Peptidyl-2-amino-1-hydroxyalkanesulfonic acid inhibitors of cysteine proteases, methods for making these, compounds, and methods for using the same are disclosed.
Numerous cysteine proteases have been identified in human tissues. A xe2x80x9cproteasexe2x80x9d is an enzyme which degrades proteins into smaller components (peptides). The term xe2x80x9ccysteine proteasexe2x80x9d refers to proteases which are distinguished by presence therein of a cysteine residue which plays a critical role in the catalytic process. Mammalian systems, including humans, normally degrade and process proteins via a variety of enzymes including cysteine proteases. However, when present at elevated levels or when abnormally activated, cysteine proteases are involved in pathophysiological processes.
For example, calcium-activated neutral proteases (xe2x80x9ccalpainsxe2x80x9d) comprise a family of intracellular cysteine proteases which are ubiquitously expressed in mammalian tissues. Two major calpains have been identified; calpain I and calpain II. While calpain II is the predominant form in many tissues, calpain I is thought to be the predominant form in pathological conditions of nerve tissues. The calpain family of cysteine proteases has been implicated in many diseases and disorders, including neurodegeneration, stroke, Alzheimer""s disease, amyotrophy, motor neuron damage, spinal cord trauma, traumatic brain injury, acute central nervous system injury, muscular dystrophy, bone resorption, platelet aggregation, cataracts and inflammation. Calpain I has been implicated in excitatory amino-acid induced neurotoxicity disorders including ischemia, spinal cord trauma, traumatic brain injury, hypoglycemia and epilepsy.
The lysosomal cysteine protease cathepsin B has been implicated in arthritis, inflammation, myocardial infarction, tumor metastasis, and muscular dystrophy. Other lysosomal cysteine proteases include cathepsins C, H, L and S. Interleukin-1xcex2 converting enzyme (ICE) is a cysteine protease which catalyzes the formation of interleukin-1xcex2. Interleukin-1xcex2 is an immunoregulatory protein implicated in inflammation, diabetes, septic shock, rheumatoid arthritis, and Alzheimer""s disease. ICE has also been linked to apoptotic cell death of neurons which is implicated in a variety of neurodegenerative disorders including Parkinson""s disease, ischemia, and amyotrophic lateral sclerosis (ALS).
Cysteine proteases are also produced by various pathogens. The cysteine protease clostripain is produced by Clostridium histolyticum. Other proteases are produced by Trpanosoma cruzi, malaria parasites Plasmodium falciparum and P.vinckei and Streptocococcus. Viral cysteine proteases such as HAV C3 are essential for processing of picornavirus structural proteins and enzymes.
Given the link between cysteine proteases and various debilitating disorders, compounds which inhibit these proteases would be useful and would provide an advance in both research and clinical settings.
The present invention is directed to novel cysteine protease inhibitors which we refer to as peptidyl-2-amino-1-hydroxyalkanesulfonic acids. Exemplary compounds are represented by Formula I: 
Constituent members and preferred embodiments are defined infra.
The compounds of the invention are useful for the inhibition of cysteine proteases. Beneficially, these compounds find utility in a variety of settings. For example, in a research arena, the subject compounds can be used as standards for screening in the discovery of agents for treating disorders associated with abnormal and/or aberrant activity of cysteine proteases. In a therapeutic arena, the compounds can be used to alleviate, mediate, reduce, and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine proteases. One particular advantage of the subject compounds is that they have unexpectedly excellent solubility in aqueous media, approximately between 1 and 300 mg/mL. Thus, the compounds can be easily administered parenterally, for example intravenously, intrathecally or supradurally by bolus or infusion in the clinical or laboratory setting in media such as aqueous saline buffer.
Also disclosed are methodologies for making the peptidyl-2-amino-1-hydroxyalkanesulfonic acids. These and other features of the invention are set forth in more detail below.
Novel cysteine protease inhibitors have been discovered which are represented by Formula I: 
wherein:
* denotes the xcex1-carbon of an xcex1-amino acid residue having the L configuration;
o denotes a carbon having either stereochemical configuration, or a mixture thereof;
A is selected from the group consisting of lower alkyl, aryl having from 6 to about 14 carbons, heterocyclyl having from about 5 to about 14 ring atoms, heterocycloalkyl having from about 5 to about 14 ring atoms, aralkyl having from about 7 to about 15 carbons, and heteroarylalkyl, said alkyl, aryl, heterocyclyl, heterocycloalkyl, aralkyl and heteroarylalkyl groups being optionally substituted with J;
B is selected from the group consisting of C(xe2x95x90O), OC(xe2x95x90O) S(xe2x95x90O) S (xe2x95x90O)2, and NR4C(xe2x95x90O), where R4 is H or lower alkyl;
each Aaa is independently an amino acid which optionally contains one or more blocking groups;
n is 0, 1, 2, or 3;
G is selected from the group consisting of H, C(xe2x95x90O)NR5R6, C(xe2x95x90O)OR5, CF3, CF2R5, P(xe2x95x90O)(R5)(OR6) and P(xe2x95x90O)(OR5)(OR6);
J is selected from the group consisting of halogen, lower alkyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, aryl substituted with aralkyloxy, C(xe2x95x90O)OR7, OC(xe2x95x90O)R7, NR8C(xe2x95x90O)OR7, OR7, CN, NO2, NR7R8, Nxe2x95x90C(R7)R8, SR7, S(xe2x95x90O)R7, S(xe2x95x90O)2R7, and C(xe2x95x90NR7)NHR8;
R5 and R6 are independently selected from the group consisting of H, lower alkyl, aralkyl, heterocyclic, and heterocycloalkyl, said lower alkyl, aralkyl, heterocyclic, and heterocycloalkyl groups being optionally substituted with one or more hydroxy, alkoxy, aryloxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, amino, monoalkylamino, dialkylamino, monoarylamino, diarylamino, or halogen groups;
R1, R2, and R3, independently, are selected from the group consisting of H, lower alkyl, aryl, and heterocyclyl, said lower alkyl, aryl and heterocyclyl groups being optionally substituted with one or more J groups;
or R2 and R3, may be taken together with the carbon and nitrogen atoms to which they are attached to form a 4-8 membered ring which is optionally substituted with one or more J groups;
R7 and R8, independently, are selected from the group consisting of H, lower alkyl, aralkyl, heterocyclic, and heterocycloalkyl, said lower alkyl, aralkyl, heterocyclic, and heterocycloalkyl groups being optionally substituted with one or more hydroxy, alkoxy, aryloxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, amino, monoalkylamino, dialkylamino, monoarylamino, diarylamino, or halogen groups;
M is a pharmaceutically acceptable cation selected from the group consisting of sodium, lithium, potassium, calcium, magnesium, zinc, aluminum, ammonium, mono-, di-, tri-, or tetraalkylammonium, morpholinium, piperidinium, and megluminium; and
with the proviso that R2 and R3 taken together is other than xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94.
In some preferred embodiments of the compounds of Formula I R1 is lower alkyl or lower alkyl substituted with J.
In some preferred embodiments, J is cycloalkyl, aryl, or aryl substituted with aralkyloxy. In more preferred embodiments, R1 is ethyl, isopropyl, benzyl, isobutyl, cyclohexylmethyl, or 4-benzyloxybenzyl.
In other preferred embodiments, R2 is alkyl or cycloalkyl. In more preferred embodiments, R2 is isobutyl, isopropyl, or cyclopentyl.
In some preferred embodiments, R3 is H. In other preferred embodiments, R2 and R3 are taken together with the carbon and nitrogen atoms to which they are attached to form a 4-8 membered ring which is optionally substituted with one or more J groups.
In some preferred embodiments, G is H or C(xe2x95x90O)NHEt. More preferably, G is H.
In further preferred embodiments, A is alkyl, aryl, aralkyl, or tetrahydroisoquinolin-2-yl. In especially preferred embodiments, A is methyl, tolyl, naphthyl, benzyl, or tetrahydroisoquinolin-2-yl. Most preferably, A is benzyl.
In still other preferred embodiments, B is C(xe2x95x90O), OC(xe2x95x90O), or S(xe2x95x90O)2. In more preferred embodiments, B is C(xe2x95x90O) or OC(xe2x95x90O). In especially preferred embodiments, A is benzyl and B is OC(xe2x95x90O).
In further preferred embodiments, R3 is H, n is 0, 1, or 2, and M is sodium. Most preferably, n is 0.
In still further preferred embodiments, Aaa is independently Ala, Phe, or Leu.
As used herein, the term xe2x80x9calkylxe2x80x9d includes straight-chain, branched and cyclic hydrocarbon groups such as, for example, ethyl, isopropyl and cyclopentyl groups. Preferred alkyl groups have 1 to about 10 carbon atoms. xe2x80x9cCycloalkylxe2x80x9d groups are cyclic alkyl groups. xe2x80x9cArylxe2x80x9d groups are aromatic cyclic compounds including but not limited to phenyl, tolyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl. Preferred aryl groups include phenyl and naphthyl. The term xe2x80x9ccarbocyclicxe2x80x9d, as used herein, refers to cyclic groups in which the ring portion is composed solely of carbon atoms. The term xe2x80x9clower alkylxe2x80x9d refers to alkyl groups of 1 to about 6 carbon atoms. The term xe2x80x9chalogenxe2x80x9d refers to F, Cl, Br, and I atoms. The term xe2x80x9caralkylxe2x80x9d denotes alkyl groups which bear aryl groups, for example, benzyl groups. As used herein, xe2x80x9calkoxyxe2x80x9d groups-are alkyl groups linked through an oxygen atom. Examples of alkoxy groups include methoxy (xe2x80x94OCH3) and ethoxy (xe2x80x94OCH2CH3) groups. In general, the term xe2x80x9coxyxe2x80x9d when used as a suffix denotes attachment through an oxygen atom. Thus, alkoxycarbonyl groups are carbonyl groups which contain an alkoxy substituent, i.e., groups of general formula xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R, where R is alkyl. The term xe2x80x9caryloxyxe2x80x9d denotes an aryl group linked through an oxygen atom, and the term xe2x80x9caralkyloxyxe2x80x9d denotes an aralkyl group linked through an oxygen atom.
The terms xe2x80x9cheterocyclexe2x80x9d, xe2x80x9cheterocyclylxe2x80x9d, and xe2x80x9cheterocyclicxe2x80x9d refer to cyclic groups in which a ring portion includes at least one heteroatom such as O, N or S. Heterocyclic groups include xe2x80x9cheteroarylxe2x80x9d as well as xe2x80x9cheteroalkylxe2x80x9d groups. xe2x80x9cHeterocycloalkylxe2x80x9d denotes a heterocycle attached through a lower alkyl group. The term xe2x80x9cheteroarylxe2x80x9d denotes aryl groups having one or more hetero atoms contained within an aromatic ring. The term xe2x80x9cheteroarylalkylxe2x80x9d denotes a heteroaryl group attached through an alkyl group. The term xe2x80x9cheteroalkylxe2x80x9d denotes a heterocyclic group which contains at least one saturated carbon atom in a heterocyclic ring. Examples of heteroalkyl groups include piperidine, dihydropyridine, and tetrahydroisoquinolin-2-yl groups.
As used herein, the term xe2x80x9camino acidxe2x80x9d denotes a molecule containing both an amino group and a carboxyl group. As used herein the term xe2x80x9cL-amino acidxe2x80x9d denotes an xcex1-amino acid having the L-configuration around the xcex1-carbon, that is, a carboxylic acid of general formula CH(COOH)(NH2)xe2x88x92(side chain), having the L-configuration. The term xe2x80x9cD-amino acidxe2x80x9d similarly denotes a carboxylic acid of general formula CH(COOH) (NH2)xe2x88x92(side chain), having the D-configuration around the xcex1-carbon. Side chains of L-amino acids include naturally occurring and non-naturally occurring moieties. Nonnaturally occurring (i.e., unnatural) amino acid side chains are moieties that are used in place of naturally occurring amino acid sidechains in, for example, amino acid analogs. See, for example, Lehninger, Biochemistry, Second Edition, Worth publishers, Inc, 1975, pages 73-75. One representative amino acid side chain is the lysyl side chain, xe2x80x94(CH2)4xe2x80x94NH2. Other representative xcex1-amino acid side chains are shown below in Table 1.
The amino acid substituents denoted xe2x80x9cAaaxe2x80x9d in the compounds of Formula I can be identical to each other, or can be different from each other.
Compounds of Formula I contain a moiety of formula: 
wherein the symbol xe2x80x9coxe2x80x9d denotes a carbon atom which can be in either stereochemical configuration (i.e., R or S). Thus, included within the present invention are compounds of Formula I wherein the carbon atom denoted by xe2x80x9coxe2x80x9d has the R configuration, compounds of Formula I wherein the carbon atom denoted by xe2x80x9coxe2x80x9d has the S configuration, and any mixtures thereof.
Functional groups present in the compounds of Formula I may contain blocking groups. Blocking groups are known per se as chemical functional groups that can be selectively appended to functionalities, such as hydroxyl groups, amino groups, thio groups, and carboxyl groups. Protecting groups are blocking groups which can be readily removed from functionalities. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention. Examples of such protecting groups are the benzyloxycarbonyl (Cbz; Z), t-butoxycarbonyl, methyl ester, and benzyl ether groups. Other preferred protecting groups according to the invention may be found in Greene, T. W. and Wuts, P. G. M., xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d 2d. Ed., Wiley and Sons, 1991.
Further blocking groups useful in the compounds of the present invention include those that bear acyl, aroyl, lower alkyl, alkanesulfonyl, aralkanesulfonyl, or arylsulfonyl substituents on their amino groups.
The peptidyl-2-amino-1-hydroxyalkanesulfonic acids of the invention have the unexpected advantage of excellent solubility in aqueous media, preferably approximately between 1 and 300 mg/mL, and preferably at temperatures between approximately 0xc2x0 and 40xc2x0 C., with 25xc2x0 C. being especially preferred. The compounds of the invention are preferably soluble at concentrations greater than about 5 mg/mL. More preferably, the compounds of the invention are soluble at concentrations above about 20 mg/mL. Thus, the compounds can be easily administered parenterally, for example intravenously, by bolus or infusion in the clinical or laboratory setting in media such as optionally buffered water, aqueous saline, or aqueous saline buffers at approximately pH 5 to 8, preferably pH 6.5 to 7.5. Examples of these media include water, optionally buffered 0.85% (145 mM) sodium chloride, phosphate buffered saline (10 mM phosphate, 120 mM NaCl, 2.7 mM KCl, pH 7.4), and Locke""s buffer (10 mM HEPES, 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 5 uM glycine, 20 mM glucose, 1 mM sodium pyruvate, pH 7.4). The enhanced aqueous solubility of the compounds of the invention is a significant improvement over the solubilities of many other cysteine protease inhibitors. For example, the aqueous solubility of the analogous dipeptide aldehyde and xcex1-ketoamide inhibitors are typically less than 1 mg/mL. See Harbeson et al. J. Med. Chem. 1994, 37, 2918-2929.
Because the peptidyl-2-amino-1-hydroxyalkanesulfonic acids of the invention inhibit cysteine proteases, they can be used in both research and therapeutic settings. Inhibition of cysteine protease activity can be measured using compounds of the invention. Thus, in a research environment, preferred compounds having defined attributes can be used to screen for natural and synthetic compounds which evidence similar characteristics in inhibiting protease activity. The compounds of the invention also can be used in the refinement of in vitro and in vivo models for determining the effects of inhibition of particular proteases on particular cell types or biological conditions. In a therapeutic setting, given the connection between cysteine proteases and certain defined disorders, compounds of the invention can be utilized to alleviate, mediate, reduce and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine proteases.
In preferred embodiments, compositions are provided for inhibiting a cysteine protease comprising a compound of the invention. In other preferred embodiments, methods are provided for inhibiting cysteine proteases comprising contacting a protease selected from the group consisting of cysteine proteases with an inhibitory amount of a compound of the invention.
The disclosed compounds of the invention are useful for the inhibition of cysteine proteases. As used herein, the terms xe2x80x9cinhibitxe2x80x9d and xe2x80x9cinhibitionxe2x80x9d mean having an adverse effect on enzymatic activity. An inhibitory amount is an amount of a compound of the invention effective to inhibit a cysteine protease. The term xe2x80x9creversiblexe2x80x9d, when used to modify xe2x80x9cinhibitxe2x80x9d and xe2x80x9cinhibitionxe2x80x9d, means that such adverse effect on catalytic activity can be reversed. The term xe2x80x9cirreversiblexe2x80x9d, when used to modify xe2x80x9cinhibitxe2x80x9d and xe2x80x9cinhibitionxe2x80x9d, means that such adverse effect on catalytic activity cannot be reversed.
Compounds provided herein can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers. As noted above, such compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; or oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, via, for example, trans-dermal patches; or prepared in other suitable fashions for these and other forms of administration as will be apparent to those skilled in the art.
The composition may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington""s Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils and vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration. Formulations for transdermal patches are preferably lipophilic emulsions.
The materials of this invention can be employed as the sole active agent in a pharmaceutical or can be used in combination with other active ingredients. The concentrations of the compounds described herein in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 30% w/v compound for parenteral administration. Typical dose ranges are from about 1 xcexcg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage of drug to be administered is likely to depend on such-variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, and formulation of the compound excipient, and its route of administration.
As used herein, the term xe2x80x9ccontactingxe2x80x9d means directly or indirectly causing at least two moieties to come into physical association with each other. Contacting thus includes physical acts such as placing a compound of the invention together with a protease in a container, or administering a compound of the invention to a patient. Thus, for example, administering a compound of the invention to a human patient evidencing a disease or disorder associated with abnormal and/or aberrant activity of such proteases falls within the scope of the definition of the term xe2x80x9ccontactingxe2x80x9d.
The invention is further illustrated by way of the following examples which are intended to elucidate the invention. These examples are not intended, nor are they to be construed, as limiting the scope of the disclosure.