This invention relates to enzymatic inhibitors, particularly calpain inhibitors, and more particularly, calpain inhibition by HIV protease inhibitors.
Calpains and Calpain Inhibitors
Calpains are mammalian calcium-dependent neutral cysteine proteases involved in programmed cell death (apoptosis). Calpains are referred to as cysteine proteases because they include a cysteine residue that plays a critical role in the catalytic process. In the presence of calcium, a cysteine protease catalytic triad forms when three amino acid residues (Cys 105, His262, and Asn 286) are brought together in the active site.
Two major classes of calpains are known, xcexc-calpain and m-calpain, which differ in their sensitivities toward calcium. Some tissue-specific forms of calpain also have been identified. Substrates of calpain include cellular proteins such as cytoskeletal proteins, membrane-bound receptors, calmodulin binding proteins, myofibrillar proteins, enzymes, and transcription factors.
Calpain can become activated under ischemic conditions. It has been proposed that ischemia overactivates cellular membrane receptors, which causes an influx of calcium ions into the cell. This influx activates calcium-dependent enzymes, including calpains, and the calpains begin digesting cellular proteins, which contributes to cell death.
Inhibition of calpain has provided therapeutic possibilities for a number of different diseases, including cerebral ischemia (particularly strokes), traumatic brain injury, subarachnoid hemorrhage, chronic neurodegeneration (e.g., Huntington""s disease, Parkinson""s disease, and amyotrophic lateral sclerosis), Alzheimer""s disease, cardiac ischemia (particularly myocardial infarction), muscular dystrophy, cataracts, thrombotic platelet aggregation, restenosis, and joint inflammation (particularly arthritis).
A more detailed overview of calpains and calpain inhibitors can be found in Wang, et al., Advances in Pharmacology, 37:117-52 (1997). Patents disclosing calpain inhibitors include: Zimmerman, et al., U.S. Pat. No. 5,374,623; Wang, et al., U.S. Pat. No. 5,760,048; Munoz, et al., U.S. Pat. No. 5,872,101; Munoz, et al., U.S. Pat. No. 5,969,100; and Spruce, et al., U.S. Pat. No. 6,004,933.
HIV Protease and Inhibitors
The human immunodeficiency virus (HIV) is a retrovirus that causes immunosuppression in humans and leads to a disease complex known as acquired immunodeficiency syndrome (AIDS). HIV protease is an aspartyl protease that plays a central role in viral processing by cleaving the HIV GAG and GAG-POL polypeptides to produce mature viral core proteins and virus-specific enzymes. The HIV protease is called an aspartyl protease due to an important aspartyl residue in the active site that hydrolyzes the peptide bond of its substrate. Coffin et al., Retroviruses (Cold Spring Harbor Laboratory Press, 1997), provides a more detailed explanation of HIV protease.
Commercially available HIV protease inhibitors include ritonavir, saquinavir, indinavir, nelfinavir, and amprenavir. Numerous other HIV protease inhibitors are currently being tested in humans, but have not yet been approved by the United States Food and Drug Administration (FDA) for use against HIV, including ABT378, BMS232632, DMP450, GW433908, L-756,423, and timpranavir. Other HIV protease inhibitors have been disclosed in the patent literature, including: Martin et al., U.S. Pat. No.5,196,438; Vacca et al., U.S. Pat. No. 5,413,999; Dressman et al., U.S. Pat. No. 5,484,926; Kempf et al., U.S. Pat. No. 5,541,206; WO 97/0139 and WO 00/04016 by Abbott Laboratories; and WO 98/56781 by Glaxo Group Limited.
A variety of calpain inhibitors are disclosed, as well as methods of using these inhibitors to treat conditions associated with calpain activation. In some embodiments, HIV inhibitors are used to inhibit calpain activity. In other embodiments, the calpain inhibitor is an analog of or compound related to an HIV protease inhibitor.
A calpain may be inhibited by contacting the calpain with a disclosed inhibitor, such as an HIV protease inhibitor, analog, or related compound. In some embodiments, the calpain inhibitor is administered in therapeutically effective amounts to inhibit calpains within cells of subjects. In other embodiments, the calpain inhibitor is a compound having one or more of the following structures or clinical names, without reference to the ability of that compound to inhibit HIV protease activity:
(1) compounds of the formula, 
xe2x80x83wherein R1 is monosubstituted thiazolyl, monosubstituted oxazolyl, monosubstituted isoxazolyl, or monosubstituted isothiazolyl; and wherein the substituent is selected from: lower alkyl; lower alkenyl; cycloalkyl; cycloalkylalkyl; cycloalkenyl; cycloalkenylalkyl; heterocyclic, wherein the heterocyclic is selected from aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl and wherein the heterocyclic is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy, and thioalkoxy; (heterocyclic)alkyl, wherein the heterocyclic is defined as above; alkoxyalkyl; thioalkoxyalkyl; alkylamino; dialkylamino; phenyl, wherein the phenyl ring is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy and thioalkoxy; phenylalkyl, wherein the phenyl ring is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy and thioalkoxy; dialkylaminoalkyl; alkoxy; and thioalkoxy;
n is 1, 2 or 3;
R2 is hydrogen or a lower alkyl;
R3 is a lower alkyl;
R4 and R5 are independently selected from phenyl, thiazolyl, and oxazolyl, wherein the phenyl, thiazolyl or oxazolyl ring is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy, and thioalkoxy;
R6 is hydrogen or lower alkyl;
R7 is thiazolyl, oxazolyl, isoxazolyl or isothiazolyl, wherein the thiazolyl, oxazolyl, isoxazolyl or isothiazolyl ring is unsubstituted or substituted with lower alkyl;
X is hydrogen and Y is xe2x80x94OH, or X is xe2x80x94OH and Y is hydrogen, with the proviso that X is hydrogen and Y is xe2x80x94OH when Z is xe2x80x94N(R8)xe2x80x94 and R7 is unsubstituted, and with the proviso that X is hydrogen and Y is xe2x80x94OH when R3 is methyl and R7 is unsubstituted; and
Z is absent, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94 or xe2x80x94N(R8)xe2x80x94 wherein R8 is a lower alkyl, cycloalkyl, xe2x80x94OH or xe2x80x94NHR9, wherein R9 is hydrogen, lower alkyl or an N-protecting group; or
(2) compounds of the formula, 
xe2x80x83wherein R is benzyloxycarbonyl or 2-quinolylcarbonyl; or,
(3) any of: ritonavir; saquinavir; indinavir; nelfinavir; or amprenavir.
Subjects at risk of suffering calpain-mediated physiological damage may be identified and administered an HIV protease inhibitor, or another compound disclosed herein, following an event associated with activation of calpain, such as ischemia in the cardiovascular (including the neurovascular) system, or the myocardial or neural tissues which these systems perfuse.
Some embodiments include treating or preventing calpain-mediated physiological damage in a subject. In these embodiments, calpain-mediated physiological damage is treated by administering to a subject a therapeutically effective amount of a pharmaceutical composition having at least one protease inhibitor. Particular examples of pharmaceutical compositions include those with plural HIV protease inhibitors; pharmaceutically compatible carriers, agents, counterions, adjuvants, or vehicles; and/or other calpain inhibitors (in addition to the compounds disclosed herein). Treatments may be prophylactic or reparative.
Particular examples of therapeutically effective amounts of compounds include amounts that provide an inhibition constant, Ki, for inhibition of calpain less than or equal to about 11 xcexcM. Additionally, therapeutically effective doses of compounds may be administered to a subject for a limited period of time, such as about a month, a week, or even less than about 72 hours.
The pharmaceutical compositions can be used to treat a variety of conditions or diseases associated with calpain activation, including cardiovascular diseases. In particular embodiments, the treated condition is ischemia, such as myocardial or neural ischemia, resulting from decreased oxygenation of these tissues, as may occur in myocardial infarction, coronary stenosis or vasospasm, cerebrovascular accidents, such as ischemic or hemorrhagic stroke, and neurological trauma.
An xe2x80x9canimalxe2x80x9d is a living multicellular vertebrate organism, a category which includes, for example, mammals and birds.
The article xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d includes both the singular or plural, unless the context of its use clearly indicates otherwise.
The term xe2x80x9caminoxe2x80x9d refers to a chemical functionality xe2x80x94NR1R2 where R1 and R2 are independently hydrogen, alkyl, or aryl.
The term xe2x80x9cactivated ester derivativexe2x80x9d refers to acid halides, such as acid chlorides, and activated esters including, but not limited to, formic and acetic acid derived anhydrides, anhydrides derived from alkoxycarbonyl halides (such as isobutyloxycarbonylchloride), N-hydroxysuccinimide derived esters, N-hydroxyphthalimide derived esters, N-hydroxybenzotriazole derived esters, N-hydroxy-5-norbomene-2,3-dicarboxamide derived esters, and 2,4,5-trichlorophenol derived esters and the like.
An xe2x80x9canalogxe2x80x9d is a molecule that is structurally similar to another molecule but which differs slightly in chemical structure from a parent compound, for example by the replacement of one atom by an atom of a different element, or by the substitution of one functional group for another. Examples include, but are not limited to, a homolog (which differs by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, or a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington: The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28. A derivative is a biologically active molecule derived from the base molecular structure. A mimetic is a biomolecule that mimics the activity of another biologically active molecule. Biologically active molecules can include both chemical structures and peptides that mimic the calpain inhibition activities of the compounds disclosed herein, regardless of whether they also inhibit HIV protease activity.
The term xe2x80x9calkylaminoxe2x80x9d refers to a lower alkyl radical appended to an xe2x80x94NH radical.
The term xe2x80x9calkoxyxe2x80x9d refers to a substituted or unsubstituted alkoxy, where an alkoxy has the structure xe2x80x94Oxe2x80x94R, where R is a substituted or unsubstituted alkyl. In an unsubstituted alkoxy, the R is an unsubstituted alkyl. The term xe2x80x9csubstituted alkoxyxe2x80x9d refers to a group having the structure xe2x80x94Oxe2x80x94R, where R is alkyl which is substituted with a non-interfering substituent. xe2x80x9cLower alkoxyxe2x80x9d refers to any alkoxy in which R is a lower alkyl. xe2x80x9cThioalkoxyxe2x80x9d refers to xe2x80x94Sxe2x80x94R, where R is substituted or unsubstituted alkyl.
The term xe2x80x9calkoxyalkylxe2x80x9d refers to an alkoxy group appended to a lower alkyl radical.
The term xe2x80x9calkylxe2x80x9d refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, which, unless otherwise described, contains one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can be unsubstituted or substituted with one or more substituents, for example halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term xe2x80x9carylxe2x80x9d refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can optionally be unsubstituted or substituted with, for example, halogen, alkyl, alkoxy, mercapto (xe2x80x94SH), alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
xe2x80x9cCalpain mediated physiological damagexe2x80x9d refers to pathological conditions mediated by calpain. Such conditions can include a variety of ischemic events (such as myocardial or cerebral ischemia), as well as non-ischemic disorders (such as Alzheimer""s disease or muscular dystrophy).
xe2x80x9cCarbonyl containing groupxe2x80x9d refers to any substituent containing a carbon-oxygen double bond, including substituents based on xe2x80x94COR where R is an alkyl, lower alkyl, hydroxyl, or a secondary, tertiary, or quaternary amine. The term also encompasses oximes and hydrazones. Alternatively, xe2x80x9ccarbonyl-containing groupxe2x80x9d refers to xe2x80x94R1COR2 groups wherein R2 is alkyl, lower alkyl, hydroxyl, or secondary, tertiary, or quaternary amine and R1 is alkylene, such as methylene (xe2x80x94CH2xe2x80x94). Examples include xe2x80x94COOH, xe2x80x94CH2COOH, xe2x80x94CH2COOCH3, xe2x80x94CH2CONH2, and xe2x80x94CH2CON(CH3)2.
xe2x80x9cCarboxylxe2x80x9d refers to the radical xe2x80x94COOH, and substituted carboxyl refers to xe2x80x94COR where R is alkyl, lower alkyl or a carboxylic acid or ester.
The term xe2x80x9ccycloalkylxe2x80x9d refers to an aliphatic ring having 3 to 7 carbon atoms including, but not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.
The term xe2x80x9ccycloalkylalkylxe2x80x9d refers to a cycloalkyl group appended to a lower alkyl radical, including, but not limited to, cyclohexylmethyl.
The term xe2x80x9ccycloalkenylxe2x80x9d refers to an aliphatic ring having 5 to 7 carbon atoms and also having one carbon-carbon double bond including, but not limited to, cyclopentenyl and cyclohexenyl.
The term xe2x80x9ccycloalkenyalkylxe2x80x9d refers to a cycloalkenyl group appended to a lower alkyl radical including, but not limited to, cyclopentenylmethyl and cyclohexenylmethyl.
The term xe2x80x9cdialkylaminoxe2x80x9d refers to xe2x80x94Nxe2x80x94R1xe2x80x94R2 wherein R1 and R2 are independently selected from lower alkyl groups.
The term xe2x80x9cdialkylaminoalkylxe2x80x9d refers to xe2x80x94Nxe2x80x94R1xe2x80x94R2, which is appended to a lower alkyl radical, wherein R1 and R2 are independently selected from lower alkyl groups.
The term xe2x80x9chaloxe2x80x9d or refers to fluoro, bromo, chloro and iodo substituents. The term xe2x80x9chalogenxe2x80x9d refers to fluorine, bromine, chlorine, and iodine.
A xe2x80x9cheart attackxe2x80x9d is a general term encompassing cardiac traumas or diseases, such as myocardial infarction, angina, or cardiac arrhythmia. It is often, but not invariable, the result of cardiovascular disease, such as that seen in atherosclerosis.
The term xe2x80x9cheterocyclexe2x80x9d (or xe2x80x9cheterocyclicxe2x80x9d) refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g. benzyl, morpholino, pyridyl, or furyl), or multiple condensed rings (e.g. naphthyl, quinolinyl, indolizinyl or benzo[b]thienyl), and having at least one heteroatom, (defined as N, O, P, or S) within the ring. A heterocycle can optionally be unsubstituted or substituted with, for example, halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality. Examples include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.
The term xe2x80x9c(heterocyclic)alkylxe2x80x9d as used herein refers to a heterocyclic group appended to a lower alkyl radical including, but not limited to, pyrrolidinylmethyl and morpholinylmethyl.
xe2x80x9cHIV diseasexe2x80x9d refers to a well recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by a human immunodeficiency virus (HIV), as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T-helper cells.
xe2x80x9cHIV protease inhibitorxe2x80x9d refers to compounds which inhibit the enzymatic activity of an HIV protease. Examples include, but are not limited to, the HIV protease inhibitors disclosed in Table 1.
xe2x80x9cHydroxylxe2x80x9d (or xe2x80x9chydroxyxe2x80x9d) refers to xe2x80x94OH.
xe2x80x9cHydroxyalkylxe2x80x9d refers to xe2x80x94Rxe2x80x94OH, wherein R is alkylene, especially lower alkylene (for example in methylene, ethylene or propylene). A hydroxyalkyl group may be either linear or branched, such as 1-hydroxyisopropyl.
xe2x80x9cIschemiaxe2x80x9d refers to a low oxygen state of a cell or tissue usually due to obstruction of the arterial blood supply or inadequate blood flow leading to hypoxia. Causes of ischemia include, but are not limited to, vasoconstriction or blockage of a blood vessel. An xe2x80x9cischemic eventxe2x80x9d is a discrete occurrence producing ischemia, such as a thrombosis, cardiac arrhythmia, or myocardial infarction.
The term xe2x80x9clower alkylxe2x80x9d refers to a cyclic, branched or straight chain monovalent alkyl radical of one to five carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), sec-butyl, n-pentyl, cyclopropylmethyl, i-amyl, n-amyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl. Lower alkyl groups can be unsubstituted or substituted. One specific example of a substituted alkyl is 1,1-dimethyl propyl.
The term xe2x80x9clower alkenylxe2x80x9d refers to a straight or branched chain alkyl radical containing from 2 to 6 carbon atoms and also having one carbon-carbon double bond including, but not limited to, vinyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, and 5-hexenyl.
A xe2x80x9cmammalxe2x80x9d includes both human and non-human mammals.
The term xe2x80x9cN-protecting groupxe2x80x9d or xe2x80x9cN-protectedxe2x80x9d refers to those chemical groups intended to protect the N-terminus of an amino acid or peptide, or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, Protective Groups In Organic Synthesis (John Wiley and Sons, New York (1981)). N-protecting groups include: acyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, xcex1-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, and 4-nitrobenzoyl; sulfonyl groups, such as benzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups, such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, xcex1,xcex1-dimethyl-3,5-dime thoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl; alkyl groups, such as benzyl, triphenylmethyl, and benzyloxymethyl; and silyl groups, such as trimethylsilyl.
The term xe2x80x9cO-protecting groupxe2x80x9d refers to a substituent which protects hydroxyl groups against undesirable reactions during synthetic procedures, such as those O-protecting groups disclosed in Greene, Protective Groups In Organic Synthesis (John Wiley and Sons, New York (1981)). O-protecting groups include: substituted methyl ethers, such as methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, t-butyl, benzyl and triphenylmethyl; tetrahydropyranyl ethers; substituted ethyl ethers, such as 2,2,2-trichloroethyl; silyl ethers, such as trimethylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl; and esters prepared by reacting the hydroxyl group with a carboxylic acid, such as acetate, propionate, and benzoate.
A xe2x80x9cpharmaceutical agent,xe2x80x9d xe2x80x9cpharmaceutical composition,xe2x80x9d or xe2x80x9cdrugxe2x80x9d refers to a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. The pharmaceutically acceptable salts of the compounds disclosed herein include, but are not limited to, those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,Nxe2x80x2-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any compound disclosed herein may alternatively be administered as a pharmaceutically acceptable salt thereof.
The term xe2x80x9cphenylxe2x80x9d refers to a phenyl group, which may be unsubstituted or substituted with a substituent selected from lower alkyl, alkoxy, thioalkoxy, hydroxy and halo.
The term xe2x80x9cphenylalkylxe2x80x9d refers to a phenyl group appended to a lower alkyl radical including, but not limited to, benzyl, 4-hydroxybenzyl, 4-chlorobenzyl, and 1-naphthylmethyl.
The term xe2x80x9cprodrugxe2x80x9d refers to a compound that is converted within the body to a more active form that has medicinal or therapeutic effects.
The term xe2x80x9cstable compoundxe2x80x9d refers to a compound that is sufficiently stable to survive isolation to a useful degree of purity from a reaction mixture and formulation into a therapeutic dosage form suitable for administration.
The term xe2x80x9csubjectxe2x80x9d includes both human and veterinary subjects such as primates, canines, felines, and rodents.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d refers to an amount or dose sufficient to inhibit the enzymatic activity of a calpain and is capable of relieving symptoms associated with calpain activation.
The term xe2x80x9cthioalkoxyalkylxe2x80x9d refers to a thioalkoxy group appended to a lower alkyl radical.
Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (1985) and The Condensed Chemical Dictionary (1981).
All chemical compounds include both the (+) and (xe2x88x92) stereoisomers, as well as either the (+) or (xe2x88x92) stereoisomer.
HIV protease inhibitors may be divided into five general classes:
1. hydroxyethylene or dihydroxyethylene isotere state analogs;
2. hydroxyethylamine isotere analogs;
3. C2 symmetry-based inhibitors;
4. (R)-(hydroxyethyl) urea isotere analogs; and
5. nonpeptide HIV protease inhibitors.
Particular, cornmonly known HIV protease inhibitors are listed in Table 1.
The term xe2x80x9cHIV protease inhibitorxe2x80x9d includes analogs of compounds disclosed herein (such as ritonavir and related compounds disclosed in U.S. Pat. No. 5,541,206; saquinavir and related compounds disclosed in U.S. Pat. No. 5,196,438; indinavir and related compounds disclosed in U.S. Pat. No. 5,413,999; nelfinavir and related compounds disclosed in U.S. Pat. No. 5,484,926; and amprenavir and related compounds disclosed in WO 98/56781). Analogs of HIV protease inhibitors include those compounds that inhibit calpain activity, regardless of their activities against HIV protease. Therefore, the term xe2x80x9cHIV protease inhibitor analogxe2x80x9d describes a class of compounds, and does not provide a functional limitation on any compound described herein.
Ritonavir
The chemical structures of ritonavir and related compounds, including ritonavir analogs, are disclosed in Kempf et al.""s U.S. Pat. No. 5,541,206, herein incorporated by reference.
Ritonavir and related compounds include compounds of the formula: 
wherein
R1 is monosubstituted thiazolyl, monosubstituted oxazolyl, monosubstituted isoxazolyl, or monosubstituted isothiazolyl; and wherein the substituent is selected from: lower alkyl; lower alkenyl; cycloalkyl; cycloalkylalkyl; cycloalkenyl; cycloalkenylalkyl; heterocyclic, wherein the heterocyclic is selected from aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl; and wherein the heterocyclic is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy, and thioalkoxy; (heterocyclic)alkyl, wherein the heterocyclic is defined as above; alkoxyalkyl; thioalkoxyalkyl; alkylamino; dialkylamino; phenyl, wherein the phenyl ring is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy and thioalkoxy; phenylalkyl, wherein the phenyl ring is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy and thioalkoxy; dialkylaminoalkyl; alkoxy; or thioalkoxy;
n is 1, 2 or 3;
R2 is hydrogen or a lower alkyl;
R3 is lower alkyl;
R4 and R5 are independently selected from phenyl, thiazolyl, and oxazolyl, wherein the phenyl, thiazolyl or oxazolyl ring is unsubstituted or substituted with a substituent selected from halo, lower alkyl, hydroxy, alkoxy, or thioalkoxy;
R6 is hydrogen or lower alkyl;
R7 is thiazolyl, oxazolyl, isoxazolyl or isothiazolyl, wherein the thiazolyl, oxazolyl, isoxazolyl or isothiazolyl ring is unsubstituted or substituted with lower alkyl;
X is hydrogen and Y is xe2x80x94OH, or X is xe2x80x94OH and Y is hydrogen, with the proviso that X is hydrogen and Y is xe2x80x94OH when Z is xe2x80x94N(R8)xe2x80x94 and R7 is unsubstituted; and with the proviso that X is hydrogen and Y is xe2x80x94OH when R3 is methyl and R7 is unsubstituted; and
Z is absent, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94 or xe2x80x94N(R8)xe2x80x94 wherein R8 is a lower alkyl, cycloalkyl, xe2x80x94OH or xe2x80x94NHR9, wherein R9 is hydrogen, lower alkyl, or an N-protecting group.
Ritonavir, specifically, is chemically designated as 10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5-thiazolylmethyl ester, [5S-(5R*,8R* 10R* 11R*)], has a molecular formula of C37H48N6O5S2, and can be represented by structural formula presented in Table 1. Ritonavir is available from Abbott Laboratories, Inc., under the product name NORVIR.(copyright)
Saquinavir
The chemical structures of saquinavir and related compounds, including saquinavir analogs, are disclosed in Martin et al.""s U.S. Pat. No. 5,196,438, herein incorporated by reference.
Saquinavir and related compounds include compounds of the formula: 
wherein R is benzyloxycarbonyl or 2-quinolylcarbonyl. Saquinavir and related compounds may have stereochemical conformations according to the formula: 
wherein R is benzyloxycarbonyl or 2-quinolylcarbonyl.
Saquinavir, specifically, is chemically designated as N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide with a molecular formula C38H50N6O5, and can be represented by structural formula presented in Table 1. Saquinavir is available from Roche Laboratories, under the product name FORTOVASE.(copyright)
Indinavir
The chemical structures of indinavir and related compounds, including analogs, are disclosed in Vacca et al.""s U.S. Pat. No. 5,413,999, herein incorporated by reference.
Indinavir is chemically designated as [1(1S,2R),5(S)]-2,3,5-trideoxy-N-(2,3-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(1,1-dimethylethyl)amino]carbonyl)-4-(3-pyridinylmethyl)-1-piperazinyl)-2-(phenylmethyl)-D-erythro-pentonamide sulfate (1:1) salt, with a molecular formula C36H47N5O4.H2SO4, and can be represented by the structural formula presented in Table 1. Indinavir can be purchased from Merck and Co., Inc., as CRIXIVAN.(copyright)
Nelfinavir
The chemical structures of nelfinavir, nelfinavir mesylate, and related compounds, including nelfinavir analogs, are disclosed in Dressman et al.""s U.S. Pat. No. 5,484,926, herein incorporated by reference.
Nelfinavir is chemically designated as [3S-[2(2S*,3s*),3xcex14axcex28axcex2]]-N-(1,1-dimethylethyl)decahydro-2-[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolinecarboxamide. The structural formula of nelfinavir is presented in Table 1, and is commercially available from Agouron Pharmaceuticals, Inc., under the product name VIRACEPT.(copyright)
Amprenavir
The chemical structures of amprenavir and related compounds, including amprenavir analogs, are disclosed in Glaxo Group Limited""s PCT application WO 98/56781, herein incorporated by reference.
Amprenavir is chemically designated as [3S-[3R*(1R*,2S*)]]-[3-[[(4-aminophenyl)sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-tetrahydro-3-furanyl ester. Amprenavir can be represented by the structural formula presented in Table 1, and may be purchased from Glaxo Wellcome, Inc., under the product name AGERNASE.(copyright)
Calpain can be inhibited by contacting at least one calpain with an effective amount of an HIV protease inhibitor, an analog, or other compound disclosed herein, including compounds that inhibit calpain even if HIV protease inhibition is reduced or lacking. These methods may be carried out in vivo or in vitro with cells, tissues, or whole animals; with one or more calpains; and with one or more HIV protease inhibitors, analogs, or related compounds disclosed herein. Additionally, these methods may employ a disclosed compound in combination with another therapeutic agent, such as another calpain inhibitor.
Some embodiments involve inhibiting calpains within particular subjects. In some embodiments, a subject at risk of suffering calpain-mediated physiological damage is first identified and then provided with a calpain inhibitor, such as an HIV protease inhibitor. Identification of an at-risk subject can include diagnosing a subject with a condition associated with calpain induced physiological damage. For example, as a prophylactic measure, a human subject demonstrating signs of an impending stroke may be administered a calpain inhibitor (such as an HIV protease inhibitor) disclosed herein. Identification of an at-risk subject can also include choosing an individual research subject for experimental purposes. For example, a rat or dog may be selected to receive treatment intended to induce a stroke and then administered a calpain inhibitor disclosed herein.
In other embodiments, an HIV protease inhibitor, analog, or related compound is administered to a subject following an actual event implicating activation of calpain, thus putting the subject at risk of suffering calpain-mediated physiological damage. For example, a human subject who recently suffered a cardiovascular ischemic event (e.g., heart attack or stroke) may be administered a therapeutically effective amount of pharmaceutical composition that includes a calpain inhibitor disclosed herein. In many embodiments, a composition is administered within several hours of the event precipitating calpain mediated pathologies.
Other embodiments include treating or preventing calpain-mediated physiological damage in a subject animal, for example, a human. In these embodiments, calpain-mediated physiological damage is treated by providing to a subject a therapeutically effective amount of a pharmaceutical composition. The pharmaceutical composition may include one or more of the calpain inhibitors disclosed herein; pharmaceutically compatible carriers, agents, counterions, adjuvants, or vehicles; another calpain inhibitor (in addition to the calpain inhibitors disclosed herein); or combinations thereof. Although the treatment can be used prophylactically in any patient in a demographic group at risk for calpain-mediated physiological damage, subjects also may be selected using more specific criteria, such as a definitive diagnosis of a particular disease or condition associated with calpain-activation (e.g., angina, cataract, myocardial infarction, stroke), or recognition of calcium activation of calpain within the subject.
Providing a pharmaceutical composition to a subject includes methods of administering that composition. Routes of administration include, but are not limited to, oral and parenteral routes, such as intravenous (IV), intraperitoneal (IP), rectal, topical, ophthalmic, nasal, and transdermal. If orally bioavailable HIV protease inhibitors are used, the pharmaceutical compositions are generally provided or administered in the form of a unit dose in solid, semi-solid, or liquid dosage forms such as tablets, pills, powders, liquid solutions, or liquid suspensions. However, the drugs also may be administered intravenously in any conventional medium for intravenous injection, such as an aqueous saline medium, or in a blood plasma medium. The medium also may contain conventional pharmaceutical adjunct materials, such as pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers (e.g., cyclodextrins), proteins (e.g., serum albumin), hydrophilic agents (e.g., methyl cellulose), detergents, buffers, preservatives and the like. A more complete explanation of acceptable pharmaceutical carriers can be found in Remington: The Science and Practice of Pharmacy (19th Edition, 1995) in chapter 95.
Therapeutically effective amounts of compounds can be determined in many different ways, depending on the toxicity of the individual HIV protease inhibitor. In some embodiments, the effective amount provides an inhibition constant, for inhibition of calpain, less than or equal to about 11 xcexcM under the following conditions: (1) using supernatant extracted from PC12 cells, (2) using N-succinyl-leu-ter 7-amino-4-methylcoumarin as the substrate for calpain activity, with (3) fluorescence measured with a 380 nm excitation filter and 480 nm emission filter, and (4) inhibition determined using enzyme kinetic equations solved using linear regression. One example determination of an effective amount under these conditions is provided below in Example #1xe2x80x94Ritonavir Inhibition.
The therapeutically effective amounts of compounds disclosed herein also may be measured in comparison to other calpain inhibitors. For example, ritonavir inhibits calpain about 20 times more effectively than the orally bioavailable calpain inhibitor, PD150606.
Additionally, certain embodiments can be distinguished from methods of treating chronic HIV infection. The treatment of the present invention can be given to a person who does not need a protease inhibitor, for example someone who is not infected with HIV. Moreover, patients suffering chronic HIV infections who take HIV protease inhibitors generally follow a prolonged treatment regimen. For example, the recommended dosage for NORVIR(copyright), an orally administered ritonavir solution produced by Abbott Laboratories, North Chicago, Ill., is 600 mg b.i.d. and, if used in combination with saquinavir, an optimum dosage of 400 to 600 mg b.i.d. As another example, the recommended dosage for INVIRASE(copyright), an orally administered saquinavir mesylate capsule produced by Roche Laboratories, Inc., Nutley, N.J., is 600 mg t.i.d., taken in combination with a nucleoside analog. Since HIV disease is not yet curable, HIV-positive persons taking these HIV protease inhibitors must follow a prolonged, often life-long, course of daily antiviral therapy.
In contrast, therapeutically effective doses of the calpain inhibitors disclosed herein can be provided to a subject for a much shorter period of time. This period of time may be measured after a diagnosis that the subject is at risk for calpain-mediated physiological damage, or after a particular ischemic event, such as a cardiovascular ischemic event. The duration of treatment may be, for example, less than about a month, two weeks, one week, or even less than about 72 hours. For example, a patient suffering a stroke can be provided a therapeutically effective dose of an HIV protease inhibitor for about 72 hours or less. Alternatively, the therapeutically effective dosage may be provided for a period of time from about 6 to about 72 hours. However, the duration of therapy with the calpain inhibitors disclosed herein can also be prolonged, for example, in the treatment of chronic angina or recurrent transient ischemic attacks (TIA""s). Moreover, administration can be repeated, but intermittent (for example, following an episode of angina or TIA), even though intermittent or episodic administration would be avoided in an antiviral treatment because it could lead to the development of viral drug resistance.
The specific dose level, frequency of dosage, and duration of treatment for any particular subject may be varied and will depend upon a variety of factors, including: the activity of the specific pharmaceutical composition; the metabolic stability and length of action of that composition; the age, body weight, general health, gender, diet, and other characteristics of the subject; mode and time of administration; the rate of excretion; drug combination parameters; and severity of the condition of the subject undergoing treatment.
The pharmaceutical compositions can be used in the treatment of a variety of conditions or diseases associated with calpain activation, including conditions or diseases caused by or mediated by enzymatically active calpains. Examples of conditions or diseases associated with calpain-mediated physiological damage include, but are not limited to, neurological conditions such as cerebral trauma, spinal cord trauma, subarachnoid hemmorrhage, Alzheimer""s disease, alcohol-induced brain damage, muscular dystrophy; as well as non-neurological conditions, such as cataract, coronary atherosclerosis, restenosis, and arthritis. Post-ischemic calpain activation also may be associated with a cardiovascular ischemic event such as, for example, myocardial infarction, angina, cardiac trauma, arhythmia, stroke, thrombosis, or thrombotic platelet aggregation. The trauma or condition leading to the calpain-mediated physiological damage may be induced or accidental.
USPTO Backgrounds
USPTO Backgrounds