Irreversible cysteine protease inhibitors containing vinyl...

Details Irreversible cysteine protease inhibitors containing vinyl... Irreversible cysteine protease inhibitors containing vinyl...

1999-08-18

2001-09-11

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S184000, C514S018700, C514S019300

Reexamination Certificate

active

06287840

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to novel cysteine protease inhibitors. The inhibitors are specific to cysteine proteases and do not inhibit serine, aspartyl or zinc protease.
BACKGROUND OF THE INVENTION
Cysteine or thiol proteases contain a cysteine residue at the active site responsible for proteolysis. Since cysteine proteases have been implicated in a number of diseases, including arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, malaria, and other parasite-borne infections, methods for selectively and irreversibly inactivating them provide opportunities for new drug candidates. See, for example, Meijers, M. H. M. et al., Agents Actions (1993), 39 (Special Conference Issue), C219; Machleidt, W. et al, Fibrinolysis (1992), 6 Suppl. 4, 125; Sloane, B. F. et al., Biomed. Biochim. Acta (1991), 50, 549; Duffy, M. J., Clin. Exp. Metastasis (1992), 10, 145; Rosenthal, P. J., Wollish, W. S., Palmer, J. T., Rasnick, D., J. Clin. Investigations (1991), 88, 1467; Baricos, W. H. et al, Arch. Biochem. Biophys. (1991), 288, 468; Thornberry, N. A. et al., Nature (1992), 356, 768.
Low molecular weight inhibitors of cysteine proteases have been described by Rich, Proteinase Inhibitors (Chapter 4, “Inhibitors of Cysteine Proteinases”), Elsevier Science Publishers (1986). Such inhibitors include peptide aldehydes, which form hemithioacetals with the cysteine of the protease active site. The disadvantage of aldehydes is their in vivo and chemical instabilities.
Aldehydes have been transformed into &agr;,&bgr;-unsaturated esters and sulfones by means of the Wadsworth-Emmons-Horner modification of the Wittig reaction (Wadsworth, W. S. and Emmons, W. D. (J. Am. Chem. Soc. (1961), 83, 1733: Equation 1).
where
R=alkyl, aryl, etc.
EWG=COOEt, SO
2
Me, etc.
&agr;,&bgr;-unsaturated esters (Hanzlik et al., J. Med. Chem., 27(6):711-712 (1984), Thompson et al., J. Med. Chem. 29:104-111 (1986), Liu et al., J. Med. Chem., 35(6):1067 (1992)) and an &agr;,&bgr;-unsaturated sulfones (Thompson et al., supra, Liu et al., supra) were made using this method and tested as inhibitors of two cysteine proteases, papain and dipeptidyl amino-peptidase I (also called cathepsin C). However, the inhibition of papain by these &agr;,&bgr;-unsaturated compounds showed poor inhibition, evidenced by second order rate constants from less than 1 M
−1
sec
−1
to less than 70 M
−1
sec
−1
for the &agr;,&bgr;-unsaturated esters, and from less than 20 M
−1
sec
−1
to less than 60 M
−1
sec
−1
for the sulfone.
In addition, this chemistry has not been demonstrated with derivatives of &agr;-amino acids other than those corresponding to glycine, or in the case of the ester, phenylalanine. Thus the chirality of these compounds is non-existent for the glycine derivatives and unclear for the phenylalanine derivatives. This is significant since inhibition of an enzyme generally requires a chiral compound.
Additional methods for selectively and irreversibly inhibiting cysteine proteases have relied upon alkylation by peptide &agr;-fluoromethyl ketones (Rasnick, D., Anal. Biochem. (1985), 149, 416), diazomethylketones (Kirschke, H., Shaw, E. Biochem. Biphys. Res. Commun. (1981), 101, 454), acyloxymethyl ketones (Krantz, A. et al., Biochemistry, (1991), 30, 4678; Krantz, A. et al., U.S. Pat. No. 5,055,451, issued Oct. 8, 1991), and ketosulfonium salts (Walker, B., Shaw, E., Fed. Proc. Fed. Am. Soc. Exp. Biol., (1985), 44, 1433). The proposed mechanism of inactivation relies upon irreversible inactivation of the active site thiol group via alkylation, as depicted in Equation 2.
where
PG=protecting group
R
1
, R
2
=amino acid side chains
X=Cl, F, N
2
, OC(O)R, (+)S(CH
3
)
3
Other families of cysteine protease inhibitors include epoxysuccinyl peptides, including E-64 and its analogs (Hanada, K. et al., Agric. Biol. Chem (1978), 42, 523; Sumiya, S. et al., Chem. Pharm. Bull. ((1992), 40, 299 Gour-Salin, B. J. et al., J. Med. Chem., (1993), 36, 720), &agr;-dicarbonyl compounds, reviewed by Mehdi, S., Bioorganic Chemistry, (1993), 21, 249, and N-peptidyl-O-acyl hydroxamates (Bromme, D., Neumann, U., Kirschke, H., Demuth, H-U., Biochim. Biophys. Acta, (1993), 1202, 271.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide novel cysteine protease inhibitors that function irreversibly, resulting in large second order rate constants for the overall inhibition reaction. Accordingly, it is an object to provide these novel cysteine protease inhibitors that can be used to inhibit cysteine proteases selectively, and are thus useful in a variety of therapeutic applications.
In accordance with the foregoing objects, the present invention provides cysteine protease inhibitors comprising a targeting group linked to an alkene bond electronically conjugated with an electron withdrawing group (EWG). The second order rate constant for inhibition of a cysteine protease with the inhibitor, expressed as k
irr
/K
I
, preferably is at least about 1000 M
−1
sec
−1
.
An additional aspect of the present invention is to provide chiral cysteine protease inhibitors comprising a targeting group linked to an alkene bond conjugated with an EWG. Additionally provided are these chiral cysteine protease inhibitors wherein the second order rate constant for inhibition of a cysteine protease with the inhibitor, expressed as k
irr
/K
I
, is at least about 1000 M
−1
sec
−1
.
In a further aspect of the present invention, a cysteine protease inhibitor is provided with the formula:
In this formula, R
10
is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid residue with or without an amino end blocking group or a label. X and R
11
are amino side chains, with either (R) or (S) configuration. The A—B linkage is a peptide or peptidomimetic linkage, and EWG is an electron withdrawing group. Also provided are cysteine protease inhibitors wherein the second order rate constant for inhibition of a cysteine protease with the inhibitor, expressed as k
irr
/K
I
, is at least about 1000 M
−1
sec
−1
.
Also provided are labelled cysteine protease inhibitors, and cysteine protease inhibitors that contain additional targeting groups linked to the EWG.
In a further aspect of the present invention, cysteine protease inhibitors are provided wherein the EWG is a moiety or group that will work in the Wadsworth-Emmons reaction for olefin synthesis when directly attached to a methylenephosphonate species. Thus the EWG group, when functionally included as a component of the cysteine protease inhibitor, may be selected from the group consisting of vinylogous esters, vinylogous sulfones, vinylogous carboxylates, vinylogous amides, vinylogous phosphonates, vinylogous ketones, vinylogous nitriles, vinylogous sulfoxides, vinylogous sulfonamides, vinylogous sulfinamides, vinylogous sulfonates and vinylogous sulfoximines.
An additional aspect of the present invention relates to methods for making a cysteine protease inhibitor. The method comprises: a) a protected &agr;-amino aldehyde is coupled with a Wadsworth-Emmons reagent containing an EWG to form a cysteine protease inhibitor intermediate; b) the cysteine protease inhibitor intermediate is the n deprotected at the N-terminus; and c) the deprotected cysteine protease inhibitor intermediate is then coupled to N-terminally protected amino acids.
The invention also includes a method for inhibiting a cysteine protease, comprising irreversibly binding an cysteine protease inhibitor to the protease.
The invention further provides a method of treating cysteine proteane-associated disorders, comprising administering a therapeutically effective dose of a cysteine protease inhibitor to a patient. Thus, pharmaceutical compositions of cysteine protease inhibitors are also provided.
Additionally, the invention provides methods of detecting a cysteine protease in a sample, comprising assaying the sample in th

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