Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Tripeptides – e.g. – tripeptide thyroliberin – etc.
Reexamination Certificate
1999-12-02
2003-04-22
Low, Christopher S. F. (Department: 1609)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Tripeptides, e.g., tripeptide thyroliberin , etc.
C530S330000, C514S018700
Reexamination Certificate
active
06552168
ABSTRACT:
BACKGROUND OF THE INVENTION
Apoptotic cell suicide is a fundamentally important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis, however, underlies the etiology of many of the most intractable of human diseases. In only the last few years, many of the molecules that participate in a conserved biochemical pathway that mediates the highly ordered process of apoptotic cell suicide have been identified. At the heart of this pathway are a family of cysteine proteases, the ‘caspases’, that are related to mammalian interleukin-1&bgr; converting enzyme (ICE/caspase-1) and to CED-3, the product of a gene that is necessary for apoptotic suicide in the nematode
C. elegans
(Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The role of these proteases in cell suicide is to disable critical homeostatic and repair processes as well as to cleave key structural components, resulting in the systematic and orderly disassembly of the dying cell.
The central importance of caspases in these processes has been demonstrated with both macromolecular and peptide-based inhibitors (which prevent apoptosis from occurring in vitro and in vivo) as well as by genetic approaches. Inhibition of apoptosis via attenuation of caspase activity should therefore be useful in the treatment of human diseases where inappropriate apoptosis is prominent or contributes to disease pathogenesis. Caspase inhibitors would thus be useful for the treatment of human diseases including, but not limited to, acute disorders such as cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, as well as chronic disorders such as neurodegenerative diseases (e.g. Alzheimer's, polyglutamine-repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging.
Ten caspases have so far been identified in human cells. Each is synthesized as a catalytically dormant proenzyme containing an amino-terminal prodomain followed by the large and small subunits of the heterodimeric active enzyme. The subunits are excised from the proenzyme by cleavage at Asp-X junctions (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The strict requirement by caspases for Asp in the P
1
position of substrates is consistent with a mechanism whereby proenzyme maturation can be either autocatalytic or performed by other caspases. The three dimensional crystal structures of mature caspase-1 and -3 show that the large subunit contains the principle components of the catalytic machinery, including the active site Cys residue which is harbored within the conserved pentapeptide motif, QACxG,
1
and residues that stabilize the oxyanion of the tetrahedral transition state (Wilson et al., 1994, Nature 370:270-75; Walker et al., 1994, Cell 78:342-52; Rotonda et al., 1996, Nat Struct Biol 3:619-25). Both subunits contribute residues which stabilize the P
1
Asp of substrates while the small subunit appears to contain most of the determinants that dictate substrate specificity and, in particular, those which form the specificity-determining S
4
subsite. One distinctive feature of these proteases is the absolute requirement for an aspartic acid residue in the substrate P
1
position. The carboxylate side chain of the substrate P
1
Asp is tethered by four residues in caspase-1 (Arg
179
, Gln
238
from p20 and Arg
341
, Ser
347
from p10) that are absolutely conserved in all caspase family members. Catalysis involves a typical cysteine protease mechanism involving a catalytic dyad, composed of His
237
and Cys
285
(contained within an absolutely conserved QACxG pentapeptide) and an ‘oxyanion hole’ involving Gly
238
and Cys
285
. Inhibitors bind, however, in an unexpected non-transition state configuration (which raises important considerations for inhibitor design) with the oxyanion of the thiohemiacetal being stabilized by the active site His
237
.
Members of the caspase family can be divided into three functional subgroups based on their substrate specificities which have been defined by a positional-scanning combinatorial substrate approach. The principle effectors of apoptosis (group II caspases, which include caspases-2, -3 and -7 as well as
C. elegans
CED-3) have specificity for [P
4
]DExD[P
1
], a motif found at the cleavage site of most proteins known to be cleaved during apoptosis. On the other hand, the specificity of group III caspases (caspases-6, -8, -9 and -10, as well as CTL-derived granzyme B) is [P
4
](I,V,L)ExD[P
1
] which corresponds to the activation site at the junction between the large and small subunits of other caspase proenzymes including group II (effector) family members. This and other evidence indicates that group III caspases function as upstream activators of group II caspases in a proteolytic cascade that amplifies the death signal. The role of group I caspases (caspases-1, -4 and -5) appears to be to mediate cytokine maturation and their role in apoptosis, if any, has not been substantiated.
A tetrapeptide corresponding to the substrate P
4
-P
1
residues is sufficient for specific recognition by caspases and as a consequence has formed the basis for inhibitor design. In addition to the requirement for a P
1
Asp, the P
4
residue in particular appears to be most important for substrate recognition and specificity. Caspase-1, for example, prefers a hydrophobic residue such as Tyr in P
4
(which corresponds to its YVHD cleavage site within proIL-1&bgr;) whereas caspase-3 (and other group II enzymes) has a preference for an anionic Asp residue (which corresponds to the DXXD cleavage sites within most polypeptides that are cleaved by these enzymes during apoptosis). Peptide aldehydes, nitriles and ketones are potent reversible inhibitors of these proteases while compounds that form thiomethylketone adducts with the active site cysteine (e.g. peptide (acyloxy)methylketones) are potent irreversible inhibitors. For example, the tetrapeptide aldehyde Ac-YVAD-CHO (SEQ ID NO: 24) (which was designed to mimic the YVHD caspase-1 recognition sequence within proIL-1&bgr;) is a potent inhibitor of caspase-1 (K
i
<1 nM) but a poor inhibitor of caspase-3 (K
i
=12 &mgr;M) (Thornberry et al., 1992, Nature 356:768-74). In contrast, the Ac-DEVD-CHO (SEQ ID NO: 25) tetrapeptide aldehyde (which was designed to mimic the caspase-3 recognition site) is a very potent inhibitor of caspase-3 (K
i
<1 nM) although it is also a weaker but reasonable inhibitor of caspase-1, presumably owing to promiscuity in the S
4
subsite of this enzyme (Nicholson et al., 1995, Nature 376:37-43).
Several features plague these peptide-derived inhibitors as a platform for drug design. In addition to their metabolic instability and membrane impermeability, the slow-binding time-dependent inhibition of activity (e.g. k
on
caspase-1:Ac-YVAD-CHO (SEQ ID NO: 24)=3.8×10
5
M
−1
s
−1
; k
on
caspase-3:Ac-DEVD-CHO (SEQ ID NO: 25)=1.3×10
5
M
−1
s
−1
) precludes them from the rapid inhibition characteristics that may be necessary to abolish enzymatic activity in vivo. The present patent application describes the resolution of this issue with the discovery of several novel ketones that make highly suitable caspase inhibitors.
SUMMARY OF THE INVENTION
The invention encompasses the novel class of compounds represented by formula I:
or a pharmaceutically acceptable salt thereof, wherein:
R is selected from the group consisting of:
(a) H and
(b) C(O)R
1
;
R
1
is selected from the group consisting of:
(a) hydrogen,
(b) C
1-6
alkoxy,
(c) NR
6
R
7
,
(d) benzyloxy or mono- or disubstituted benzyloxy, wherein the substituent is selected from the group consisting of:
(1) methyl,
(2) halogen,
(3) methoxy and
(4) cyano,
(e) C
1-6
alkyl or substituted C
1-6
alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) C
1-3
alkoxy,
(4) C
Aspiotis Renee
Bayly Christopher I.
Black Shawn
Grimm Erich L.
Renaud Johanne
Low Christopher S. F.
Lukton David
Merck Frosst Canada & Co.
Rose David L.
Yuro Raynard
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