Gamma-ketoacid dipeptides as inhibitors of caspase-3

Details Gamma-ketoacid dipeptides as inhibitors of caspase-3 Gamma-ketoacid dipeptides as inhibitors of caspase-3

2001-09-07

2003-02-25

Raymond, Richard L. (Department: 1624)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S564000, C544S315000, C546S152000, C546S168000, C548S131000, C562S437000

Reexamination Certificate

active

06525025

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 rodomain followed by the large and small subunits of the heterodimeric active enzyme. The subunits are excised from the proenzyme by cleavage at Asp-X unctions (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The strict requirement by caspases for Asp in the P1 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, QAC×G,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 P1 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 S4 subsite. One distinctive feature of these proteases is the absolute requirement for an aspartic acid residue in the substrate P1 position. The carboxylate side chain of the substrate P1 Asp is tethered by four residues in caspase-1 (Arg179, Gln238 from p20 and Arg341, Ser347 from p10) that are absolutely conserved in all caspase family members. Catalysis involves a typical cysteine protease mechanism involving a catalytic dyad, composed of His237 and Cys285 (contained within an absolutely conserved QAC×G pentapeptide) and an ‘oxyanion hole’ involving Gly238 and Cys285. 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 His237.
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 [P4]DE×D[P1], a motif found at the cleavage site of most proteins known to be cleaved during apoptosis. On the other hand, the specificity of group mH caspases (caspases-6, -8, -9 and -10, as well as CTL-derived granzyme B) is [P4](I,V,L)E×D[P1] 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 P4-P1 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 P1 Asp, the P4 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 P4 (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 (which was designed to mimic the YVHD caspase-1 recognition sequence within proIL-1) is a potent inhibitor of caspase-1 (Ki<1 nM) but a poor inhibitor of caspase-3 (Ki=12 &mgr;M) (Thornberry et al., 1992, Nature 356:768-74). In contrast, the Ac-DEVD-CHO tetrapeptide aldehyde (which was designed to mimic the caspase-3 recognition site) is a very potent inhibitor of caspase-3 (Ki<1 nM) although it is also a weaker but reasonable inhibitor of caspase-1, presumably owing to promiscuity in the S4 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. kon caspase-1:Ac-YVAD-CHO=3.8×105 M-1s-1; kon caspase-3:Ac-DEVD-CHO=1.3×105 M-1s-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 gamma-ketoacids that make highly suitable caspase inhibitors.
SUMMARY OF THE INVENTION
This invention encompasses the novel compounds of Formula I:
or a pharmaceutically acceptable salt, ester or hydrate thereof, wherein:
W is a bond, —CH
2
—, —C(O)— or —C(O)CH
2
—;
Z is selected from the group consisting of:
(1) H,
(2) C
1-11
alkyl,
(3) C
3-11
cycloalkyl or a benzofused analog thereof,
(4) phenyl or naphthyl, and
(5) HET
1
, wherein HET
1
represents a 5- to 10-membered mono- or bicyclic, aromatic or non-aromatic ring, or a benzofused analog thereof, containing 1-3 heteroatoms selected from O, S and N,
groups (2), (3) and (5) above are optionally substituted with 1-2 oxo groups,
groups (2)-(5) above are further optionally substituted with 1-3 substituents independently selected from the group consisting of:
(a) halo
(b) nitro,
(c) hydr

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