The invention pertains to certain peptide-like and peptidomimetic compounds that advantageously inhibit the enzymatic activity of picornaviral 3C proteases, especially rhinovirus 3C proteases (RVPs), and that retard viral growth in cell culture. The invention also relates to the use of such compounds in pharmaceutical compositions and therapeutic treatments for rhinoviral infections. The invention further relates to processes for synthesizing such compounds and compounds useful in such syntheses.
The picomaviruses are a family of tiny non-enveloped positive-stranded RNA-containing viruses that infect humans and other animals. These viruses include the human rhinoviruses, human polioviruses, human coxsackieviruses, human echoviruses, human and bovine enteroviruses, encephalomyocarditis viruses, meningitis virus, foot and mouth viruses, hepatitis A virus, and others. The human rhinoviruses are a major cause of the common cold. To date, there are no effective therapies on the market that cure the common cold, only treatments that relieve the symptoms.
Picomaviral infections may be treated by inhibiting the proteolytic 3C enzymes. These enzymes are required for the natural maturation of the picornaviruses. They are responsible for the autocatalytic cleavage of the genomic, large polyprotein into the essential viral proteins. Members of the 3C protease family are cysteine proteases, where the sulfhydryl group most often cleaves the glutamine-glycine amide bond. Inhibition of 3C proteases is believed to block proteolytic cleavage of the polyprotein, which in turn can retard the maturation and replication of the viruses by interfering with viral particle production. Therefore, inhibiting the processing of this cysteine protease with selective small molecules that are specifically recognized should represent an important and useful approach to treat and cure viral infections of this nature and, in particular, the common cold.
Some small-molecule inhibitors of the enzymatic activity of picornaviral 3C proteases (i.e., antipicornaviral compounds) have been recently discovered. See, for example: U.S. Pat. No. 5,856,530, issued Jan. 5, 1999, to Webber et al.; U.S. patent application Ser. No. 08/991,282, filed Dec. 16, 1997, by Dragovich et al.; U.S. patent application Ser. No. 08/991,739, filed Dec. 16, 1997, by Webber et al.; and U.S. patent application Ser. No. 09/301,977, filed Apr. 29, 1999, by Dragovich et al. See also: Dragovich et al., xe2x80x9cStructure-Based Design, Synthesis, and Biological Evaluation of Irreversible Human Rhinovirus 3C Protease Inhibitors . . . ,xe2x80x9d J. Med. Chem. (1999), vol. 42, no. 7, 1203-1212, 1213-1224; and Dragovich et al., xe2x80x9cSolid-phase Synthesis of Irreversible Human Rhinovirus 3C Protease Inhibitors . . . ,xe2x80x9d Bioorg. and Med. Chem. (1999), vol. 7, 589-598. There is still a desire, however, to discover small-molecule compounds that are especially potent antipicornaviral agents.
Inhibitors of other related cysteine proteases such as cathepsins have been described in, e.g., U.S. Pat. No. 5,374,623, issued Dec. 20, 1994, to Zimmermnan et al.; U.S. Pat. No. 5,498,616, issued Mar. 12, 1996, to Mallamo et al.; and WIPO International Publication Nos. WO 94/04172, WO 95/15749, WO 97/19231, and WO 97/49668. There yet remains a need for inhibitors targeting the picornaviral 3C cysteine protease with desirable pharmaceutical properties, such as high specificity.
Thus, an object of this invention is to discover small-molecule compounds that inhibit picornaviral 3C proteases and are especially potent antipicornaviral agents. A further object of the invention is to provide intermediates useful for the synthesis of protease-inhibiting compounds and synthetic methods useful for such syntheses. A yet further object of the invention is to achieve pharmaceutical compositions that are effective for treating maladies mediated by inhibition of picornaviral 3C proteases, such as the common cold.
Such objects have been attained through the discovery of compounds of the following general formula 1: 
wherein:
Y is xe2x80x94N(Ry)xe2x80x94, xe2x80x94C(Ry)(Ry)xe2x80x94, or xe2x80x94Oxe2x80x94, where each Ry is independently H or lower alkyl; R1 is unsubstituted or substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or xe2x80x94C(O)R16, where R16 is unsubstituted or substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocycloalkoxy, aryloxy, heteroaryloxy, or amine;
R2 and R8 are each independently H, F, or unsubstituted or substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
R3 and R9 are each independently H or unsubstituted or substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, xe2x80x94OR17, xe2x80x94SR17, xe2x80x94NR17R18, xe2x80x94NR19NR17R18, or xe2x80x94NR17OR18, where R17, R18, and R19 are each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or acyl;
R4 is a suitable organic moiety;
R5, R6and R7 are each independently H, F, or lower alkyl;
m is 0 or 1;
p is an integer of from 0 to 5;
A1 is CH or N;
each A2 present is independently C(R10)(R11), N(R12), S, S(O), S(O)2, or O, where each R10, R11 and R12 is independently H or lower alkyl;
each A3 present is independently C(R10)(R11), N(R12), S, S(O), S(O)2, or O, where each R10, R11 and R12 is independently H or lower alkyl;
when p is 1, 2, 3, or 4, A4 is N(R13), C(R10)(R11), or O, and when p is 0 (i.e., A3 is not present), A4 is N(R13)(R14), C(R10)(R11)(R12), and O(R14), provided that when p is 0 and A4 is O(R14), A1 is not CH, where each R10, R11 and R12 is independently H or lower alkyl, each R13 is H, alkyl, aryl, or acyl, and each R14 is H, alkyl, or aryl
provided that no more than two heteroatoms occur consecutively in the above-depicted ring formed by A1, (A2)m, (A3)p, A4, and Cxe2x95x90O, where each dotted line in the ring depicts a single bond when A2 is present (i.e., m=1) and a hydrogen atom when A2 is absent (i.e., m=0).
In addition to compounds of the formula I, antipicomaviral agents of the invention include prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts and solvates of such compounds.
In a preferred embodiment of formula I, R2 and R8 are not both hydrogen, and R3 and R9 are not both hydrogen. In another preferred embodiment of formula I: R2 is benzyl optionally substituted with a halogen; R3 is a lower alkyl; R4 is Cbz; and R7, R8, and R9 are each H.
In a preferred embodiment, R1 is a substituted methylene group, for example, xe2x80x94CH2NR20R21, xe2x80x94CH2OR20, xe2x80x94CH2OC(O)R20, xe2x80x94CH2ONR20R21, or xe2x80x94CH2SR20, where R20 and R21 are each independently selected from H, optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and xe2x80x94C(O)R22, where R22 is selected from optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocycloalkoxy, aryloxy, heteroaryloxy, and amine, and optionally any two of R20, R21, and R22, together with the atoms to which they are bound, form a 4- to 7-membered ring. In an alternative preferred embodiment, R1 is xe2x80x94CR23xe2x95x90CR24R25 or xe2x80x94Cxe2x89xa1CR26, where R23, R24, R25 and R26 are each independently selected from H and optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In yet another preferred embodiment, R1 is a xe2x80x94C(O)R16 group, where R16 is xe2x80x94NR27R28, wherein R27 and R28 are each independently selected from H and optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, or R27 and R28 together with the nitrogen to which they are bound form a 4- to 7-membered heterocyclic ring. In another preferred embodiment, R1 is a mono- or bi-cyclic heteroaryl or aryl group.
Especially preferred compounds are depicted by formula I-a: 
wherein R1 through R6, A1 through A4, m, p, and Y are as defined above.
In preferred embodiments of compounds of the formula I-a, R1 is selected from monocyclic and bicyclic heteroaryl and aryl groups.
R2 in formula I-a is preferably selected from unsubstituted and substituted benzyl groups, preferably benzyl, mono-substituted benzyl, and disubstituted benzyl, where the substituents are independently selected from lower alkyl, lower alkoxy, and halogen.
R3 is preferably an optionally substituted alkyl (e.g., 2-propyl, 2-methyl-2-propyl, or 2-methyl-1-propyl) or arylmethyl (e.g., unsubstituted or substituted phenylmethyl or naphthylmethyl).
R4 is preferably a suitable organic moiety selected from xe2x80x94[C(O)]nxe2x80x94R15, where n is 0 or 1 and R15 is optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, alkoxy, cycloalkoxy, aryloxy, or heteroaryloxy. In especially preferred embodiments, R4 is benzyloxycarbonyl, arylcarbonyl, or heteroarylcarbonyl, more preferably heteroarylcarbonyl, where the heteroaryl moiety is a five-membered heterocycle having from one to three heteroatoms selected from O, N, and S, more preferably a five-membered heterocycle having at least one nitrogen heteroatom and at least one oxygen heteroatom (e.g., unsubstituted or substituted 1,2-oxazolyl (i.e., isoxazolyl), 1,3-oxazolyl (i.e., oxazolyl), or oxadiazolyl (1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, or 1,2,5-oxadiazolyl). When the heteroaryl moiety is oxadiazolyl, unsubstituted and monomethyl-substituted 1,2,4-oxadiazolyl are preferred. In especially preferred embodiments, the heteroaryl moiety is 3-isoxazolyl or 5-isoxazolyl, either unsubstituted or substituted with one or two methyl groups and/or halogens (F, Cl, Br or I), with chlorine and fluorine being preferred.
Preferably, the moiety: 
is selected from xe2x80x94CH2CH2C(O)NH2; xe2x80x94CH2CH2C(O)NH-alkyl; xe2x80x94CH2NHC(O)CH3; and 
where n is 1 or 2. The moiety is more preferably 
In preferred embodiments, the compounds, prodrugs, pharmaceutically acceptable salts, and pharmaceutically active metabolites and solvates have an antipicomaviral activity with an EC50 less than or equal to 100 xcexcM in the H1-HeLa cell culture assay.
The invention is also directed to intermediates of formula II, preferably of the subformula II-a, which are useful in the synthesis of certain compounds of formula I: 
wherein:
q is an integer of from 0 to 5, preferably 1 or 2;
A11 is C,CH or N;
A12 and each A13 are each independently selected from C(R61)(R62), N(R63), S, S(O), S(O)2, and O, where each of R61, R62 and R63 is independently H or lower alkyl;
A14 is NR64, where R64 is H, alkyl, aryl, or acyl, and R64 is preferably a suitable protecting group for amide nitrogen;
provided that no more than two heteroatoms occur consecutively in the above-depicted ring in formula II formed by A11, A12, (A13)p, A14, and Cxe2x95x90O;
R141 and R142 are each independently H, F or lower alkyl, or R142 is absent;
the dotted line depicts an optional valence bond, and when such bond is present, R142 is absent and A11 is C;
R51 is H, alkyl, aryl, or acyl, preferably a protecting group for amide nitrogen;
R52, R53, and R54 are each independently selected from H, hydroxyl, alkyl, acyl, aryl, heteroaryl, suitable protecting groups for carbonyl or hydroxy, OR57, and NR57R58 where R57 is selected from alkyl, aryl and Si(R59)3, and R58 is selected from alkyl, aryl, alkoxy, aryloxy, and Si(R59)3, where each R59 is independently alkyl or aryl; or any two of R52, R53, and R54 together form xe2x95x90O; and
R55 and R56 are each independently H or a suitable protecting group for nitrogen.
Formula II compounds where at least one of R52, R53, and R54 is NR57R58 are preferred. In preferred formula II-a embodiments, R52 and R53 together form xe2x95x90O and R54 is selected from alkyl, acyl, aryl, heteroaryl, OR57, and NR57R58, where R57 and R58 are as defined above. In other preferred embodiments, R52 is H, R53 is OH, and R54 is selected from alkyl, acyl, aryl, heteroaryl, OR57, and NR57R58 where R57 and R58 are as defined above.
The invention is also directed to pharmaceutically acceptable salts of the compounds of formulae II and II-a.
The invention also relates to pharmaceutical compositions containing a therapeutically effective amount of at least one compound of the formula I, or a prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite, or solvate thereof (collectively, xe2x80x9cagentsxe2x80x9d). Additionally, the invention relates to methods of inhibiting picornaviral 3C protease by administering a therapeutically effective amount of at least one such agent.
In accordance with a convention used in the art, 
is used in structural formulas herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.
As used herein, the term xe2x80x9calkyl groupxe2x80x9d is intended to mean a straight- or branched-chain monovalent radical of saturated and/or unsaturated carbon atoms and hydrogen atoms, such as methyl (Me), ethyl (Et), propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, pentenyl, butenyl, propenyl, ethynyl, butynyl, propynyl, pentynyl, hexynyl, and the like. Unless otherwise indicated, such groups may be unsubstituted (i.e., containing only carbon and hydrogen) or substituted by one or more suitable substituents (e.g., one or more halogens, such as F, Cl, Br, or I, with F and Cl being preferred). A xe2x80x9clower alkyl groupxe2x80x9d is intended to mean an alkyl group having from 1 to 4 carbon atoms in its chain.
A xe2x80x9ccycloalkyl groupxe2x80x9d is intended to mean a non-aromatic monovalent monocyclic, bicyclic, or tricyclic radical containing from 3 to 14 carbon ring atoms, each of which may be saturated or unsaturated. Unless otherwise indicated, such groups may be unsubstituted or substituted by one or more suitable substituents. Illustrative examples of cycloalkyl groups include the following moieties: 
A xe2x80x9cheterocycloalkyl groupxe2x80x9d is intended to mean a non-aromatic monovalent monocyclic, bicyclic, or tricyclic radical, which is saturated or unsaturated, containing from 3 to 18 ring atoms, which includes from 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise indicates, such radicals may be unsubstituted or substituted by one or more suitable substituents. Illustrative examples of heterocycloalkyl groups include the following moieties: 
An xe2x80x9caryl groupxe2x80x9d is intended to mean an aromatic monovalent monocyclic, bicyclic, or tricyclic radical containing from 6 to 18 carbon ring atoms. Unless otherwise indicates, such radicals may be unsubstituted or substituted by one or more suitable substituents. Illustrative examples of aryl groups include the following moieties: 
A xe2x80x9cheteroaryl groupxe2x80x9d is intended to mean an aromatic monovalent monocyclic, bicyclic, or tricyclic radical containing from 4 to 18 ring atoms, including from 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise indicated, such radicals may be unsubstituted or substituted by one or more suitable substituents. Illustrative examples of heteroaryl groups include the following moieties: 
A xe2x80x9cheterocyclexe2x80x9d is intended to mean a heteroaryl or heterocycloalkyl group.
An xe2x80x9cacyl groupxe2x80x9d is intended to mean a xe2x80x94C(O)xe2x80x94R radical, where R is a suitable substituent.
A xe2x80x9cthioacyl groupxe2x80x9d is intended to mean a xe2x80x94C(S)xe2x80x94R radical, where R is a suitable substituent.
A xe2x80x9csulfonyl groupxe2x80x9d is intended to mean a xe2x80x94SO2R radical, where R is a suitable substituent.
A xe2x80x9chydroxy groupxe2x80x9d is intended to mean the radical xe2x80x94OH.
An xe2x80x9caminexe2x80x9d or xe2x80x9camino groupxe2x80x9d is intended to mean the radical xe2x80x94NH2. An xe2x80x9coptionally substitutedxe2x80x9d amines refers to xe2x80x94NH2 groups wherein none, one or two of the hydrogens is replaced by a suitable substituent. Disubstituted amines may have substituents that are bridging, i.e., form a heterocyclic ring structure that includes the amine nitrogen.
An xe2x80x9calkylamino groupxe2x80x9d is intended to mean the radical xe2x80x94NHRa, where Ra is an alkyl group.
A xe2x80x9cdialkylamino groupxe2x80x9d is intended to mean the radical xe2x80x94NRaRb, where Ra and Rb are each independently an alkyl group.
An xe2x80x9calkoxy groupxe2x80x9d is intended to mean the radical xe2x80x94ORa, where Ra is an alkyl group. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, and the like. xe2x80x9cLower alkoxyxe2x80x9d groups have alkyl moieties having from 1 to 4 carbons.
An xe2x80x9calkoxycarbonyl groupxe2x80x9d is intended to mean the radical xe2x80x94C(O)ORa, where Ra is an alkyl group.
An xe2x80x9calkylsulfonyl groupxe2x80x9d is intended to mean the radical xe2x80x94SO2Ra, where Ra is an alkyl group.
An xe2x80x9calkylaminocarbonyl groupxe2x80x9d is intended to mean the radical xe2x80x94C(O)NHRa, where Ra is an alkyl group.
A xe2x80x9cdialkylaminocarbonyl groupxe2x80x9d is intended to mean the radical xe2x80x94C(O)NRaRb, where Ra and Rb are each independently an alkyl group.
A xe2x80x9cmercapto groupxe2x80x9d is intended to mean the radical xe2x80x94SH.
An xe2x80x9calkylthio groupxe2x80x9d is intended to mean the radical xe2x80x94SRa, where Ra is an alkyl group.
A xe2x80x9ccarboxy groupxe2x80x9d is intended to mean the radical xe2x80x94C(O)OH.
A xe2x80x9ccarbamoyl groupxe2x80x9d is intended to mean the radical xe2x80x94C(O)NH2.
An xe2x80x9caryloxy groupxe2x80x9d is intended to mean the radical xe2x80x94ORc, where Rc is an aryl group.
A xe2x80x9cheteroaryloxy groupxe2x80x9d is intended to mean the radical xe2x80x94ORd, where Rd is a heteroaryl group.
An xe2x80x9carylthio groupxe2x80x9d is intended to mean the radical xe2x80x94SRc, where Rc is an aryl group.
A xe2x80x9cheteroarylthio groupxe2x80x9d is intended to mean the radical xe2x80x94SRd, where Rd is a heteroaryl group.
The term xe2x80x9csuitable organic moietyxe2x80x9d is intended to mean any organic moiety recognizable, such as by routine testing, to those skilled in the art as not adversely affecting the inhibitory activity of the inventive compounds. Illustrative examples of suitable organic moieties include, but are not limited to, hydroxy groups, alkyl groups, oxo groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups, alkoxy groups, carboxy groups, amino groups, alkylamino groups, dialkylamino groups, carbamoyl groups, arylthio groups, heteroarylthio groups, and the like.
The term xe2x80x9csubstituentxe2x80x9d or xe2x80x9csuitable substituentxe2x80x9d is intended to mean any suitable substituent that may be recognized or selected, such as through routine testing, by those skilled in the art. Illustrative examples of suitable substituents include hydroxy groups, halogens, oxo groups, alkyl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups, alkoxy groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, carboxy groups, amino groups, alkylamino groups, dialkylamino groups, carbamoyl groups, aryloxy groups, heteroaryloxy groups, arylthio groups, heteroarylthio groups, and the like.
The term xe2x80x9coptionally substitutedxe2x80x9d is intended to expressly indicate that the specified group is unsubstituted or substituted by one or more suitable substituents, unless the optional substituents are expressly specified, in which case the term indicates that the group is unsubstituted or substituted with the specified substituents. Various groups may be unsubstituted or substituted (i.e., they are optionally substituted) as indicated.
A xe2x80x9cprodrugxe2x80x9d is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active.
A xe2x80x9cpharmaceutically active metabolitexe2x80x9d is intended to mean a pharmacologically active compound produced through metabolism in the body of a specified compound.
A xe2x80x9csolvatexe2x80x9d is intended to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates include compounds of the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
A xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is intended to mean a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, xcex3-hydroxybutyrates, glycollates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
If an inventive compound is a base, a desired salt may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.
If an inventive compound is an acid, a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine; ammonia; primary, secondary, and tertiary amines; and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
In the case of compounds, salts, or solvates that are solids, it is understood by those skilled in the art that the inventive compounds, salts, and solvates may exist in different crystal forms, all of which are intended to be within the scope of the present invention and specified formulas.
The inventive compounds may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the broad scope of the present invention. Preferably, however, the inventive compounds are used in optically pure form.
As used herein, the term xe2x80x9coptically purexe2x80x9d is intended to mean a compound comprising at least a sufficient amount of a single enantiomer to yield a compound having the desired pharmacological activity. Preferably, an optically pure compound of the invention comprises at least 90% of a single isomer (80% enantiomeric excess), more preferably at least 95% (90% e.e.), even more preferably at least 97.5% (95% e.e.), and most preferably at least 99% (98% e.e.).
Preferably, the compounds of formula I and their pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates have antipicornaviral activity, more preferably antirhinoviral activity, corresponding to an EC50 less than or equal to 100 xcexcM in the H1-HeLa cell culture assay.
In preferred embodiments, the formula I compounds are of sub-formula I-a as defined above. Especially preferred embodiments of the invention have formula I-b: 
where R1 through R4 are as defined above. In especially preferred embodiments of formula I-b, R1 is mono- or bi-cyclic heteroaryl. Preferably, R2 is selected from unsubstituted, mono-substituted, and disubstituted benzyl groups, where the substituents are independently selected from lower alkyl, lower alkoxy, and halogen. R3 is preferably alkyl (e.g., 2-propyl, 2-methyl-2-propyl, or 2-methyl-1-propyl) or arylmethyl (e.g., unsubstituted or substituted phenylmethyl or naphthylmethyl). The variable R4 is preferably benzyloxycarbonyl, arylcarbonyl, or heteroarylcarbonyl, more preferably heteroarylcarbonyl, where the heteroaryl moiety is a five-membered heterocycle having from one to three heteroatoms selected from O, N, and S. More preferably R4 is a five-membered heterocycle having at least one nitrogen heteroatom and at least one oxygen heteroatom (e.g., unsubstituted or substituted 1,2-oxazolyl (i.e., isoxazolyl), 1,3-oxazolyl (i.e., oxazolyl), or oxadiazolyl (1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, or 1,2,5-oxadiazolyl); preferred oxadiazolyls are unsubstituted and monomethyl-substituted 1,2,4-oxadiazolyl. In especially preferred embodiments, the heteroaryl moiety is 3-isoxazolyl or 5-isoxazolyl, either unsubstituted or substituted with one or two substituents selected from methyl and halogens, with chlorine and fluorine being preferred halogen substituents.
Varying the group Y in formula I gives rise to formulae I-c (peptides), I-d (depsipeptides), and I-e (ketomethylenes), where all variables are as defined previously: 
Preferred embodiments of formula I-a are shown in formulae I-f, I-g and I-h below: 
In the compounds of formulae I-c, I-e, I-f, and I-h, Ry is preferably H or methyl. Preferred specific compounds include those of the Examples below, especially: 
In another aspect, the invention is directed to intermediates of formula 11, preferably of the sub-formula II-a, which are useful in the synthesis of various compounds of formula I: 
wherein all variables are as defined above. Preferred R55 and R56 groups are H and suitable protecting groups for nitrogen, for example, Boc (t-butyloxycarbonyl), Cbz (benzyloxycarbonyl), FMOC (fluorene-9-methyloxycarbonyl), other alkyloxycarbonyls (e.g., methyloxycarbonyl), and trityl (triphenylmethyl). Other suitable nitrogen-protecting groups may be readily selected by artisans (see, e.g., Greene and Wuts, Protecting Groups in Chemical Synthesis (3rd ed.), John Wiley and Sons, NY (1999)). Preferred groups for R52, R53, and R54 are H, alkoxy, hydroxy, carbonyl, OR59, and suitable protecting groups for carbonyl or hydroxy. A preferred protecting group for hydroxy is t-butyldimethylsilyl (TBS).
Preferred examples of the formula II useful as intermediates include the following: 
and pharmaceutically acceptable salts thereof.
The present invention is also directed to a method of inhibiting picomaviral 3C protease activity, comprising contacting the protease with an effective amount of a compound of formula I, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite, or solvate thereof. For example, picomaviral 3C protease activity may be inhibited in mammalian tissue by administering a compound of formula I or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite, or solvate thereof. More preferably, the present method is directed at inhibiting rhinoviral protease activity.
xe2x80x9cTreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d is intended to mean at least the mitigation of a disease condition in a mammal, such as a human, that is alleviated by the inhibition of the activity of one or more picornaviral 3C proteases, such as human rhinoviruses, human poliovirus, human coxsackieviruses, encephalomyocarditis viruses, meningitis virus, and hepatitis A virus, and includes: (a) prophylactic treatment in a mammal, particularly when the mammal is found to be predisposed to having the disease condition but not yet diagnosed as having it; (b) inhibiting the disease condition; and/or (c) alleviating, in whole or in part, the disease condition.
The activity of the inventive compounds as inhibitors of picomaviral 3C protease activity may be measured by any of the suitable methods known to those skilled in the art, including in vivo and in vitro assays. An example of a suitable assay for activity measurements is the antiviral H1-HeLa cell culture assay described herein.
Administration of the compounds of the formula I and their pharmaceutically acceptable prodrugs, salts, active metabolites, and solvates may be performed according to any of the generally accepted modes of administration available to those skilled in the art. Illustrative examples of suitable modes of administration include oral, nasal, parenteral, topical, transdermal, and rectal. Intranasal delivery is preferred.
An inventive compound of formula I or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof may be administered as a pharmaceutical composition in any pharmaceutical form recognizable to the skilled artisan as being suitable. Suitable pharmaceutical forms include solid, semisolid, liquid, or lyophilized formulations, such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols. Pharmaceutical compositions of the invention may also include suitable excipients, diluents, vehicles, and carriers, as well as other pharmaceutically active agents, depending upon the intended use or mode of administration. In preferred embodiments, the inventive pharmaceutical compositions are delivered intranasally in the form of suspensions.
Acceptable methods of preparing suitable pharmaceutical forms of the pharmaceutical compositions may be routinely determined by those skilled in the art. For example, pharmaceutical preparations may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural, and/or rectal administration.
Solid or liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions. Illustrative solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid. Illustrative liquid carriers include syrup, peanut oil, olive oil, saline solution, and water. The carrier or diluent may include a suitable prolonged-release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or a nonaqueous or aqueous liquid suspension.
A dose of the pharmaceutical composition contains at least a therapeutically effective amount of the active compound (i.e., a compound of formula I or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof), and preferably is made up of one or more pharmaceutical dosage units. The selected dose may be administered to a mammal, for example, a human patient, in need of treatment mediated by inhibition of picomaviral 3C protease activity, by any known or suitable method of administering the dose, including: topically, for example, as an ointment or cream; orally; rectally, for example, as a suppository; parenterally by injection; or continuously by intravaginal, intranasal, intrabronchial, intraaural, or intraocular infusion.
A xe2x80x9ctherapeutically effective amountxe2x80x9d is intended to mean the amount of an inventive agent that, when administered to a mammal in need thereof, is sufficient to effect treatment for disease conditions alleviated by the inhibition of the activity of one or more picomaviral 3C proteases, such as human rhinoviruses, human poliovirus, human coxsackieviruses, encephalomyocarditis viruses, menigovirus, and hepatitis A virus. The amount of a given compound of the invention that will be therapeutically effective will vary depending upon factors such as the particular compound, the disease condition and the severity thereof, the identity of the mammal in need thereof, which amount may be routinely determined by artisans.
By way of illustration, a formulation for nasal delivery of the inventive compounds for treatment of rhinoviral infections may include a compound of formula I that is micronized to a reduced particle size in a suspension containing a final concentration of from about 0.01% to about 2% of the active compound, preferably about from 0.2% to 2%.
An exemplary nasal formulation is as follows: 2.0 weight percent of micronized compound of formula I-a; 1.2 weight percent of a mixture of microcrystalline cellulose and carboxymethyl cellulose sodium (e.g., Avicel RC/CL); 0.1 weight percent of polysorbate 80; 0.01 weight percent of disodium ethylenediamine tetraacetate (EDTA); 0.02 weight percent of benzalkonium chloride solution (50 wt. % BzCl); 5.0 weight percent of dextrose (anhydrous); and balance of purified water.
General Syntheses
The inventive compounds of formula I may be advantageously prepared by the methods of the present invention, including the general methods described below. In each of these general methods, the variables are as defined above.
When stereochemistry is not specified in chemical structures, either stereocenter may be utilized. The following abbreviations also apply: Boc (tert-butoxycarbonyl), Ac (acetyl), Cbz (benzyloxycarbonyl), and Tr (triphenylmethyl). 
In this general synthesis scheme, an amino acid A (prepared by standard peptide coupling conditions and/or methods known in the art), where P1 is an appropriate protecting group for nitrogen (e.g., Boc or Ac) and R is a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-), is transformed into Weinreb amide B. Compound B is treated with an excess of an organometallic reagent (e.g., an alkyllithium or Grignard reagent) to provide product C. At this point, the P1 nitrogen protecting group present in C may be exchanged for an alternate if necessary (e.g., Boc exchanged for Ac). 
An alternate method of preparing product C is depicted above. In this general method, amino acid D (prepared by known methods), where P1 is an appropriate protecting group for nitrogen (e.g., Boc or Ac) and P2 is an appropriate orthogonal protecting group for nitrogen (e.g., Cbz), is transformed into Weinreb amide E. Compound E is treated with an excess of an organometallic reagent (e.g., an alkyllithium or Grignard reagent) to provide intermediate F. The P2 protecting group present in F is then removed, and the resulting amine G (or salt thereof) is derivatized (coupled) with a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-) to afford product C. As described above, the P1 nitrogen protecting group present in C may be exchanged at this point for an alternate if necessary (e.g., Boc exchanged for Ac). 
In this general process, an amino acid H (either commercially available or prepared by standard peptide coupling conditions and/or methods known in the art), where P3 is an appropriate protecting group for the amide nitrogen (e.g., Tr) and R is any suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-), is transformed into Weinreb amide I. Compound I is treated with an excess of an organometallic reagent (e.g., an alkyllithium or Grignard reagent) to provide product J. If necessary, the P3 protecting group present in J is then removed to afford product K. 
An alternate method of preparing products J and K is shown above. In this general method, amino acid L (either commercially available or prepared by a known method), where P3 is an appropriate protecting group for the amide nitrogen (e.g., Tr) and P2 is an appropriate orthogonal protecting group for nitrogen (e.g., Boc or Cbz), is transformed into Weinreb amide M. Compound M is treated with an excess of an organometallic reagent (e.g., an alkyllithium or Grignard reagent) to provide intermediate N. The P2 protecting group present in N is then removed and the resulting amine O (or salt thereof) is derivatized (coupled) with a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-) to afford product J. As described above, the P3 protecting group present in J is then removed to afford product K if necessary. 
In this general method, amino alcohol P (prepared by known methods) where P3 is an appropriate protecting group for the amide nitrogen (e.g., 2,4-dimethoxybenzyl) and P2 is an appropriate orthogonal protecting group for nitrogen (e.g., Boc or Cbz) is oxidized to aldehyde Q. This intermediate is then further oxidized to carboxylic acid R, which is subsequently transformed into Weinreb amide S. Compound S is treated with an excess of an organometallic reagent (e.g., an alkyllithium or Grignard reagent) to provide intermediate T. The P3 protecting group present in T is then removed and the resulting amide U is further deprotected (by removal of the P2 protecting group) to afford amine (or salt thereof) V. Intermediate V is then derivatized (coupled) with a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-) to afford product W. 
An alternate method of preparing product W is depicted above. In this general process, intermediate T (described above), where P3 is an appropriate protecting group for the amide nitrogen (e.g., 2,4-dimethoxybenzyl) and P2 is an appropriate orthogonal protecting group for nitrogen (e.g., Boc or Cbz), is deprotected by removal of the P2 protecting group to give amine (or salt thereof) X. Compound X is then derivatized (coupled) with a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-) to afford intermediate Y. The P3 protecting group present in Y is then removed to provide product W. 
An additional alternate scheme for preparing product W is depicted above. Intermediate T (prepared above) where P3 is an appropriate protecting group for the amide nitrogen (e.g., 2,4-dimethoxybenzyl) and P2 is an appropriate orthogonal protecting group for nitrogen (e.g., Boc or Cbz) is reduced to alcohol Z. The P2 protecting group present in Z is then removed and the resulting amine (or salt thereof) AA is derivatized (coupled) with a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-) to afford intermediate BB. At this point, BB may be further derivatized if necessary by removing any protecting groups present (other than P3) and coupling any and/or all unprotected reactive functional groups (e.g., amines or alcohols) with suitable organic moieties to afford additional BB intermediates. When all appropriate derivatizations of BB have been completed, an oxidation is performed to give ketone V. The P3 protecting group present in V is then removed to provide product W. 
In this method, an amino acid A (prepared by standard peptide coupling conditions and/or methods), where P1 is an appropriate protecting group for nitrogen (e.g., Boc or Ac) and R is any suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-), is transformed into diazo compound CC. Compound CC, in turn, is converted to the bromide DD. This intermediate is subjected to a displacement reaction employing a carboxylic acid moiety to afford product EE. At this point, the P1 nitrogen protecting group present in EE may be exchanged for an alternate if necessary (e.g., Boc exchanged for Ac). 
In the above-illustrated method, an amino acid L (either commercially available or prepared by known methods), where P3 is an appropriate protecting group for the amide nitrogen (e.g., Tr) and P2 is an appropriate orthogonal protecting group for nitrogen (e.g., Boc or Cbz), is transformed into diazo compound FF. Compound FF, in turn, is converted to the chloride GG. This intermediate is subjected to a displacement reaction employing a carboxylic acid moiety to afford intermediate HH. The P2 protecting group present in HH is then removed and the resulting amine (or salt thereof) II is derivatized (coupled) with a suitable organic moiety (e.g., Cbz-L-Leu-L-Phe-) to afford intermediate JJ. The P3 protecting group present in JJ is then removed to provide product KK.
To illustrate, the specific syntheses of the compounds of Examples 21 and 28 are summarized below. 
To prepare W1 (compound 21), alcohol P1 (prepared as described in Dragovich et al., J. Med. Chem. (1999), vol. 42, 1213) is oxidized to give aldehyde Q1 which, in turn, is transformed into carboxylic acid R1. This intermediate may be converted without purification to Weinreb amide S1. Exposure of S1 to an excess of 2-lithiobenzothiazole (generated from nBuLi and benzothiazole) provides ketone T1. The 2,4-dimethoxybenzyl nitrogen protecting group is subsequently removed from T1 to give U1. The Boc protecting group present in U1 is removed under acidic conditions and the resulting amine salt (not shown) is coupled with commercially available Cbz-L-Leu-L-Phe-OH to afford W1 (compound 21).
The synthesis of specific compound W8 is as follows: 
Ketone T1 (prepared as described above) is reduced to alcohol Z1 (isolated as a 1:1 mixture of diastereomers). The Boc protecting group present in Z1 is removed under acidic conditions and the resulting amine salt (not shown) is coupled with Boc-L-Val-L-Phe(4-F)-OH (prepared using standard peptide coupling techniques) to afford intermediate BB1 (isolated as a 1:1 mixture of diastereomers). The Boc protecting group present in BB1 is also removed under acidic conditions and the resulting amine salt (not shown) is derivatized with commercially available 5-methylisoxazole-3-carboxyl chloride to give intermediate BB2 (isolated as a 1:1 mixture of diastereomers). Oxidation of BB2 provides ketone Y1, and subsequent removal of the 2,4-dimethoxybenzyl nitrogen protecting group from Y1 affords W2 (compound 28).
Detailed procedures used to make compounds 21 and 28 and other exemplary compounds of formula I are set forth in the following illustrative examples.