INHIBITORS OF PICORNAVIRUS PROTEASES

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: INHIBITORS OF PICORNAVIRUS PROTEASES Description Technical Field This invention relates to the fields of virology and proteases. More specifically, the invention relates to small compounds useful as inhibitors of picornavirus pro¬ tease enzymes, and their use in the treatment of viral disease. Background of the Invention Picornaviruses are very small RNA-containing viruses which infect a broad range of animals, including humans. The Picomaviridae include human polioviruses, human cox- sackieviruses, human echoviruses, human and bovine enteroviruses, rhinoviruses, encephalomyocarditis viruses, foot-and-mouth disease viruses (FMDV) , and hepa¬ titis 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 paralysis. Although severe infection is rare in the Western world, occasional cases still occur. Only palliative therapy is currently available. Coxsackieviruses and echoviruses are related entero¬ viruses causing a diverse variety of diseases, including herpangina, pleurodynia, aseptic meningitis, myocardiop- athy, acute hemorrhagic con unctivitis, and acute diarrhea. Aseptic meningitis and myocardiopathy are par¬ ticularly serious, and may be fatal. Rhinoviruses are the most important etiologic agents of the common 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 infectious hepatitis. Although it rarely causes chronic hepatitis, there is no current vaccine or effective treatmen . FMDV is considered to be the most serious single pathogen affecting livestock, and thus is a commercially 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 all exposed animals be destroyed, or vaccinated and sequestered until all animals are free of symptoms for 30 days. The dis- 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 specific- 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 signifi¬ cantly from the host. As a result, only a few antiviral drugs are presently available, and most present serious side effects. Current antiviral drugs, such as acyclovir and gan- ciclovir, target the viral polymerase. These drugs are nucleic acid analogs, and rely on the fact that the viral polymerase is less discriminating than eukaryotic pol- ymerases: 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 for treatment of AIDS and AIDS-related infections such as cytomegalovirus infection in immunocompromised patients. Another strategy is to block the virus's means for entering the host cell. Viruses typically bind to a par¬ ticular 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 theoret¬ ically 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 by viruses due to the large num¬ bers of receptors. Even if successful, blocking such receptors could have other adverse effects due to inter¬ ference 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, constrained to a particular primary (and possibly second ¬ ary) structure. Thus, it may be possible to design com¬ pounds which mimic the cleavage/recognition site of a viral protease, inhibiting the protease and interfering with the viral replication cycle. Moiling et al., EP 373,576 disclosed peptides which mimic the recognition site for an HIV 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 as follows: • • • P - " -?~ ~-?2~-?l~Pi —~?2 * P 3 • • • where cleavage occurs between P-. and P . Proteases hav¬ ing low specificity may be constrained only by the iden- tity of the residues in the P α and P x ' positions, cleav¬ ing all polypeptides containing that dipeptide regardless of the more removed residues. However, most specific proteases require that at least some of the residues P 4 - P 4 ' be limited to certain amino acids (or a small set of certain amino acids) . The picornaviral cysteine pro¬ teases generally require Gin at the P x position. A general form of protease inhibitor includes enough polypeptide sequence to induce binding to the protease to be inhibited, but substitutes an electrophillic anchoring group for the P -P, portion. Upon recognition by the protease, the anchor group binds to the essential resi¬ dues in the active site, such as the active site nucleo- phile, and inhibits further proteolytic activity. How¬ ever, it is difficult to prepare peptide protease inhib- itors which end with Glu or Gin, due to the tendency of these residues to cyclize and reduce the concentration of the anchoring moiety (which significantly decreases bind¬ ing to the protease) . Disclosure of the Invention We have now invented a class of cysteine protease inhibitors which are useful in the therapeutic treatment of infection by picornaviridae such as Hepatitis A virus, rhinovirus, coxsackieviruses, and the like. We have found that P x Gin residues may be replaced with Gin ana¬ logs which retain side chain carbonyl group, with reten¬ tion of protease binding activity. The inhibitors of the invention are compounds of formula I: R 2 - Formula I wherein Rj is -OR 3 or -NR 3 R 4 , where R 3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R 4 is H or lower alkyl; R 2 is H or lower acyl; X is an anchor group selected from the group consisting of -CHO, -C≡N, -COCH 2 F, -COCHjCl. -COCH 2 N 2 , -CH=N-NH-C(=S)-NH 2 , or -COCOR 5 where R 5 is lower alkyl, lower alkoxy, lower aryl, aryl- lower alkyl or aryl-lower alkoxy; and (aa) n indicates a polypeptide of 2-40 amino acids which is recognized spe¬ cifically by the particular protease selected. Another aspect of the invention is a method for treating picornaviral 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 pre¬ paring the compounds of formula I. Brief Description of the Drawings Figure 1 is a graph depicting the inhibition of HAV C3 protease as a function of inhibitor concentration for the inhibitors Ac-LRTE(OMe)-CHO, Ac-TPLSTE(OMe)-CHO, and Ac- RTQ(NMe 2 )-CHO. Modes of Carrying Out The Invention A. Definitions The term "lower alkyl" as used herein refers to straight and branched chain hydrocarbon 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 carbon atoms, preferably phenyl or naphthyl. "Aryl-lower alkyl" 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, formyl, benzoyl, and the like. The term "picornaviral 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 picornaviral cysteine protease is preferably an enzyme essential to the replication and/or infectivity of the virus, particularly a protease respon¬ sible for cleaving the viral polyprotein into its consti- tuent proteins. The term "anchor" as used herein refers to a radical which, when introduced into the active site of a pro¬ tease, binds to the protease reversibly or irreversibly and inhibits the proteolytic activity of the enzyme. Presently preferred anchors include aldehyde (-CHO) , nitrile (-C≡N), α-keto esters (-COCOR 5 ) , halo-methyl- ketones (-COCH 2 F, -C0CH 2 C1), diazomethylketones (-COCH 2 N 2 ), and thiosemicarbazones (-CH=N-NH-C(=S)-NH 2 ) . The most effective anchor group may vary from protease to protease. The 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 patho¬ gens, inhibiting or preventing the release of toxins by pathogens, killing pathogens, and preventing the estab¬ lishment of infection (prophylaxis) . The precise effec¬ tive 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. How¬ ever, the effective amount for a given situation can be determined by routine experimentation based on the infor- mation provided herein. The term "pharmaceutically acceptable" refers to compounds and compositions which may be administered to mammals without undue toxicity. Exemplary pharmaceutic¬ ally acceptable salts include mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as ace¬ tates, propionates, malonates, benzoates, and the like. The term "amino acid" refers generally to those nat¬ urally-occurring amino acids commonly found as constit- uents of proteins and peptides: L-alanine (A), L-cys- teine (C) , L-aspartic acid (D) , -glutamic acid (E) , L- phenylalanine (F) , glycine (G) , -histidine (H) , L-iso- leucine (I) , L-lysine (K) , -leucine (L) , L-methionine (M) , -asparagine (N) , -proline (P) , L-glutamine (Q) , - arginine (R) , -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, homologs such as norleu- cine, phenylglycine, N,N'-dimethyl-D-arginine, and the like. Preferred amino acids are common, naturally-occur¬ ring amino acids. The phrases "specific inhibition" and "specifically inhibiting" refer to the reduction or blockage of the proteolytic activity of a selected protease, without sub¬ stantial 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 pro- tease will bind and cleave only peptides having a partic¬ ular amino acid sequence: peptides having such a sequence are "recognized" by the protease. B. General Method The 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 example J. Sambrook et al, "Molecular Cloning; A Laboratory 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 & 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 Molec ¬ ular Cloning" (1984) ; the series, "Methods In Enzymol- ogy" (Academic Press, Inc.); "Gene Transfer Vectors For Mammalian Cells" (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Meth Enzvmol (1987) 154 and 155 (Wu and Grossman, and Wu, eds., respectively); Mayer & Walker, eds. (1987), "I munochemical Methods In Cell And Molecular Biology" (Academic Press, London) ; Scopes, "Protein Purification: Principles And Practice", 2nd Ed (Springer-Verlag, N.Y., 1987); and "Handbook Of Experimental Immunology", volumes I-IV (Weir and Black- well, eds, 1986) . The amino acid sequences of picornaviral substra es 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 contain¬ ing the cleavage site and incubation with the viral pro¬ tease. Once the native recognition site has been estab¬ lished, the inhibitor is- prepared by the methods des¬ cribed 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 picornaviral proteases share similar substrate sequence requirements. In general, P j should be Gin, and P 4 should be aliphatic ( e . g. , Leu, lie, Val, and the like) . In HAV 3C protease, P 2 should bear a hydroxyl side chain (e.g., Ser, Thr, hydroxypro- line, and the like) . The P 3 and P 5 residues do not appear to contribute to protease specificity. The mini¬ mal substrate recognitions sites for picornaviral pro ¬ teases are currently believed to be polio: ALFQ(GPL) ; HRV 14: PVWQ(GP); HAV: LRTQ(SFS); where P n ' residues are in parentheses. Alternatively, a "library" of inhibitors may be syn¬ thesized following the methods disclosed in U.S. Pat. No. 5,010,175, and copending application USSN 07/652,194 filed 16 February 1991, both incorporated herein by ref¬ erence in full. Briefly, one prepares a mixture of pep¬ tides, 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, acti¬ vated 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 coup- 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 par¬ ticular 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) thioester derivative such as Glu(OMe)-thioester. After deprotection and conversion of the thioester to an aldehyde, the mixture of inhibitors is screened for bind¬ ing to (or inhibition of) the selected picornaviral pro- 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 correspond¬ ing 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 second amino acid. In this manner, each reaction may be easily driven to com¬ pletion. Additionally, one may maintain separate "sub- pools" by treating portions in parallel, rather than co - bining all resins at each step. This simplifies the pro¬ cess of determining which inhibitors are responsible for any observed activity. The '175 and '194 methods may be used even in instances where the natural substrate for the protease is unknown or undetermined. 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 oligo¬ mers were cleaved) . One may, in fact, employ the '194 method to determine inhibitors of particular viruses even in cases where the viral proteases have not been identi- 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 resynthe- sized as a group of smaller subpools (sub-subpools) con- taining, e.g., 20-100 candidates, and reassayed. Posi ¬ tive sub-subpools may be resynthesized as individual com ¬ pounds, and assayed finally to determine the active inhibitors. The methods described in '194 enable the preparation of such pools and subpools by automated tech ¬ niques in parallel, such that all synthesis and resynthe- sis 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 rel- evant literature. Protease inhibitors are screened using any available method. The methods described herein are presently pre¬ ferred. In general, a substrate is employed which mimics the enzyme's natural substrate, but which provides a quantifiable signal when cleaved. The signal is prefer¬ ably detectable by colorimetric or fluorometric means: however, other methods such as HPLC or silica gel chroma- tography, 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, tempera- 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 inhib¬ itors capable of binding irreversibly to the protease active site may be added in concentrations on the order of the enzyme concentration. It is presently preferred to mix the substrate with the candidate inhibitors in varying concentrations, fol¬ lowed by addition of the protease. Aliquots of the reac¬ tion mixture are quenched at periodic time points, and assayed for extent of substrate cleavage. The presently preferred technique is to add TNBS (trinitrobenzene sul- fonate) to the quenched solution, which reacts with the free amine generated by cleavage to provide a quantifi¬ able yellow color. The protease inhibitors of the invention may be administered by a variety of methods, such as intraven¬ ously, orally, intramuscularly, intraperitoneally, bron- chially, intranasally, and so 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 intranas¬ ally. Inhibitors may sometimes be administered orally if well absorbed and not substantially degraded upon inges- tion. However, most inhibitors are expected to be sensi¬ tive to digestion, and must generally be administered by parenteral routes. The inhibitors may be administered as pharmaceutical compositions in combination with a pharma¬ ceutically acceptable excipient. Such compositions may be aqueous solutions, emulsions, creams, ointments, sus¬ pensions, gels, liposomal suspensions, and the like. Thus, suitable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG) , phosphate, acetate, gelatin, collagen, Carbopol®, vegetable oils, and the like. One may additionally include suitable preservatives, stabi¬ lizers, antioxidants, antimicrobials, and buffering agents, for example, BHA, BHT, citric acid, ascorbic 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 implanta¬ tion near the site to be treated locally. Other devices include indwelling catheters and devices such as the Alzet® minipump. Further, one may provide the inhibitor in solid form, especially as a lyophilized powder. Lyo- philized 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 Reming¬ ton's Pharmaceutical Sciences (Mack Pub. Co. ) . C. Examples The examples presented below are provided as a fur¬ ther guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the inven¬ tion in any way. Example 1 (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) 2j3:3787) . The peptide was cleaved from the resin using 10% HOAc in CH 2 C1 2 for two hours. Commercially available t-Boc-glutamate methyl ester (2.5 g) 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 fc-Boc protecting groups were removed by reaction with 100 mL of 25% trifluoroacetic acid ("TFA") in CH 2 C1 2 for 30 minutes at room temperature to provide ethyl glutamate thioester. The protected peptide (41.5 mg) was coupled to the ethyl glutamate thioester (117.5 mg, 3 eq) using HOBt (3 eq, 77 mg) and BOP (3 eq, 252 mg) in DMF (1.14 mL) . The fc-butyl protecting groups were then removed by treating the peptide (20 mg) with 50% TFA in CH 2 C1 2 for two hours at room temperature to provide the peptide thioester. The peptide (Ac-TPLSTE(OMe)-SEt) was then reduced by treating the peptide (2 mg) with triethylsilane (40 eq, 70 mg) and palladium (1.4 eq, 13.9 mg) in CH 2 C1 2 (1 mL) for one hour at room temperature. The product, Ac-TPLST- E(OMe)-CHO, was filtered through Celite, concentrated by rotary evaporation under high vacuum to remove volatile material, and purified by C 18 -HPLC. Structure of the peptide was confirmed by H-NMR and mass spectrometry (calculated M+H = 687.3; observed = 687.4). B. Ac-LRTE(OMe)-CHO The compound Ac-LRTE(OMe) -CHO was prepared analog¬ ously to the compound of part A above, substituting Ac- LR(Pmc)T(t-Bu)-OH for Ac-T(t-Bu)PLS(t-Bu)T(t-Bu) -OH. The structure of the product was confirmed by ^- MR and mass spectrometry (calculated M+H = 558.3; observed = 558.5). C. Other Anchors Inhibitors having other anchoring groups are pre¬ pared as described above, with modification of the alde¬ hyde by standard chemical techniques. For example, the -CHO group may be converted to an amide, followed by dehydration (e.g., using SOCl 2 ) to provide the nitrile. Alpha-keto esters are prepared by treating the aldehyde with KCN to form an α-hydroxy acid, followed by esterif- ication. Diazomethylketo analogs are prepared by con¬ verting the aldehyde to an acyl halide, followed by reac¬ tion with diazomethane. Thiosemicarbazones are prepared from the aldehyde by simple addition. Halomethylketo groups are prepared following the method described in J, Med Chem (1990) 23:394-407. Example 2 (Synthesis of Glutamate Dialkylamine Aldehyde Inhibitors) A. Ac-LRTE(NMe,)-CHO Commercially available t-Boc-glutamate α-O-benzyl ester (3 g) was mixed with dimethylamine-ΗC1 (2 eq, 1.46 g) and BOP (1.1 eq, 4.33 g) in the presence of triethyl- amine (1.1 eq, 1 g) for two hours at room temperature to provide t-Boc-glutamate-α-O-benzyl-χ-dimethylamide. The benzyl group was removed by hydrogenolysis over Pd (0.69 g) in MeOH (19 mL) and HOAc (1 mL) to yield t-Boc-gluta- ate γ-dimethylamide. One equivalent of .t-Boc-glutamate γ-dimethylamide (200 mg) was treated with EtSH (10 eq, 440 mg) and ethyl chloroformate (3.6 eq, 285 mg) in the presence of tri- ethylamine (3.6 eq, 266 mg) and DMAP (0.1 eq, 9 mg) for one hour at 0°C, followed by removal of the t-Boc group using TFA in CH 2 C1 2 (25%, 100 mL) for 30 minutes at room temperature to provide t-Boc-glutamate γ-dimethylamide thioester. Ac-LR(Pmc)T(t-Bu)-OH (170 mg) was coupled with t- Boc-glutamate γ-dimethylamide thioester (3 eq, 137 mg) using HOBt (3 eq, 85 mg) and BOP (3 eq, 278 mg) . Pmc and fc-butyl protecting groups were removed by treating the peptide (50 mg) with 50% TFA in CH 2 C1 2 (100 mL) for two hours at room temperature to afford the peptide thio¬ ester, which was then reduced to the aldehyde by treating 2 mg with triethylsilane (20 eq, 70 mg) and Pd (0.6 eq, 16 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 C 18 -HPLC. The structure of the product, Ac-LRTE(NMe 2 ) - CHO, was verified by α H-NMR and mass spectrometry (calcu¬ lated M+H = 571.3; observed = 571.3) . Example 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 mM inhibitor was added to eight 63 μL solutions of reaction buffer (6 mM Na citrate, 94 mM Na phosphate, 2 mM EDTA, 3.5 mM substrate LRTESFS, pH 7.6) to provide a final reaction volume of 80 μL having inhibitor at a concentration of 60, 20, 6.0, 2.0, 0.6, 0.2, 0.06, and 0.02 μM. The reaction was initiated by adding 8 μL of purified HAV 3C protease (3.7 μM) , and was incubated at room temperature. Cleavage of the substrate was halted by transferring 8 μL aliquots from each reac- tion vial into 50 μL 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 (trinitro- benzene sulfonate) . TNBS (10 μL, 35 mg/mL) in borate (0.25 M) was added to each well and incubated for 20 min¬ utes. The resulting yellow color was stabilized by add¬ ing 225 μL sodium sulfite (19 mg/50 mL, 0.4 M KH 2 P0 4 ) , and the optical density of the resulting solution recorded at 405 nm. The results are depicted in Figure 1 and Table 1 below: TABLE 1; Compound IC C n ( UM) Ac -LRTE ( OMe ) -CHO 0 .3 Ac-TPLSTE ( OMe ) -CHO 0 .3 Ac -LRTQ ( NMe 2 ) -CHO 0 .3

Claims: WHAT IS CLAIMED: 1. A compound of Formula I useful for spe¬ cifically inhibiting the proteolytic activity of a sel¬ ected protease: Formula I wherein Rj is -OR 3 or -NR 3 R 4 , where R 3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R 4 is H or lower alkyl; R 2 is H or lower acyl; n is an integer from 2 to 40 inclusive; X is an anchor group selected from the group con¬ sisting of -CHO, -C≡N, -COCH 2 F, -COCH 2 Cl, -COCH 2 N 2 , -CH=N-NHC(=S)NH 2 , or -COCOR 5 where R 5 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 by said selected protease. 2. The compound of claim 1 wherein R : is -OR 3 . 3. The compound of claim 2 wherein R 3 is methyl. 4. The compound of claim 2 wherein R 3 is ethyl. 5. The compound of claim 1 wherein R 2 is acetyl 6. The compound of claim 1 wherein (aa) n comprises Leu-Arg-Thr. 7. The compound of claim 1 wherein (aa) n comprises Thr-Pro-Leu-Ser-Thr. 8. A composition for treating viral infec¬ tion, comprising: an effective amount of a compound of Formula I: R Formula I wherein R x is -OR 3 or -NR 3 R 4 , where R 3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R 4 is H or lower alkyl; R 2 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, -COCH 2 F, -COCH 2 Cl, -COCH 2 N 2 , -CH=N-NHC(=S)NH 2 or -COCOR 5 where R 5 is lower alkyl, lower alkoxy, lower aryl, aryl-lower alkyl or aryl-lower alkoxy; and aa indicates an amino acid; wherein (aa). is an amino acid sequence recognized specif¬ ically 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: administering to said subject an effective amount of a compound of Formula I: R 2 -(aa) Formula I wherein R j is -OR 3 or -NR 3 R 4 , where R 3 is lower alkyl, hydroxy, lower alkoxy, or aryl-lower alkyl, and R 4 is H or lower alkyl; R 2 is H or lower acyl; n is an integer from 2 to 40 inclusive; X is an anchor group selected from the group con¬ sisting of -CHO, -C≡N, -COCH 2 F, -COCH 2 Cl, -COCH 2 N 2 , -CH=N-NHC(=S)NH 2 or -COCOR 5 where R 5 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 sel¬ ected protease. 10. The method of claim 9 wherein said virus is hepatitis A virus. 11. The method of claim 9 wherein said virus is poliovirus. 12. The method of claim 9 wherein said virus is rhinovirus . 13. The method of claim 9 wherein said virus is selected from the group consisting of coxsackie- viruses, echoviruses, enteroviruses, encephalomyocarditis viruses, and foot-and-mouth disease viruses.

Inventors: MALCOLM BRUCE, YANG CHI CHING, MALCOLM, BRUCE, YANG, CHI, CHING
Applicants: CHIRON CORP, CHIRON CORPORATION
Application year: 1992
Main classification: C07K7
IPC codes: A61K38, A61P31, C07K5, C07K7, C07K14