Organic compounds -- part of the class 532-570 series – Organic compounds – Amino nitrogen containing
Reexamination Certificate
2000-03-21
2002-09-17
Raymond, Richard L. (Department: 1624)
Organic compounds -- part of the class 532-570 series
Organic compounds
Amino nitrogen containing
C564S502000
Reexamination Certificate
active
06452050
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to non-peptidic &agr;-ketoamide compounds and molecular arrays of potential protease inhibitors and the uses thereof.
BACKGROUND OF THE INVENTION
Proteolytic enzymes, or proteases, are proteins that catalyze the degradation of peptide bonds in protein and peptide substrates. Proteases are typically categorized into four major classes (i.e., serine, aspartyl, metallo, and cysteine), classified according to the catalytic site chemical group that facilitates peptide bond hydrolysis. Proteases are involved in a wide variety of physiological and pathological processes including blood coagulation, protein turnover, complement activation, hormone processing, and cancer cell invasion.
Cysteine proteases, for example, are utilized by living organisms to perform a variety of key cellular functions, and thus are potential targets for drug discovery. For example, cathepsin B has been studied for its role in the progression of normal tissue to cancerous tissue, and the protease cruzain is believed to be essential for the parasitic infection in Chagas' disease (a major public health problem in South and Central America, affecting about 25% of the population of those regions).
The interaction of a protease with a substrate is a highly specific binding event that is driven by, for example, favorable molecular shape recognition (i.e., between the protease and the substrate) and electrostatic e.g., charge-charge, dipolar, or van der Waals) interactions that occur upon binding. Recognition and binding typically involves 3 to 4 amino acid residues of the substrate on either side of an enzyme's catalytic site. Although the kinetics of all proteolytic events are not fully understood, most protease-mediated catalysis occurs because the catalytic site stabilizes a transition-state, structural intermediate in the pathway to peptide-bond cleavage.
Inhibitors of proteolytic activity typically interact with a protease at its active site, preventing interaction (e.g., recognition, binding, or reaction) of enzyme and substrate. However, inhibition via allosteric change (i.e., conformational or other structural change) and co-factor binding inhibition are some other possible modes of inhibition. Potent and specific synthetic inhibitors can: 1) interact with the enzyme's binding pocket with high affinity, and 2) interact with the catalytic site to mimic the transition state structure.
Modulation, e.g., inhibition or enhancement, of protease activity can profoundly influence biological systems, and, therefore, proteases are often chosen as targets for drug discovery. In the design of protease inhibitors, researchers have generally identified chemical structures that interact with the catalytic chemical group at the active site of the protease and find structures that mimic the transition state of the catalytic reaction. These identified structures are then linked to a di- or tri-peptide sequence that specifically binds to the active site substrate binding pockets. Peptide-based inhibitors, mimicking the primary sequence of the natural substrate, often show very high potency against the target; however, orally administered peptides generally exhibit poor bioavailability due to hydrolysis by nonspecific proteolytic enzymes in the digestive system. Substitution of the peptide portion of protease inhibitors with small organic molecules that mimic the molecular shape and charge. interactions of the peptides frequently results in improved bioavailability and oral absorption for that inhibitor.
SUMMARY OF THE INVENTION
The invention is based on new methods for making and using compounds and arrays of novel &agr;-ketoamides, and the arrays and compounds made by these methods. These novel compounds are potential inhibitors of proteolytic enzymes, particularly cysteine proteases such as cruzain. Application of the new methods has led-to the identification of a number of new inhibitors, from amongst an array of about 38,000 &agr;-ketoamide derivatives, having specific activity against two cysteine proteases: cruzain and cathepsin B. These compounds and other compounds identified by the methods described herein can be useful, for example, in developing pharmaceutical agents for the treatment of diseases (e.g., Chagas' disease) associated with these proteases. Although the disclosed compounds have specific activity for cruzain and cathepsin B, the methods described herein can also be used to identify inhibitors of other proteases.
In one embodiment, the invention features a method for preparing a monoacylated diamine compound. The method includes the step of reacting a diamine with an &agr;-ketoester compound, under conditions such that a monoacylated diamine is prepared. The diamine can be represented, for example, by the structure B—NH—Y—NH—C, where Y is a linker moiety (i.e., a divalent alkyl, carbocyclic, or aryl groups), and B and C can independently be hydrogen, an alkyl group, a carbocyclic group, or an aryl group. The &agr;-ketoester can be represented, for example, by the structure:
where A and R
2
can independently be an alkyl group, a carbocyclic group, or an aryl groups.
A functionalized &agr;-ketoamide compound can also be formed, for instance, by preparing a monoacylated diamine by the above method, then reacting the monoacylated diamine with an electrophile (e.g., an alkoxymethylene oxazolone, an acid halide, an isocyanate, an isothiocyanate, an anhydride, a halotriazine, a Michael acceptor, an aldehyde, or a ketone).
In another embodiment, the invention features a method for preparing a plurality (e.g., 100 or more, or 1000 or more) of &agr;-ketoamide compounds. The method includes the step of reacting a plurality of diamine compounds with a plurality of &agr;-ketoester compounds, under conditions whereby a plurality of &agr;-ketoamide compounds is prepared.
The plurality of diamines can include, for example, a diamine that can be represented by the structure B—NH—Y—NH—C, where Y, B, and C are defined as above. Each of the &agr;-ketoester compounds can be, for example, represented by the structure:
wherein A and R
2
are defined as above.
The method can also include the step of reacting the plurality of &agr;-ketoamide compounds with a plurality of electrophiles (alkoxymethylene oxazolones, acid halides, isocyanates, isothiocyanates, anhydrides, halotriazines, Michael acceptors, aldehydes, ketones, or combinations thereof), such that a plurality of functionalized &agr;-ketoamide compounds is prepared.
The plurality of compounds can, for example, be arranged in a spatially addressable array format. An array produced by this method is contemplated.
Still another embodiment of the invention features an array of &agr;-ketoamide compounds. Each of the &agr;-ketoamide compounds can be represented, for example, by the formula:
where A can be an alkyl group, a carbocyclic group, or an aryl group; B and C independently can be hydrogen, an alkyl group, a carbocyclic group, or an aryl group; Y can be a divalent alkyl group, carbocyclic group, or aryl group; and D can be hydrogen, an alkyl group, a carbocyclic group, an aryl group, or —C(X)—Z—W, where X is O or S, Z is a single bond or NR; and R and W independently can be hydrogen, an alkyl group, a carbocyclic group, or an aryl group.
The array can include, for example, at least about 100 compounds, or at least about 1000 compounds.
D can be, for example, the triazolinyl moiety:
where X and Z independently can be an unsubstituted, monosubstituted, or disubstituted amino group, a thioalkyl group, a thioaryl group, an alkoxy group, an aryloxy group, a halogen (e.g., F, Cl, Br, or I), an alkyl group, a carbocyclic group, or an aryl group.
Alternatively, D can be the oxazolinyl moiety:
where X can be an alkyl group, a carbocyclic group, or an aryl group.
A method of identifying a compound having a specific characteristic (e.g., inhibition of an enzyme, or other biological activity or interaction) is also contemplated. The method includes screening any of the arrays described above with an assay capab
Baldino Carmen M.
Cheng Hong
Chipman Stewart D.
Coffen David L.
ArQule, Inc.
Balasubramanian Venkataraman
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