CA2111471A1 - 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

-~092/2~570 PCT/US92/~5167 INHIBITORS OF PICORNAVIRUS PROTEASES
. "

escription Technical Field ~ ~ :
This invention relates to the fields of virology:ancl :
proteases. More specifically, the invention relates to small compounds useful as inhibitors Qf picornavirus:Qro-tease enzymes, and their use in the treatment of viral~
disease.

~
Picornaviruses are very small RNA-contai~ing vlruses : which lnfect a~broad range of~animals~, :includi;ng~humans.
The Picorna~iridae in~lude~human poli:oviruses, human cox-sackieviruses, human echoviruses,: human:and bovine :entero~iruses, rhinoviruses,~encephalomuoca:r~itis : : ~:viruses, foot-and-mouth~disease~vi~uses :(F ~ V),~and:hepa~
:: ~ : titis:A virus~(HAV), among~others~
Polio~irus~is:~an~acid-sta~l~e ~irus which~i~nfects~
humans.~ The vlrus:~enters.~:b~ oral~`ingestion,~m~ltipl~ies~
5 ~ in the gastrointestinal~tra:¢tl a~d invades~:the:~n~rvous system. Poliovirus may spread along~nerve~fibers~:unt:
it reaches the centxal~nervous~sys~t~em`,~whereupon~it~
attacks the motor nerves~, spinal cord, and brain stem.
Advanced infection may result~in-paralysis.~Al~though~
~: 30 ~ severe infection~ls rare in the~Western~world,~occaslonal~
cases still occur. ~Only pa~llia~ive therapy is~currently available.
~oxsacklevlruses and~:~echoviruses:are related ent~ero~
v~ruses causing a diverse:variety of dlseases, includl-g:

W092/22570 PCT/US92/051~-herpangina, pleurodynia~ a~eptic meningitis, myocardiop-athy, acute hemorrhagic conjunctivitis, 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 treatment.
FMDV is considered to be the most serious single pathogen affecting livestock, and thus is a commercially significant virus. It is highly con~agious~ and may reach mortality rates as high as 70%. Control of the virus in the U.S. generally mandates that all exposed animals be destr~yed, or vaccinated and sequestered unti].
all animals are free of symptoms for 30 days. The dis-ease may be pas~.ed to humans ~y contact.
Treatment of infection, in general, relies upon the premise that tlh~! infecting organism employs a metabolic system distinct from its host. Thus, antibiotics are used to combat bacterial infection becaùse~they specific--ally (or preferentially) inhibit or disrupt some aspect of the bacterium's life cycle. The fact that bacterial enzymes are str~Lcturally different from eukaryotic le.g., human) enzymes makes it possible to find compounds which - inactivate or disable a bacterial enz~me without untoward effPct 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 ~ W092~22570 PCT/US92/05167 drugs are presently available, and most present serious side effects.
Current ant:iviral drugs, such as acyclovir and gan-ciclovir, targel: ~he viral polymerase. The~e 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 cytomegalo~irus 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 ~riral entry tand thus replication and infection) by blocking the receptor u~ed for entry. An example of this approach is the use of soluble CD4 to inhibit ~ntry oi HIV. However,;~it~would be~difficult to block,all receptors used by~rirus~es~due to the large num-bers,of receptors.-t Eyeniif successful,~ blocking such ; ~ receptors cauld~have other adverse effects due to inter~
ference with the receptor's normal~ function.
~n alternat:e stratesy relies~upon~the protein expression system peculiar to some viruses. In some viruses, the entire viral genome i 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, ox a combination of the two. The viral protease may reguire a very specific cleavage site, W092~2257~ PCT/US92/~167 constrained to a particular primary (and possibly second-ary) structureO Thus, it may be possible to design com-pounds which mimic ~he cleavage/recognition site of a viral protease, inhibiting the protease and in~erfering with the viral replication cycle. Molling 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 competiti~e binding.
The residues surrounding a protease recognition site within a peptide are generally designated as follows:
~ ~ ~ P4 P3 P2-Pl Pl P2 P3 ~ -where cleavage occurs between P~ and P,'. Pro~eases hav-ing low specificity may be cons~rained only by the iden-tity of the residues in the P~ and P1' 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~-P4 ' be limitçd to certain amino acids (or a small set of certain amino acids). The~picornaviral cysteine pro-teases generally require Gln at the~P~ position.
A g~neral form of protease inhibi~r includes enough polypeptide-;sequence-ko induce bindlng to the;'protease to ` be`inhibited~,~ but ~su~sti~ut~s;ian~electro`phillic anchoring group for the P~-Px~ 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 inhi~its further prote~olytic acti~ity. How-ever, it is difficult to prepare peptide protease inhib-itors which end with Glu or Gln, due to the tendency of these residue9 to cyclize and~reduce the concentration of the anchoring moiety twhich significantly decreases bind-ing to the protease).

~W092/22570 PCT/IJS92/~5167 2 1 ~

Disclosure of the Invention We have now inve:nted a class of cysteine protease inhibitors wh.ic:h are useful in the therapeutic treatment of infection by picornaviridae such as He~atitis A virus, rhinovirus, coxsackieviruses, and the like. We have found that P1 Gln res.Ldues may be replaced with Gln 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:
l~2-(aa)n-NH ~X Formula I

COR

.
wherein R, is -C)R3 or -NR3R4, where R3 is lower alkyl, hydroxy, lower alkoxy, or aryl~lower alkyl, and R4 is:H
2S or lower al.kyl; R2 ie~ H or lower acyl; X is an anchor group selected from t:he group conslsting of -CHO, -C-N, : -COCH2F, -COCH2Cl, -COCH2N2,~-CH=N-NH-C;(~=S~)-NH2, or -COCOR5 :
` where Rs is lowe~r ial~l,``lower alkoxy, lower aryl,: aryl~
~ ~lowër alkyl or i~ryl~-1Owèr alkoxy;~ and (aa)n indicates a;
:~ 30 polypeptide of 2-40 almino acids which~is recognized spe-~
cifically by thé part:icular protease selected.~ ;
Another aspect of the invention is a method~for treating picorna~iral infection by administering an effective amount of a compound of formula I to a subject - 35 in need thereof.
Another:as]?ect of the invention is a method for pre-paring the compounds of formula I.

W092/22570 PCT~US92/05167 71.' ~rief Desc-l~tion of lhe i~rawln~s __ Figure 1 i.~3 a graph depicting the inhibition of HAV
C3 protease as a function of inhibitor concentration for the inhibitors i~c-LRTE(OMe)-CHO, Ac-TPLSTE(OMe)-CHO, and Ac-LRTQ(NMe2)-C~[O.

Modes of Carryi.~ ~ The Invention A. Definitions The term ":Lower alkyl" as used herein refers to straight and br.anched chain hydrocarbon radicals having from 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-bulyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like. I'Lower alkoxy" refers to radicals of the formula -OR, where R is lower alkyl as defined a~ove.
"Aryl" refers t:o aromcltic hydrocarbons having up to 14 carbon atoms, p]-eferably phenyl or naphthyl. "Aryl-lower alkyl~ refers to radicals of the form Ar-R-, where Ar is aryl and R is lt)wer a]kyl.
The term ":Lower acyl" refers to a radical of the formula RCO-, i.n which R is H, lower alkyl as defined above, phenyl or benzyl. Exemplary lower acyl groups include acetyl, propionyl, formyl, benzoyl, and the li:ke.
The term Upicornc~viral cysteine protease". refers to an enzyme encoded within the genome of a picornavirus, which contains ~ cysteine residue wit~hin the active sit.e of the enzyme. T:he pi.cornaviral cysteine protease is preferably an enæyme essential to the replication and/or infectivity oE t:he virus, particularly a protease respon-sible for clea~ing the viral polyprotein into its consti-tuent protein~.
The term "z.lnchor''l as used herein refers to a radical which, when in~roducecl into the active site of a pro tease, binds to the protease reversibly or irreversibly and inhibits th.e proteolytic acti~ity of the enzyme.

7.1 Presently prefe:rred anchors include aldehyde (-CHO), nitrile (-C-N), a-keto esters (-COCOR~), halo-methyl-ketones (-COCH2F, -COCH2C1), diazomethylketones (-COCH2N2), and thiose:micarbazones (-CH-N-NH-C(=S) -NH2 ) .
The most effect::ive anchor group may vary from protease to protease.
The term "effect:ive amount" refers to an amount of compound suffic:ient ts~ exhibit a detectable therapeutic effect. The therap~ulic effect may include, for exampl~, without limitat:ion, 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-~ive 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 ~xact ef.fective~amount in advance. How-ever, the effect:ive amount for a given situation can be determined by routine experimentation based on the infor-mation pro~ided herein.
The term l~)harmac:eutically acceptable" refers to compounds and composit:io~s which m~y be administered to : mammals without undue toxicity. Exemplary pharmaceutic-ally acceptable salts include mineral:acid:salts such as hydrochlorides, hydrobromides, phosphates, ~ulfates,:and the like; and t:he salts of organic acids such as ace-t:ates, propionat.es, malonates, benzoate!s, and the like.
The term "c~mino clcid" refers generally to those nat-urally-occurrin~ amino aclds commonly ~ound as constit-uents of prsteins and peptides: L alanine tA), L~Cys-teine (C), L~as~artiC acid (D), L-glut:amic acid ~E), L-phenylalanine tF`), ~ cine (G), L-histidine (H), L-iSo-leucine ( I ), IJ~ 1YSine (K), L-1eUCine (L), L-methiOnine (M), L-asparagine (N), L-proline IP), L-glutamine ~Q), L-WO~/22570 PCT/US92/05167 4 7 l ;

arginine (R), L-serine (s), L-threonine (T), L-valine ~V), L-tryptopha:n tW), and L-tyrosine ~Y~. However, other analogous compounds may be substituted if they do not adversely a:Efect recognition of the inhibitor by the selected protease. Exemplary analogs include D- isomers of the above-li.sted amino acids, homologs such as norleu-cine, phenylgly~ine, N,N'-dimethyl-D-arginine, and the like. Preferred amino acids are common, naturally-occur-ring amino acids.
The phrases "specific inhibition'l and "specifically inhibiting" refer to t;he reduction or blockage of the proteolytic acti^vity o:f a selected protease, without sub-stantial effect on pro!teolytic enzymes having a different substrate speci~E:icity. Thus, protease inhibitors of the invention preferably include enough of the specifying sequence (typically 4-'7 amino acids upstream from the cleavage site) '30 that only the selected picornaviral protease recogn:iæes an~ is inhibited by the compound. In this regard, "recognlze" refers to ~he fact that the pro-tease will bind and clleave only peptides having a partic-ular amino acid sequenl-e: peptides having such a sequ~nce are "recognized" b~ the protease.

~ B. eneral Meth~d~
The practi~ of t~he pres nt-invention generally employs convent:ional techni.~ues of molecular biology, micro~iology, rerombinant 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, "Mole illar Clonin~; A Laboratory Manual (1989); "DNA Cloning"~, Vol. I and II (D.N Glover ed.:
1985); 'IOligonllcleotide Synthesis" (M.J. Gait ed, 1984);
IlNucleic Acid ~bridization" (B.D. Hames & S.J. Higgins eds. 1984); 'lTranscril?tion And Translation" (B.D. Hames ~..'"~ .",'"~J,1",,.,..~,`," ~

~W092/22570 PCT/US92~05167 7 i g & S.J. Higgins eds. 1984); ~Animal Cell Culture~ ~R.I.
Freshney ed. 1986); "]:mmobilized Cells ~ d Enzymes" (IRL
Press, 1986); B. Perbal, ~A Practîcal Guide To Molec-ular Cloning" (lC184); the series, "Metho~s~In Enzymol-o~y" (Academic Press, ]:nc.); "Gene Transfer Vectors For Mammalian Cells" (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Meth EnzYmol (1987) 154 and 155 (Wu and Grossman, and Wu, eds., respectively);
Mayer & Walker, eds. (1987), ~Immunochemical Methods In Cell And Molecular Biol.ogy" (Academic P~ess, London);
Scopes, "Protei:n Puri~i.cation: Principles And Practice", 2nd Ed (Springer-Verlag, N.Y., 1987); a~d "Handbook Of Experimental Immunology", volumes I-IV (Weir and Black-well, eds, 1986).
The amino acid se~uences of picornaviral substra~es may be determined. by examination of the ~iral genome and comparison to the termini:of viral: proteins. By aligning the ~iral proteins with. the genomic nucleic acid sequence, one can ascertain the~puta~tive cleavage sites, which:may be confirmed:~by synthesis of a peptide contain-ing:the cleavage si:~e and:incubation with the viral pro-tease. Once the~native recognition site has~been~estab-~ . lished, the inh:ibitor is prepared by~-~the~methods-des-: : cri~ed.herein.: ~The (~a)D~portion o~f~the~inhibitor may be ;
altered.systematically to optimize~activity. The~most effective inhibitor will not necess~arily exhibit a : sequence identical to the native substrate, although it~
is expected that any ~ariation will be minor (less than three amino acids:difference).
We have foland that the picornaviral proteases share~
similax substrate sequence requireme`n~s.~ In general, P
should be Gln, and P4 should:be aliphatic (e.~., Leu, Ile,-~aI, and the like). In HAV 3C protease, P2 should bear a hydroxyl side chain (e.g., Ser, Thr, hydroxypro-W092/22S70 PCT/US92~05167 4~ ~

line, and the like). The P3 and P5 residues do not appear to contribllte to protease specificity. The mini-mal substrate rec~gnitic~ns sites for picornaviral pro-teases are currently be:Lieved to be polio: JALFQ(GPL);
HRV 14: PVVVQ~GP); HA~I: LRTQ(SFS); where Pn~ residues are in parenthese~.
Alternatively, a "]Library9' of inhibitors may be syn-thesized following the methods disclosed in U.S. Pat. No.
S,010,175, and copendincl application USSN Q7/652,194 filed 16 February 1991, both incorporated herein by ref-erence in full. Briefly, one prepares a mixture of pep-tides, which i5 then screened to determine the peptides exhibiting the de~.ired ZLCtiVity . In the '175 method, a suitable peptide synthe,:;s support (e.g., a resin) is coupl~d to a mixture of approp:riately protected, acti-vated amino acids. The concen~tration of each amino acid in the reaction mi.xture ~s balanced or adjusted in inverse proportion to its coupling reac~ion rate so that the product is an equimc~lar mixture of amino acids coup-led to the starti~g resin. The bound amino acids are then deprotected, and re!acted with another balanced amino acid mixture to form an equimolar mixture of all possible : dipeptides. Thiq proces~ is rep~ated until a mixture of :
pep~ides;of;the desired:length (e.g., hexamers~ is~
2S 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 ac:id 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 alde~yde, the mixture of inhibitors is screened for bind-ing to (or inhibitlon of) the selected picornaviral pro-:

W092/22570 PC~/US92/~5167 ~111471 tease. Inhibitors exhibiting satisfactory activity are then isolated and sequenced.
The method described ln '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 indi~idually to its portion of resin. The resin portions are ~hen 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 ~eparate "sub-pools" by treating portions in parallel, rather than com-bining all resins at each s~eE~. This simplifies t~le pro-cess of determining which inhibitors are responsible for any observed acti~ity.
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 inhi~itors may: be assayed or bindin g to protease in the absence of the natural substrate. ~Alternatively, one may determlne~the:~substrates by using the '175~and '194 ~
: ~metho~s ~(i.e., ~y preparing a mixture~of all~possible oligomers, contacting:the mixtuxe~with the enz~me, and assayiny 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 bee~ 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 ~esynthe-sized as a group of smaller subpools (sub-subpools) con-W092~2570 P~r/US92~5l67 '7 ~

taining, e.g., 20-100 candidates, and reassayed. Posi-tive sub-subpools may b~ resyn~hesized as individual com-pounds, and assayed finally to determine the active inhibitors. l'he methods described in '194 enable the S preparation of.- such pools and subpools by automated tech-ni~ues in parallel, such that all synthesis and resynthe-sis may be performed in a matter of days. In general, it is preferred t:o 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, ~ut which provides a quantifiable signal when cleaved. The signal is pre~er-ably detectable by colorimetric or fluorometric means:
howe~er, other methods such as HPLC or silica gel chroma-tography, GC-M~, nuclear magnetic resonance, and the like may also be useful. ~fter optimum substrate and enzyme concentrations are determined, a candidate protease inhibitor is a!dded to the reaction mixture at a range of concentrations. The assay conditlons ideally should resemble the:conditions~-under wh.i~h the protease is to be : inhibited in-~i~o,~: i.e.~,~u~der ph~siologic 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 e~ual to or greater than the substrate concentration, while inhib-itors capa~le of binding irreversibIy to the protease active site ma~ be added in concentrations on the order of the enzyme concentration.

.. ~V092/Z2~70 ~ 7 ~ PCT/U~92/05~67 It is presently preferred to mix the sllbstrate wi~h the candidate inhibitors in varying concentrations, fol-lowed by addit:ion of the protease. Ali~uots of the reac-tion mixture axe quenched at periodic tim~ ~oints, and assayed or ext:ent of substrate clea~age. 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 ye~low color.
The protease inhibitors of the invention may be administered b~ 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 th~
inhibitor a~ the pathogen to:be :treated. For example, inhibitors adm:;nistered for the treatment of rhinovirus infection will most preferably be administered:intranas-ally. Inhibit~rs 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 di~est.ion, and must generally be~administere~d by parenteral rout;es. The inhibitors may be administered as pharmaceutical compositions in combination with a pharma-ceutically acc~ptable excipient. Such compositions may be aqueous solutions,:emulsions,;~ creams, ointments,:sus-pensions, gels, Iiposomal suspensions;:, and the like.
: Thus, suitable excipients include water, ~saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextra~, mannose, mannitoI, sorbitol~, polyethylene glycol:(PEGj, phosphate, acetate, gelatin, coll~geni Carbopol~, ve~etable oils, and the like. One may additionally include suitable preservatives, stabi-lizers, antioxidants, antimicrobials, and buffering agents, for example, BHA, BHT, citric acid, ascorbic W092/22570 PCT/US~2/051,67 7 ~

acid, tetracycl:ine, and the like. Cream or ointment bases useful in formulation include lanolin, 5ilvadene~
~Marion), Aquaphor~ ~Duke Laboratories), and the like.
Other topical formuIations include aerosolsr 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 thle like. A tho:rough discussion of pharma-ceutically acceptable excipients is availab~e in Remina:-~ (Mack Pub. Co.).

C. Examples The examples pres~nted below ~re~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.

~Synthesis of Glutamate Ester Aldehyde Inhibitorsj :~
A. ~s~ M~ -CHQ
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). The p~ptide was cleaved from the resin using 10% HOAc in CH2C12 for two hours.

7 l ' Commercially available t-soc-glutamate methyl ester ~2.5 g) was reacted with ethane thiol tlO eq, 7.16 g) and ethyl ch.l.oroformate (3.6 eq, 4.5 g) in the presence of triethylamine (7.1 eq, 8.39 g) and DMAP (O.~.eg, 0.14 g) at 0C for one hour. The t-Boc prot~cting groups were remo~ed by react:ion with 100 mL of 25~ trifluoroacetic acid ("TFA") in CH2Cl2 for 30 minutes at room temperature to provide ethyl glutamate thioester.
The protect:ed 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 t-butyl protecti.ng groups were t~en removed by treating the peptide (20 mg) with 50% TFA in CH2Cl2 for two hours at room temperat;ure to provide the peptide thioes~er.
The peptide (Ac--TPLSTE(~Me)-SEt) was then reduced by treating the p~)tide (2 mg3 with triethylsilane (40 eq, 70 mg) and palladium (1.4 e~, 13.9 mg~ in CH2Cl2 ~1 mL) for one hour at room temperature. The pro~uct, Ac-TP~ST-E(OMe)-CHO, was filtered ~hrou~h Celite, concentrated by rotary evaporation under high vacuum to remove volatil material, and purified by Ci8-HPLC.: Structure of~the peptide was con~ixmed by:~H-NMR and mass sp:~ctrometry ~cal~ulated ~= 687.3; observed~ 687.~4;3.~

The compourld Ac LRTE(OMe)-CHO was prepared ànalog-ously to the coDnpound of part A above, substituting~ Ac- :
LR(Pmc) T ~ t -Bu ) -OH f or Ac-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; observe~ = 558.~5).
C. oeh.r ~rch~lrs ~:
Inhibitors having other anchoring groups are pre-pared as descrihe~d above, with modification of th alde-hyde by standar~ chemical ~echni~ues. For examplej the -CHO group may~e converted to an amide, followed by W092/22570 PCT/US92/051~7 7 ~

dehydration (e g., using SOCl2) to provide the nitrile.
Alpha-keto esters are prepared by treating the aldehyde with KCN to form an a-hydroxy acid, followed by esterif-ication. Diazometh~lketo analogs are pre~ared by con-verting the alclehyde 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) 33:394-407.
Example 2 (Synthesis of Glutamate Dialkylamine Aldehyde Inhibitors) A. Ac-LRTE(N~Ie~-CHO
Commercially a~ailable t-Boc-glutama~e a-o-benzyl ester (3 g) was mixed with dimethylamine-HCl (2 eq, 1.4^6 g) and BOP ll.l eq, 4.33 g) in the presence of triethyl-amine (1.1 eg, 1 g) for two hours at room temperature to pro~ide t-Boc- glutamate-a-O-benzyl-x-dimethylamide. me benzyl group was removed by hydrogeno~ysls over Pd (O.69 g) in MeOH ~19 mL) and HOAc (1 mL) to yiel~d t-Boc-gluta-mate y-dimethylamide.
One equivalent of t-Boc-glutamate ~-dimethylamide~
~200 mg) was treated with EtSH (l0 e~, 440 mgj and ethyl c~loroformate ~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 0C, followed by removal of the t-Boc group using TFA in CH2Cl2 (25%, 100 mL) for 30 minutes at room temperature to provide t-Boc-glutamate ~-dimethylamide thioester.
Ac-LR(Pmc)T(t-Bu)-OH (170 mg) was coupled with t-~
Boc-glutamat2 y-dimethylamide thioester (3 eq, 137 mg) using HOBt (3 e~, 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 CH2Cl2 (100 mL) for two .~W092/22~70 PCT/US92/0~1~7 7 ~.

hours at room temperature to afford ~he peptide thio-ester, which was then reduced to the aldehyde by treating 2 mg with triethylsilane (20 2q, 70 mg) and Pd (0.6 eq, 16 mg) in anhydrous acetone (1 mL) for one...hour at room S temperature. The crude product was filtered through Celite, concentrated by rotary evaporation, and purified by Cl8-HPLC. I~he structure of the product, Ac-LRTE(MMe2)-CHO, was verified by 'H-NMR and mass spectrometry (calcu-lated M+H = 571.3; observed = 571.3).
Example 3 ~Demonstration of Protease Inhibition) The inhibltors prepared in Examples 1 and 2 were assayed for inhibition of HAV 3C protease on 96-well microtiter plates.
~n aliquot of 0.6 mM inh:ibitor was added to eight 63 ~L solutions of reaction buffer ~6 mM Na:citrate, 94 mM
Na phospha~e, 2 mM EDTA, 3.5 mM subs~rate LRTESFS, pH
. 7.6) to provide a final reaction:volume of 80 ~L having inhibitor at a concentratlon of~ 60, 20, 6.0, 2.0, 0.6, : 0.2, 0.0~, and 0.02 ~M. The~reaction was initiated by :~ adding 8 ~L of purified KAV 3C protease (3.7 ~M?, and was incuba~ed at room temperature. Clea~age of the substrate was halted ~y trans~erring 8 ~L aliquots from each reac-: 25 tion vial into 50 ~L of quench solution (0.24 M borate, 0.125 M NaOH) in a m.icrotiter plate well at five minute intervals.
The degree of substrate cleavage is determined by reaction of the resulting free amine with TNBS (trinitro-benzene sulfonat:e). TNBS (10 ~L, 35 mg/mL) in borate (0.25 M3 was added to each well and incubated for 20 min-utes. The resulting yellow color was stabilized by àdd-lng 225 ~L sodium sulfite (19 mg/50 mL, 0.4 M KH2PO4), W092/22570 PCT/US92/05~67 , 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:
Com~ound ~5~n ( UM ) Ac-LRTE(OMe)-CHO 0.3 Ac-TPLSTE~OMe)-CHO 0.3 Ac-LRTQ(MMe2)-CHO 0.3 , ~: : :
:

.:

Claims (13)

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 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 intege from 2 to 40 inclusive;
X is an anchor group selected from the group con-sisting 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 by said selected protease.

2. The compound of claim 1 wherein R1 is

3. The compound of claim 2 wherein R3 is methyl.

4. The compound of claim 2 wherein R3 is ethyl.

5. The compound of claim 1 wherein R2 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:

Formula I
wherein 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 axyl-lower alkoxy: and aa indicates an amino acid; wherein (aa)n 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:
Formula I

wherein 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 con-sisting of -CHO, -C?N, -COCH2F, COCH2Cl, -COCH2N2, -CH=N-NHC(=S)NH,2 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 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. me 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.