IE921941A1 - Inhibitors of picornavirus proteases - Google Patents

Abstract

Compounds of formula (I) inhibit the proteolytic activity of picornaviral proteases, and are thus effective antiviral agents. In formula (I) R1 is -OR3 or -NR3R4, where R3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R4 is H or lower alkyl; R2 is H or lower acyl; n is an integer from 2 to 40 inclusive; X is an anchor group selected from the group consisting of -CHO, -C=N, -COCH2F, -COCH2Cl, -COCH2N2, -CH=N-NHC(=S)NH2 or -COCOR5 where R5 is lower alkyl, lower alkoxy, lower aryl, aryl-lower alkyl or aryl-lower alkoxy; and aa indicates an amino acid; wherein (aa)n is an amino acid sequence recognized specifically by said selected protease.

Description

This invention relates to the fields of virology and proteases. More specifically, the invention relates to small compounds useful as inhibitotSL.rof picornavirus protease enzymes, and their use in the treatment of viral disease.

Background_of.the Invention Picoroaviruses are very small RNA-containing viruses which infect a broad range'of' animals, including humans. The Picornaviridae include human polioviruses, human coxsackieviruses, human echo viruses, human and bovine enteroviruses, rhinoviruses, encephalonyocerditis viruses, foot-and-mouth disease viruses (EMDV), and hepatitis A virus (HAV), among others.

Poliovirus is an acid-stable virus which infects humans. The virus enters by oral ingestion, multiplies in the gastrointestinal tract, and invades the nervous system. Poliovirus may spread along nerve fibers until it reaches the central nervous system, whereupon it attacks the motor nerves, spinal cord, and brain stem. Advanced infection may result in paralysiB. Although severe infection is rare in the Western world, occasional cases still occur, only palliative therapy is currently available.

Coxsackieviruses and echovi ruses are related enteroviruses causing a diverse variety of diseases, including 0166.100 - 2 ~ herpangina, pleurodynia, aseptic meningitis, myocardiopathZ/ acute hemorrhagic conjunctivitis, and acute diarrhea. Aseptic meningitis and ayocardiopathy are particularly serious, and may be fatal.

Rhinoviruses are the most important etiologic agents of the consnon cold, and infect nearly every human at some point during his or her lifetime. There is no current treatment approved.

HAV is a highly transmissible etiologic cause of 10 infectious hepatitis. Although i,t rarely causes chronic hepatitis, there is no current vaccine/5 γα£ feet ive treatment.

FMDV is considered to be the most serious single pathogen affecting livestock, and thus is a ccumercially significant virus. It is highly contagious, and may reach mortality rates as high as 70%. Control of the virus in the U.S. generally mandates that ail exposed animals be destroyed/ or vaccinated and sequestered until all animals are free of symptoms for 30 days. The dis20 ease may be passed to humans by contact.

Treatment of infection, in general, relies upon the premise that the infecting organism employs a metabolic system distinct from its host. Thus, antibiotics are used to combat bacterial infection because they specific25 ally (or preferentially) inhibit or disrupt some aspect of the bacterium's life cycle. The fact that bacterial enzymes are structurally different from eukaryotic (e.g., human) enzymes makes it possible to find compounds which inactivate or disable a bacterial enzyme without untoward effect on the eukaryotic counterpart. However, viruses rely on local host enzymes and metabolism to a large extent: thus it is difficult to treat viral infection because viruses present few targets which differ significantly from the host. As a result, only a few antiviral 0166.100 - 3 drugs are presently available, and most present serious side effects.

Current antiviral drugs, such as acyclovir and ganciclovir, target the viral polymerase. These drugs are nucleic acid analogs, and rely on the fact that the viral polymerase is less discriminating than eukaryotic polymerases: the drug is incorporated into replicating viral DNA by the polymerase, which is then unable to attach additional bases. The viral replication is then incomplete and ineffective. However, these drugs present serious side effects, and are currently used only fgr, treatment of AIDS and AIDS-related infections such as cytomegalovirus infection in insnunoconpromised patients.

Another strategy is to block the virus's means for entering the host cell. Viruses typically bind to a particular cell surface receptor and enter the cell, either by internalization of the receptor by the host, or by membrane fusion with the host. Thus, one could theoretically prevent viral entry (and thus replication and infection) by blocking, the receptor used for entry. An example of this approach is the use of soluble CD4 to inhibit entry of HIV. However, it would be difficult to block all receptors used hy viruses due to the large numbers of receptors. Even if successful, blocking such receptors could have other adverse effects due to interference with the receptor's normal function.

An alternate strategy relies upon the protein expression system peculiar to some viruses. In some viruses, the entire viral genome is expressed as one long polyprotein*, which is then cleaved into the structural and non-structural viral proteins. The cleavage may be accomplished by specific viral proteases or endogenous host cell proteases, or a combination of the two. The viral protease may require a very specific cleavage site. 0166.100 - 4 constrained to a particular primary (and possibly secondary) structure. Thus, it may be possible to design compounds which mimic the cleavage/recognition site of a viral protease, inhibiting the protease and interfering with the viral replication cycle. Moiling et al., BP 373,576 disclosed peptides which mimic the recognition site for an Hrv protease. The peptides contain only one uncommon amino acid (5-oxoproline), and thus presumably act by competitive binding.

The residues surrounding a .protease recognition site within a peptide are generally designated/«s follows. ...Ρ,-Ρ,-Ρ,-Ρ^ΡΖ-ΡΖ-Ρ/... where cleavage occurs between Px and P/. Proteases having low specificity may be constrained only by the iden15 tity of the residues in the Pj and P/ positions, cleaving all polypeptides containing that dipeptide regardless of the more removed residues. However, most specific proteases require that.at least some of the residues p4p/ be limited to' certain amino acids (or a small set of certain amino acids). The picoraaviral cysteine proteases generally require Gin at the Pt position.

A general form of protease inhibitor includes enough polypeptide sequence to induce binding to the protease to be inhibited, but substitutes an electrpphillic anchoring group for the P/-P,' portion. Upon recognition hy the protease, the anchor group binds to the essential residues in the active site, such as the active site nucleophile, and inhibits further proteolytic activity. However, it is difficult to prepare peptide protease inhib30 itors which end with Glu or Gin, due to the tendency of these residues to cyclise and reduce the concentration of the anchoring moiety (which significantly decreases binding to the protease). 0166.100 - 5 Disclosure of the Invention We have now invented a class of cysteine protease inhibitors which are useful in the therapeutic treatment of infection by picoraaviridae such as Hepatitis A virus, rhinovirus, coxsackieviruses, and the like, we have found that Pj Gin residues may be replaced with Gin analogs which retain side chain carbonyl group, with retention of protease binding activity. Ihe inhibitors of the invention are compounds of formula I: i2-(aa)a-KH --fornela J wherein Rj is -ORj or -NR,R«, where R, is lower alkyl, .1 hydroxy, lower alkoxy, or aryl-lower alkyl, and R, is H 25 or lower alkyl? Ri is H or lower acyl; X is an anchor group selected from the group consisting of -CHO, -CsN, -COCHjF, -COCHjCl, -COCHjNj, -CH«N-NH-C(=S)-NHj, or -COCORS where Rs is lower alkyl, lower alkoxy, lower aryl, aryllower alkyl or aryl-lower alkoxy; and (aa), indicates a polypeptide of 2-40 amino acids which is recognized specifically by the particular protease selected.

Another aspect of the invention is a method for treating pieornaviral infection by administering an effective amount of a compound of formula I to a subject in need thereof.

Another aspect of the invention is a method for preparing the compounds of formula I. 0166.100 - 6 Brief Description of the Drawings Figure 1 is a graph depicting the inhibition of KAV C3 protease as a function of inhibitor concentration for the inhibitors Ac-LRTE(OMe)-CHO, Ac-TPLSTE(OMe)-CHO, and Ac-LRTQ (NMeJ -CHO.

Modes of Carrying Out The Invention A. Definitions The term 'lower alkyl* as used herein refers to 10 straight and branched chain hydrQparbon_radicals having from 1 to 8 carbon atoms, such as methyl,-'ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl. n-pentyl, n-hexyl, and the like. 'Lower alkoxy' refers to radicals of the formula -OR, where R is lower alkyl as defined above.

Aryl' refers to aromatic hydrocarbons having up to 14 «μΛολ atoms, rr-^forably phenvl or naphthyl. 'Aryl-lower alkyl1 refers to radicals “of the form Ar-R-, where Ar is aryl and R is lower alkyl.

The term 'lower acyl' refers to a radical of the formula RCO-, in which R is H, lower alkyl as defined above, phenyl or benzyl. Exemplary lower acyl groups include acetyl, propionyl, fomyl, benzoyl, and the like.

The term 'picomaviral cysteine protease' refers to an enzyme encoded within the genome of a picornavirus, which contains a cysteine residue within the active site of the enzyme. The picomaviral cysteine protease is preferably an enzyme essential to the replication and/or inf activity of the virus, particularly a protease responsible for cleaving the viral polyprotein into its consti30 tuent proteins.

The term 'anchor* as used herein refers to a radical which, when introduced into the active site of a protease, binds to the protease reversibly or irreversibly and inhibits the proteolytic activity of the enzyme.

IE 921941 0166.100 - 7 Presently preferred anchors include aldehyde (-CHO), nitrile α-keto esters (-COCORj), halo-methylketones (-COCHjF, -COCffjCl), diazomethylketones ί-COCHjNj), and thiosemicarbazones (-CH=N-NH-c(=S)-NHj).

The most effective anchor group may vary from protease to proteaseThe term effective amount refers to an amount of compound sufficient to exhibit a detectable therapeutic effect. The therapeutic effect may include, for example, without limitation, inhibiting the replication of pathogens, inhibiting or preventing the-release-of toxins by pathogens, killing pathogens, and preventing the establishment of infection (prophylaxis). The precise effective amount for a subject will depend upon the subject's size and health, the nature of the pathogen, the severity of the infection, and the like. Thus, it is not possible -» to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation based on the infor20 mation provided herein.

The term pharmaceutically acceptable* refers to compounds and conpositions which may be administered to mammals without undue toxicity. Exemplary pharmaceutically acceptable salts include mineral acid salts such as hydrochlorides, hydrobrcanides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

The term amino acid refers generally to those naturally-occurring amino acids commonly found as constit30 uents of proteins and peptides: L-alanine (A), L-cysteine (C), L-aspartic acid (D), L-glutamic acid (E), t,phenylalanine (F), glycine (G1, L-histidine (H), b-ieoleucine (I), b-lysine (K), b-leucine (L), L-methionine (M), L-asparagine (N), t-proline (P), b-glutamine (Q), bIE 921941 0166.100 - 8 arginine (R), L-serine (S), L-threonine (T), L-valine (V), L-tryptophan (W), and L-tyrosine (Y). However, other analogous compounds may be substituted if they do not adversely affect recognition of the inhibitor by the selected protease. Exemplary analogs include d- isomers of the above-listed amino acids, hcmologs such as norleucine, phenylglycine, N,N'-dimethyl-D-arginine, and the like. Preferred amino acids are common, naturally-occurring amino acids.

The phrases specific inhibition and specifically inhibiting refer to the reduction er^b lockage of the proteolytic activity of a selected protease, without substantial effect on proteolytic enzymes having a different substrate specificity. Thus, protease inhibitors of the invention preferably include enough of the specifying sequence (typically 4-7 amino acids upstream from the cleavage site) so that only the selected picornaviral protease recognizes and is inhibited by the compound. In this regard, recognize refers to the fact that the pro20 tease will bind and cleave only peptides having a particular amino acid sequence: peptides having such a sequence are recognized by the protease.

B- General Method lhe practice of the present invention generally employs conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See for exanqple J.

JU Scwibxuuk cL al, Molocrulax Cloning·.· λ T.ahnratory Manual (1989); DNA Cloning, Vol. I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.j. Higgins eds. 1984); Transcription And Translation (B.D. Hames 0166.100 - 9 & S.J. Higgins eds. 1984); ‘Animal Cell Culture (R.I. Freshney ed. 1986); 'Immobilized Cells And Enzymes* (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning' (1984); the series, 'Methods In EnzymolS ogy' (Academic Press, Inc.); 'Gene Transfer Vectors For Mammalian Cells' (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratoiy); Meth Enzvmol (1987) 154 and 155 (Wu and Grossman, and Wu, eds., respectively); Mayer 6 walker, eds. (1987), ·immunochemical Methods in Cell And Molecular Biology* (Academic Press, London); Scopes, Protein Purification; PrineiplesAnd practice', 2nd Ed (Springer-Verlag, M.Y., 1987); and Handbook Of Experimental Immunology, volumes I-IV (Weir and Blackwell, eds, 1986).

The amino acid sequences of picomaviral substrates may be determined by examination of the viral genome and comparison to the termini of viral proteins. By aligning the viral proteins with the genomic nucleic acid sequence, one can ascertain the putative cleavage sites, which may be confirmed by synthesis of a peptide containing the cleavage site and incubation with the viral protease. Once the native recognition site has been established, the inhibitor is prepared by the methods described herein. The (aa)n portion of the inhibitor may be altered systematically to optimize activity. The most effective inhibitor will not necessarily exhibit a sequence identical to the native substrate, although it is expected that any variation will be minor (less than three amino acids difference).

We have found that the picomaviral proteases share similar substrate sequence requirements. In general, Pt should be Gin, and P« should be aliphatic (e.g., Leu, lie, Val, and the like). In HAV 30 protease. P2 should bear a hydroxyl side chain (e.g., Ser, Thr, hydroxyproIE 921941 0166.100 - 10 line, and the like) . The Pj and Ps residues do not appear to contribute to protease specificity. The minimal Bubstrate recognitions sites for picomaviral proteases are currently believed to be polio: ALFQ(GPL); HRV 14: PVWQ(GP); HAV: LRTQ(SFS); where P/ residues are in parentheses.

Alternatively, a ·library of inhibitors may be synthesized following the methods disclosed in U.S. Fat. No. 5,010,175, and copending application USSN 07/652,194 filed 16 February 1991, both incorporated herein by reference in full. Briefly, one prepares a-mixture of peptides, which is then screened to determine the peptides exhibiting the desired activity. In the '175 method, a suitable peptide synthesis support (e.g., a resin) is coupled to a mixture of appropriately protected, activated amino acids. The concentration of each amino acid . in the reaction mixture is balanced or adjusted in inverse proportion .to its coupling reaction rate so that the product is an equimolar mixture of amino acids coup20 led to the starting resin, The bound amino acids are then deprotected, and reacted with another balanced amino acid mixture to form an equimolar mixture of all possible dipeptides. This process is repeated until a mixture of peptides of the desired length (e.g., hexamers) is formed. Note that one need not include all amino acids in each step: one may include only one or two amino acids in some steps (e.g., where it is known that a particular amino acid is essential in a given position), thus reducing the complexity of the mixture. In the present case, the final amino acid added would be a Gln(X) tbioester derivative such as Glu(OMe)-thioester. After deprotection and conversion of the thioester to an aldehyde, the mixture of inhibitors is screened for binding to (or inhibition of) the selected picomaviral proIE 921941 0166.100 - 11 tease. Inhibitors exhibiting satisfactory activity are then isolated and sequenced.

The method described in '194 is similar. However, instead of reacting the synthesis resin with a mixture of activated amino acids, the resin is divided into twenty equal portions (or into a number of portions corresponding to the number of different amino acids to be added in that step), and each amino acid is coupled individually to its portion of resin. The resin portions are then combined, mixed, and again divided into.a number of equal portions for reaction with the secdnd^amiifo acid. In this manner, each reaction may be easily driven to completion. Additionally, one may maintain separate subpools· hy treating portions in parallel, rather than ccoi15 bining all resins at each step. This simplifies the process of determining which inhibitors are responsible for any observed activity.

The- J175 and '194 methods may be used even in instances where the natural substrate for the protease is unknown or undetermined.1 The mixtures of candidate inhibitors may be assayed for binding to protease in the absence of the natural substrate. Alternatively, one may determine the substrates by using the '175 and '194 methods (i.e., by preparing a mixture of all possible oligomers, contacting the mixture with the enzyme, and assaying the reaction products to determine which oligomers were cleaved). One may, in fact, employ the '194 . method to dot.rm-irfl 4 nhi Hi cnrs nf particular viruses even in cases where the viral proteases have not been identi30 fied or isolated. In such cases, the virus is cultured on host cells in a number of wells, and is treated with subpools containing, e.g., 1-2,000 candidates each. Each subpool that produces a positive result is then resynthesized as a group of smaller subpools (sub-subpools) conIE 921941 0166.100 - 12 taining, e.g., 20-100 candidates, and reessayed. Positive sub-subpools may be resynthesized as individual compounds, and assayed finally to determine the active inhibitors. The methods described in '194 enable the preparation of such pools and subpools by automated techniques in parallel, such that all synthesis and resynthesis may be performed in a matter of days. In general, it is preferred to employ viral proteases in purified form. Such proteases may usually be found reported in the rel10 evant literature. ... __ Protease inhibitors are screenedusing any available method. The methods described herein are presently preferred. In general, a substrate is employed which mimics the enzyme's natural substrate, but which provides a quantifiable signal when cleaved, lhe signal is preferably detectable by colorimetric or fluorometric means: however, other methods such as HPLC or silica gel chromatography, GC-MS, nuclear magnetic resonance, and the like may also be useful. After optimum substrate and enzyme concentrations are determined, a candidate protease inhibitor is added to the reaction mixture at a range of concentrations. The assay conditions ideally should resemble the conditions under which the protease is to be inhibited in vivo, i.e., under physiologic pH, tempera25 ture, ionic strength, etc. Suitable inhibitors will exhibit strong protease inhibition at concentrations which do not raise toxic side effects in the subject. Inhibitors which compete for binding to the protease active site may require concentrations equal to or greater than the substrate concentration, while inhibitors capable of binding irreversibly to the protease active site may be added in concentrations on the order of the enzyme concentration. 0166.100 - 13 It is presently preferred to mix the substrate with the candidate inhibitors in varying concentrations, followed by addition of the protease. Aliquots of the reaction mixture are quenched at periodic time points, and assayed for extent of substrate cleavage. The presently preferred technique is to add TUBS (trinitrobenzene sulfonate) to the quenched solution, which reacts with the free amine generated by cleavage to provide a quantifiable yellow color.

The protease inhibitors of the invention may be administered by a variety of methods77'rsuc3i-a> intravenously, orally, intramuscularly, intraperitoneally, bronchially, intranasally, and ao forth. The preferred route of administration will depend upon the nature of the inhibitor and the pathogen to be treated. For example, inhibitors administered for the treatment of rhinovirus infection will most preferably be administered intranasally. Inhibitors may sometimes be administered orally if well absorbed and. not substantially degraded upon inges20 tion. However, most inhibitors are expected to be sensitive to digestion, and mi>st generally be administered by parenteral routes. The inhibitors may be administered as pharmaceutical compositions in combination with a pharmaceutically acceptable excipient. Such compos it ions may be aqueous solutions, emulsions, creams, ointments, suspensions, gels, liposomal suspensions, and the like.

Thus, suitable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannoee, mannitol, sorbitol, polyethylene glycol (PBG), phosphate, acetate, gelatin, collagen, Carbopol®, vegetable oils, and the like. One may additionally include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, for example, BHA, BRT, citric acid, ascorbic 0166.100 - 14 acid, tetracycline, and the like. Cream or ointment bases useful in formulation include lanolin, Silvadene® (Marion), Aquaphor® (Duke Laboratories), and the like. Other topical formulations include aerosols, bandages, sustained-release patches, and the like. Alternatively, one may incorporate or encapsulate the inhibitor in a suitable polymer matrix or membrane, thus providing a sustained-release delivery device suitable for implantation near the site to be treated locally. Other devices include indwelling catheters anddevices such as the Alzet® minipump. Further, one may~provide the inhibitor in solid form, especially as a lyophilized powder. Lyophilized formulations typically contain stabilizing and bulking agents, for example human serum albumin, sucrose mannitol, and the like. A thorough discussion of pharma ceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co.).

C. Examples ; lhe examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

(Synthesis of Glutamate Ester Aldehyde Inhibitors) A. Ac-TPLSTE(OMe) -CHO A protected peptide having the sequence Ac-T(t-Bu)P-L-S(t-Bu)-T(t-Bu)-OH was synthesized by the standard solid-phase Fmoc method using Rink resin as support (H.

Rink, Tetrahedron Lett (1987) 28;3787). tte peptide was cleaved from the resin using 10% HOAc in CH,C1, for two hours. 0156.100 - 15 Commercially available fc-Boc-glutamate methyl ester (2,5 ¢) was reacted with ethane thiol (10 eq, 7.16 g) and ethyl chloroformate (3.6 eq, 4.5 g) in the presence of triethylamine (7.1 eq, 8.39 g) and DMAP (0.1 eq, 0.14 g) at 0°C for one hour. The t-Boe protecting groups were removed by reaction with 100 mL of 25% trifluoroacetic acid (TFA) in CH2C13 for 30 minutes at room temperature to provide ethyl glutamate thioester.

The protected peptide (41.5 mg) was coupled to the 10 ethyl glutamate thioester (117.5-mg, 3 eq) using HOBt (3 eq, 77 mg) and BOP (3 eq, 252 mg) in 35ΜΡΪΙ.Τ4 mL), ’ The t-butyl protecting groups were then removed hy treating the peptide (20 mg) with 50% TFA in CHjClj for two hours at room tenperature to provide the peptide thioester.

The peptide (Ac-TPLSTE(OMe) -SBt) was then reduced hy treating the peptide (2 mg) with triethylsilana (40 eq, mig) and palladium (1.4 eq, 13.9 mg) in CH2C12 (1 mL) for one hour at foam temperature. The product, Ac-TPLSTE(CMe)-CHO, was filtered through Celite, concentrated by rotary evaporation under high vacuum to remove volatile material, and purified by Cie-HPLC. Structure of the peptide was confirmed by ^-NMR and mass spectrometry (calculated M+H = 687.3; observed = 687.4).

B. AC-LRTE(OMe)-CBQ The compound Ac-LRTE(CWe)-CHO was prepared analogously to the compound of part A above, substituting AcLR(Pmc)T(fc-Bu)-OH for Ac-T(t-Bu)PLS(t-Bu)T(t-Bu, -OH. The structure of the product was confirmed by lH-NMR and mass spectrometry (calculated M+H = 558.3; observed = 558.5).

C. Other Anchors Inhibitors having other anchoring groups are prepared as described above, with modification of the aldehyde by standard chemical techniques. For example, the -CHO group may be converted to an amide, followed by ,E 921941 0166.100 - 16 dehydration (e.g., using SOClj) to provide the nitrile. Alpha-keto esters are prepared by treating the aldehyde with KCN to form an α-hydroxy acid, followed by esterification. Diazomethylketo analogs are prepared by con5 verting the aldehyde to an acyl halide, followed by reaction with diazomethane. ihiosemicarbazones are prepared from the aldehyde by simple addition. Halomethylketo groups are prepared following the method described in £ Med Chem (1990) 33:394-407.

— - „ Example 2 ~-2?cr (Synthesis of Glutamate Dialkylamine Aldehyde Inhibitors) a. A? CTQ Commercially available t-Boc-glutamate α-0-benzyl 15 ester (3 g) was mixed with dimethylamine «HCI (2 eg, 1.46 g) and BOP (1.1 eg, 4.33 g) in the presence of triethylamine (1.1 eg, 1 g, for two hours at room temperature to provide t-Boc-glutamate-a-O-benzyl-X-dimethylamide. The benzyl group was removed by hydrogenolysis over Pd (0.69 g, in MeOH (19 mb) and HOAc (1 mL) to yield t-Boc-glutamate γ-dimethyl amide.

One equivalent of t-Boc-glutamate γ-dimethylamide (200 mg) was treated withEtSH (10 eq, 440 mg) and ethyl chloroformate (3.6 eq, 285 mg) in the presence of tri25 ethylamine (3.6 eq, 266 mg) and DMAP (0.1 eq, 9 mg) for one hour at 0°C, followed bz removal of the t-Boc group using TFA in CH,Clj (25%, 100 mL) for 30 minutes at room ten^erature to provide t-Boc-glutamate γ-dimethylamide thioester.

Ac-LR(Pmc)T(t-Bu)-OH (170 mg) was coupled with tBoc-glutamate γ-dimethylamide thioester (3 eq, 137 mg) using HOBt (3 eq, 85 mg) and BOP (3 eq, 278 mg). Pmc and t-butyl protecting groups were removed by treating the peptide (50 mg) with 50% TFA in CHjC12 (100 mL) for two 0166.100 - 17 hours at room temperature to afford the peptide thioester, which was then reduced to the aldehyde by treating 2 mg with triethylsilane (20 eq, 70 mg) and Pd (0.6 eq, mg) in anhydrous acetone (1 mL) for one hour at room temperature. The crude product was filtered through Celite, concentrated by rotary evaporation, and purified by CU-HPLC. The structure of the product, Ac-LRTE(NMe2) CHO, was verified by ’H-NMR and mass spectrometry (calculated M+H · 571.3» observed « 571.3).

(Demonstration of Protease Inhibition) The inhibitors prepared in Examples 1 and 2 were assayed for inhibition of HAV 3C protease on 96-well microtiter plates.

An aliquot of 0.6 iM inhibitor was added to eight 63 gL solutions of reaction buffer (6 mM Na citrate, 94 fflM Na phosphate, 2 mM EDTA, 3.5 mM substrate LRTESFS, pH 7.6) to provide a final reaction volume of 80 gL having inhibitor at a concentration of 60, 20, 6.0, 2.0, 0.6, 0.2, 0.06, and 0.02 μΜ. Hie reaction was initiated by adding 8 gL of purified HAV 3C protease (3.7 μΜ), and was incubated at room temperature. Cleavage of the substrate was halted by transferring 8 μί, aliquots from each reaction vial into 50 μί, of quench solution (0.24 M borate, 0.125 M NaOH) in a microtiter plate well at five minute intervals.

The degree of substrate cleavage is determined by reaction of the resulting free amine with TNBS (trinitrobenzene sulfonate), WBS (10 gL, 35 mg/mL) in borate (0.25 M) was added to each well and incubated for 20 minutes. The resulting yellow color was stabilized by adding 225 gL sodium sulfite (19 mg/50 mL, 0.4 K KH2PO4), 0166.100 - 18 and the optical density of the resulting solution recorded at 405 mn.

The results are depicted in Figure 1 and Table 1 below: TABLE 1: Compound_ Ac-LRTE(QMe)-CHO AC-TPLSTE(OMe)-CHO Ac-LRTQ (NMe2)-CHO IC.n (UM) 0.3 0.3 0.3 0166.100 - 19

Claims (17)

1. A compound of Formula I useful for specifically inhibiting the proteolytic activity of a selected protease: ij’C·») Foranla I COB j 15 wherein Rj is -0¾ or -NRjR», where R 3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R, is H or lower alkyl; Rj is H or lower acyl; n is an integer from 2 to 40 inclusive; 20 X is an anchor group selected from the group consisting of -CHO, -CsN; -COCHjF, -COCHjCl, -OOCHjNj, -CH=N-NHC(=S)NH 2 , or -0000¾ where R5 is lower alkyl, lower alkoxy, lower aryl; aryl-lower alkyl or aryl-lower alkoxy; and 25 aa indicates an amino acid; wherein (aa)„ is an amino acid sequence recognized by said selected protease. »

2. lhe conpound of claim 1 wherein R t is -0¾.

3. The compound of claim 2 wherein ¾ is methyl.

4. The conpound of claim 2 wherein R 3 is 35 ethyl.

5. The conpound of claim 1 wherein ¾ is acetyl 0166.100 - 20

6. The compound of claim 1 wherein (aa)„ comprises Leu-Arg-Thr.

7. The compound of claim 1 wherein (aa), comprises Thr-Pro-Leu-Ser-Thr. tion,

8. A composition for treating viral infecconprising: an effective amount of a compound of Formula I: Porault I CORj wherein Ri is -OR 3 or -NR 3 R 4 , where R 3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower 20 alkyl, and R, is H or lower alkyl; . Rj is H or lower acyl; n is an integer from 2 to 40 inclusive; X is an anchor group selected from the 25 group consisting of -CHO, -cmN, -COCHjF, -COCHjCl, -COCHjNj, -CH=N-NHC(=S)NHj or -COCOR S where R s is lower alkyl, lower alkoxy, lower aryl, aryl-lower alkyl or aryl-lower alkoxy; and aa indicates an amino acid; wherein 30 (aa), is an amino acid sequence recognized specifically by said selected protease; and a pharmaceutically acceptable excipient.

9. A method for treating a subject for a viral infection wherein said virus includes a cysteine protease, comprising: Έ921941 0166.100 - 21 administering to said subject an effective amount of a compound of Formula I: Forsuit I wherein R x is -ORj or -NRjR,, where Rj is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R 4 is H or lower alkyl; __ R t is H or lower acyl; 15 n is an integer from 2 to 40 inclusive; X is an anchor group selected from the group consisting Of -CHO, -C«N, -COCH,,F, -COCH 2 C1, -COCHjNj, -CH»N-NHC (»S)NHj or -COCORs where R, is lower alkyl, lower alkoxy, lower aryl, aryl-lower alkyl or aryl-lower 20 alkoxy; and aa indicates an amino acid; wherein (aa) e is an amino acid eequence recognized specifically by said selected protease. .

10. The method of is hepatitis A virus.

11. .The method of is poliovirus.

12. . The method of is rhinovirus. claim 9 wherein said virus claim 9 wherein said virus claim 9 wherein said virus

13. The method of claim 9 wherein said virus is selected from the group consisting of coxsackieviruses, ecboviruses, enteroviruses, encephalomyocarditis viruses, and foot-and-mouth disease viruses. - 22

14. A compound of Formula I given and defined in claim 1, substantially as hereinbefore described and exemplified.

15. A process for preparing a compound of Formula I given and defined in claim 1, substantially as hereinbefore described and exemplified.

16. A compound of Formula I given and defined in claim 1, whenever prepared by a process claimed in claim 15.

17. A composition according to claim 8, substantial ly as hereinbefore described.