Cysteinyl protease inhibitors

Details Cysteinyl protease inhibitors Cysteinyl protease inhibitors

1999-06-22

2003-09-09

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Tripeptides, e.g., tripeptide thyroliberin , etc.

C514S018700, C514S019300

Reexamination Certificate

active

06617426

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to protease inhibitor compounds useful in treatments of neurodegenerative, cardiovascular, pulmonary, neuromuscular, musculovascular and autoimmune diseases characterized by tissue damage, induction of apoptosis, cell death and/or the accumulation of amyloid proteins in deposits, plaques, fibrils and tangles.
BACKGROUND OF INVENTION
Cysteinyl proteases are implicated in important cellular processes including endocytosis and degradation of proteins, immune recognition, apoptosis and processing of amyloid precursor protein. Cysteinyl proteases are localized in the endosomal lysosomal compartment, possibly the endoplasmic reticulum and cytosol. While many of the molecular details are at present uncertain, the importance of cysteinyl proteases at the cellular level and at an organism level cannot be overstated.
In Alzheimer's disease (AD), amyloid precursor protein (APP; i.e., APP
695
) is proteolytically processed in neural cells and A&bgr; cleavage fragments are secreted into cerebral extracellular perivascular matix. Plaques containing A&bgr; act as a pathological landmark of AD (2-5) and in vitro studies have shown to that A&bgr; can aggregate into insoluble fibrils which ultrastructurally resemble amyloid plaques (7). Aggregated in a fibrillar beta-pleated sheet configuration, A&bgr; is thought to form a nidus for binding other proteins contributing to Alzheimer's dementia and pathology. A&bgr; fibrils are reportedly toxic for cultured neurons in vitro (8), possibly through induction of apoptosis (9), initiation of free radical formation (10) or increases in peroxide (H
2
O
2
) levels in cells (11).
APP, synthesized by neuronal cells, full length APP may be degraded in lysosomes (21,22), resulting in non-amyloidogenic fragments, or it can be proteolytically processed to polypeptides of 110 kDa, i.e., sAPP (16-17); 9-10 kDa (17); and 4.2 kDa, i.e., A&bgr; (18-19). Characterization of fragments and protease inhibitor profiles suggest three intracellular enzymes, termed secretases, differing as follows: namely,
Alpha-secretase (&agr;-secretase) mediates cleavage of APP
695
and APP
751
producing sAPP (110 kDa) and a 9-10 kDa carboxy-terminal-fragment (CTF; i.e., APP
611-695
). The CTF fragment may undergo further degradation in lysosomes (17). Products of lysosomal and &agr;-secretase cleavages are generally considered to be non-amyloidogenic;
Beta-secretase (&bgr;-secretase) mediates cleavage of APP
695
in the carboxy-terminal region (i.e., at APP Met
595
-Asp
596
; A&bgr; Asp
1
; 23), producing a 11.4-11.8 kDa fragment, 100 amino acids in length (CTF100), and containing the A&bgr; domain; and,
Gamma-secretase (&ggr;-secretase) mediates cleavage in the C-terminal transmembrane domain of APP, and possibly also CTF100, to release the A&bgr;
1-39
, A&bgr;
1-40
and/or A&bgr;
1-42
peptides. Both A&bgr;
1-40
and A&bgr;
1-42
are reportedly amyloidogenic; with A&bgr;
1-42
being more active than A&bgr;
1-40
(7,24,25).
Sensitivity of secretases to protease inhibitors has been studied in cell-based assays, i.e., by treating APP producer cells with protease inhibitors. Results have varied in different cells and under differing conditions of culture. In certain reports, APP cleavage is reportedly inhibited by serine protease inhibitors; in others by inhibitors of chymotrypsin-like enzymes; and in others by inhibitors of cysteinyl proteases such as cathepsins B, D, H, L and S and calpain. Studies with leupeptin (26,27), E64 and Z-Phe-Ala-CHN
2
(28), i.e., lysosomal enzyme inhibitors, have failed to implicate known lysosomal hydrolases in production of A&bgr;. Similarly, although calpain inhibitor I, a caspase inhibitor, was initially reported effective in reducing A&bgr; levels in vitro (29), subsequent findings seem at odds with an effect on a putative &ggr;-secretase (30). Dipeptidyl-aldehyde inhibitor, Z-L-Val-L/D-Phe-aldehyde (MDL 28170) and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] were also once thought to inhibit A&bgr;
1-42
production (32). Since non-specific acidotropic agents such as NH
4
Cl and chloroquine, or Golgi inhibitors such as brefeldin A and monensin (19,27), reportedly affect APP processing it is possible that it takes place in the endoplasmic reticulum (ER) and/or Golgi apparatus. Specific and targeted inhibitors of secretase enzymes have (to date) proven elusive.
In apoptosis, an intracellular cascade of cysteinyl proteases, termed caspases, is activated by radiation damage to cellular DNA or structures (e.g., mitochondria), or by removal of one or more critical growth factors (e.g., EGF, PGF, TGF&bgr;, NGF, BDNF), glucose or serum. In addition, a variety of different receptor ligand interactions at the cell surface may trigger apoptosis in vitro: e.g., anti-Fas antibody or Fas ligand binding to Fas receptor; IL-1 binding to IL-1R&bgr;; TNF&agr; binding to TNFR1 and TNFR2; Apo 3 ligand binding to Death Receptor 3; glucocorticoids binding to cell surface glycocorticoid receptors; TRAIL, TRANCE and NOC18 binding to yet other cell surface “death” receptors. Important cysteinyl proteases activated in this manner are thought to include caspases 3, 8 and 9.
Apoptosis has recently been implicated in mechanisms by which tissue damage is triggered following traumatic injury, vascular insufficiency and stroke.
Using knockout mice, Cathepsin S is reportedly involved in class I MHC antigen processing, i.e., of self and viral antigens, while Cathepsin L is involved in class II processing of foreign proteins.
Thus, protease inhibitors find a variety of important medical and veterinary uses in treatments of diseases, as well as, uses in the food, dairy, agriculture, chemical and biotechnology industries.
SUMMARY OF THE INVENTION
Cysteinyl protease inhibitors, pharmaceutical compositions and methods of treating protein processing disorders, trauma, stroke, autoimmunity, neurodegenerative and cognitive disorders including Alzheimer's disease, are disclosed. The di- and tri-peptide analogues with unnatural &agr;-amino side chain residues and derivatized amino and carboxyl terminal residues constitute highly effective inhibitors of cysteinyl proteases including e.g. cathepsins, caspases, calpains and secretases. Methods and pharmaceutical compositions are provided for modulating activities of cysteinyl proteases involved in diseases including methods for altering secretase-mediated processing of APP in Alzheimer's disease and aberrant cathepsin S-mediated processing of self proteins for MHC class I presentation in autoimmune diseases. The instant compounds include vinyl sulfides, sulfoxides, sulfones, amines, amine oxides, phosphines, phosphine oxides, phosphinates and phosphonates of peptidyl analogs of di- and tri-peptides, where the vinyl substituent in the peptidyl analog preferably occupies the carboxyl-terminal position and consists of a substituted alkenyl or alkynyl side chain.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Embodiments of the invention provide cysteinyl protease inhibitors. The instant compounds are vinyl heterocyclic alkyls and aryl adducts of peptidyl analogs of natural &agr;-amino acid containing cysteinyl protease substrates; according to the following FORMULA I, II or III: namely,
wherein R
1
is an unnatural amino acid side chain, preferably a lower alkenyl or lower alkynyl that is unsubstituted, or alternatively, substituted with W,
W, in turn, is halo, hydroxy, alkyl, aryl, heterocycle, heteroaryl, alkoxy, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, heterocycle-carbonyl, nitro, haloalkyl; preferably, trifluoromethyl, cyano, amino or aminocarbonyl; and most preferably, a side chain not present in a natural amino acid;
X is —CH═CH—(CH═CH)
k
—(CH
2
)
j
or —CH
2
CH
2
—(CH
2
)
b
where “k” is 0-9, preferably 0-3, and most preferably 0; “j” is 0-4, preferably 0-2, most preferably 0; and, “b” is 0-6, preferably 3-6, most preferably 3 or 4;
Y is S, SO, SO
2
, NR
2
O, —N(O)(R
20
)—, —PR
20
, —P(O)(R
20
)—, —P(O)O— or —P(O)(

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