Tricyclic compounds for the inhibition of the ICE/ced-3 protease family of enzymes

This invention is directed to novel tricyclic ICE/ced-3 family inhibitor compounds. The invention is also directed to pharmaceutical compositions of such tricyclic compounds, plus the use of such compositions in the treatment of patients suffering inflammatory, autoimmune and neurodegenerative diseases, and for the prevention of ischemic injury.

BACKGROUND OF THE INVENTION 
The present invention relates to novel classes of compounds which are 
inhibitors of interleukin-1.beta. converting enzyme and related proteases 
("ICE/ced-3 family of cysteine proteases"). This invention also relates to 
pharmaceutical compositions comprising these compounds and to methods of 
using such pharmaceutical compositions. The compounds, pharmaceutical 
compositions and methods of this invention are particularly well suited 
for inhibiting the protease activity of the ICE/ced-3 family and 
consequently, may be advantageously used as agents against interleukin-1 
("IL-1") mediated diseases, including inflammatory diseases, autoimmune 
diseases and neurodegenerative diseases and for inhibiting unwanted 
apoptosis in various disease states such as ischemic injury to the heart 
(e.g., myocardial infarction), brain (e.g., stroke), and kidney (e.g., 
ischemic kidney disease). 
Interleukin 1 ("IL-1") is a major pro-inflammatory and immunoregulatory 
protein that stimulates fibroblast differentiation and proliferation, the 
production of prostaglandins, collagenase and phospholipase by synovial 
cells and chondrocytes, basophil and eosinophil degranulation and 
neutrophil activation. Oppenheim, J. H. et al., Immunology Today, 7:45-56 
(1986). As such, it is involved in the pathogenesis of chronic and acute 
inflammatory and autoimmune diseases. IL-1 is predominantly produced by 
peripheral blood monocytes as part of the inflammatory response. Mosely, 
B. S. et al., Proc. Nat. Acad. Sci., 84:4572-4576 (1987); Lonnemann, G. et 
al., Eur. J. Immunol., 19:1531-1536 (1989). 
IL-1.beta. is synthesized as a biologically inactive precursor, 
proIL-1.beta.. ProIL-1.beta. is cleaved by a cysteine protease called 
interleukin-1.beta. converting enzyme ("ICE") between Asp-116 and Ala-117 
to produce the biologically active C-terminal fragment found in human 
serum and synovial fluid. Sleath, P. R. et al., J. Biol. Chem., 
265:14526-14528 (1992); A. D. Howard et al., J. Immunol., 147:2964-2969 
(1991). 
ICE is a cysteine protease localized primarily in monocytes. In addition to 
promoting the pro-inflammatory and immunoregulatory properties of 
IL-1.beta., ICE, and particulary its homologues, also appear to be 
involved in the regulation of cell death or apoptosis. Yuan, J. et al., 
Cell, 75:641-652 (1993); Miura, M. et al., Cell, 75:653-660 (1993); 
Nett-Giordalisi, M. A. et al., J. Cell Biochem., 17B:117 (1993). In 
particular, ICE or ICE/ced-3 homologues are thought to be associated with 
the regulation of apoptosis in neurogenerative diseases, such as 
Alzheimer's and Parkinson's disease. Marx, J. and M. Baringa, Science, 
259:760-762 (1993); Gagliardini, V. et al., Science, 263:826-828 (1994). 
Thus, disease states in which inhibitors of the ICE/ced-3 family of 
cysteine proteases may be useful as therapeutic agents include: infectious 
diseases, such as meningitis and salpingitis; septic shock, respiratory 
diseases; inflammatory conditions, such as arthritis, cholangitis, 
colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and 
reperfusion injury, ischemic diseases such as the myocardial infarction, 
stroke and ischemic kidney disease; immune-based diseases, such as 
hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone 
diseases; and certain neurodegenerative diseases, such as Alzheimer's and 
Parkinson's disease. 
ICE inhibitors represent a class of compounds useful for the control of the 
above-listed disease states. Peptide and peptidyl inhibitors of ICE have 
been described. However, such inhibitors have been typically characterized 
by undesirable pharmacologic properties, such as poor oral absorption, 
poor stability and rapid metabolism. Plattner, J. J. and D. W. Norbeck, in 
Drug Discovery Technologies, C. R. Clark and W. H. Moos, Eds. (Ellis 
Horwood, Chichester, England, 1990), pp. 92-126. These undesirable 
properties have hampered their development into effective drugs. 
Accordingly, the need exists for compounds that can effectively inhibit the 
action of the ICE/ced-3 family of proteases, for use as agents for 
preventing unwanted apoptosis and for treating chronic and acute forms of 
IL-1 mediated diseases, such as inflammatory, autoimmune or 
neurodegenerative diseases. 
The compounds of this invention incorporate a conformationally constrained 
dipeptide mimetic. This mimetic exhibits improved properties relative to 
their peptidic counterparts, for example, such as improved absorption and 
metabolic stability resulting in enhanced bioavailability. 
SUMMARY OF THE INVENTION 
One aspect of this invention is compounds of the formula: 
##STR1## 
wherein: n is 1 or 2; 
m is 1 or 2; 
A is R.sup.2 CO--, R.sup.3 --O--CO--, or R.sup.4 SO.sub.2 --; 
a group of the formula: 
##STR2## 
further wherein: R.sup.1 is a hydrogen atom, alkyl or phenylalkyl; 
R.sup.2 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.3 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl or 
(substituted phenyl)alkyl; 
R.sup.4 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.5 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.6 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or 
(substituted phenyl)alkyl; 
R.sup.7 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.8 is an amino acid side chain chosen from the group consisting of 
natural and unnatural amino acids; 
B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, 
(cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted 
phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or a halomethyl group; 
a group of the formula: 
EQU --CH.sub.2 XR.sup.9 ; 
wherein R.sup.9 is phenyl, substituted phenyl, phenylalkyl, (substituted 
phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a 
sulfur atom; 
a group of the formula: 
EQU --CH.sub.2 --O--CO--(aryl); 
a group of the formula: 
EQU --CH.sub.2 --O--CO--(heteroaryl); 
a group of the formula: 
EQU --CH.sub.2 --O--PO--(R.sup.10) R.sup.11 ; 
wherein R.sup.10 and R.sup.11 are independently selected from a group 
consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, 
and (substituted phenyl)alkyl; 
or a pharmaceutically-acceptable salt thereof. 
A further aspect of the instant invention are pharmaceutical compositions 
comprising a compound of the above Formula 1 and a 
pharmaceutically-acceptable carrier therefor. 
Another aspect of this invention involves a method for treating an 
autoimmune disease comprising administering an effective amount of a 
pharmaceutical composition discussed above to a patient in need of such 
treatment. 
Yet another aspect of the instant invention is a method of treating an 
inflammatory disease comprising administering an effective amount of a 
pharmaceutical composition discussed above to a patient in need of such 
treatment. 
A further aspect of the instant invention is method of treating a 
neurodegenerative disease comprising administering a pharmaceutically 
effective amount of a pharmaceutical composition discussed above to a 
patient in need of such treatment. 
Yet another aspect of the instant invention is a method of preventing 
ischemic injury to a patient suffering from a disease associated with 
ischemic injury comprising administering an effective amount of a 
pharmaceutical composition discussed above to a patient in need of such 
treatment. 
DETAILED DESCRIPTION 
One aspect of the instant invention is compounds of the Formula 1: 
##STR3## 
wherein: n is 1 or 2; 
m is 1 or 2; 
A is R.sup.2 CO--, R.sup.3 --O--CO--, or R.sup.4 SO.sub.2 --; 
a group of the formula: 
##STR4## 
further wherein: R.sup.1 is a hydrogen atom, alkyl or phenylalkyl; 
R.sup.2 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.3 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or 
(substituted phenyl)alkyl; 
R.sup.4 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.5 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.6 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or 
(substituted phenyl)alkyl; 
R.sup.7 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, 
substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or 
(heteroaryl)alkyl; 
R.sup.8 is an amino acid side chain chosen from the group consisting of 
natural and unnatural amino acids; 
B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, 
(cycloalkyl)alkyl, phenyl, phenylalkyl, (substituted)phenyl, 
(substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; 
a group of the formula 
EQU --CH.sub.2 XR.sup.9 ; 
wherein R.sup.9 is phenyl, phenylalkyl, substituted phenyl, (substituted 
phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a 
sulfur atom; 
a group of the formula: 
EQU --CH.sub.2 --O--CO--(aryl); 
a group of the formula: 
EQU --CH.sub.2 --O--CO--(heteroaryl); 
a group of the formula: 
EQU --CH.sub.2 --O--PO--(R.sup.10) R.sup.11 ; 
wherein R.sup.10 and R.sup.11 are independently selected from a group 
consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, 
and (substituted phenyl)alkyl; 
or a pharmaceutically-acceptable salt thereof. 
As used in the above formula, the term "alkyl" means substituted or 
unsubstituted a straight chain or branched C.sub.1 to C.sub.8 carbon chain 
such as methyl, ethyl, tert-butyl, iso-propyl, n-octyl, and the like. 
Suitable substituents include carboxy, protected carboxy, amino, protected 
amino, halo, hydroxy, protected hydroxy, nitro, cyano, monosubstituted 
amino, protected monosubstituted amino, disubstituted amino, C.sub.1 to 
C.sub.1 alboxy, C.sub.1 to C.sub.7 acyl, C.sub.1 to C.sub.7 acyloxy, and 
the like. 
The term "cycloalkyl" means a mono-, bi-, or tricyclic saturated ring that 
is fully saturated or partially unsaturated. Examples of such a group 
included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 
adamantyl, cyclooctyl, cis- or trans decalin, bicyclo[2.2.1]hept-2-ene, 
cyclohex-1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like. 
The term "(cycloalkyl)alkyl" means the above-defined alkyl group 
substituted for one of the above cycloalkyl rings. Examples of such a 
group include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl, 
5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like. 
The term "substituted phenyl" specifies a phenyl group substituted with one 
or more, and preferably one or two, moieties chosen from the groups 
consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, 
trifluoromethyl, C.sub.1 to C.sub.7 alkyl, C.sub.1 to C.sub.7 alkoxy, 
C.sub.1 to C.sub.7 acyl, C.sub.1 to C.sub.7 acyloxy, carboxy, protected 
carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected 
hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected 
(monosubstituted)amino, (disubstituted)amino, carboxamide, protected 
carboxamide, N--(C.sub.1 to C.sub.6 alkyl)carboxamide, protected 
N--(C.sub.1 to C.sub.6 alkyl)carboxamide, N,N-di(C.sub.1 to C.sub.6 
alkyl)carboxamide, trifluoromethyl, N--((C.sub.1 to C.sub.6 
alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, substituted or 
unsubstituted, such that, for example, a biphenyl or naphthyl group 
results. 
Examples of the term "substituted phenyl" includes a mono- or 
di(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl, 
2,5-dichlorophenyl, 3,4-dichlorophenyl, 2,3 or 4-bromophenyl, 
3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl and the 
like; a mono or di(hydroxy)phenyl group such as 2, 3, or 4-hydroxyphenyl, 
2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and the 
like; a nitrophenyl group such as 2, 3, or 4-nitrophenyl; a cyanophenyl 
group, for example, 2,3 or 4-cyanophenyl; a mono- or di(alkyl)phenyl group 
such as 2, 3, or 4-methylphenyl, 2,4-dimethylphenyl, 2, 3 or 
4-(iso-propyl)phenyl, 2, 3, or 4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl 
and the like; a mono or di(alkoxy)phenyl group, for example, 
2,6-dimethoxyphenyl, 2, 3 or 4-(isopropoxy)phenyl, 2, 3 or 
4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 
4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protected 
carboxy)phenyl group such as 2, 3 or 4-carboxyphenyl or 2,4-di(protected 
carboxy)phenyl; a mono- or di(hydroxymethyl)phenyl or (protected 
hydroxymethyl)phenyl such as 2, 3 or 4-(protected hydroxymethyl)phenyl or 
3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or 
(protected aminomethyl)phenyl such as 2, 3 or 4-(aminomethyl)phenyl or 
2,4-(protected aminomethyl)phenyl; or a mono- or 
di(N-(methylsulfonylamino))phenyl such as 2, 3 or 
4-(N-(methylsulfonylamino))phenyl. Also, the term "substituted phenyl" 
represents disubstituted phenyl groups wherein the substituents are 
different, for example, 3-methyl-4-hydroxyphenyl, 
3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 
4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl 
and the like. 
The term "(substituted phenyl)alkyl" means one of the above substituted 
phenyl groups attached to one of the above-described alkyl groups. 
Examples of include such groups as 2-phenyl-1-chloroethyl, 
2-(4'-methoxyphenyl)ethyl, 4-(2',6'-dihydroxy phenyl)n-hexyl, 
2-(5'-cyano-3'-methoxyphenyl)n-pentyl, 3-(2', 6'-dimethylphenyl)n-propyl, 
4-chloro-3-aminobenzyl, 6-(4'-methoxyphenyl)-3-carboxy(n-hexyl), 
5-(4'-aminomethylphenyl)-3-(aminomethyl)n-pentyl, 
5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like. 
The terms "halo" and "halogen" refer to the fluoro, chloro, bromo or iodo 
groups. There can be one or more halogen, which are the same or different. 
Preferred halogens are chloro and fluoro. 
The term "aryl" refers to aromatic five and six membered carbocyclic rings. 
Six membered rings are preferred. 
The term "heteroaryl" denotes optionally substituted five-membered or 
six-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur 
and/or nitrogen atoms, in particular nitrogen, either alone or in 
conjunction with sulfur or oxygen ring atoms. These five-membered or 
six-membered rings are fully unsaturated. 
Furthermore, the above optionally substituted five-membered or six-membered 
rings can optionally be fused to a aromatic 5-membered or 6-membered ring 
system. For example, the rings can be optionally fused to an aromatic 
5-membered or 6-membered ring system such as a pyridine or a triazole 
system, and preferably to a benzene ring. 
The following ring systems are examples of the heterocyclic (whether 
substituted or unsubstituted) radicals denoted by the term "heteroaryl": 
thienyl, furyl, pyrrolyl, imidazolyl, isoxazolyl, triazolyl, thiadiazolyl, 
oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, 
pyrazinyl, pyridazinyl, oxazinyl, triazinyl, thiadiazinyl tetrazolo, 
1,5-[b]pyridazinyl and purinyl, as well as benzo-fused derivatives, for 
example, benzoxazolyl, benzthiazolyl, benzimidazolyl and indolyl. 
Substituents for the above optionally substituted heteroaryl rings are from 
one to three halo, trihalomethyl, amino, protected amino, amino salts, 
mono-substituted amino, di-substituted amino, carboxy, protected carboxy, 
carboxylate salts, hydroxy, protected hydroxy, salts of a hydroxy group, 
lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, 
substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, 
phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl. 
Substituents for the heteroaryl group are as heretofore defined, or in the 
case of trihalomethyl, can be trifluoromethyl, trichloromethyl, 
tribromomethyl, or triiodomethyl. As used in conjunction with the above 
substituents for heteroaryl rings, "lower alkoxy" means a C.sub.1 to 
C.sub.4 alkoxy group, similarly, "lower alkylthio" means a C.sub.1 to 
C.sub.4 alkylthio group. The term "substituted alkyl" means the above 
defined alkyl group substituted from one to three times by a hydroxy, 
protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl, 
mono-substituted amino, di-substituted amino, lower alkoxy, lower 
alkylthio, carboxy, protected carboxy, or a carboxy, amino, and/or hydroxy 
salt. As used in conjunction with the substituents for the heteroaryl 
rings, the terms "substituted (cycloalkyl)alkyl" and "substituted 
cycloalkyl" are as defined above substituted with the same groups as 
listed for a "substituted alkyl" group. The term "(monosubstituted)amino" 
refers to an amino group with one substituent chosen from the group 
consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, 
C.sub.1 to C.sub.7 acyl, C.sub.2 to C.sub.7 alkenyl, C.sub.2 to C.sub.7 
substituted alkenyl, C.sub.2 to C.sub.7 alkynyl, C.sub.7 to C.sub.16 
alkylaryl, C.sub.7 to C.sub.16 substituted alkylaryl and heteroaryl group. 
The (monosubstituted)amino can additionally have an amino-protecting group 
as encompassed by the term "protected (monosubstituted)amino." The term 
"(disubstituted)amino" refers to amino groups with two substituents chosen 
from the group consisting of phenyl, substituted phenyl, alkyl, 
substituted alkyl, C.sub.1 to C.sub.7 acyl, C.sub.2 to C.sub.7 alkenyl, 
C.sub.2 to C.sub.7 alkynyl, C.sub.7 to C.sub.16 alkylaryl, C.sub.7 to 
C.sub.16 substituted alkylaryl and heteroaryl. The two substituents can be 
the same or different. The term "heteroaryl(alkyl)" denotes an alkyl group 
as defined above, substituted at any position by a heteroaryl group, as 
above defined. 
The term "pharmaceutically-acceptable salt" encompasses those salts that 
form with the carboxylate anions and includes salts formed with the 
organic and inorganic cations such as those chosen from the alkali and 
alkaline earth metals, (for example, lithium, sodium, potassium, 
magnesium, barium and calcium); ammonium; and the organic cations (for 
example, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, 
bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium, 
dibenzylethylenediammonium, and like cations.) Other cations encompassed 
by the above term include the protonated form of procaine, quinine and 
N-methylglucosamine, the protonated forms of basic amino acids such as 
glycine, ornithine, histidine, phenylglycine, lysine, and arginine, and 
acetic acid-like counter-ions such as acetate and trifluoroacetate. 
Furthermore, any zwitterionic form of the instant compounds formed by a 
carboxylic acid and an amino group is referred to by this term. A 
preferred cation for the carboxylate anion is the sodium cation. 
Furthermore, the term includes salts that form by standard acid-base 
reactions with basic groups (such as amino groups) and includes organic or 
inorganic acids. Such acids include hydrochloric, sulfuric, phosphoric, 
acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, 
pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, 
lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic, sorbic, 
picric, benzoic, cinnamic, and the like acids. 
The compounds of Formula 1 may also exist as solvates and hydrates. Thus, 
these compounds may crystallize with, for example, waters of hydration, or 
one, a number of, or any fraction thereof of molecules of the mother 
liquor solvent. The solvates and hydrates of such compounds are included 
within the scope of this invention. 
The term "carboxy-protecting group" as used herein refers to one of the 
ester derivatives of the carboxylic acid group commonly employed to block 
or protect the carboxylic acid group while reactions are carried out on 
other functional groups on the compound. Examples of such carboxylic acid 
protecting groups include t-butyl, 4-nitrobenzyl, 4-methoxybenzyl, 
3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 
2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, 
benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 
2-phenylpropyl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 
2,2,2-trichloroethyl, .beta.-(trimethylsilyl)ethyl, 
.beta.-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl, 
4-nitrobenzylsulfonylethyl, allyl, cinnamyl, 
1-(trimethylsilylmethyl)-propenyl and like moieties. The species of 
carboxy-protecting group employed is not critical so long as the 
derivatized carboxylic acid is stable to the conditions of subsequent 
reaction(s) and can be removed at the appropriate point without disrupting 
the remainder of the molecule. Further examples of these groups are found 
in C. B. Reese and E. Haslam, "Protective Groups in Organic Chemistry," J. 
G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, 
respectively, and T. W. Greene and P. G. M. Wuts, "Protective Groups in 
Organic Synthesis," 2nd ed., John Wiley and Sons, New York, N.Y., 1991, 
Chapter 5, each of which is incorporated herein by reference. A related 
term is "protected carboxy," which refers to a carboxy group substituted 
with one of the above carboxy-protecting groups. 
The term "hydroxy-protecting group" refers to readily cleavable groups 
bonded to hydroxyl groups, such as the tetrahydropyranyl, 
2-methoxyprop-2-yl, 1-ethoxyeth-1-yl, methoxymethyl, 
.beta.-methoxyethoxymethyl, methylthiomethyl, t-butyl, t-amyl, trityl, 
4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, benzyl, 
allyl, trimethylsilyl, (t-butyl)dimethylsilyl, 
2,2,2-trichloroethoxycarbonyl groups and the like. 
Further examples of hydroxy-protecting groups are described by C. B. Reese 
and E. Haslam, "Protective Groups in Organic Chemistry," J. G. W. McOmie, 
Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4, respectively, 
and T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic 
Synthesis," Second Edition, John Wiley and Sons, New York, N.Y., 1991, 
Chapters 2 and 3. A preferred hydroxy-protecting group is the tert-butyl 
group. The related term "protected hydroxy" denotes a hydroxy group bonded 
to one of the above hydroxy protecting groups. 
The term "amino-protecting group" as used herein refers to substituents of 
the amino group commonly employed to block or protect the amino 
functionality while reacting other functional groups of the molecule. The 
term "protected (monosubstituted)amino" means there is an amino-protecting 
group on the monosubstituted amino nitrogen atom. 
Examples of such amino-protecting groups include the formyl ("For") group, 
the trityl group, the phthalimido group, the trichloroacetyl group, the 
trifluoroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetyl 
groups, urethane-type blocking groups, such as t-butoxycarbonyl ("Boc"), 
2-(4-biphenylyl)propyl-2-oxycarbonyl ("Bpoc"), 
2-phenylpropyl-2-oxycarbonyl ("Poc"), 2-(4-xenyl)isopropoxycarbonyl, 
1,1-diphenylethyl-1-oxycarbonyl, 1,1-diphenylpropyl-1-oxycarbonyl, 
2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl ("Ddz"), 
2-(p-toluyl)propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl, 
1-methylcyclopentanyloxycarbonyl, cyclohexanyloxy-carbonyl, 
1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 
2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl, 
2-(triphenylphosphino)-ethoxycarbonyl, 9-fluorenylmethoxycarbonyl 
("Fmoc"), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 
1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl, 
5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 
2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl, 
cyclopropylmethoxycarbonyl, isobornyloxycarbonyl, 1-piperidyloxycarbonyl, 
benzyloxycarbonyl ("Cbz"), 4-phenylbenzyloxycarbonyl, 
2-methylbenzyloxy-carbonyl, .alpha.-2,4,5,-tetramethylbenzyloxycarbonyl 
("Tmz"), 4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 
4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 
2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 
4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 
4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, 
4-(decyloxy)benzyloxycarbonyl and the like; the benzoylmethylsulfonyl 
group, 2,2,5,7,8-pentamethylchroman-6-sulfonyl group ("PMC") 
dithiasuccinoyl ("Dts"), the 2-(nitro)phenylsulfenyl group ("Nps"), the 
diphenyl-phosphine oxide group and like amino-protecting groups. The 
species of amino-protecting group employed is not critical so long as the 
derivatized amino group is stable to the conditions of the subsequent 
reaction(s) and can be removed at the appropriate point without disrupting 
the remainder of the molecule. Preferred amino-protecting groups are Boc, 
Cbz and Fmoc. Further examples of amino-protecting groups embraced by the 
above term are well known in organic synthesis and the peptide art and are 
described by, for example, T. W. Greene and P. G. M. Wuts, "Protective 
Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New York, 
N.Y., 1991, Chapter 7, M. Bodanzsky, "Principles of Peptide Synthesis," 
1st and 2nd revised ed., Springer-Verlag, New York, N.Y., 1984 and 1993, 
and J. M. Stewart and J. D. Young, "Solid Phase Peptide Synthesis," 2nd 
ed., Pierce Chemical Co., Rockford, Ill., 1984, E. Atherton and R. C. 
Shephard, "Solid Phase Peptide Synthesis--A Practical Approach" IRL Press, 
Oxford, England (1989), each of which is incorporated herein by reference. 
The related term "protected amino" defines an amino group substituted with 
an amino-protecting group discussed above. 
The terms "natural and unnatural amino acids" (.alpha.-amino acid) refers 
to both the naturally occurring amino acids and other "non-protein" 
.alpha.-amino acids commonly utilized by those in the peptide chemistry 
arts when preparing synthetic analogues of naturally occurring peptides, 
including D and L forms. The naturally occurring amino acids are glycine, 
alanine, valine, leucine, isoleucine, serine, methionine, threonine, 
phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, 
aspartic acid, asparagine, glutamic acid, glutamine, 
.gamma.-carboxyglutamic acid, arginine, ornithine and lysine. Examples of 
"non-protein" alpha-amino acids include hydroxylysine, citrulline, 
kynurenine, (4-aminophenyl)alanine, 3-(2'-naphthyl)alanine, 
3-(1'-naphthyl)alanine, methionine sulfone, (t-butyl)alanine, 
(t-butyl)glycine, 4-hydroxyphenylglycine, aminoalanine, phenylglycine, 
vinylalanine, propargyl-gylcine, 1,2,4-triazolo-3-alanine, thyronine, 
6-hydroxytryptophan, 5-hydroxytryptophan, 3-hydroxykynurenine, 
3-aminotyrosine, trifluoromethyl-alanine, 2-thienylalanine, 
(2-(4-pyridyl)ethyl)cysteine, 3,4-dimethoxy-phenylalanine, 
3-(2'-thiazolyl)alanine, ibotenic acid, 1-amino-1-cyclopentane-carboxylic 
acid, 1-amino-1-cyclohexanecarboxylic acid, quisqualic acid, 
3-(trifuoromethylphenyl)alanine, (cyclohexyl)glycine, thiohistidine, 
3-methoxytyrosine, elastatinal, norleucine, norvaline, alloisoleucine, 
homoarginine, thioproline, dehydroproline, hydroxyproline, homoproline, 
.alpha.-amino-n-butyric acid, cyclohexylalanine, 2-amino-3-phenylbutyric 
acid, phenylalanine substituted at the ortho, meta, or para position of 
the phenyl moiety with one or two of the following: a (C.sub.1 to C.sub.4) 
alkyl, a (C.sub.1 to C.sub.4)alkoxy, halogen or nitro groups or 
substituted with a methylenedioxy group; .beta.-2- and 3-thienylalanine, 
.beta.-2- and 3-furanylalanine, .beta.-2-, 3- and 4-pyridylalanine, 
.beta.-(benzothienyl-2- and 3-yl)alanine, .beta.-(1- and 
2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or 
tyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulfate, 
O-phosphate and O-carboxylate esters of tyrosine, 3-(sulfo)tyrosine, 
3-(carboxy)tyrosine, 3-(phospho)tyrosine, the 4-methane sulfonic acid 
ester of tyrosine, 4-methane phosphonic acid ester of tyrosine, 
3,5-diiodotyrosine, 3-nitrotyrosine, .epsilon.-alkyl lysine, and 
delta-alkyl ornithine. Any of these .alpha.-amino acids may be substituted 
with a methyl group at the alpha position, a halogen at any aromatic 
residue on the .alpha.-amino side chain, or an appropriate protective 
group at the O, N, or S atoms of the side chain residues. Appropriate 
protective groups are discussed above. 
Depending on the choice of solvent and other conditions known to the 
practitioner skilled in the art, compounds of this invention may also take 
the hemi-ketal, hemi-acetal, ketal or acetal form, which forms are 
included in the instant invention. 
In addition, it should be understood that the equilibrium forms of the 
compounds of this invention may include tautomeric forms. All such forms 
of these compounds are expressly included in the present invention. 
Also, it will be understood by those skilled in the art that when B in 
Formula 1 is a hydrogen atom, a semicarbazone may be formed with the 
resulting aldehyde. Such semicarbazones are also included as compounds of 
Formula 1, as well as the pharmaceutical compositions containing those 
compounds. Such semicarbazones include, for example, semicarbazone 
derivative of the optimal groups and embodiments of the 4-oxo butanoic 
acid derivatives of the oxoazepino indole and oxo-azepino quinoline 
compounds set forth below. 
An optimal group of compounds of Formula 1 exists when in the above Formula 
n is 1, and more so when m is 1. These compounds will be referred to 
herein as the "oxoazepino indole" compounds. 
An optimal group of oxoazepino indole compounds exists when B in Formula 1 
is a hydrogen atom, compounds referred to herein as "4-oxobutanoic 
derivatives". One embodiment of note of such 4-oxobutanoic acid 
derivatives occurs when A in Formula 1 is a group of the formula R.sup.2 
CO--, more so when R.sup.2 is 2-(carboxy)eth-1-yl, 2-(phenyl)eth-1-yl, 
methyl, napth-1-yl or phenyl, and especially so when R.sup.1 is a hydrogen 
atom. Another embodiment of 4-oxobutanoic acid derivatives exists when A 
is a group of the formula R.sup.5 --CO--NH--CHR.sup.8 --CO--, more so when 
R.sup.5 is a methyl group and R.sup.8 is a group of the formula --CH.sub.2 
COOH, and especially so when R.sup.1 is a hydrogen atom. Yet another 
embodiment of 4-oxobutanoic acid derivatives of note has A as a group of 
the formula R.sup.6 --O--CO--NH--CHR.sup.8 --CO--, further wherein R.sup.6 
is a benzyl group and R.sup.8 is a group of the formula --CH.sub.2 --COOH, 
and especially so when R.sup.1 is a hydrogen atom. Still another 
embodiment of the 4-oxobutanoic derivatives occurs when A is a group of 
the formula R.sup.3 --O--CO-- wherein R.sup.3 is benzyl, and especially so 
when R.sup.1 is a hydrogen atom. 
Another optimal group of oxoazepino indole compounds occurs when B in 
Formula 1 is a monofluoromethyl group and are thus referred to as 
"monofluoromethyl derivatives." A noteworthy embodiment of 
monofluoromethyl derivatives has A as a group of the formula R.sup.3 
--O--CO--, more so when R.sup.3 is a benzyl group, and especially so when 
R.sup.1 is a hydrogen atom. 
A further optimal group of oxoazepino indole compounds occurs when B in 
Formula 1 is a group of the formula --CH.sub.2 --O--PO(R.sup.10)R.sup.11. 
This group of compounds is referred to herein as "phosphinyloxy 
derivatives". A group of noteworthy phosphinyloxy derivatives has R.sup.10 
and R.sup.11 each as phenyl groups. An embodiment of such diphenyl 
phosphinyloxy compounds exists wherein A in Formula 1 is a group of the 
formula R.sup.3 --O--CO-- wherein R.sup.3 is benzyl, and especially so 
when R.sup.1 is a hydrogen atom. 
Another optimal group of compounds of Formula 1 exists when in the above 
Formula n is 2, and more so when m is 1. These compounds will be referred 
to herein as the "oxoazepino quinoline" compounds. 
An exemplary group of oxoazepino quinoline compounds occurs when B in 
Formula 1 is a hydrogen atom, more so when A is a group of the formula 
R.sup.3 --O--CO-- wherein R.sup.3 is benzyl, and especially so when 
R.sup.1 is a hydrogen atom. 
It should be understood that the compounds of this invention may be 
modified by appropriate functionalities to enhance selective biological 
properties. Such modifications are known in the art and include those 
which increase biological penetration into a given biological system 
(e.g., blood, lymphatic system, central nervous system), increase oral 
availability, increase solubility to allow administration by injection, 
alter metabolism and alter rate of exertion. In addition, the compounds 
may be altered to pro-drug form such that the desired compound is created 
in the body of the patient as the result of the action of metabolic or 
other biochemical processes on the pro-drug. Some examples of pro-drug 
forms include ketal, acetal, oxime, and hydrazone forms of compounds which 
contain ketone or aldehyde groups, especially where they occur in the A 
group or the modified aspartic or glutamic residues attached to the 
tricyclic nucleus of the compounds of this invention. 
The compounds of Formula 1 of this invention may be synthesized using 
conventional techniques. Advantageously, these compounds are conveniently 
synthesized from readily available starting materials. 
Thus, compounds of the instant invention can be synthesized in general by 
combining a tricyclic nucleus set forth below in Formula 2: 
##STR5## 
with the modified aspartic and glutamic acid residues of Formula 3a-d: 
##STR6## 
in the presence of a standard peptide coupling agents such as 
dicyclohexylcarbodiimide(DCC)/1-hydroxybenzotriazole (HOBt), BOP reagent, 
pyBOP, TBTU, EEDQ, 
1-ethyl(3,3'-dimethyl-1'-aminopropyl)carbodiimide(EDAC)/HOBt, and the 
like, as discussed in J. Jones, "Amino Acid and Peptide Synthesis," Steven 
G. Davis ed., Oxford University Press, Oxford, pp. 25-41 (1992), herein 
incorporated by reference. In the above formula, APG is an amino 
protecting group. The amino protecting group is then removed and the 
resulting amine is combined with the substituted acyl group of Formula 4: 
EQU R.sup.C --CO--X FORMULA 4 
or the sulfonyl group of Formula 5: 
EQU R.sup.4 SO.sub.2 --X. FORMULA 5 
In the above formulas, R.sup.1 is as defined above, and R.sup.C is R.sup.2, 
R.sup.3 --O , R.sup.4, or any of the side chains containing R.sup.8 as 
defined for group A in Formula 1. Of course, such moieties would have any 
hydroxy, carboxy or amino groups in the protected form so as not to 
interfere with the coupling reaction (Formula 3a-d), the acylation 
reaction (Formula 4) or the sulfonation reaction (Formula 5). X in the 
above Formulas represents a facile leaving group for the acylation or 
sulfonation reactions. 
In the case where the coupling reaction was carried out with the amino 
alcohol of Formula 3c, the alcohol moiety must be oxidized to the 
corresponding carbonyl compound prior to removal of the protecting groups. 
Preferred methods for the oxidation reaction include Swern oxidation 
(oxalyl chloride-dimethyl sulfoxide, methylene chloride at -78.degree. C. 
followed by triethylmine; and Dess-Martin oxidation (Dess-Martin 
periodinane, t-butanol, and methylene chloride.) The protecting groups 
contained in substructures of the Formula 2 and 3a-d are removed by 
methods well known in the art. 
The tricyclic nucleus of Formula 2 is synthesized by methods known in the 
art. For example, see D. S. Karanewsky, U.S. Pat. No. 5,504,080 issued 
Apr. 2, 1996; J. A. Robl et al., Tetrahedron Letters, 36:1593-1596 (1995); 
and S. De Lombaert et al., Tetrahedron Letters, 35:7513-7516 (1994), all 
of which are incorporated herein by reference. 
The modified aspartic or glutamic acid for Formula 3a-d can be elaborated 
by methods well known in the art. See, for example, European Patent 
Application 519,748 (corresponding to U.S. Pat. No. 5,434,248); PCT Patent 
Application No. PCT/US91/06595 (corresponding to WO93/05071 and U.S. Pat. 
Nos. 5,416,013 and 5,756,465); PCT Patent Application No. PCT/US91/02339 
(corresponding to WO91/15577); European Patent Application No. 623,592; 
PCT Patent Application No. PCT/US94/08868 (corresponding to WO95/05192); 
European Patent Application No. 623,606 (corresponding to U.S. Pat. Nos. 
5,462.939 and 5,585,486); European Patent Application No. 618,223; 
European Patent Application No. 533,226; European Patent Application No. 
528,487; European Patent Application No. 618,233; PCT Patent Application 
No. PCT/EP92/02472 (corresponding to WO93/09135); PCT Patent Application 
No. PCT/US93/03589 (corresponding to WO94/03480 and U.S. Pat. No. 
5,739.279); and PCT Patent Application No. PCT/US93/00481 (corresponding 
to WO94/14777 and U.S. Pat. No. 5.612,356), all of which are herein 
incorporated by reference. 
The acyl group of Formula 4 and the corresponding R.sup.4 SO.sub.2 groups 
are also synthesized by methods well known in the art. See, for example, 
U.S. Pat. No. 5,504,080, issued Apr. 2, 1996, herein incorporated by 
reference. While this group can be elaborated once bonded to the tricyclic 
nucleus, it is preferable that it be intact before being attached to the 
nucleus. 
Once the side chains of Formula 3 and Formula 4 or Formula 5 are bonded to 
the tricyclic nucleus of Formula 2, one skilled in the art would usually 
remove any amino, hydroxy, or carboxy-protecting groups to enhance the 
activity of the synthesized molecule. 
Pharmaceutical compositions of this invention comprise any of the compounds 
of Formula 1, and pharmaceutically acceptable salts thereof, with any 
pharmaceutically acceptable carrier, adjuvant or vehicle (hereinafter 
collectively referred to as "pharmaceutically-accetable carriers"). 
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be 
used in the pharmaceutical compositions of this invention include, but are 
not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum 
proteins, such as human serum albumin, buffer substances such as the 
various phosphates, glycine, sorbic acid, potassium sorbate, partial 
glyceride mixtures of saturated vegetable fatty acids, water, salts or 
electrolytes, such as protamine sulfate, disodium hydrogen phosphate, 
potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal 
silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based 
substances, polyethylene glycol, sodium carboxymethylcellulose, 
polyarylates, waxes, polyethylene-polyoxypropylene-block polymers, 
polyethylene glycol and wool fat. 
An optimal group of pharmaceutical compositions of Formula 1 exists when in 
the above Formula n is 1, and more so when m is 1. These compositions will 
be referred to herein as the "oxoazepino indole" compostions. 
An optimal group of oxoazepino indole compositions exists when B in Formula 
1 is a hydrogen atom, compositions referred to herein as "4-oxobutanoic 
derivatives". One embodiment of note of such 4-oxobutanoic acid 
derivatives occurs when A in Formula 1 is a group of the formula R.sup.2 
CO--, more so when R.sup.2 is 2-(carboxy)eth-1-yl, 2-(phenyl)eth-1-yl, 
methyl, napth-1-yl or phenyl, and especially so when R.sup.1 is a hydrogen 
atom. 
Another embodiment of 4-oxobutanoic acid derivatives exists when A is a 
group of the formula R.sup.5 --CO--NH--CHR.sup.8 --CO--, more so when 
R.sup.5 is a methyl group and R.sup.8 is a group of the formula --CH.sub.2 
COOH, and especially so when R.sup.1 is a hydrogen atom. Yet another 
embodiment of 4-oxobutanoic acid derivatives of note has A as a group of 
the formula R.sup.6 --O--CO--NH--CHR.sup.8 --CO--, further wherein R.sup.6 
is a benzyl group and R.sup.8 is a group of the formula --CH.sub.2 --COOH, 
and especially so when R.sup.1 is a hydrogen atom. Still another 
embodiment of the 4-oxobutanoic derivatives occurs when A is a group of 
the formula R.sup.3 --O--CO-- wherein R.sup.3 is benzyl, and especially so 
when R.sup.1 is a hydrogen atom. 
Another optimal group of oxoazepino indole compositions occurs when B in 
Formula 1 is a monofluoromethyl group and are thus referred to as 
"monofluoromethyl derivatives." A noteworthy embodiment of 
monofluoromethyl derivatives has A as a group of the formula R.sup.3 
--O--CO--, more so when R.sup.3 is a benzyl group, and especially so when 
R.sup.1 is a hydrogen atom. 
A further optimal group of oxoazepino indole compositions occurs when B in 
Formula 1 is a group of the formula --CH.sub.2 --O--PO(R.sup.10)R.sup.11. 
This group of compositions is referred to herein as "phosphinyloxy 
derivatives". A group of noteworthy phosphinyloxy derivatives has R.sup.10 
and R.sup.11 each as phenyl groups. An embodiment of such diphenyl 
phosphinyloxy compositions exists wherein A in Formula 1 is a group of the 
formula R.sup.3 --O--CO-- wherein R.sup.3 is benzyl, and especially so 
when R.sup.1 is a hydrogen atom. 
Another optimal group of pharmaceutical compositions of Formula 1 exists 
when in the above Formula n is 2, and more so when m is 1. These 
compositions will be referred to herein as the "oxoazepino quinoline" 
compositions. 
An exemplary group of oxoazepino quinoline compositions occurs when B in 
Formula 1 is a hydrogen atom, more so when A is a group of the formula 
R.sup.3 --O--CO-- wherein R.sup.3 is benzyl, and especially so when 
R.sup.1 is a hydrogen atom. 
The pharmaceutical compositions of this invention may be administered 
orally, parenterally, by inhalation spray, topically, rectally, nasally, 
buccally, vaginally or via an implated reservoir. Oral and parenteral 
administration are preferred. The term "parenteral" as used herein 
includes subcutaneous, intracutaneous, intravenous, intramuscular, 
intraarticular, intrasynovial, intrasternal, intrathecal, intralesional 
and intracranial injection or infusion techniques. 
The pharmaceutical compositions may be in the form of a sterile injectable 
preparation, for example, as a sterile injectable aqueous or oleaginous 
suspension. This suspension may be formulated according to techniques 
known in the art using suitable dispersing or wetting agents (such as, for 
example, Tween 80) and suspending agents. The sterile injectable 
preparation may also be a sterile injectable solution or suspension in a 
non-toxic parenterally-acceptable diluent or solvent, for example, as a 
solution in 1,3-butanediol. Among the acceptable vehicles and solvents 
that may be employed are mannitol, water, Ringer's solution and isotonic 
sodium chloride solution. In addition, sterile, fixed oils are 
conventionally employed as a solvent or suspending medium. For this 
purpose, any bland fixed oil may be employed including synthetic mono- or 
diglycerides. Fatty acids, such as oleic acid and its glyceride 
derivatives are useful in the preparation of injectables, as are natural 
pharmaceutically-acceptable oils, such as olive oil or castor oil, 
especially in their polyoxyethylated versions. These oil solutions or 
suspensions may also contain a long-chain alcohol diluent or dispersant 
such as Ph. Helv or a similar alcohol. 
The pharmaceutical compositions of this invention may be orally 
administered in any orally acceptable dosage form including, but not 
limited to, capsules, tablets, and aqueous suspensions and solutions. In 
the case of tablets for oral use, carrier which are commonly used include 
lactose and corn starch. Lubricating agents, such as magnesium stearate, 
are also typically added. For oral administration in capsule form useful 
diluents include lactose and dried corn starch. When aqueous suspension 
are administered orally, the active ingredient is combined with 
emulsifying and suspending agents. If desired, certain sweetening and/or 
flavoring and/or coloring agents may be added. 
The pharmaceutical compositions of this invention may also be administered 
in the form of suppositories for rectal administration. These compositions 
can be prepared by mixing a compound of this invention with a suitable 
non-irritating excipient which is solid at room temperature but liquid at 
the rectal temperature and therefore will melt in the rectum to release 
the active components. Such materials include, but are not limited to, 
cocoa butter, beeswax and polyethylene glycols. 
Topical administration of the pharmaceutical compositions of this invention 
is especially useful when the desired treatment involves areas or organs 
readily accessible to topical application. For application topically to 
the skin, the pharmaceutical composition should be formulated with a 
suitable ointment containing the active components suspended or dissolved 
in a carrier. Carriers for topical administration of the compounds of this 
invention include, but are not limited to, mineral oil, liquid petroleum, 
white petroleum, propylene glycol, polyoxyethylene, polyoxypropylene 
compound, emulsifying wax and water. Alternatively, the pharmaceutical 
composition can be formulated with a suitable lotion or cream containing 
the active compound suspended or dissolved in a carrier. Suitable carriers 
include, but are not limited to, mineral oil, sorbitan monostearate, 
polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, 
benzyl alcohol and water. The pharmaceutical compositions of this 
invention may also be topically applied to the lower intestinal tract by 
rectal suppository formulation or in a suitable enema formulation. 
Topically-transdermal patches are also included in this invention. 
The pharmaceutical compositions of this invention may be administered by 
nasal aerosol or inhalation. Such compositions are prepared according to 
techniques well-known in the art of pharmaceutical formulation and may be 
prepared as solutions in saline, employing benzyl alcohol or other 
suitable preservatives, absorption promoters to enhance bioavailability, 
fluorocarbons, and/or other solubilizing or dispersing agents known in the 
art. 
The pharmaceutical compositions of this invention may be employed in a 
conventional manner for the treatment of diseases which are mediated by 
IL-1 in mammals. Such methods of treatment, their dosage levels and 
requirements may be selected by those of ordinary skill in the art form 
available methods and techniques. For example, a pharmaceutical 
composition of this invention may be administered to a patient suffering 
from an IL-1 mediated disease in a pharmaceutically acceptable manner and 
in an amount effective to lessen the severity of that disease. 
In addition, the compounds of this invention may be used in combination 
with either conventional anti-inflammatory agents or with matrix 
metalloprotease inhibitors, lipoxygenase inhibitors and antagonists of 
cytokines other than IL-1.beta.. 
The compounds of this invention can also be administered in combination 
with immunomodulators (e.g., bropirimine, anti-human alpha interferon 
antibody, IL-2, GM-CSF, methionine enkephalin, interferon alpha, 
diethyldithiocarbamate, tumor necrosis factor, naltrexons and rEPO) or 
with prostaglandins, to prevent or combat IL-1-mediated disease symptoms 
such as inflammation. 
When the compounds of this invention are administered in combination 
therapies with other agents, they may be administered sequentially or 
concurrently to the patient. Alternatively, pharmaceutical compositions 
according to this invention may be comprised of a combination of a 
compound of Formula 1 and another therapeutic or prophylactic agent. 
The disease states which may be treated or prevented by the instant 
pharmaceutical compositions include, but are not limited to, inflammatory 
diseases, autoimmune diseases and neurodegenerative diseases, and for 
inhibiting unwanted apoptosis involved in ischemic injury, such as 
ischemic injury to the heart (e.g., myocardial infarction), brain (e.g., 
stroke), and kidney (e.g., ischemic kidney disease). Methods of 
administering an effective amount of the above-described pharmaceutical 
compositions to mammals, also referred to herein as patients, in need of 
such treatment (that is, those suffering from inflammatory diseases, 
autoimmune diseases, and neurodegenerative diseases,) are additional 
aspects of the instant invention. 
Yet another aspect of the instant invention is a method of preventing 
ischemic injury to a patient suffering from a disease associated with 
ischemic injury comprising administering an effective amount of a 
pharmaceutical composition discussed above to a patient in need of such 
treatment. 
Also, each of the methods directed to methods for treating inflammatory 
diseases, autoimmune diseases, neurodegenerative disease, and preventing 
ischemic injury emcompass using any of the optimal groups and embodiments 
of pharmaceutical compositions set forth above. 
Inflammatory disease which may be treated or prevented include, for 
example, septic shock, septicemia, and adult respiratory distress 
syndrome. Target autoimmune diseases include, for example, rheumatoid, 
arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, 
Graves' disease, autoimmune gastritis, insulin-dependent diabetes 
mellitus, autoimmune hemolytic anemia, autoimmune neutropenia, 
thrombocytopenia, chronic active hepatitis, myasthenia gravis and multiple 
sclerosis. Target neurodegenerative diseases include, for example, 
amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, 
and primary lateral sclerosis. The pharmaceutical compositions of this 
invention may also be used to promote wound healing. Target diseases 
associated with harmful, thus unwanted apoptosis, in other words, those 
associated with ischemic injury, includes myocardial infarction, stroke, 
and ischemic kidney disease. The pharmaceutical compositions of this 
invention may also be used to treat infectious diseases, especially those 
involved with viral infections. 
The term "effective amount" refers to dosage levels of the order of from 
about 0.05 mg to about 140 mg per kilogram of body weight per day for use 
in the treatment of the above-indicated conditions (about 2.5 mg to about 
7 gms. per patient per day). For example, inflammation may be effectively 
treated by the administration of from about 0.01 to 50 mg of the compound 
per kilogram of body weight per day (about 0.5 mg to about 3.5 gms per 
patient per day). 
The amount of the compounds of Formula 1 that may be combined with the 
carrier materials to produce a single dosage form will vary depending upon 
the host treated and the particular mode of administration. For example, a 
formulation intended for the oral administration of humans may contain 
from 0.5 mg to 5 gm of a compound of Formula 1 compounded with an 
appropriate and convenient amount of carrier material which may vary from 
about 5 to about 95 percent of the total composition. Dosage unit forms 
will generally contain between from about 1 mg to about 500 mg of an 
active compound of Formula 1. 
It will be understood, however, that the specific "effective amount" for 
any particular patient will depend upon a variety of factors including the 
activity of the specific compound employed, the age, body weight, general 
health, sex, diet, time of administration, route of administration, rate 
of excretion, drug combination and the severity of the particular disease 
undergoing therapy. 
Although this invention focuses on the use of the compounds disclosed 
herein for preventing and treating IL-1-mediated diseases, the compounds 
of this invention can also be used as inhibitory agents for other cysteine 
proteases. 
The compounds of this invention are also useful as commercial reagents 
which effectively bind to the ICE/ced-3 family of cysteine protease or 
other cysteine proteases. As commercial reagents, the compounds of this 
invention, and their derivatives, may be used to block proteolysis of a 
target peptide or may be derivatized to bind to a stable resin as a 
tethered substrate for affinity chromatography applications. These and 
other uses which characterize commercial cystine protease inhibitors will 
be evident to those of ordinary skill in the art. 
In order that this invention be more fully understood, the following 
examples are set forth. These examples are for the purpose of illustration 
only and are not to be construed as limiting the scope of the invention in 
any way. 
In the following Examples, proton NMR spectra were obtained at 300 MHZ; 
chemical shifts are quoted downfield from internal tetramethylsilane. 
##STR7## 
Preparation of 
(2S-cis)-5-Benzyloxycarbonylamino-1,2,4,5,6,7-Hexahydro-4-Oxoazepino[3,2,1 
-hi]indole-2-Carboxylic Acid, Ethyl Eater 
To a solution of 
(2S-cis)-5-amino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carb 
oxylic acid, ethyl ester (0.437 g, 1.73 mmol, prepared as described in 
Tetrahedron Letters 36, pp. 1593-1596 (1995) and U.S. Pat. No. 5,504,080 
(Apr. 2, 1996)) in methylene chloride (4 mL) stirring at 0.degree. C. was 
added benzyl chloroformate (0.370 mL, 2.6 mmol) and triethylamine (0.724 
mL, 5.2 mmol) and the resulting mixture was stirred under nitrogen for 45 
minutes. The reaction was quenched with water then partitioned between 
ethyl acetate and 5% aqueous potassium bisulfate solution. The aqueous 
layer was back-extracted two times with ethyl acetate, then the combined 
organic layers were washed with saturated sodium chloride solution, dried 
over sodium sulfate and evaporated to dryness. Purification of the crude 
product by flash chromatography on silica gel (S/P brand silica gel 60 
.ANG., 230-400 mesh ASTM) eluting with ethyl acetate-hexane (2:1) gave 
0.558 g (68%) of crude product. Trituration with ethyl acetate-hexane 
(1:4) gave 0.480 g of the title compound as white solid; m.p.: 
139-140.degree. C. TLC (ethyl acetate-hexane, 2:1): R.sub.f =0.6; .sup.1 
H-NMR (300 MHz, CDCl.sub.3): .delta.7.35-7.30 (m, 5H), 7.02-6.94 (m, 3H), 
6.17 (d, J=5.4 Hz, 1H), 4.15 (q, J=7.1 Hz, 2H), 3.46 (dd, J=11.0, 16.7 Hz, 
1H), 3.29 (m, 1H), 3.10 (d, J=116.5, 2H), 2.35 (m, 1H), 2.16 (m, 1H), 1.23 
(t, J=7.2 Hz, 3H). 
##STR8## 
Preparation of 
(2S-cis)-5-Benzyloxycarbonylamino-1,2,4,5,6,7-Hexahydro-4-Oxoazepino[3,2,1 
-hi]indole-2-Carboxylic Acid 
To a solution of 
(2S-cis)-5-benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1 
-hi]indole-2-carboxylic acid, ethyl ester, (0.428 g, 1.05 mmol) in 
1,4-dioxane (7.5 mL) and water (2.5 mL) was added 1M aqueous lithium 
hydroxide (1.6 mL, 1.6 mmol) and the resulting mixture was stirred at room 
temperature under nitrogen for 30 minutes. The reaction mixture was 
acidified to pH 3 with a 5% aqueous potassium bisulfate sodium chloride 
solution. The aqueous layer was back-extracted two times with ethyl 
acetate, and the combined organic layers were dried over sodium sulfate 
and evaporated to dryness to yield 0.395 g (99%) of title compound as a 
fine white solid; m.p.: 188-189.degree. C. TLC (methylene 
chloride-methanol-acetic acid, 9:1:1): R.sub.f =0.55; .sup.1 H-NMR (300 
MHz, CDCl.sub.3) .delta.7.34-7.26 (m, 5H), 7.07-6.97 (m, 3H), 6.08 (d, 
J=5.7 Hz, 1H), 5.25 (dd, J=3.2, 9.8 Hz, 1H), 5.10 (s, 2H), 4.30 (m, 1H), 
3.36 (m, 1H), 3.26 (m, 2H), 3.06 (d, J=12.0 Hz, 1H), 2.36 (m, 1H), 2.09 
(m, 1H). 
##STR9## 
Preparation of 
(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepi 
no[3,2,1-hi]quinoline-3-carboxylic acid, methyl ester 
To a solution of 
(3R,S-cis)-6-Amino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quino 
line-3-carboxylic acid, methyl ester (0.570 g, 2.1 mmol, prepared as 
described in Tetrahderon Letters 36, pp. 1593-1596 (1995) and U.S. Pat. 
No. 5,504,080 (Apr. 2, 1996)) in methylene chloride (6 mL) stirring at 
0.degree. C. was added benzyl chloroformate (0.6 mL, 4.2 mmol) and 
triethylamine (1.2 mL, 8.4 mmol) and the resulting mixture was stirred 
under nitrogen for 30 minutes. The reaction was quenched with water then 
partitioned between ethyl acetate and 5% aqueous potassium bisulfate 
solution. The aqueous layer was back extracted two times with ethyl 
acetate, then the combined organic layers were washed with saturated 
sodium chloride solution, dried (sodium sulfate) and evaporated to 
dryness. Purification of the crude product by flash chromatography on 
silica gel (S/P brand silica gel 60 .ANG., 230-400 mesh ASTM) eluting with 
ethyl acetate-hexane (2:1) gave 0.643 g (76%) of the title compound as a 
white foam. TLC (methylene chloride-methanol, 95:5) R.sub.f =0.8. .sup.1 
H-NMR (300 MHz, CDCl.sub.3) .delta.7.36-7.25 (m, 5H), 7.13-7.02 (m, 3H), 
5.67 (d, J=7.8 Hz, 1H), 5.02 (t, J=9.15, 18.3 Hz, 2H), 4.34 (m, 1H), 3.70 
(s, 3H), 3.16 (m, 1H), 2.69-2.56 (m, 5H), 2.06 (m, 1H). Mass spectrum: m/z 
408 (M+H). 
##STR10## 
Preparation of (3R, 
S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3 
,2,1-hi]quinoline-3-carboxylic acid 
To a solution of 
(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepi 
no[3,2,1-hi]quinoline-3-carboxylic acid, methyl ester (0.622 g, 1.53 mmol) 
in 1,4-dioxane (10.5 mL) and water (3.5 mL) was added 1M aqueous lithium 
hydroxide (2.3 mL, 2.3 mmol) and the resulting mixture was stirred at room 
temperature under nitrogen for 1 hour. The reaction mixture was acidified 
to ca. pH 2 with a 5% aqueous potassium bisulfate solution, then 
partitioned between ethyl acetate and saturated sodium chloride solution. 
The aqueous layer was back extracted two times with ethyl acetate, and the 
combined organic layers were dried (sodium sulfate) and evaporated to 
yield 0.670 g of the title compound. TLC (methylene 
chloride-methanol-acetic acid, 32:1:1) R.sub.f =0.35. .sup.1 H-NMR (300 
MHz, CDCl.sub.3) .delta.7.38-7.28 (m, 5H), 7.13-7.04 (m, 3H), 5.72 (d, 
J=8.1 Hz, 1H), 5.03 (s, 2H), 4.35 (m, 1H), 3.77-3.67(m, 5H), 3.10 (m, 1H), 
2.72-2.52 (m, 5H), 2.07 (m, 1H), 1.70 (m, 1H). 
##STR11## 
Preparation of N-(Benzyloxycarbonyl)-L-(N'-Methyl-N'-Methoxy)asparatamide 
.beta.-(tert-Butyl Ester) 
To a solution of N-(benzyloxycarbonyl)-L-aspartic 
acid-.beta.-(tert-butyl)ester (14.65 g, 45.3 mmol, Bachem) in CH.sub.2 
Cl.sub.2 (150 mL) at 0.degree. C. (ice bath) under a nitrogen atmosphere 
was added 1-hydroxybenzotriazole hydrate (7.29 g, 47.6 mmol, Aldrich) 
followed by 1-ethyl-3-(3',3'-dimethyl-1'-aminopropyl)carbodiimide 
hydrochloride (9.55 g, 49.8 mmol, Sigma). After stirring at 0.degree. C. 
for 15 min., N,O-dimethylhydroxylamine hydrochloride (5.10 g, 52.3 mmol, 
Aldrich) and N-methylmorpholine (5.8 mL, 53 mmol, Aldrich) were added. The 
mixture was allowed to warm to room temperature over 3 hours then stirred 
at room temperature for 16 hours. The solution was concentrated under 
vacuum and the residue partitioned between ethyl acetate-5% KHSO.sub.4 
(200 mL each). The organic phase was washed in turn with 5% KHSO.sub.4, 
saturated sodium bicarbonate and saturated sodium chloride solutions; 
dried over anhydrous sodium sulfate and evaporated to an oil. The oil was 
crystallized from hexane to give the title product (16.10 g, 97% yield) as 
a fluffy white crystalline solid. TLC (ethyl acetate), single spot (UV and 
PMA): R.sub.f =0.37. 
A similar procedure to the one above, starting with 29.3 g of 
N-(benzyloxycarbonyl)-L-aspartic acid-.beta.-(tert-butyl)ester (2-fold 
scale up) gave 31.18 g (94% yield) of the title product. 
##STR12## 
Preparation of N-(Benzyloxycarbonyl)-L-Aspartic Acid Semicarbazone 
.beta.-(tert-Butyl)Ester 
To a solution of N-(benzyloxycarbonyl)-L-(N'-methyl-N'-methoxy)aspartamide 
.beta.-(tert-butyl ester) (15.50 g, 42.3 mmol) in anhydrous ether (400 mL) 
at 0.degree. C. (ice bath) under a nitrogen atmosphere was added dropwise 
to a 1.0 M solution of LiAlH.sub.4 in ether (22.0 mL, 22.0 mmol, Aldrich) 
at such a rate as to keep the reaction solution temperature between 
0-5.degree. C. (addition time 15-20 min). After the addition of the 
lithium aluminum hydride reagent was complete, the mixture was stirred at 
0-5.degree. C. for 1 hr, then quenched by the dropwise addition of 0.3 N 
KHSO.sub.4 solution (100 mL). The resultant mixture was transferred to a 
separatory funnel adding sufficient 5% KHSO.sub.4 solution (75 mL) to 
dissolve the solids. The organic phase was separated and the combined 
aqueous washes back-extracted with ether (100 mL). The combined ether 
extracts were washed with saturated NaCl solution, dried over anhydrous 
sodium sulfate and concentrated in vacuo with minimal heating. TLC (ethyl 
acetate): streaky spot (UV and PMA) R.sub.f =0.48. TLC (methanol/methylene 
chloride, 1:9) major spot (UV and PMA): R.sub.f =0.75. 
The crude aldehyde was immediately taken up in aqueous ethanol (45 mL 
water/105 mL alcohol), placed in an ice bath and treated with sodium 
acetate (3.82 g, 46.6 mmol) and semicarbazide hydrochloride (5.20 g, 46.6 
mmol, Aldrich). The mixture was stirred at 0.degree. C. (ice bath) under a 
nitrogen atmosphere for 3 hrs, allowed to warm to room temperature, and 
stirred overnight (16 hrs). Most of the ethanol was removed under vacuum 
and the residue partitioned between ethyl acetate and water (100 mL each). 
The organic phase was washed sequentially with 5% KHSO.sub.4 , saturated 
sodium bicarbonate and saturated sodium chloride solutions; dried over 
anhydrous sodium sulfate and evaporated to dryness. The crude product of 
this reaction was combined with that of two similar procedures starting 
with 15.40 g and 4.625 g of 
N-(benzyloxycarbonyl)-L-(N'-methyl-N'-methoxy)aspartamide 
.beta.-(tert-butyl ester) (total: 35.525 g, 97 mmol) and these combined 
products were purified by flash chromotagraphy on silica gel eluting with 
acetone/methylene chloride (3:7) then methanol-acetone-methylene chloride 
(0.5:3:7) to give pure title product (27.73 g, 78.5%) as a colorless foam. 
TLC (MeOH--CH.sub.2 Cl.sub.2, 1:9): single spot (UV and PMA), R.sub.f 
=0.51. 
##STR13## 
Preparation of L-Aspartic Acid Semicarbazone .beta.-(tert-Butyl) Ester, 
p-Toluenesulfonate Salt 
To a solution of N-(benzyloxycarbonyl)-L-aspartic acid semicarbazone 
.beta.-(tert-butyl)ester (13.84 g, 38.0 mmol) in absolute ethanol (250 mL) 
was added 10% Pd/C (1.50 g, Aldrich) and the resulting mixture stirred 
under an atmosphere of hydrogen (balloon) until TLC (methanol/methylene 
chloride, 1:9) indicated complete consumption of the starting material (60 
min). Note: It is important to follow this reaction closely since the 
product can be over-reduced. The mixture was filtered though Celite and 
evaporated to an oil. The oil was chased with methylene chloride 
(2.times.75 mL) then with methylene chloride/toluene (1:1, 75 mL) to give 
the crude amine as a white crystalline solid. TLC 
(EtOAc-pyridine-AcOH--H.sub.2 O; 60:20:5:10) single spot (UV and PMA) 
R.sub.f =0.24. 
Note: In this TLC system, any over-reduced product will show up immediately 
below the desired product, R.sub.f =0.18 (PMA only). 
The crude amine was taken up in CH.sub.3 CN (60 mL) and treated with a 
solution of p-toluenesulfonic acid monohydrate (7.22 g, 38.0 mmol) in 
acetonitrile (60 mL). The crystalline precipitate was collected, washed 
with acetonitrile and ether, and air-dried to give the title compound 
(13.95 g, 92% yield) as a white, crystalline solid. 
The optical purity of this material was checked by conversion to the 
corresponding Mosher amide [1.05 equiv 
(R)-(-)-.alpha.-methoxy-.alpha.-(trifluoromethyl)phenylacetyl chloride, 
2.1 equivalents of i-Pr.sub.2 NEt in CH.sub.2 Cl.sub.2, room temperature, 
30 min]. The desired product has a doublet at 7.13 ppm (1H, d, J=2.4 Hz, 
CH.dbd.N) while the corresponding signal for its diastereomer is at 7.07 
ppm. The optical purity of the title compound obtained from the above 
procedure is typically &gt;95:5. 
PREATION 8 
Assay for Inhibition of ICE/ced-3 Protease Family Activity 
A. Determination of IC.sub.50 Values 
Fluorescence enzyme assays detecting the activity of the compounds of 
Formula 1 utilizing the recombinant ICE and CPP32 enzymes were performed 
essentially according to Thornberry et al. (Nature, 356:768:774 (1992)) 
and Nicholson et al. (Nature, 376:37-43 (1995)) respectively, (herein 
incorporated by reference) in 96 well microtiter plates. The substrate is 
Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC) for the ICE assay and 
Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin for the CPP32, Mch2, Mch3 
and Mch5 assays. Enzyme reactions were run in ICE buffer (25 mM HEPES, 1 
mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) containing 2 mM DTT at room 
temperature in duplicate. The assays were performed by mixing the 
following components: 
50 .mu.L ICE, Mch2, Mch5, CPP32 (18.8, 38, 8.1 and 0.153 nM concentrations, 
respectively) or Mch3 (1 unit) enzyme in ICE buffer containing either 8.0 
(ICE, Mch2, Mch3, CPP32) or 20 (Mch5) mM DTT; 
50 .mu.L compound of Formula 1 or ICE buffer (control); and 
100 .mu.L of 20 .mu.M substrate. 
The enzyme and the compound of Formula 1 to be assayed were allowed to 
preincubate in the microtitre plate wells for 30 minutes at room 
temperature prior to the addition of substrate to initiate the reaction. 
Fluorescent AMC product formation was monitored for one hour at room 
temperature by measuring the fluorescence emission at 460 nm using an 
excitation wavelength of 360 nm. The fluorescence change in duplicate 
(control) wells were averaged and the mean values were plotted as a 
function of inhibitor concentration to determine the inhibitor 
concentration producing 50% inhibition (IC.sub.50). The results of this 
assay are set forth below in Table 1. 
The reference compound for this assay was Cbz-ValAlaAsp-H and the values 
are denoted in Table 1 as "Reference". 
TABLE 1 
______________________________________ 
Example mICE CPP32 MCH-2 MCH-3 MCH-5 
No. IC.sub.50 (.mu.M) 
IC.sub.50 (.mu.M) 
IC.sub.50 (.mu.M) 
IC.sub.50 (.mu.M) 
IC.sub.50 (.mu.M) 
______________________________________ 
3 0.019 1.03 40.1 6.96 &gt;10 
7 0.694 0.0014 6.47 0.145 2.09 
10 0.571 0.245 1.81 2.83 7.98 
13 0.096 0.00052 ND 0.084 1.19 
16 0.045 0.780 &gt;10 32.6 18.7 
19 3.07 3.87 &gt;10 &gt;50 &gt;50 
25 0.159 8.77 &gt;50 &gt;50 4.63 
22 0.10 2.91 &gt;50 12.3 1.09 
28 0.26 0.437 32.0 1.11 2.06 
Ref. 0.064 47.0 &gt;10 &gt;10 2.96 
______________________________________ 
B. Determination of the dissociation constant Ki and irreversible rate 
constant k.sub.3 for irreversible inhibitors 
For the irreversible inhibition of a ICE/ced-3 Family Protease enzyme with 
a competitive irreversible inhibitor; using the model represented by the 
following formulas: 
##STR14## 
The product formation at time t may be expressed as: 
##EQU1## 
where E, I, EI and E-I denote the active enzyme, inhibitor, non-covalent 
enzyme-inhibitor complex and covalent enzyme-inhibitor adduct, 
respectively. The K.sub.i value is the overall dissociation constant of 
the reversible binding steps, and k.sub.3 is the irreversible rate 
constant. The [S] and K.sub.s values are the substate concentration and 
dissociation constant of the substrate bound to the enzyme, respectively. 
[E].sup.T is the total enzyme concentration. 
The above equations were used to determine the K.sub.i and k.sub.3 values 
of a given inhibitor bound to a ICE/ced-3 family protease. Thus, a 
continuous assay was run for sixty minutes at various concentrations of 
the inhibitor and the substrate. The assay was formulated essentially the 
same as described above for generating the data in Table 1, except that 
the reaction was initiated by adding the enzyme to the substrate-inhibitor 
mixture. The K.sub.i and k.sub.3 values were obtained by simulating the 
product AMC formation as a function of time according to Equation I. The 
results of this second assay are set forth below in Table 2. 
The reference compound for this assay was Cbz-ValAlaAsp-CH.sub.2 F and the 
values are denoted in Table 2 as "Reference". 
TABLE 2 
______________________________________ 
Example 31 Reference 
Enzyme Ki (.mu.M) 
k.sub.3 /Ki (.mu.M) 
Ki (.mu.M) 
k.sub.3 /Ki (M.sup.-1 s.sup.-1) 
______________________________________ 
mICE 0.0005 12,000,000 0.015 214,000 
CPP32 0.012 960,000 0.820 12,200 
MCH-2 0.033 25,000 0.594 2,950 
MCH-5 0.022 98,000 0.018 83,300 
______________________________________