLC, Rf = 0.77, C; 0.78, K.
Z-Leu-Leu-Abu-EE. This compound was prepared from Z-Leu-Leu-Abu-OH in 62%
yield by the general p;ocedure described above. Purification by flash chrornatography on silica gel. Element CHC13:MeOH = 50:1 v/v. Single spot on TLC, Rf = 0.89. A; 0.75, M.
Z-Leu-Leu-Ph~-EE. This compound was preparcd from Z-Leu-Leu-Phe-OH in 60~o 30 yield by the general procedure described above. Purification by flash chromatography on silica gel. Eluent CHC13:.~1eOH = j0: I v/v. Single spot on TLC, Rf = 0,80, K; 0.70. M. 2-NapS02-Leu-Leu-Abu-EE. This compound was prepared from 'apSO,-Leu-Abu-OH in 73% yield by ~he general procedure descnbed above. Purification by flash chromatography on silica gel. Eluent AcOEt. single spot on TLC. Rf = 0.71, K; 0.54.
C.
2-.~VapS02-Leu-Leu-Abu-EE. This compound was prepared from '-.'~iapSO,,-Leu-Leu-Abu-OH in 74% yield by the general procedure described above.
Purification by flash chromatographv on silica gel. Eluent AcOEt AcOH = 200:1 v/v. Single spot on TLC. Rf = 0.69, K.

SlJBSTlTUTE SHEET

WO 92/12140 PCr/US91/09801 q",~ 48-Z-Leu-Phe-COOEt. Single Aminoacid. Di-and Tripepride-keroesters ~General Procedure). To a stirred solution of 8 53 g (15.0 mmol) of Z-Leu-Phe-EE in 40 ml anhydrous ethanol at room temperature was added dropwise a solution of sodium ethoxide (0.204 g, 3 0 mmol) in 20.0 ml anhydrous ethanol. The color of the reaction mixD change from colorless or pale yellow to deep yellow or orange dependent on enol-ester. Then the reaction mixture was stirred at room temperature for 4-5 hours, the ethanol was then evaporated in vacuo (rotavapora~or) and the residue trcated 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~O, 2 x 75 ml sat~ NaCI. decolorized with carbon and dried over MgS04. After evaporation of solvent, the crude product 6.09 g (89.7%) was flash chromatographed on silica gel using CHCl3: MeOH = 50: l vlv. Evapot~ion of solvent give pure Z-Leu-Phe-COOEt (4.08 g;
58.0%) as a thick oil. Single spot on TLC, Rf = 0.60, A; 0~47, M. Mass spec~um, FB-MS
[ (M+ I )/Zl = 469.

2-Leu.;~va.COOEt. This was prepared by the preceding general procedure.
Purification by Slash chromatography on silica gel, eluent CHC13: MeOH = l00: l v/v, yield 86.6%, thicl;, colorless oil. single spot on TLC, Rf = 0.49, A; 0.37, M. Mass spectrum FB-~IS [(M+l)IZ] = '~21.

Z-Len-Abu-COOEt. This was prepared by the preceding general procedure.
Puri~lcation by flash chromatogtaphy on silica gel, eluent CHCl3, yield 82%, thick, pale yellow oil, single spot on ll,C, Rf = 0.66, A. Mass spectrum, CI-MS [(M+l)/Z~ = 407.

PhCO-Abu-COOEt. This was prepared by the p~ceding general procedure.
'5 Purificauon by flash chromatography on silica gel. eluent CHa3:MeOH = 50: l v/v, yield 83%, oil, single spot on ll C. Rf = 0.44, M. Mass spectrum. M/Z 263 (M+); CI-MS, 264 ((M+l)n).
EXA~LE 3 l (CH3)2CH(CH2)2CO-Abu-COOEt. This was prepared by the preceding general procedure. Purification by flash chromatography on silica gel. eluent AcOEt, yield 43%. oil, single spot on ll C. Rf = 0.S6. C. Mass spectrum EI-MS M/Z 257 ~Mt); FB-MS. [(M+ l ?/Zl = 258.
EXAMPLE 3' CH3CH,CH),CHCO-Abu-COOEt. This was prepared by the preceding general procedure. Purificauon by flash chromatography on silica gel, eluent CHC13:MeOH = 50: 1 v/v, thick, yellowish oil. yield 66%. single spot on TLC, Rf = 0.80, C; 0.66. M. ~ass spectrum EI-MS M/Z = ~85 (M~); CI-MS, [(Mt I )/Z] = 286.

SUBSTITUTE S~ ET

WO 92/12140 2 ~ 9 ~ 7 0 2 Pcr/usg1/09801 Ph(cH2)6co~Abu-cooEt~ This was prepared by the preceding general procedure. Purification by flash chromatography on silica gel, eluem CHC13:.~eOH = 50:1 v/v, yield 64%, pale yellow oil. single spot on TLC. Rf = 0.29, M. Mass spectrum EI-MS
~I/Z = 347 (M'), FB-MS, [(M+l~/Z~ = 348.

Z-Leu.4-CI-Phe-COOEt. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent AcOEt. yield 100%, colorless oil, single spot on TLC. Rf = 0.71, C. Mass spectrum FB-MS M/Z = 503(M~).

~-Leu-Leu-Abu-COOEt. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CHC13:MeOH = 50:1 v/v, yield 79.2%, very thick, colorless oil, single spot on TLC, Rf = 0.28. M. Mass spectrum FB-MS, [(M+l)/Zl = 520.
EXA~MPLE 36 Z-Leu-Leu-Phe-COOEt. This was prepared by the pxceding general procedure.
Purification by flash chromatography on silica gel, eluent CHa3:MeOH = 50:1 v/v, yield 33%, oil, single spot on TLC, Rf = 0.56, M. Mass spectrum, FB-MS, [(M+l)/Zl = 582.

2-NapS02-Leu Abu-COOEt. This was prepared by the preceding general procedure. Pu~lcation by flash chromatography on silica gel, eluent CHC13:MeOH = 50:l v/v, yield 38%, thick oil, single spot on TLC, Rf = 0.71, K; 0.54, A. Mass spectrum FB-MS, [(M+ I )/Zl = 463.

2.NapS02-Leu.Leu-Abu-COOEt. This was prepared by the preceding general '5 procedure. Puriflcation by flash chromatography on silica gel. eluent AcOEt:AcOH = 200: l v/v, yield 61%, semi-solid, single spot on TLC, Rf = 0.67, K. Mass spectrum FB-MS, [(M+l)/ZI = 576.

Z-Leu-Met-C02Et. This compound was prepared by the above procedure. Yellow oil, single spot on TLC, Rf = 0.52 (CHC13:CH30H=S0: 1), yield 46% (from dipep~ide), MS
(FAB) 454 (m+ 1).

Z.Leu-NLeu-CO2Et. This compound was prepared by the above procedure. Pale yellow oil. single spot on TLC, Rf = 0.57 (CHC13:CH30H = 30: l ), yield 53% (from 33 dipepude), .~IS (FAB) 434 (m+ 1).
EXA~LE 4 1 Synrhesis o~n-Buryl Oxalyl Chloride. This was prepared bv a literature procedure[Warren and Ma~cc, J. Chromar. 64, 219-222 ( 1972)]. .~-Butanol (0.1 mol. 7.41 g) was added dropwise to oxalyl chloride (0.5 mol. 63.S g) at -10 C. After the addition was SUBSTITUTE SHEE~r wo 92/12140 PCr/US91/09801 q~ 0~
completed. thc reaction m xture was stirred for 20 min at r.t. and distilled. giving 15 0 g (91.18 mol. 91 %) of the product n-butyl oxalyl chloride, bp 58-60 C (0.6 mrn Hg) Z-Leu-Phe-C02Bu. This compound was prepared from Z-Leu-Phe-OH and butyl oxalyl chloride in 43% yield by the procedurc described for the synthesis of Z-Leu-Phe-CO~Et.
5 except that butyl oxalyl chloride was used in placc of ethyl oxalyl chloride and sodium butyloxide in butanol was used for enol ester hydrolysis. Single spot on TLC. Rf = 0.54 (CHC13:CH30H = 50:1) MS(FAB) m/e = 497 (m+l), lH NMR (CDC13) ok.
EXA~LE 42 Z-Leu-Abu-C02Bu. This compound was prepared by the above procedure. Single spot on TLC, Rf = 0.53 (CHC13:CH30H = 50:1), yield = 36%, pale yellow oil, MS (FAB) m/e = 435 (M+l), lH N~ (CDC13) ok.

S-nthesis of Benzvl Oxalyl Chloride. Benzyl alcohol (0.15 mol. 16 g) was added dropwise to oxalyl chloride (0.75 mol. 95 g) at 5-10 C. After the addition was complete, the reaction was stirred for 20 min. at r.t. The excess oxalyl chloride was distilled and recycled.
Then the mixture was distilled under vacuo, giving 26 g (0.12 mol. 86%) of benzyl oxalyl chloride. bp. 110-112 C (0.6 mm-Hg). HlNMR (CDC13) 7.39 (s, 5H), 5.33 (s, 2H).
Z-Leu-Phe-C02Bzl. This compound was prcpared from Z-Leu-Phe-OH and benzyl oxalyl chloridc in 17% yicld by the procedurc dcseribcd h thc synthesis of Z-Leu-Phc-C02Et~ cxcept that bcnzyl oxalyl chloridc was uscd in placc of cthyl oxalyl chloride and sodium benzyloxide in bcnzyl alcohol was uscd for cnol cster hydrolysis. Single spot on TLC, Rf = 0.63 (CHa3:CH30H = 50:1). Pale ycllow solid, mp 117-119 C. MS(FAB) m/c = 532 (m+l). HlNMR ok.
EXA~LE 44 '5 Z-Leu-Abu-C02Bzl. This compound was preparcd by the above procedure.
Single spot on TLC. Rf = O.5i (CHC13:CH30H = 50:1), palc yellow oil, MS(FAB) m/e = 469 (m+ I ), yield = 26%.
EXA~MPLE 45 Z-Leu-Phe COOH. Dipcptide Ketoacids (General Procedure). To a stirred solution of 0.53g (1.13 mmol) Z-Lcu-Phe-COOEt in 6.0 ml methanol was added 1.27 rnl (1.27 mmol) IM NaOH. The color of thc reaction mixture turned dark yellow and a small amount of solid was dcpositcd. The rcaction was run at room tempcrature and progress of the hydrolysis was checked on TLC. After 24 h. no more substrate was detecte~ The reaction mixture was chilled in one ice bath at 5 C. acidified with IM HCI to pH = 3 and extracted with AcOEt (2 x 50 mL). The organic extract were washed with 2 x 50 rnl H~O and if necessarv. decolorized with carbon and dried over MgS04. After evaporation of the solvent (rotavaporator), the residue (thick oil) were titurated with 2 x 25 ml n-hex~ne and dried in vacuo. Yield 0.39 g (78%) of colorless. very thick oil, TLC. rnain spot at Rf = 0.24, trace of impurity at Rf =
0.78.1. Mass spectrum, FB-MS [(M+l)/Z~ = 441.

S'~E~S~TUTE SHEET

wo 92/12140 2 0 9 ~ 7 O ~ PCltUS91/09801 EXA~LE 46 Z-Leu-Abu-COOH. This compound was prepared from Z-L-Leu-Abu-COOEt in 83% yield by the general procedurc as describcd above: TLC. main spot at Rf = 0.14. trace of impurity at Rf = 0.73.1. Mass spectrum, FB-MS [(M+l)IZ~ = 379.
S Example 47 Z-Leu-Phe-CONH-Et. To a stirred solution of Z-Lcu-Phe-OH (20 g, 48.5 =olc), 4-dimethylaminopyridine (0.587 g, 4.8 =ole),and pyridine (15.7 ml, 194 =ole) in anhydrous lHF (100 ml) was addcd ethyl oxalyl chlonde (11.4 ml, 101.8 mmole) at a rate sufficient to initiate refluxing. The rnixture was gently refluxed for 4 hours, cooled to room temperature. and water (80 ml) was adde~ The reaction mixture was sti~ed vigorously for 30 min, and extracted with ethyl acetate (3 x 100 ml). The combined organic layers were washed with water (2 x 100 ml), saturated sodium chloride (2 x 100 ml), decolorized with dccolorizing carbon, dried over magncsium sulfate, and concenerated, leaving a dark orange oil.
Chromato~raphy on a silica gel column with CHC13/CH30H (50:1 v/v) afforded 14.63 g (y =
53 %) of Z-Lcu-Phe-enolester. The product was a yellow oil. Single spot on TLC, Rf = 0.77 (CHCL3/CH30H 50:1). NMR (CDC13) ok.
To a stirred pale yellow solution of the Z-Lcu-Phe-enolester (14.63 g, 25.73 mmole) in anhydrous ethanol (50 ml) was addcd a solution of sodium ethoxide (0.177 g, 2.6 =ole) in ethanol (5 ml). The orange solution was stirred for 3 hours at room temperature, then the ethanol was evaporated and the residue was tseated with ethyl ether (300 ml). The ether layer was washed with water (2 x 100 ml), saturated sodium chloride (2 x 100 ml), dried over magnesium sulfatc. and concentrated, leaving a orange oil. Chromatography on a silica gel column with CHC13/CH30H (50: 1 v/v) afforded 7.76 g (y = 64 %) of the a-ketoester Z-Leu-Phe-COOEt. The product was a yellow oil. Single spot on TLC, Rf = 0.44 (CHC131CH30H
"5 50:1). NMR (CDC13) ok. MS (FAB, calcd. for C26H32N206: 468.6), m/e = 469 (MT 1).
The -carbonyl group of Z-Leu-Phe-COOEt was protectcd by following procedure. A
solution of Z-Leu-Phe-COOEt (1 g, 2.13 mmolc) in 5 ml of CH2CI~ was added 1,2-ethanedithiol (0.214 ml. 2.55 mmolc), followed by 0.5 ml of boron trifluoride etherate. Ihe solution was sti~ed ovenught at room temperatu~. Watcr (20 ml) and cthyl ether (20 ml) were added. The organic layer was separated, washed with waur (2 x 10 ml), saturated sodium chloride (2 x 10 ml). dried over magnesium sulfate~ and evaporated to afford 0.98 g (y = 84 %) yellow semisolid.
The protected -keloester (0.98 g, 1.8 mrnole) was dissolved in cthanol (5 ml), cooled to O-S C in a ice bath. and ethylaminc was bubbled through the solution until 2.43 g (54 :nmolc) had been added. The reaction mix~ure was allowed to warm to room temperature slowly, and sti~Ted ovemight. The mixmre was filtered. a white precipitate was removed, leaving a yellow semisolid. Chromatography on a silica gel column with CHC13/CH30H (30:1 wv) afford 0.63 g (y = 75 ~o) of Z-Leu-Phe-CONH-Et. The product was a pale yellow solid.
Single spoton TLC. Rf = û.60 (CHC13/CH30H 20:1); mp 145-147 C. Anal. calcd. for ~suBsTlT~JTE S~EET

9~ -52-C~6H33N3Os 467.56; C. 66.79; H. 711; N,8.99; found: C. 66.59: H. 7.09; ~', 8.95. ~rMR
(CDC13) ok. ~IS (FAB) m/e = 468 (M+l).
Example 48 Z-Leu-Phe-CONH-nPr. This compound was synthesized from the protectcd a-ketoester and propylamine in 92 % yield by the proccdure describcd in Exarnple 47. Single spot on TLC, Rf = 0.50 (CHC13/CH30H 50:1); mp 152-153 C. Anal. calcd. for C~7H3sN3Os: 481.57: C. 67.33; H. 7.33; N, 8.72. Found: C, 67.'1; H. 7.38; N, 8.64.
NMR (CDC13) ok. MS (FAB) m/e = 482 (M+l).
Exarnple 49 Z-Leu-Phe-CONH-nBu. This compound was synthesized from the protcctcd a-ketoes~er and butylamine in 67 % yield by the procedure described in Examplc 47. Singlc spot on TLC. Rf = 0.50 (CHC13/CH30H 50:1); mp 152-153 C. Anal. calcd. for C2gH37N3Os:
195.59: C, 67.85; H, 7.52: N, 8.48. Found: C, 67.70; H, 7.57; N, 8.43. ~ (CDC13) ok.
~IS (FAB) rrue = 496 (M+l).
Example 50 Z-Leu-Phe-CONH-iBu. This compound was synthcsized from the protectcd a-ketoester and isobutylamine in 53 % yield by thc proccdurc describcd in Examplc 47. Singlc spot on TLC. Rf = 0.54 (CHC13/CH3OH 50:1); mp 152 C. Anal. calcd. for C2gH37N3Os:
495.59: C, 67.85; H, 7.52; N, 8.48. Found: C, 67.77; H, 7.56: N, 8.40. NMR (CDC13) o~.
'O ~lS (FAB) m/c = 496 (M+l).
Example 5 1 Z-Leu-Phe-CONH-Bzl. This compound was synthesized from thc protcetcd a-ketoester and benzylamine in 40 % yicld by the proccdurc describcd in Exarnple 47. Aftcr reacting overnight, ethyl acetate (60 rnl) was added. The mixture was filtered to remove a white '5 precipitate. The solution was washed with cooled I N HCI (3 x 25 rnl), water ( I x 20 ml), saturatcd sodium chloride (2 x 20 ml), and dricd ovcr magncsium sulfate. The solution was evaporated leaving a ycllow solid. Chromatography on a silica gcl column with CHC13/CH30H 30:1 v/v) afforded a yellow solid. Single spot on TLC, Rf = 0.45 (CHC13/CH30H 30:1); mp 160-162 C. Anal. calcd. for C31H3sN3Os: 529.61; C, 70.30; H,

6.66: N. 7.93. Found: C, 70.18: H. 6.67; N, 7.99. NMR (CDC13) ok. MS (FAB) m/e = 530 (~1+1).
Examlple S 2 Z-Leu-Phe-CONH-(CH~)2Ph. This compound was synthesized from the protccted -kewcster and phenethylarnine in 50 % yield by the procedure described in Exarnple ^5 51. SinglespotonTLC,Rf=O.SO(CHC13/CH30H30:1);mp 151-153C. Anal.calcd.for C3 ~H37N3Os: 543.66: C. 70.70: H. 6.86; N. 7.73. Found: C. 70.54: H. 6.88; ~, 7.74.
~IR (CDC13) ok. .~S ~FAB) mJe = 544 (M+1).
E~wnple 53 SUBSTiTUTE SHEET

WO 92/12140 2 0 9 ~ 7 0 2 PCl/US91/09801 -~3-Z-Leu-Abu-CONH-Et. This compound was synthesized from protected c~-ketoester derived from Z-Leu-Abu-CO~Et and ethylarsune in 64 % yield by the procedure descnbed in Example 47. Single spot on 11 C, Rf = 0.36 (CHC13tCH30H 50:1); mp 130-13' ~C. ~nal. calcd. for C21H31N3Os: 405.45; C, 62.20; H. 7.71; N. 10.36. Found: C. 61.92;
H, 7.62: .~T, 10.31. NMR (CDC13) ok. MS (FAB) mle = 406 (M+l).
Example 54 Z-Leu-Abu-CONH-nPr. This compound was synthesized from the corresponding protecsed ct-ketoester and propylamine in 47 % yield by the procedure described in Example 47. Single spot on l~C, Rf = 0.28 (CHC131CH30H 50:1); mp 134-135 C. Anal. calcd. for C22H33N3OS: 419.50; C, 62.98: H. 7.93: N, 10.02. Found: C, 62.84: H. 7.97 ~, 9 94 NMR (CDC13) ok. MS (FAB) m/e = 420 (M+l).
Example 55 Z-Leu-Abu-CONH-nBu. This compound was synthesized from the corresponding protected c~-ketoester and butylars, ne in 42 % yield by the procedure described in Example 47.
Single spot on TLC, Rf = 0.54 (CHC13/CH3OH 50:1); mp 135-136 C. Anal. calcd. for C~3H3sN3Os: 433.53; C, 63.71; H, 8.13; N, 9.69. Found: C, 63.48; H, 8.07: N. 9.67.
NMR (CDC13) ok. MS (FAB) mle = 434 (M+l).
Example 56 Z-Leu-Abu-CONH-iBu. This compound was synthesized from thc corresponding protected ~-ketoester and isobutylamine in 65 % yield by the procedure described in Example 47. Single spot on ll C, Rf = 0.25 (CHC131CH30H 50:1); mp 133-135 C. Anal. calcd. for C~3H3sN3Os: 433.52; C, 63.72: H, 8.14: N, 9.69. Found: C. 63.46; H. 8.10: N, 9.60.
NMR (CDC13) ok. MS (FAB) mle = 434 (M+ 1).
Example 57 Z-Leu-Abu-CONH-Bzl. This compound was synthesized from the corresponding protected c~-ketoester and benzylamine in 29 % yield by the procedure described in Example 51. Single spot on TLC, Rf = 0.56 (CHC131CH30H 30:1); mp 140-141 C. Anal. calcd. for C~6H33N3Os: 467.54: C, 66.79: H, 7.11: N, 8.99. Found: C, 66.65: H, 7.07. N. 8.93.
NMR (CDC13) ok. MS (FAB) mle = 468 (M+l).
Example 58 Z-Leu-Abu-CONH-(CH2)2Ph. This compound was synthesized from the corresponding protected c~-ketoester and phenethylamine in S I % yield by the procedure descnbed in Example 51. Single spot on ll.C. Rf = 0.44 (CHC131CH30H 30:1): mp 156- lS7 C. Anal. calcd. for C27H3sN3Os: 481.59: C. 67.34: H, 7.33: N, 8.72. Found: C. 67.38:
H. ?.33; N, 8.78. N~R (CDC13) ok. MS (FAB) m/e = 482 (M+l).
Example S9 Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)2O. lllis compound was synthesized from protected ~x-ketoester and 4(3-aminopropyl)morpholine in 33 % yield by the procedure descnbed in Example 47. After reacting overnlght. ethvl acetate (80 ml) was added. The WO 92/l2140 PCr/U$91/09801 ?JQ~$j ~I'J -j4 mixture was filtered to remove a white preeipitate, The solution was washed with water ~3 x 0 ml). saturated sodium chlo}ide (2 x 20 ml), and dried over magnesium sulfate, The solution was evaporated leaving a yellow oil, Chromatography on a silica ~el eolumn with CHC13/CH3OH ( 10:1 v/v) afforded a yellow semisolid. whieh was recrystallized from ethyl 5 acetate/hexane to obtain a pale yellow solid. Single SpOt on TLC, Rf = 0.42 (CHa3lCH3OH
10:1): mp 125-126 C. Anal. calcd. for C26H40N4o6: 504.63: C. 61.88; H. 7.99: ~'. 11,10, Found: C, 61.69: H, 7.95; ~, 11.07. NMR (CDC13) ok. ~IS (FAB) m/e = 505 (M+l).
Example 60 Z Leu-Abu-CONH-(CH2)7CH3. This compound was synthesized-from the 10 corresponding proteeud a-ketoester and octylamine in 67 % yield by the procedure described in Example 51. It was white solid. Single spot on TLC, Rf = 0.55 (CHC13/CH3OH 30:1); mp 134-135 C. Anal. ealcd. for C27H43N3Os: 489.66: C, 66.23; H, 8.85; N, 8.58. Found: C.
66.19: H, 8.81; N, 8.61. NMR (CDC13) ok. MS (FAB) m/e = 490 (M+l).
Example 6 1 Z-Leu Abu-CONH-(CH232OH. This compound was synthesized from the corresponding proteeted a-ke~oester and ethanolamine in 29 % yield by the procedure described in Example 59. The product was a white stieky solid. Single spot on TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 151-li3 C. Anal: ealcd. for C21H31N3O6: 421.49; C, 59.84: H.

7.41; .~1, 9.97. Found: C, 59.11; H. 7.44; N, 9.81. NMR (CDC13) ok. MS (FAB) m/e = 422 (M+l).
Example 62 Z-Leu-Abu-CONH-(CH2)2O(CH2)2OH. This compound was synthesized from the eorresponding proteeted a-ketoester and 2-(2-aminoethoxy)ethanol in 34 % yield by the procedure deseribed in Exa nple 59. The produet was white sticky solid. Single spot on TLC, Rf z 0.42 (CHC13/CH30H 10:1); mp 103-105 C. Anal.: ealed. for C23H3sN3O7:
465.55; C, 59.34; H. 7.58; N, 9.03. Found: C, 59.23; H. 7.58; N. 9.01. NMR (CDC13) ok.
.~S (FAB) nve = 466 (M~l).
Example 63 Z-Leu-Abu-CONH-(CH2)17CH3. This eompound was synthesized from the corresponding proteeted a-ketoester and ocudecylamine in I % yield by the procedure described in Example 51. The produe~ was a pale vellow solid. Single spot on TLC. Rf =
0.54 (CHC13/CH30H 30:1 ); mp 134-136 C. Anal: ealed. for C37H63N3Os: 629.92; C.70.55; H, 10.08: N, 6.67. Found: C. 70.71; H. 10.14: N. 6.~5. ~MR (CDC13) ok. MS(FAB) mle = 630,~ (M+l).
Example 64 Z-Leu-Abu-CO~H CH- C6H3(OCH3)2. This compound was syn~hesized from the eorresponding proteeted a-ketoester and 3,5-dimethox,vbenzylamine in 45 % yield by the proeedure deseribed in Example 51. The produet was yellow stieky solid. Single spot on TLC, Rf z 0,44 (CHCl3/CH30H 30:1); mp 153-155 C. Anal.: calcd. for C~gH37N3O7:

SUBSTITUT~ SHEET

WO 92/12140 2 0 9 3 7 0 2 PCr/US91/09801 -j5-S27.62; C, 63.74; H. 7.07; N. 7.96. Found: C. 63 66: H. 7.09~ '. 7 92. ~ (CDC13) ok ~vlS (FAB) m/e = 528.8 (M+l).
Examplc 65 Z-Leu Abu-CONH-CH~-C4H4N. This compound was syn~hcsizcd from thc S corrcsponding protccted -kctoester and 4-(aminomethyl)pyridine in 45 % yicld by thc procedure described in Example 59. Thc product was grccnish yellow solid. Singlc spot on ll C, Rf = 0.55 (CHCl3/CH30H 10:1); mp 124-126 C. Anal: calcd. for C2sH32N40s:
468.55; C, 64.08; H. 6.88; N, 11.96. Found; C. 63.88; H, 6.87; N, 11.96. NMR (C~C133 ok. MS (FAB) m/c =469 (M+1).
It is obvious that those skillcd in the art may make modifications ~o thc invcntion without dcparting from thc spirit of the invention or the scopc of the subjoincd claims and thcir equivalcnts.

SUBSTITUTE SHE~T

WO 92/12140 PCltUS91/09801 V?~ -~6-Table I. Inhibition of~enne proteases by peptidc ketoesters and keIoacids a Compounds Kl (~M) ~E PPECathepin GChymotrypsin _ Bz-DL-Phe-COOEt 58 0.28 Bz-DL-Ala-COOEt 640 590 8z-DL-Ala-COOH 3100 ~3200 Bz-DL-Ala-COOBzl l9 23 Bz-DL-Ala-COO n-Bu 260 NIb Bz-DL-Ala-COOCH2-C6H4-CF3 (para) 81C llc Z-Ala-DL-Ala-COOEt 100 210 Z-Ala-DL-Ala-COO n-Bu 250 80 Z-Ala-DL-Ala-COOBzl 46 l l MleO-Suc-Ala-DL-Ala-COOMe 470C 520C
Z-Ala-Ala-DL-Ala-COOEt 1.3 0.65 Z-Ala-Ala-DL-~va-COOEt 0.52 0.36 Z-Ala-Pro-DL-Ala-COOEt 2.8 2.4 Z-Ala-Ala-DL-Abu-COOEt 0.12 0.15 Z-Ala-Ala-DL-Abu-COOBzl 0.09 0.08 Z-Ala-Ala-DL-Abu-COOCH2-C6H4-CF3 0.08 0.33 (para) MeO-Suc-Val-Pro-DL-Phc-COOMe l.l J 26 Z-Ala-Ala-Ala-DL-Ala-COOEt 0.3 0.14 MeO-Suc-Ala-Ala-Pro DL-Abu-COOMe 0.42 0.93 . _ _ aInhibition constants werc measured in 0. I M Hepes. 0.5 M NaCI. pH 7.5 buffer. 9 ~ Me2SO and a 25 C.
bNo inhibition.
C!~ioncompetitivc inhibition.

SUE~STITUTE SHEET

WO 92/12140 ~ 17 !~ ~ PCI'/US91/09801 E u ~

E 5 ~ ~ <

1 1~
ia c ¦ 3 C ,~ 1 ~0 ~0 ~0 V ~0 Z O ¦ V~
o~

o ¦ C .`' I O `O O O O O O 5 ~ ' E ~ c a ~ I . I ~

o i e ~ ~r ~ i o ~J, ,, - i ~ a C J~ 1 5 `O -- X ~ ~ ~ 1, V ~

U ~o v , . E

~ Q ~ ~ ~ ~
, 'S _ ~ ~ ~ i < ; C ~ -- C
;~ o V _ ~ O ~ --~UBSTl~UTE SHEET

wO 92/12140 PCr/US91/09801 Table III. Inhibition of cysteine proteases by peptide ke~oestcrs and ke~oacias, Compounds K~

Papaina Cathepsin Bb Calpain lC Calpain ~c Z-Leu-Abu-COOE~ 0.04 0.4 Z-Leu-Phe-COOEt 0.23 0.4 Z-Leu-Me-COOEt 0.12 0. l 8 Bz-DL-Phe-COOEt 500d 64 Z-Phe-DL-Phe-COOEt 1.8 0.1 Z-Phe-DL-Ala-COOE~ 3 . 6 3 . ' Z-Ala-Ala-DL-Ala-COOE~ 1.5 2.' '00 Z-,'.la-Ala-DL-Abu-COOEt 0.9 10 50 200 Z-Ala-Ala-DL-Abu-COOBzl 30 60 Z-Ala-Ala-DL-Nva-COOEt 30 0.1 Z-Ala-P~DL-Ala-COOEI 26 66 ~leO-Suc-Val-Pro DL-Phe-COOMe 1.10.1 2.9d Z-Ala-Ala-Ala-DL-Ala-COOEt 2. l 10.0 ~leO-Suc-Ala-Ala-Pro-Abu-COOMe 0.76.0 100 aInhibition constants were measured in 0.05 M Tris-HCI. pH 7.5 buffer . containing 2 mM
EDTA. 5 mM cystcine (freshly prcpared). l % Me2SO. and at 25 C. ~'-Benzoyl-Arg-AMC was used as a substrate.
blnhibition constants werc measured in 88 mM KH2P04, 12 m.~l ~'a~HPO4, pH 6.0 buffer, containing 1.33 mM EDTA. '.7 mM cys~eine (freshly prepared). and al '5 C. Z-Arg-Arg-AFC
was used as a substratc.
Clnhibiuon constants were mcasured in 20 mM Hepes. pH 7.2 buffer ~ containing 10 rrLM CaCI~, 10 m.~l ~-mercap~oethanol, and at 25 C. Suc-Leu-Tyr-.4.MC was used as a substrate dlnhibition constants were measured in i0 rnM Tris HCI, pH 7.5 buffer, con~aining '0 rnM
EDTA. i mM cys~cine. 9 % Me~SO. and a~ '5 C. ~ -Benzoyl-Arg-~'A was used as a substrate.

SUBSTITUTE SHEET

WO 92/12140 2 ~ ~ ~ 7 0 2 PCI`/US91/09801 -- ~ o o c ~ 3 ~ A A A A A
. _ ~ _ X
~ O V) C O O O O C O
C A A A A A ~ A

0 '~) C ~ C oo -- O ~

. _, C
O

C
~. ,Q ~ Q vl 1-- x x o u~ ~ ~ o ~, A ~ A
-O O O O ~ O Vl O O O O O O
c ~r C ~ O ~I ~ ~ O O o o 1~ _ G~ O ~ ~ ~ V- C~ -- ~ X ~ ~r ~

-- ~ ~r er ~o ~ o ~ o ~ ~ -- ~ o o o o -- ,.- o j ~ -- O -- O C j C j O C O C

'C
Y _ r~
.= ~ X V~ X O ~ ~
~ _ ~ _ ~ ~ O O C
-C

", , --- 8 ~ ~ c _ _ ~ ~ _ C_) tJ _ _ _ _ _ ~ ~ 8 6 ~ _ ^ ^` ~ U t_) _ _ ~ -~ A~ _ -- 5~ Z Z O C ~ O O ~ C ~

> ~ 6 6 ~ ~ -- ~ ~ 6 c 6 6 6 6 D6 6 6 D
c ~, , V ~ 4 4 SUE~STlTUTE SHET

WO 92/12140 PCI-/US91/~)98U1 o o o o -- ~A ~A ~A ~A
-v 3~ r~ r~ 00 ~ -- o o o o o o ~ ~ ~o y A A A A A A

~ A~
O
C ~ r~ ~ X ~ ~ C O -- --C C ~ _ _ t ~ ~. ~ ~ ~r c V
y c1~ ~ ~ ~ ~ 1~ ~ ~ E

V
, o V~o o o C o o o o ~ ~ C~-_ o o o V~ ~ o o o C _ .c A A A A A A A A ^ ^ E
C
--~ E
~ ~'O O -- O ~ ~ X

. _ ~ _,,, . _ _:~O ~r7 ~ rJ ~r ~O ~ ~ ~ O ~ ~ O C
rJ ~ v~

X ~ ~O
~ . rJ X ._ O ~ O C O O O ~
~) C C------OOOOOOOO----O~
~l ,. .__ x o ~r ~r g o o ~ o o J ~ j _ ~ _ o ~ ~ -- -- V
._ , C~) ^
A ~ .=
C ~ _ . _ ~ rJ V
~ , _ -- _ ~ ~-- _ ._ ,; rJ ~ t _ ^ = _ rJ , P ~ ~ ~ c C 8 C C C Z Z Z ~ C C u ~
Z V _ _ A~ V V ~ A~ ~ V A~ a _ ~ V
> ., ~ C ^ ^ ^ C Z Z ~ ~r v A~ -- = _ = Z Z Z ~ ~ Z Z Z = i Z i Z
--V ,, , ,------------____--SlJBS~lTlJTE SHEEl

Claims

What is claimed is:

1. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl subsututed with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H. amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl,and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutarnic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine. and hexafluoroleucine;
R2 is selected from the group consisting of C1-8 branched and unbranched alkyl, C1-8 branched and unbranched cyclized alkyl, and C1-8 branched and unbranched fluoroalkyl;
R3 and R4 are selected independently from the group consisting of H, C1-20 alkyl, C1-20 cyclized alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, C1-20 cyclized alkyl with an attached phenyl group, C1-20 alkyl with an attached phenyl group substituted with K, C1-20 alkyl with an attached phenyl group disubstituted with K, C1-20 alkyl with an attached phenyl group trisubstituted with K. C1-20 cyclized alkyl with an attached phenyl group substituted with K. C1-10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C1-20 alkyl with an OH group attached to the alkyl, -CH2CH2OCH2CH2OH. C1-10 with an attached 4-pyridyl group, C1-10 with an attached 3-pyridyl group, C1-10 with an attached 2-pyridyl group, C1-10 with an attached cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).

2. A compound of the formula:

or a pharmaceutically acceptable salt. wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J. C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl subsututed with K, phenyl disubstituted with K. phenyl trisubstituted with K, naphthyl, naphthyl subsututed with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, C1-10 alkyl with two attached phenyl groups subsuituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy,C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is seleeted from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA1 is a side chain blocked or unblocked amino acid with the L configuration. D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic aeid, Iysine. arginine. hisuidine, phenylglyeine. beta-alanine. norleucine. norvaline. alpha-aminobutyric acid. epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine. indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-melhylserine, O-ethylserine, S-methylcysteine. S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH.
NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine. tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine. norleucine, nor raline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, C1-20 alkyl, C1-20 cyclized alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, C1-20 cyclized alkyl with an attached phenyl group, C1-20 alkyl with an attached phenyl group substituted with K, C1-20 alkyl with an attached phenyl group disubstituted with K, C1-20 alkyl with an attached phenyl group trisubstituted with K, C1-20 cyclized alkyl with an attached phenyl group substituted with K, C1-10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C1-20 alkyl with an OH group attached to the alkyl, -CH2CH2OCH2CH2OH, C1-10 with an attached 4-pyridyl group, C1-10 with an attached 3-pyridyl group, C1-10 with an attached 2-pyridyl group, C1-10 with an attached cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).

3. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl subsututed with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl subsututed with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl subsututed with K, naphthyl disubstituted with K, naphthyl trisubstiruted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups. C1-10 alkyl with an attached phenyl group substituted with K, C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblockcd amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutarnine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylsenne, O-ethylserine. S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected intependently from the group consisting of H, C1-20 alkyl, C1-20 cyclized alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, C1-20 cyclized alkyl with an attached phenyl group, C1-20 alkyl with an attached phenyl group substituted with K, C1-20 alkyl with an attached phenyl group disubstituted with K, C1-20 alkyl with an attached phenyl group trisubstituted with K, C1-20 cyclized alkyl with an attached phenyl group substituted with K, C1-10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C1-20 alkyl with an OH group attached to the alkyl, -CH2CH2OCH2CH2OH, C1-10 with an attached 4-pyridyl group, C1-10 with an attached 3-pyridyl group, C1-10 with an attached 2-pyridyl group, C1-10 with an attached cyclohexyl group. -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).
A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine.
valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), amethylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH.
NH2 CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, C1-20 alkyl, C1-20 cyclized alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, C1-20 cyclized alkyl with an attached phenyl group, C1-20 alkyl with an attached phenyl group substituted with K, C1-20 alkyl with an attached phenyl group disubstituted with K, C1-20 alkyl with an attached phenyl group trisubstituted with K, C1-20 cyclized alkyl with an attached phenyl group substituted with K, C1-10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C1-20 alkyl with an OH group attached to the alkyl, -CH2CH2OCH2CH2OH, C1-10 with an attached

4-pyridyl group, C1-10 with an attached 3-pyridyl group. C1-10 with an attached 2-pyridyl group, C1-10 with an attached cyclohexyl group. -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).

5. A compound of the formula:

or a pharmaceutically acceptable salt wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl tnsubsituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K.
C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, C1-10 alky with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine. C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H. amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine. norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine.
homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, C1-20 alkyl, C1-20 cyclized alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, C1-20 cyclized alkyl with an attached phenyl group, C1-20 alkyl with an attached phenyl group substituted with K, C1-20 alkyl with an attached phenyl group disubstituted with K, C1-20 alkyl with an attached phenyl group trisubstituted with K, C1-20 cyclized alkyl with an attached phenyl group substituted with K, C1-10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1-10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C1-10 alkyl with a pyrrolidine ring attached through nitrogen to the alkyl, C1-20 alkyl with an OH group attached to the alkyl, -CH2CH2OCH2CH2OH, C1-10 with an attached 4-pyridyl group, C1-10 with an attached 3-pyridyl group, C1-10 with an attached 2-pyridyl group, C1-10 with an attached cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).

6. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroallcyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configurarion, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid.
NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH. NH2-CH(CH2-cyclohexyl)-COOH. NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents C1-8 branched and unbranched alkyl, C1-8 branched and unbranched cyclized alkyl, or C1-8 branched and unbranched fluoroalkyl;

7. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represenls H, NH2-CO-, NH2-CS-. NH2-SO2-, X-NH-CO-. X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 alkyl with an attached phenoxy group substituted with K on the phenoxy group;
J is seleeted from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C1-10 alkoxy, C1-10 alkylamine. C2-12 dialkylamine. C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA1 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleueine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methyleysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine. arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine. S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;

8. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine. O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;

9. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, Y1-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group subsituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 alkyl with an attached phenoxy group subsituted with K on the phenoxy group;
Y1 is selected from the group consisting of C2-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admanryl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leueine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine. cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline. hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine. S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cycloburyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;

10. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 alkyl with an attached phenoxy group substituted with K on the phenoxy group;
Y2 is selected from the group consisting of C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from thc group consisting of halogen, COOH, OH, CN, NO2, NH2, C1-alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine. arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;

11. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-. X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA1 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine. S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline. alpha-aminobutyric acid, epsilon-aminocaproic acid. citrulline. hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH. NH2-CH(CH2-cyclobutyl)-COOH. NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R1 is selected from the group consisuing of H, C1-20 alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, and C1-20 alkyl with an attached phenyl group substituted with K.

12. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubsuituted with K, naphthyl, naphthyl subsututed with K, naphthyl disubstituted with K, naphthyl trisubsuituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups subsututed with K, C1-10 alkyl with an attached phenoxy group, and C1-10 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, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents C1-8 branched and unbranched alkyl, C1-8 branched and unbranched cyclized alkyl, or C1-8 branched and unbranched fluoroalkyl;

R is selected from the group consisuing of H, C1-20 alkyl, C1-20 alkyl with a pheny1 group attached to the C1-20 alkyl, and C1-20 alkyl with an attached phenyl group substituted with K.

13. A compound of the formula:

or a pharmaceutically acceptable salt. wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 alkyl with an attached phenoxy group substituted with K on the phenoxy group;
T is selected from the group consisting of C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-adrnantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C2-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
R is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleueine, 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-earboxylie aeid, 2-azetidineearboxylie aeid, pipeeolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine. NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl) COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R is selccted from the group consisting of H, C2-20 alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, and C1-20 alkyl with an attached phenyl group substituted with K.

14. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl subsututed with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 alkyl with an attached phenoxy group substituted with K on the phenoxy group;
T is selected from the group consisting of C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C2-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C
10 alkoxy,C1-10 alkylamine, C1-10 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 aLkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN. OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid. citrulline, hydroxyproline. ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleueine, and hexafluoroleueine;
R2 represents C1-8 branched and unbranched alkyl, C1-8 branched and unbranched cyclized alkyl, or C1-8 branched and unbranched fluoroalkyl;
R is selected from the group consisting of H, C1-20 alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, and C1-20 alkyl with an attached phenyl group substituted with K.

15. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-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 C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group, and C1-10 alkyl with an attached phenoxy group substituted with K on the phenoxy group;
T is selected from the group consisting of C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K. phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with R, C2-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups. C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, C1-alkoxy, C1-10 alkylamine. C2-12 dialkylamine, C1-10 alkyl-O-CO-, C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2, CN, OH, CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino. C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;

AA is a side chain blocked or unblocked amino aeid with the L configuration, D
configuration. or no chirality at the .alpha.-carbon selected from the group consisting of alanine.
valine, leucine. isoleueine, proline, methionine, meshionine sulfoxide, phenylalanine, tryptophan, glycine. serine, threonine, cysteine, tyrosine, asparagine, glutamine. aspartic acid.
glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine. norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methyleysteine, S-ethylcysteine, S-benzylcysteine. NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,NH2-CH(CH2-2-napthyl)-COOH,NH2-CH(CH2-cyclohexyl)-COOH,NH2-CH(CH2-cyclopentyl)-COOH,NH2-CH(CH2-cyclobutyl)COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;;
AA4 is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the .alpha.-carbon selected from the group consisting of leucine, isoleucine, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine. beta-alanine, norleucine, norvaline, alpha-aminobutyric acid.
epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine. sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine. S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,NH2-CH(CH2-cyclohexyl)-COOH. NH2-CH(CH2-cyclopentyl)-COOH,NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH.
trifluoroleucine, and hexafluoroleucine;
R is selected from the group consisting of H, C1-20 alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, and C1-20 alkyl with an attached phenyl group subssituted with K.

16. A compound of the formula:

or a pharmaceutically acceptable salt, wherein M1 represents H. NH2-CO-. NH2-CS-, NH2-SO2-, X-NH-CO-. X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, Y-CO-. X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of C1-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituted with J, C1-10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K, C1-10 alkyl with an attached phenoxy group. and C1-10 alkyl with an attached phenoxy group substituted with K on the phenoxy group;
Y is selected from the group consisting of C6-10 alkyl, C1-10 fluoroalkyl, C1-10 alkyl substituud with J, C1-10 fluoroalkyl substituled with J, 1-admantyl, 9-fluorenyl, phenyl substituud with K, phenyl disubstituud with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-10 alkyl with an attached phenyl group, C1-10 alkyl with two attached phenyl groups, C1-10 alkyl with an attached phenyl group substituted with K, and C1-10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH,OH,CN,NO2,NH2,C1-10 alkoxy, C1-10 alkylamine, C2-12 dialkylamine, C1-10 alkyl-O-CO-,C1-10 alkyl-O-CO-NH-, and C1-10 alkyl-S-;
K is selected from the group consisting of halogen, C1-10 alkyl, C1-10 perfluoroalkyl, C1-10 alkoxy, NO2,CN,OH,CO2H, amino, C1-10 alkylamino, C2-12 dialkylamino, C1-C10 acyl, and C1-10 alkoxy-CO-, and C1-10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration. or no chirality at the .alpha.-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide. phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine. asparagine, glutamine, aspartic acid, glutarnic 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, piplecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine. NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,NH2-CH(CH2-2-napthyl)-COOH,NH2-CH(CH2-cyclohexyl)-COOH,NH2-CH(CH2-cyclopentyl)-COOH,NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R is selected from the group consisting of H, C1-20 alkyl, C1-20 alkyl with a phenyl group attached to the C1-20 alkyl, and C1-20 alkyl with an attached phenyl group substituted with K.