WO1992022570A1 - Inhibiteurs de proteases picornavirales - Google Patents

Inhibiteurs de proteases picornavirales Download PDF

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Publication number
WO1992022570A1
WO1992022570A1 PCT/US1992/005167 US9205167W WO9222570A1 WO 1992022570 A1 WO1992022570 A1 WO 1992022570A1 US 9205167 W US9205167 W US 9205167W WO 9222570 A1 WO9222570 A1 WO 9222570A1
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Prior art keywords
lower alkyl
aryl
protease
coch
compound
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PCT/US1992/005167
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English (en)
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Bruce Malcolm
Chi Ching Yang
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Chiron Corporation
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Priority to EP92914531A priority Critical patent/EP0668870A1/fr
Priority to JP5501114A priority patent/JPH06510986A/ja
Publication of WO1992022570A1 publication Critical patent/WO1992022570A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • 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.
  • infections in general, relies upon the premise that the infecting organism employs a metabolic system distinct from its host.
  • antibiotics are used to combat bacterial infection because they specific- ally (or preferentially) inhibit or disrupt some aspect of the bacterium's life cycle.
  • 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.
  • 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.
  • 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.
  • 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.
  • An alternate strategy relies upon the protein expression system peculiar to 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.
  • Proteases hav ⁇ ing low specificity may be constrained only by the iden- tity of the residues in the P ⁇ and P x ' positions, cleav ⁇ ing all polypeptides containing that dipeptide regardless of the more removed residues.
  • 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.
  • 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.
  • the inhibitors of the invention are compounds of formula I:
  • 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.
  • 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.
  • 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.
  • lower alkyl 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.
  • 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.
  • 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.
  • anchor 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.
  • 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.
  • 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.
  • 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-ty
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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) .
  • the picornaviral proteases share similar substrate sequence requirements.
  • P j should be Gin
  • P 4 should be aliphatic ( e . g. , Leu, lie, Val, and the like) .
  • 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.
  • 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.
  • 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.
  • a mixture of peptides of the desired length e.g., hexamers
  • 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.
  • the final amino acid added would be a
  • Gln(X) thioester derivative such as Glu(OMe)-thioester.
  • 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.
  • 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.
  • 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.
  • 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.
  • TNBS trinitrobenzene sul- fonate
  • 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.
  • 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.
  • 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.
  • 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.
  • Topical formulations include aerosols, bandages, sustained-release patches, and the like.
  • Other devices include indwelling catheters and devices such as the
  • Lyo- philized formulations typically contain stabilizing and bulking agents, for example human serum albumin, sucrose, mannitol, and the like.
  • stabilizing and bulking agents for example human serum albumin, sucrose, mannitol, and the like.
  • 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.
  • 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.
  • TFA trifluoroacetic acid
  • 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.
  • Inhibitors having other anchoring groups are pre ⁇ pared as described above, with modification of the alde ⁇ hyde by standard chemical techniques.
  • 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 ⁇ -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.
  • t-Boc-glutamate ⁇ -O-benzyl ester (3 g) was mixed with dimethylamine- ⁇ C1 (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- ⁇ -O-benzyl- ⁇ -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 ⁇ -dimethylamide.
  • Boc-glutamate ⁇ -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.
  • 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.
  • 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 ⁇ L having inhibitor at a concentration of 60, 20, 6.0, 2.0, 0.6, 0.2, 0.06, and 0.02 ⁇ M.
  • the reaction was initiated by adding 8 ⁇ L of purified HAV 3C protease (3.7 ⁇ M) , and was incubated at room temperature.
  • Cleavage of the substrate was halted by transferring 8 ⁇ L aliquots from each reac- tion vial into 50 ⁇ L 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 trinitro- benzene sulfonate

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Abstract

Des composés de formule (I) inhibent l'activité protéolitique de protéases picornovirales et constituent ainsi des agents antiviraux efficaces. Dans ladite formule, R1 représente -OR3 ou -NR3R4, où R3 représente alkyle inférieur, hydroxy, alcoxy inférieur, ou aryle-alkyle inférieur, et R4 représente H ou alkyle inférieur; R2 représente H ou acyle inférieur; n est un nombre entier de 2 à 40 y compris; X est un groupe d'ancrage choisi dans le groupe constitué de -CHO, -C=N, -COCH2F, -COCH2Cl, -COCH2N2, -CH=N-NHC(=S)NH2 ou -COCOR5 où R5 représente alkyle inférieur, alcoxy inférieur, aryle inférieur, aryle-alkyle inférieur ou aryle-alcoxy inférieur; et aa représente un acide aminé; (aa)n représente une séquence d'acides aminés reconnue spécifiquement par ladite protéase sélectionnée.
PCT/US1992/005167 1991-06-14 1992-06-12 Inhibiteurs de proteases picornavirales WO1992022570A1 (fr)

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EP92914531A EP0668870A1 (fr) 1991-06-14 1992-06-12 Inhibiteurs de proteases picornavirales
JP5501114A JPH06510986A (ja) 1991-06-14 1992-06-12 ピコルナウイルスプロテアーゼのインヒビター

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US714,908 1991-06-14

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0632051A1 (fr) * 1993-07-01 1995-01-04 Eli Lilly And Company Agents antiviraux contre picornavirus
WO1996030395A2 (fr) * 1995-03-31 1996-10-03 Takeda Chemical Industries, Ltd. Inhibiteur de la protease de cysteine
US5646121A (en) * 1993-09-14 1997-07-08 Bayer Aktiengesellschaft Pseudopeptides with antiviral activity
US5744451A (en) * 1995-09-12 1998-04-28 Warner-Lambert Company N-substituted glutamic acid derivatives with interleukin-1 β converting enzyme inhibitory activity
WO1998022496A2 (fr) * 1996-11-18 1998-05-28 F. Hoffmann-La Roche Ag Derives peptidiques antiviraux
US5856530A (en) * 1996-05-14 1999-01-05 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US5962487A (en) * 1997-12-16 1999-10-05 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US6020371A (en) * 1997-03-28 2000-02-01 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds compositions containing them and methods for their use
US6214799B1 (en) 1996-05-14 2001-04-10 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US6331554B1 (en) 1997-03-28 2001-12-18 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds, compositions containing them, and methods for their use
US6395897B1 (en) 1999-03-02 2002-05-28 Boehringer Ingelheim Pharmaceuticals, Inc. Nitrile compounds useful as reversible inhibitors of #9 cathepsin 5
US6534530B1 (en) 1999-08-04 2003-03-18 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis
US6632825B2 (en) 2000-06-14 2003-10-14 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis
US6756372B2 (en) 1999-09-13 2004-06-29 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
WO2005044799A1 (fr) 2003-10-30 2005-05-19 Boehringer Ingelheim Pharmaceuticals, Inc. Synthese d'analogues dipeptidiques
US6982263B2 (en) 2001-06-08 2006-01-03 Boehringer Ingelheim Pharmaceuticals, Inc. Nitriles useful as reversible inhibitors of cysteine proteases
US6995142B2 (en) 1998-04-30 2006-02-07 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis
WO2011141658A1 (fr) * 2010-05-11 2011-11-17 Universite Claude Bernard Lyon I Peptides a activite antiproteasique

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US5821331A (en) * 1993-07-01 1998-10-13 Eli Lilly And Company Anti-picornaviral agents
US5514778A (en) * 1993-07-01 1996-05-07 Eli Lilly And Company Anti-picornaviral agents
EP0632051A1 (fr) * 1993-07-01 1995-01-04 Eli Lilly And Company Agents antiviraux contre picornavirus
US5646121A (en) * 1993-09-14 1997-07-08 Bayer Aktiengesellschaft Pseudopeptides with antiviral activity
WO1996030395A2 (fr) * 1995-03-31 1996-10-03 Takeda Chemical Industries, Ltd. Inhibiteur de la protease de cysteine
WO1996030395A3 (fr) * 1995-03-31 1996-12-27 Takeda Chemical Industries Ltd Inhibiteur de la protease de cysteine
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
US5744451A (en) * 1995-09-12 1998-04-28 Warner-Lambert Company N-substituted glutamic acid derivatives with interleukin-1 β converting enzyme inhibitory activity
US5856530A (en) * 1996-05-14 1999-01-05 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US6214799B1 (en) 1996-05-14 2001-04-10 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US6362166B1 (en) 1996-05-14 2002-03-26 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US5866684A (en) * 1996-11-18 1999-02-02 Hoffmann-La Roche Inc. Peptidyl inhibitors of viral proteases
US6018020A (en) * 1996-11-18 2000-01-25 Hoffman-La Roche Inc. Amino acid derivatives
WO1998022496A3 (fr) * 1996-11-18 1998-07-16 Hoffmann La Roche Derives peptidiques antiviraux
WO1998022496A2 (fr) * 1996-11-18 1998-05-28 F. Hoffmann-La Roche Ag Derives peptidiques antiviraux
US6331554B1 (en) 1997-03-28 2001-12-18 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds, compositions containing them, and methods for their use
US6020371A (en) * 1997-03-28 2000-02-01 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds compositions containing them and methods for their use
US6649639B2 (en) 1997-03-28 2003-11-18 Peter S. Dragovich Antipicornaviral compounds, compositions containing them, and methods for their use
US5962487A (en) * 1997-12-16 1999-10-05 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and methods for their use and preparation
US6995142B2 (en) 1998-04-30 2006-02-07 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis
US6395897B1 (en) 1999-03-02 2002-05-28 Boehringer Ingelheim Pharmaceuticals, Inc. Nitrile compounds useful as reversible inhibitors of #9 cathepsin 5
US6608057B2 (en) 1999-03-02 2003-08-19 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cathepsin S
US6730671B2 (en) 1999-03-02 2004-05-04 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cathespin S
US6534530B1 (en) 1999-08-04 2003-03-18 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis
US6756372B2 (en) 1999-09-13 2004-06-29 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US6982272B2 (en) 1999-09-13 2006-01-03 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7056915B2 (en) 1999-09-13 2006-06-06 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7265132B2 (en) 1999-09-13 2007-09-04 Boehringer Ingelheim Pharmaceuticals Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7279472B2 (en) 1999-09-13 2007-10-09 Boehringer Ingelheim Pharmaceuticals Inc. Compounds useful as reversible inhibitors of cysteine proteases
US6632825B2 (en) 2000-06-14 2003-10-14 Agouron Pharmaceuticals, Inc. Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis
US6858623B2 (en) 2000-09-08 2005-02-22 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US6982263B2 (en) 2001-06-08 2006-01-03 Boehringer Ingelheim Pharmaceuticals, Inc. Nitriles useful as reversible inhibitors of cysteine proteases
WO2005044799A1 (fr) 2003-10-30 2005-05-19 Boehringer Ingelheim Pharmaceuticals, Inc. Synthese d'analogues dipeptidiques
WO2011141658A1 (fr) * 2010-05-11 2011-11-17 Universite Claude Bernard Lyon I Peptides a activite antiproteasique
FR2959992A1 (fr) * 2010-05-11 2011-11-18 Univ Claude Bernard Lyon Peptides a activite antiproteasique

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EP0668870A1 (fr) 1995-08-30
IE921941A1 (en) 1992-12-16

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