NO311573B1 - Reversible protease inhibitors - Google Patents

Description

The invention relates to new reversible protease inhibitors. The inhibitors are selective for cysteine proteases.

Cysteine or thiol proteases contain a cysteine residue in the active site that is responsible for proteolysis. Since cysteine proteases have been implicated in a number of diseases, including arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, malaria and other parasite-borne infections, methods for selective and irreversible inactivation of these will provide opportunities for new drug candidates. See e.g. Esser, R.E. et al., Arthritis & Rheumatism (1994) 37, 236; Meijers, M.H.M. et al, Agents Actions

(1993), 39 (Special Conference Issue), C219; Machleidt, W. et al., Fibrinolysis (1992), 6 Suppl. 4.125; Sloane, B.F. et al., Biomed. Biochem. Acta (1991), 50, 549; Duffy, M.J., Clin. Exp. Metastasis (1992), 10, 145; Rosenthal, P. J., Wollish, W. S., Palmer, J. T., Rasnick, D., J. Clin. Investigations (1991), 88,1467; Baricos, W.H. et al., Arch. Biochem. Biophys. (1991), 288, 468; Thornberry, N.A. et al., Nature (1992), 356, 768.

Low molecular weight inhibitors of cysteine proteases have been described by Rich, Proteinase Inhibitors (Chapter 4, "Inhibitors of Cysteine Proteinases"), Elsevier Science Publishers

(1986). Such easier ones include peptide aldehydes, which form hethioacetals with the cysteine of the protease active site. See e.g. Cheng, H., Keitz, P., and Jones, J.B., J. Org. Chem. (1994), 59, 7671. The disadvantage of aldehydes is their in vivo and chemical instabilities.

Aldehydes have now been converted to α,l3-unsaturated esters and sulfones by means of the Wadsworth-Emmons-Horner modification of the Wittig reaction, shown below (Wadsworth, W.S. and Emmons, W.D. (J. Am. Chem. Soc. (1961), 83, 1733).

where R = alkyl, aryl, etc.

EWG = COOEt, SO 2 Me, etc.

α,fl-unsaturated esters (Hanzlik et al., J. Med. Chem., 27(6): 711-712 (1984), Thompson et al., J. Med. Chem. 29: 104-111 (1986) , Liu et al., J. Med. Chem., 35(6): 1067 (1992) and

α,13-Unsaturated sulfones (Thompson et al., supra, Liu et al., supra) were tested as inhibitors of two cysteine proteases, papain and dipeptidylaminopeptidase I (also called cathepsin C). Inhibition of papain by these a,l3-unsaturated compounds showed poor inhibition, as shown by second-order rate constants from less than 1 M"V to less than 70"'s"<1> for a,G-unsaturated esters, and from less than 20 NfV<1> to less than 60 M^s"<1> for sulfones.

Additionally, this chemistry has not been demonstrated with derivatives of cc-amino acids different from the equivalent of glycine, or in the case of the ester, phenylalanine. Thus, the chirality of these compounds is non-existent for glycine derivatives and unclear for phenylalanine derivatives. This is significant since inhibition of an enzyme generally requires a chiral compound.

Alpha-amino sulfonic acids were suggested as potential inhibitory compounds and several were made, although their inhibitory effects were not reported (Mcllwain et al., J. Chem. Soc. 75 (1941)).

In addition, Mannich condensation of sulfinic acid, aldehyde and ethyl carbamate to form urethanes has been reported (Engberts et al., Recueil 84: 942 (1965).

Other methods of selective and irreversible inhibition of cysteine proteases have relied on alkylation by peptide α-fluoromethyl ketones (Rasnick, D., Anal. Biochem.

(1985), 149, 416), diazomethyl ketones (Kirschke, FL, Shaw, E. Biochem. Biphys. Res. Commun. (1981), 101, 454), acyloxymethyl ketones (Krantz, A. et al., Biochemistry,

(1991), 30, 4678; Krantz, A. et al., U.S. Patent 5,055,451, issued October 8, 1991) and ketosulfonium salts (Walker, B., Shaw, E.Fed.Proc.Fed.Am.Soc.Exp.Biol., (1985), 44, 1433).

Other families of cysteine protease inhibitors include epoxysuccinyl peptides, including E-64 and its analogs (Hanada, K. et al., Agric.Biol.Chem. (1978), 42, 523; Sumiya, S. et al., Chem. Pharm. Bull ((1992), 40,299 Gour-Salin, B.J. et al., J. Med. Chem., (1993), 36, 720), α-dicarbonyl compounds, reported by Mehdi, S., Bioorganic Chemistry, (1993), 21 , 249 and N-peptidyl-O-acyl hydroxamates (Bromme, D., Neumann, U., Kirschke, H., Demuth, H-U., Biochim. Biophys. Acta, (1993), 1202, 271. A further summary of methods for reversible and irreversible inhibition of cysteine proteases have recently been described, see Shaw, E., Advances in Enzymology and Related Areas of Molecular Biology (1990), 63, 271.

Thus, the present invention relates to a compound characterized by formula I:

where W is SO2R<2>, PO(OR<10>)2 (where R<10> is independently CiC6 alkyl) phenyl (optionally substituted with amino, Cu alkoxy or C1-C4 alkyl), COOR<2> (where R <21> is H or C16 alkyl), or CONHR5 where R<5> is benzyl, phenyl or phenyl-C14 alkyl;

n is 0 to 4;

A-B represents -C(O)NH-;

X represents -(CH2)m-, where m is 0-4:

Z is -C(R<6>)(R<7>)-, where R 6 is hydrogen or C 1 -C 1 alkyl and R 7 is as defined below; Z is -C(R )(R )-, where R is hydrogen or C 1 -C alkyl and R is defined below;

R<1> is methyl, morpholinyl, benzyloxyaminoalkyl, 0-(CH2)2COOR<22> where R<22> is H or C1-C4 alkyl, naphthyl-OC(0)(CH2)2CO2H, phenylacetyl, benzyloxycarbonyl, Ci- C6 alkoxy, phenyl-C1-C4-alkoxy, phenyl-C1-C4-alkyl (optionally substituted with OH in the phenyl ring and with -C1-C4-alkylcarbonylamino in the alkyl chain) or naphthyl, morpholinylnaphthyl;

R and R are independently hydrogen, (C mo ) alkyl (optionally substituted with a radical selected from amino, carboxy, phenyl or naphthyl (optionally substituted in the aryl ring with a radical selected from hydroxy, halo, amino or a protected thereof);

R<2> is (Ci-10) alkyl (optionally substituted with one or more radicals selected from chlorine, bromine, fluorine, iodine, phenyl, C1-C4-alkoxyphenyl or C1-C4-alkyl-silyl-tri-Ci-C4 -alkyl) or a group selected from phenyl or naphthyl or a protected form thereof;

and pharmaceutically acceptable salts, single isomers and mixtures of isomers thereof.

Advantageous embodiments of the present invention are given in the independent claims 2-28.

Figure 1 shows Scheme 1, synthesis of Formula I compounds when X is a bond. The synthetic steps are as follows: a) HCO 2 H, H 2 O; b) Hbr/acetic acid; c) 4-methylmorpholine, isobutyl chloroformate, Mu-ROH; and d) chromatographic purification. The groups are as defined here.

Figure 2 shows Scheme 2, synthesis of Formula I compounds where X is a methylene group. The 5 synthetic steps are as follows: a) 4-methylmorpholine, isobutyl chloroformate, followed by NaBH4 reduction in water/THF; b) CH3SO2Cl, triethylamine, CH2Cl2; c) RiSH, NaH, CH3OH, THF, heat; d) 4-chloroperbenzoic acid, CH2C12; e) Hcl/dioxane or p-CH3C6H4SO3H/ether; and f) Mu-ROH, 4-methylmorpholine, isobutyl chloroformate. 10 Figure 3 shows Scheme 3, synthesis of Formula 1 compounds when X is a methylene group. The synthetic steps are as follows: a) (CH3)3CH2CH2SH, NaH, MeOH, THF, heat; b) 4-chloroperbenzoic acid; c) (n-C4H9)4N<+>F", THF, followed by BrCH2Cl, heat; d) Hcl/dioxane or 4-CH3C6H5H4S03H/ether, and; e) 4-methylmorpholine, isobutyl chloroformate,

Mu-PheOH.

15

Figure 4 shows Scheme 4, synthesis of Formula II compounds. The synthetic steps are as follows: a) Cl"H2N<+>(CH3)OCH3, dicyclohexylcarbodiimide, Et3N/CH2Cl2; b) L1AIH4/THF; c) NaH/THF; d) Hcl/dioxane/CH2Cl2; e) 4- methylmorpholine,

isobutyl chloroformate/THF; and f) H2, 5% Pd/C.

20

Figure 5 shows Scheme 5, synthesis of Formula I compounds when X is an ethylene. The synthetic steps are as follows: a) (CH2O)n, HCl, dioxane, e.g. when Ar = 2 naphthyl; b)(EtO)3P; c) CH3CO3H, CH2Cl2; d) NaH, THF; e) p-CH 3 C 6 H 4 SO 3 H, Et 2 O; f) 4-methylmorpholine, isobutyl chloroformate; and g) H 2 , Pd/C.

25

Unless otherwise stated, the following terms are used in the description and requirements and are defined in connection with the application and have the following meanings as given below:

"Alkyl" as in alkyl, alkyloxy, alkylthio, alkylsulfonyl, alkylcarbamoyl,

30 dialkylcarbamoyl, heteroarylalkyl, arylalkyl and the like, means a straight or branched, saturated or unsaturated hydrocarbon radical having from 1 to 10 carbon atoms or the number of carbon atoms indicated (e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl , tert-butyl, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl,

ethynyl, 1-propynyl, 2-propynyl, etc).

35

"Aryl" means an aromatic monocyclic or polycyclic hydrocarbon radical containing 6 to 14 carbon atoms, or the number of carbon atoms specified and which

preferably a carboxyl ketone or thioketone derivative thereof, wherein the free valence carbon atom is a member of an aromatic ring, (e.g. aryl includes phenyl, naphthyl, anthracenyl, phenanthrenyl, 1,2,3,4-tetrahydro-5-naphthyl, l -oxo-1,2-dihydro-5-naphthyl, 1-thioxo-1,2-dihydro-5-naphthyl, 1-thioxo-1,2-dihydro-5-naphthyl, etc).

"Halogen" means fluorine, chlorine, bromine or iodine.

"1,2-phenylenedimethylene" means a divalent radical of formula -CH2C6H4CH2-. The group PJY-Z-A- in which Y is -N(R<5>), Z is -CH(R<7>)-, A is carbonyl and R<7> together with R<5 >forms 1,2-diphenylenedimethylene" means a group with the following formula:

and substituted derivatives and individual stereoisomers and mixtures of stereoisomers thereof. Substituted derivatives of 1,2-phenylenedimethylene divalent radical may contain a hydroxy group or any carbon in the ring system or an oxo group on one or other of the unsaturated ring carbon atoms.

"Phosphon" means radical -P(0)(OH)2.

"Methylene" as in "(C3.4)methylene" and "(C3_7)methylene" means a straight, saturated divalent radical having the number of carbon atoms as indicated; "(C3-4) methylene" includes trimethylene (-(CH2)3-) and tetramethylene (-(CH2)4-). A preferred embodiment here utilizes e.g. a proline residue as an A-B-Z group, where A-B represents CH2-NR<3> and R<3> together with either R or R form a C3 methylene. The group R -Y-Z-A in which Y is -(NR )-, Z is -CH(R<7>)-, A is carbonyl and R7 together with R<5> forms trimethylene means a group with the following formula:

and the individual stereoisomers and mixtures of stereoisomers thereof. Substituted derivatives of trimethylene and tetramethylene divalent radicals may contain a hydroxy group or a protected derivative thereof, or an oxo group on any of the ring carbon atoms. Suitable hydroxy protecting groups are defined below. "Oxa(C3-7)methylene" and "aza(C3-7)methylene" means methylene as defined above where one of the specified carbon atoms is replaced by an oxygen or nitrogen atom respectively. "oxa(C5)methylene" includes 3-oxapentamethylene (-CH2CH2OCH2CH2-) and 2-oxapentamethylene (-CH2OCH2CH2CH2-). -C(0)NR<21>R<22> means the radical 4-morpholinylcarbonyl when R21 and R<22> together form 3-oxapentamethylene and the radical 1-piperazinylcarbanoyl when R21 and R<22> together form 3-azapentamethylene. "Adjacent" as used in the expression "R7 together with an adjacent R<3>", means that the atoms to which R<7> and R<3> groups respectively are attached are in turn attached to each other. "Animals" includes humans, non-human animals (eg, dogs, cats, rabbits, cattle, horses, sheep, goats, pigs, deer, etc.) and non-mammals (eg, birds, etc.). "Disease" specifically includes any non-healthy condition of an animal or part thereof and includes any non-healthy condition that may be caused by, or cause of, medical or veterinary therapy applied to the animal, i.e. the "side effects" by such therapy. "Electron-withdrawing group" (EWG) means a functional group which, in its broadest sense, is a group capable of exerting a polarizing force on the bond between itself and the carbon to which it is attached, so that the electrons are polarized in favor of the electron-withdrawing the group. While not being bound by any particular theory, it is believed that the polarizing property enables the electron-withdrawing group to engage in hydrophobic or hydrogen bonding interactions with an active site of the cysteine protease, resulting in enzyme inhibition. In general, a moiety is suitable as an electron-withdrawing group when present in the a-position of a phosphonium ylide of the general structure Pli3P=C(R)EWG which exerts sufficient polarization to stabilize the ylide against undergoing decomposition reactions with oxygen, water, hydrohalic acid , alcohols and the like. Preferred electron-withdrawing groups are those which will similarly stabilize the ylides of general formula (RO)2P(0)C(R)EWG. Suitable electron withdrawing groups include cyan, -S(0)2R<2>, -C(0)OR<10>, -P(O)(OR<I0>)2, -S(O)(NR<10>) R<10>, C(0)R<n>, -S(0)R<n>, -C(0)NR<12>R<13>, -S(0)2NR<12>R<13 >, -C(0)NHR<14>, -S(0)2NHR<14>, phenyl and (C5.6)heteroaryl, wherein R<2>, R<10>, R<1>1, R12, R13 and R<14> are as defined in their broadest definitions as indicated under the summary of the invention. When the electron-withdrawing group is phenyl or (C 5 -heteroaryl, the ring may be substituted with one or more meta directing groups (eg, alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, phosphon, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkylsulfamoyl, dialkylsulfamoyl, alkyloxysulfonyl, disubstituted amine, trisubstituted ammonio, and the like), ortho and para directing groups (e.g. hydroxy, alkyloxy, optionally halogen-substituted alkyl, aryl, arylalkyl, halogen and the like ) and electron-withdrawing moieties (eg alkylcarbamoyl, dialkylcarbamoyl, alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyl, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkylsulfamoyl, dialkylsulfamoyl, alkyloxysulfonyl , aryl, heteroaryl and the like).

"Leaving group" has the meaning conventionally known in synthetic organic chemistry, i.e. an atom or group which can be displaced under the alkylation conditions, and also includes halogen and alkane or arenesulfonyloxy, such as mesyloxy, ethanesulfonyloxy, benzenesulfonyloxy and tosyloxy, and alkanesulfonylamine, alkanecarbonylamine, aminosulfonylamine, aminocarbonylamine and the like.

Isomerism is the phenomenon where compounds have identical molecular shapes but differ in the nature or sequence of bonding and their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of each other are termed "diastereomers" and stereoisomers that are non-superimposable mirror images are termed "enantiomers" or often "optical isomers". A carbon atom bonded to four non-identical substituents is termed a "chiral center".

A compound with a chiral center having two enantiomeric forms of opposite chirality is termed a "racemic mixture". A compound having more than one chiral center has two 2""<1> enantiomeric pairs, where n is the number of chiral centers. Compound with more than one chiral center can exist as either an individual diastereomer or a mixture of diastereomers, termed a "diastereomer mixture".

Compounds of Formulas I, II and III may exist as individual stereoisomers or mixtures of stereoisomers. The compounds with Formulas I, II and III contain e.g. a chiral center at the carbon to which the substituent R8 is attached. Furthermore, compounds of Formulas I, II and III in which Z is -C(R<6>)(R<7>) contain a chiral center on the carbon to which the R substituent is attached. For example, the compounds of Formulas I, II and III in which n is 0 and Z is -C(R )(R ) will thus have two chiral centers and may exist as four individual stereoisomers or any mixture thereof.

Individual stereoisomers can be characterized by the absolute configuration of their chiral centers. Absolute configuration refers to the spatial arrangement of substituents attached to the chiral center. The substituents attached to the chiral center are ranked according to the sequence rule of Cahn, Ingold and Prelog and then the absolute descriptor R is assigned if the three highest ranked substituents are arranged in space (where the fourth lowest ranked substituent is directed away from the observer) from high to low priority in a clockwise order and the absolute descriptor S is assigned a counter-clockwise arrangement. When an individual stereoisomer containing a chiral center is described, the absolute descriptor R or S is quoted in parentheses followed by a hyphen and the chemical name of the compound. In the context of the present invention, when an individual stereoisomer or mixture of stereoisomers containing two or more chiral centers is described, the absolute descriptor R or S is cited immediately after the appropriate location. Acyl radicals derived from naturally occurring amino acids are referred to as their amino acid radicals preceded by the descriptor L (eg, L-phenylalanine). Non-natural enantiomers of amino acid acyl radicals are preceded by the descriptor D. Amino acid side chains are preferably (S) or L-form, due to the stereospecificity of the enzyme, although D-forms may be used in some cases. When no absolute descriptor is given for a chiral center, the descriptor is meant to include both configurations and mixtures thereof, racemic or otherwise. A connection e.g. with the following formula

is named: Af2-(4-morpholinylcarbonyl^ phenylalanine amide, when R 1 is 4-morpholinylcarbonyl, R R is 2-phenylethyl and lies on the same side of the reference plane as the R<7> substituent, R<7> is benzyl and R<19> is phenylsulfonyl; iV<2->(4-morpholinylcarbonyl)-7V<1->[3-phenyl-1-(2-phenylsulfonylethyl)propyl]-L-phenylalanine amide, when R<1> is 4-morpholinylcarbonyl, R< 8> is 2-phenylethyl and lies on one or both sides of the reference plane, R<7> is benzyl and R<19> is <f>enylsulfonyl; A^<2->(4-morpholinylcarbonyl)-A^- [3-phenyl-1S-(2-phenylsulfonylethyl)propyl]-6-(2-naphthyl)-L-alanine amide, when R is 4-morpholinylcarbonyl, R is 2-phenylethyl lies on the same side of the reference plane as the R7 substituent, R7 is 2-naphthylmethyl and R<19> is phenylsulfonyl; and ethyl 4S-(iV-4-morpholinylcarbonyl-L-phenylalanylamine)-6-phenylhexanoate, when R<1> is 4-morpholinylcarbonyl, R Q is 2-phenylethyl and lies on same side of the reference plane as the R7 substituent, R7 is benzyl and R<19> is ethoxycarbonyl.

In a preferred embodiment, compositions of the invention are pure diastereomers. Alternatively, the compositions contain mixtures of diastereomers. Preferred embodiments have higher than approx. 70% of a single diastereomer, with at least approx. 90% is particularly preferred.

"Protecting group" has the same meaning as conventionally associated with it in synthetic organic chemistry, ie a group that blocks a reactive site in a compound. See e.g. Greene et al., Protective Groups in Organic Synthesis, 2nd. Ed., John Wiley & Sons, 1991, herein incorporated by reference. Examples of hydroxy protecting groups include heterocycloalkylcarbonyl such as 4-morpholinylcarbonyl and the like, aryl such as benzoyl and arylalkyl such as benzyl and the like. Examples of amine protecting groups include aryloxycarbonyl such as benzyloxycarbonyl and the like, aroyl such as benzoyl and the like, and oxycarbonyl such as ethoxycarbonyl and 9-fluorenylmethoxycarbonyl and the like. Examples of guanidine protecting groups include sulfonyl such as 2,3,5-tyrmethyl-4-methoxyphenyl-sulfonyl and the like. Examples of suitable carboxy-protecting groups that form ester parts are alkoxycarbonyl with a total of 4 to 8 carbon atoms, especially tert-butoxycarbonyl (BOC) or benzyloxycarbonyl (CBZ, Z), especially cycloalkylaminocarbonyl or oxacycloalkylaminocarbonyl with a total of 4 to 8 atoms in the ring, especially 4-morpholinecarbonyl ( Mu) and the like.

"Protected" in reference to a compound of a group means a derivative of a compound or group in which a reactive site or sites are blocked with protecting groups.

"Possible" or "optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes situations in which the event or circumstance occurs and situations in which it does not. For example, "possibly further substituted with one or more functional groups" means that the substituents may or may not be present so that the compound as described falls within the scope of the invention, and the invention includes those compounds where one or more functional groups are present and in the compounds where no functional groups are present.

By "cysteine protease-associated disorders" is meant here pathological conditions associated with cysteine protease. In some disorders, the condition is associated with increased levels of cysteine protease; e.g. arthritis, muscular dystrophy, inflammation, tumor invasion and glomerulonephritis are all associated with increased levels of cysteine protease. In other disorders or diseases, the condition is associated with the occurrence of an extra cellular cysteine protease activity that is not present in normal tissue. In other embodiments, a cysteine protease is associated with the ability of a pathogen, such as a virus, to infect or replicate in the host organism.

Specific examples of cysteine protease-related disorders include, but are not limited to, arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, malaria, Alzheimer's disease, cancer metastasis, trauma, inflammation, gingivitis, leishmaniasis, filariasis, and other bacterial and parasite-borne infections. In particular, disorders associated with interleukin lfi converting enzyme (ICE) are included.

"Pharmaceutically acceptable" is understood to be useful in the manufacture of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes being acceptable for veterinary use as well as human pharmaceutical use.

"Pharmaceutically acceptable salts" means salts which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cylopentanepropionic acid, glycolic acid, grape acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, madelic acid,

methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, laurylsulphuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like.

Pharmaceutically acceptable salts also include base addition salts which may be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.

"Therapeutically effective amount" means that amount which, when administered to an animal to treat a disease includes: (1) prevents the disease from occurring in an animal that may be predisposed to the disease but is not yet experiencing or exhibiting symptoms; on

the disease,

(2) inhibit the disease, i.e. stop its development or

(3) alleviate the disease, i.e. cause regression of the disease.

The present invention relates to new cysteine protease inhibitors. Without being bound by theory, it is believed that the inhibitors bind to cysteine proteases based on the following scheme:

It is assumed that the enzyme is thus reversibly inhibited by means of interactions between R, Y and Z parts and the inhibitor and the surface of the binding sites on the enzyme, and by means of hydrogen bond interactions between sulfone and active site amino acid side chains.

The mechanism of reversible inhibition allows specificity of the enzyme inhibitors for cysteine protease. The inhibitors of the present invention generally inhibit cysteine proteases and do not inhibit serine, aspartyl and zinc protease. In some embodiments, the protease inhibitors from the present invention may have activity against other protease types, such as serine, aspartyl or other metal proteases, but to a lesser extent.

In addition, the electron-withdrawing properties of the sulfone group in Formula I will polarize the electrons between the sulfone group and the carbon to which they are attached, thus enabling hydrogen bonding between itself and the active site residues in a cysteine protease, to allow tight binding between inhibitor and cysteine protease, as are generally described below. It is understood that there is probably additional electron attraction or electron polarization occurring between the sulfur atom and oxygen atom, which allows oxygen atoms to participate in hydrogen bonding with the active site residues of the proteases and thus contribute to further inhibition of the enzyme.

The present invention will generally provide new peptide-based and peptide-domimetric cysteine protease inhibitors for use as reversible cysteine protease inhibitors. By "cysteine protease inhibitor" is meant here an inhibitor that inhibits cysteine protease. In a preferred embodiment, the cysteine protease inhibitors are specific to cysteine protease; i.e. they do not inhibit other types of protease such as serine, aspartyl or other metalloproteases. In alternative embodiments, the cysteine protease inhibitors in the invention can inhibit other types of proteases.

By "reversible" is meant here that the inhibitor binds non-covalently to the enzyme, and must be distinguished from irreversible inhibition. See Walsh, Enzymatic Reaction Mechanisms, Freeman & Co., N.Y., 1979. "Reversible" in this context is a term known in the art. In addition, the reversible cysteine protease inhibitors are competitively easier, i.e. they compete with substrate in reversible binding to the enzyme, where the binding of inhibitor and substrate is mutually exclusive. In addition, the stoichiometry in the inhibition is 1:1; i.e. a single inhibitor molecule is sufficient to inhibit a single enzyme molecule.

The cysteine protease inhibitors here are said to bind reversibly to cysteine protease. This binding is carried out by using peptide-based or peptide-domimetic structures as targeting groups that mimic naturally occurring substrates and/or easier. "Peptide domimetic" in the context of the invention, means amino acids or peptide-like structure where one or more of the peptide bonds (i.e. -C(O)NR-) is substituted with an isostereomeric form, i.e. -CH2NR-, -C(0) CH2- or -NRC(O)- and/or where non-naturally occurring amino acid substituents are present.

"Targeted group" in the context of the invention, means a peptide or peptidomimetic residue of cysteine protease inhibitors that allows binding of the inhibitor to a cysteine protease. In a preferred embodiment, the targeting group of a cysteine protease inhibitor comprises at least two amino acid side chains or side chain analogs, linked via a peptide bond or isostere. The targeting group can comprise up to 15 amino acids or analogues, although simpler ones are generally from approx. 1 to 7 amino acids or analogs, since less easily is usually desired in therapeutic use. In Formulas I, II and III, n is thus preferably from 0 to 13, with from 0 to 5 being preferred, and from 0 to 3 being particularly preferred.

As shown in Formulas I, II and III, the targeting group may be represented by a naturally or non-naturally occurring peptide residue of formula:

where the R R and R 0 components represent naturally or non-naturally occurring amino acid analogues or substituents which are described in detail in the following. The targeting group of inhibitors may also contain additional functional moieties, as shown by R<1> and described herein.

While not being bound by any particular theory, it is believed that the amino acid substituents in the targeting group react with surface binding sites of the protease to promote binding. It is also assumed that the amino acid substituent proximal to the electron-withdrawing group (e.g. R in the formula above) will occupy the Si position of the substrate binding site and is therefore designed as the Pi residue in the inhibitor. The next adjacent amino acid substituent (e.g. R in the above-mentioned formula) will occupy the S2 position of the substrate binding site and is designated the P2 residue in the inhibitor. If present, the amino acid substituent will occupy the S3, S4, etc. positions of the substrate binding site and will be designated as P3, P4, etc. residues of the inhibitor. An additional targeting group may be attached to electron-withdrawing groups, and if present, the amino acid substituent will occupy Si', S2', etc. positions of substrate binding sites and be designated P3', P4', etc. residues of the inhibitor.

In general, the targeting groups for specific enzymes are determined by rules governing substrate specificity in cysteine proteases (see, e.g., "Proteinase Inhibitors", in Research Monographs in Cell and tissue Physiology (1986), ed. Barret et al., Vol. 12, Chapter 4: Inhibitors of Cysteine Proteinases, Daniel Rich, Elsevier, New York; and Thornberry et al., supra.. For example, Interleukin-1 converting enzyme (ICE) accepts an aspartic acid substituent (ie, 2-carboxyethyl) in Pi position and an alanine (methyl), alline (isopropyl) or histidine (4-imidazolylmethyl) substituent at the P2 position.Papain accepts an arginine, lysine, N-benzyloxycarbonyllysine (i.e. 4-benzyloxycarbonylaminobutyl), homophenylalanine (i.e. 2-phenylethyl) , Guanidino-phenylalanine (ie 4-guanidinobenzyl) or norleucine (ie butyl) substituents in the Pi position and phenylalanine, tyrosine, bi-(2-naphthyl)alanine (ie 2-naphthyl), leucine, norleucine, isoleucine or alanine substituents in the P2 position Cathepsin B accepts an arginine, lysine, 7V-benzyloxycarbonyllysine, guanidine-phenylalanine, homophenylalanine or norleucine substituents in the Pi position and phenylalanine, tyrosine, 3,5-diiodotyrosine (i.e. 3,5-diiodo-4-hydroxybenzyl), J3-(2-naphthyl)alanine, arginine, guanidine-phenylalanine or citrulline (ie 3-ureidopropyl) substituents in the P2 position. Cathepsin L and cruzain accept arginine, lysine, homophenylalanine, guanidinephenylalanine, citrulline or norleucine substituents in the Pi position and phenylalanine, tyrosine or 6-(2-naphthyl)alanine substituents in the P2 position. Cathepsin S accepts arginine, lysine, homophenylalanine, guanidine-phenylalanine, citrulline or norleucine substituents in the Pi position and phenylalanine, tyrosine, B-(2-naphthyl)anine, valine, leucine, norleucine, isoleucine or alanine substituents in the P2 position. DPP-1 accepts phenylalanine or tyrosine substituents in the Pi position and no substituents or alanine in the P2 position. Calpain accepts phenylalanine, tyrosine, methionine, B-methylsulfonylmethyl-alanine (ie 2-methylsulfonylethyl) or valine substituents in the Pi position and valine, leucine, norleucine or isoleucine substituents in the P2 position.

Preferred R 7 and R 8 groups are naturally occurring amino acid side chains and homologous derivatives. These include but are not limited to alanine (methyl), arginine (3-guanidinopropyl), asparagine (carbamoylmethyl), citrulline (3-ureidopropyl), aspartic acid (carboxymethyl), cysteine (mercaptomethyl), glutamic acid (2-carboxyethyl), glutamine (2-carbamoylethyl), glycine (hydrogen), histidine (4-imidazolylmethyl), homophenylalanine (2-phenylethyl), homoserine (2-hydroxyethyl), isoleucine ((1-methylpropyl), leucine (isobutyl), lysine (4-aminobutyl ), methionine (2-methylthioethyl), β-(1-naphthyl)alanine (1-naphthylmethyl), B-(2-naphthyl)alanine (2-naphthylmethyl), norleucine (butyl), norvaline (propyl), ornithine (3 -aminopropyl), phenylalanine (benzyl), proline (as described herein), sarcosine (methylaminomethyl), serine (hydroxymethyl), threonine (1-hydroxyethyl), tryptophan (3-indolylmethyl), tyrosine (4-hydroxybenzyl), and vanillin (isopropyl).

More preferred compounds of Formula I are those wherein n is 0 to 2; A-B represents a bond selected from -C(0)NR<3>, where R<3> is hydrogen or as defined below; / is -N(R<5>)-, where R<5> is hydrogen or as defined below; Z is -(CH2)2- or -C(R<6>)(R<7>)- (with the proviso that when n is 0, Z is not -(CH2)2-); Z<1> is -CH(R<8>)-; R < 1 > is hydrogen, (C 4.8 )alkyloxycarbonyl, (C 2.6 )alkanoyl (optionally substituted with a radical selected from carboxy, (C 1.5 )alkyloxycarbonyl and hetero(C 4.8 )cycloalkyl(C 4.6 )alkanoylamino ), - C(0)NR<21>R<22> where R<21> and R<22> together form aza(C2-6)methylene, oxa(C2.6)methylene or (C3-7)methylene, (C4.g)cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, benzoyl or dimethylaminosulfonyl; and R<8> and R<7> are independently (C 5 -cycloalkyl, (C 5 -6)cycloalkylmethyl, 3-pyridyl, 2-thienyl, 2-furyl, 4-imidazolyl, 3-indolyl, 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, (Ci.s)alkyl (optionally substituted with a radical selected from mercapto, carboxy, amine, methylthio, methylsulfonyl, carbamoyl, dimethylcarbamoyl, guanidine and hydroxy, or a protected derivative thereof), a group selected from phenyl, 1-naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted in its aryl ring with a radical selected from hydroxy, amine, chlorine, bromine and fluorine or a protected form thereof) or together with an adjacent R or R forms a divalent radical selected from (C3^)methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof or oxo).

Particularly preferred compounds of Formula I are those wherein n is 0 to 1; A-B represents a bond selected from -C(0)NR<3->; Y is -N(R<5>)-, where R<5> is hydrogen or as defined below; Z is -C(R<6>)(R<7>)-; Z<1> is -CH(R<8>)-; R<1> is hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, 3-carboxypropionyl, 3-methoxycarbonylpropionyl, biotinylaminohexanoyl, phenylacetyl, benzoyl, dimethylaminosulfonyl, benzylsulfonyl, 1-piperazinylcarbonyl, 4-methylpiperazin-1-ylcarbonyl or 4-morpholinylcarbonyl; R7 is 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, (Ci.s)alkyl (optionally substituted with a radical selected from mercapto, carboxy, amine, methylthio, methylsulfonyl, carbamoyl, dimethylcarbamoyl, guanidino and hydroxy, or a protected derivative thereof), a group selected from benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted in the aryl ring with a radical selected from hydroxy, amine, chlorine, bromine and fluorine , or a protected form thereof) or together with adjacent R<3> or R<5> forms a divalent radical selected from (C3^)methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative hence, or oxo); and R<8> is butyl, 2-phenylethyl-2-methylsulfonylethyl, 2-tert-butoxycarbonylethyl, 2-tert-butoxycarbonylmethyl, 4-tert-butoxycarbonylaminobutyl, 4-benzoylaminobutyl or benzyloxymethyl.

Particularly preferred compounds of Formula I are those wherein n is 0; A-B represents a bond selected from -C(O)NH-; Y is -NH-; Z is -CH(R<7>)-; Z<1> is -CH(R<8>)-; R<1> is hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, biotinylaminohexanoyl, benzoyl, piperizin-1-ylcarbonyl, 4-methylpiperazin-1-ylcarbonyl or 4-morpholinylcarbonyl; R7 is (C1-5)alkyl, optionally substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-pyridinylmethyl or 2-methylsulfonylethyl; and R<8> is butyl, 2-phenylethyl or 2-methylsulfonylethyl.

The most preferred compounds of Formula I are those wherein n is 0; A-B represents a bond selected from -C(O)NH-; Y is -NH-; Z is -CH(R<7>)-; Z<1> is -CH(R<8>)-; R<1> is 1-piperizinylcarbonyl, 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl; R<7> is optionally substituted benzyl, 1-naphthylmethyl or 2-naphthylmethyl; and R<8> is 2-phenylethyl.

Generally preferred compounds of Formula I are those wherein R<2> is independently (Ci-5) alkyl (optionally substituted with one or two radicals selected from amine, chlorine, bromine, fluorine, hydroxy and methoxy, or a protected derivative thereof ), perhalo(Ci-5)alkyl, (C3-7)cycloalkyl, (C3-7)cycloalkyl(Ci-5)alkyl or a group selected from phenyl, pentafluorophenyl, naphthyl and phenyl(Ci-6)alkyl (which group is optionally substituted in the aryl ring with one to two radicals selected from amine, chlorine, bromine, fluorine, hydroxy, methoxy and optionally halogen-substituted methyl, (or a protected derivative thereof) and R<4> is hydrogen, (Ci-5) alkyl or (C6-10)aryl(C1-5)alkyl More preferred compounds of Formula I are those wherein R<2> is (C1-5)alkyl (optionally substituted with one or two radicals selected from amine, chlorine, bromine, fluorine and hydroxy, or a protected derivative thereof), perfluoro(Ci-5)alkyl, (C5.6)cycloalkyl, (C5-6)cycloalkylmethyl or a group selected from phenyl, naphthyl and benzyl (which group eve ntual is substituted by a radical selected from amine hydroxy, chlorine, bromine or fluorine, (or a protected derivative thereof) and R<4> is hydrogen or methyl. Particularly preferred compounds with Formula I are those in which R<2> is methyl, trifluoromethyl, optionally substituted phenyl, 2-naphthyl or 2-phenylethyl. It is most preferred with compounds of Formula I in which R<2> is phenyl, 2-naphthyl or 2-phenylethyl, especially phenyl or 2-naphthyl, and R<4> is hydrogen.

In general, preferred cysteine protease inhibitors of the invention are those in which the absolute configuration of each chiral center present is the (^) configuration. However, preferred compounds of Formula I wherein n is 0 are those in which the absolute configuration of the chiral center to which the R<7> substituent is attached is in the (Æ) configuration Preferred compounds of Formula I include, for example: N 7 -(4-morpholinylcarbonyl)-A^ 1-(3-phenyl-1Æ-phenylsulfonylpropyl)-L-phenylalanine amide (compound 1) , 7^<2->(4-morpholinylcarbonyl)-A/<1->(3-phenyl-15-phenylsulfonylpropyl)-L-phenylalanine amide (compound 2), N -(4-morpholinylcarbonyl)-V -(3- phen<y>l-1 -phenylsulfonylpropyl)-L-phenylalanine amide (compound 3), N ")-(4-morpholinylcarbonyl)-./V 1-(3-phenyl-1-benzylsulfonylpropyl)-L-leucinamide (compound 4 ), A^<2->(4-morpholinylcarbonyl)-V-(3-phenyl-1-trifluoromethylsulfonylpropyl)-L-phenylalanine amide (compound 5), N -(4-morpholinylcarbonyl)-A^<1->(3 -phenyl-l-benzylsulfonylpropyl)-L-phenylalanine amide (pass compound 6), A^<2->(4-morpholinylcarbonyl)-V-(3-phenyl-1-phenylsulfonylpropyl)-L-leucinamide (compound 7), N -(4-morpholinylcarbonyl)-A^ -(3- phenyl-1-fluoromethylsulfonylpropyl)-L-phenylalaninamide (compound 8), N -(4-morpholinylcarbonyl)-//1 -(3 -phenyl-1S-phenylsulfonylmethylpropyl)-L-phenylalaninamide (compound 9), Ar2-(4- morpholinylcarbonyl)-A^1-{3-phenyl-15-[2-(2-phenylethylsulfonyl)ethyl]propyl}-L-phenylalanine amide (compound 10), 7^<2->(4-morpholinylcarbonyl)-V-{ 3-phenyl-15'-[2-(2-naphthylsulfonyl)ethyl]propyl}-6-(2-naphthyl)-L-alanine amide (compound 11), Ar2-phenylacetyl-V-[3-phenyl-15-( 2-phenylsulfonylethyl)propyl]-L-phenylalaninamide (compound 12), N -(A7-benzyloxycarbonyl-6-alanyl)-V-[3-phenyl-15-(2-phenylsulfonylethyl)propyl]-L-phenylalaninamide (compound 13 ), 3-{2-phenyl-1S-[3-phenyl-1S-(2-phenylsulfonylethyl)propylcarbamoyl]ethylcarbamoyl }propionic acid (compound 14), 3-{2-naphthyl-15'-[3-phenyl-15- (2-Phenylsulfonylethyl)propylcarbamoyl]-ethylcarbamoyl}propionic acid (compound 15), N/2-(4-morpholinyl carbon yl)-Δ71-{3-phenyl-15-[2-(2-naphthylsulfonyl)ethyl]propyl}-L-tyrosinamide (compound 16), methyl 3-{2-phenyl-1S-[3-phenyl-1S- (2-phenylsulfonylethyl)propylcarbamoyl]ethylcarbamoyl}propionate (compound 17), Ar2-(4-morpholinylcarbonyl)-Arl-[3-phenyl-15'-(2-phenylethylsulfonylethyl)propyl]-L-phenylalanine amide (compound 18), A72 -(6-alanyl)-A^1-(3-phenyl-15-(2-phenylsulfonylethyl)propyl]-L-phenylalanine amide (compound 19), and 5-phenylsulfonyl-3iS-{A7-[A7-(A< 7->acetyl-L-tyrosyl)-L-valyl]-L-alanylamino}valeric acid (compound 20). Preferred compounds of Formula II include: ethyl 45-(A7-benzylsulfonyl-6-(2-naphthyl)-L-alanylamino)-6-phenylhexanoate (compound 21), ethyl 4S-(N-benzylcarbamoyl-6-(2-natyl )-L-alanylamino)-6-phenylhexano (compound 22); ethyl 4S-|>J-(4-morpholinylcarbonyl)-B-2-(naphthyl-L-alanylamino]-6-phenylhexanoate (compound 23), ethyl 4S-(N-benzylcarbamoyl-L-phenylalanylamino)-6-phenylhexanoate ( compound 24); ethyl 4S[N-(4-morpholinylcarbonyl)-L-phenylalanylamino]-L-phenylalanylamino]-6-phenylhexanoate (compound 25); N<2->(4-morpholinylcarbonyl)-N<1->[ 3-phenyl-1S-(2-phenylcarbamoylethyl)propyl]-L-phenylalanine amide (compound 26); and N<2->(4-morpholinylcarbonyl)-N<1->[3-phenyl-1S-(2-benzylcarbamoylethyl )propyl]-L-phenylalanine amide (Compound 27) Preferred compounds of Formula III include: N2-(4-morpholinylcarbonyl)-N'-{3-phenyl-1S-[2-(4-aminophenyl)ethyl]propyl} -L-phenylalanine amide (compound 29).

As is known in the art, Formula I includes structures represented by preferred compounds IV as indicated below.

where M is zero, one or two carbon atoms. A-B are as defined above, R<1>, R2, R7 and R<8> are as defined above, and Q is NH or CH2. Preferred embodiments utilize the A-B bond containing nitrogen in the B position. In this embodiment, the number of carbon atoms between the carbon to which the R group is attached and the sulfur atom of the sulfone group determines whether the compound is a ct-aminosulfone, a 6-aminosulfone or a y-aminosulfone. As indicated below in the Examples, the compounds may be named as aminosulfones by using the name in amino acids by using the chemical name.

The compounds V are thus e.g. an α-amino sulfone:

Compound VI is a 6-aminosulfone:

Compound VII is a γ-aminosulfone:

Determination of dissociation constants is well known in the field. For reversible inhibition reactions such as those in the present invention, e.g. reaction scheme as follows:

Enzyme and inhibitor combine to give an enzyme-inhibitor complex, E-I. The step is believed to be rapid and reversible, with no chemical change taking place; enzyme and inhibitor are held together by non-covalent forces. In this reaction, ki is the second-order rate constant for the formation of the E-I reversible complex. k2 has the first order rate constant for disassociation of reversible E-I complex. In this reaction, Ki= k2/ki.

Measurement of the equilibrium constant Ki takes place according to techniques well known in the field, as described in the examples. For example, the assays generally use synthetic chromogenic or fluorogenic substrates.

The respective Ki values can be estimated by using the Dixon plot as described by Irwin Segel in Enzyme Kinetics: Behavior and analysis of rapid equilibrium and steady-state enzyme systems, 1975, Wiley-Interscience Publication, John Wiley & Sons, New York, or for competitive binding inhibitors from the following equation:

Equation 4:

there

v0 is the rate of substrate hydrolysis in the absence of inhibitor, and v, is the rate in the presence of competitive inhibitor.

It is understood that the dissociation constant is a particularly useful way of quantifying the effect of an enzyme with a particular substrate or inhibitor, and is often used within the field as it is. If an inhibitor exhibits a very low Ki, it is an effective inhibitor. The cysteine protease inhibitors from the present invention have dissociation constants, Ki, at most approx. 100 µM. Preferred embodiments have inhibitors that exhibit dissociation constants at most about 10 uM, where the most preferred embodiments have dissociation constants that are mostly approx. 1 uM.

CHEMISTRY

Synthesis of inhibitors from the present invention takes place as follows. Compounds of Formula I in which X represents a bond can be prepared by the method shown in Scheme 1 in Figure 1.

Treatment of tert-butyl carbamate or benzyl carbamate with a suitable aldehyde, such as isobutyraldehyde or hydrocinnamaldehyde, together with the sodium salt of a suitable sulphidic acid, such as benzene sulphidic acid (Aldrich Chemical Co.), in the presence of aqueous formic acid gives the corresponding N-protected aminomethylsulfone. Benzyloxycarbonyl-protected aminomethylsulfone is deprotected with hydrogen bromide in acetic acid. Coupling with a suitable N-protected amino acid or peptide or a peptide domimetic derivative thereof gives a compound of Formula I wherein X represents a bond. Alternatively, a suitable N-terminally protected amino acid or peptidomimetic derivative peptide thereof, such as N-(4-morpholinylcarbonyl)phenylalanine amide, is reacted with a suitable aldehyde together with the sodium salt of a suitable sulphidic acid, in the presence of aqueous formic acid to give a compound of Formula I wherein X represents a bond.

Compounds with Formula I in which X represents a methylene bond can be prepared by the method shown in Schemes 2 and 3, respectively Figures 2 and 3.

Treatment of an appropriate N-protected amino acid or peptidomimetic derivative thereof with sodium borohydride yields the corresponding B-aminoethanol. Treatment of the alcohol with methanesulfonyl chloride in the presence of triethylamine gives the corresponding mesylate. Nucleophilic replacement with the anion of a thiol, such as thiophenol, according to the method of Spaltenstein, A., Carpion, P., Miyake, F., and Hopkings, P.B., J. Org.Chem. (1987) 52, 3759, gives the corresponding B-amino sulphide. The sulfide is oxidized using 4-chloroperbenzoic acid to give the corresponding N-protected B-aminoethylsulfone. In a particular case, the mesylate is treated with the thiolation such as that derived from 2-(trimethylsilyl)ethanethiol, the synthesis of which is described by Anderson, M.B., Ranasinghe, M.B:, Palmer, J.T., Fuchs, P.L. J. Org. Chem. (1988) 53, 3125, to give the corresponding B-aminoethyl 2-trimethylsilylethyl sulfide. The 2-trimethylsilylethyl sulfide is reduced to the corresponding B-aminoethyl 2-trimethylsilylethyl sulfone, which is then subjected to fluoride-mediated cleavage, extrusion with trimethylsilyl fluoride and ethylene as a gaseous byproduct, and an intermediate sulfinate, where the sulfinate is alkylated in situ with appropriate halogen-containing compounds such as bromochloromethane to give the corresponding N-protected-B-aminoethyl halomethyl sulfone. N-protected B-aminoethylsulfone is deprotected and then coupled with a suitable N-protected amino acid or peptide or a peptidomimetic derivative thereof to give a compound of Formula I wherein X is methylene.

Compounds of Formula II and Formula I in which X represents ethylene can be prepared by methods shown in Equations 5, 6 and 7.

Equation 5

where a) is a) Cl-H.2N+(Me)0Me, dicyclohexylcarbodiimide, triethylamine; and b) lithium aluminum hydride.

Equation 6

An appropriate N-tert-butoxycarbonylamino acid or peptidomimetic derivative thereof was converted to the corresponding aminomethylaldehyde (e.g., see the method of Fehrentz, J-A. and Castro, B. (Synthesis, (1983), 676; Equation 5). The aldehyde was converted to corresponding vinyl-containing compound via a Wittig reaction or a Wadsworth-Emmons-Horner modification of the Wittig reaction (see, e.g., Wadsworth et al., J. Amer. Chem. Soc. 83; 1733 (1991); Equation 6). the compound was reduced by catalytic hydrogenation (e.g. see Equation 7) and then deprotected and coupled with a suitable N-protected amino acid or peptide or peptidomimetic derivative thereof giving the corresponding compound of Formula I or II. Alternatively, the vinyl-containing compound is deprotected and coupled with N-protected amino acid or peptide or a peptidomimetic derivative thereof to give the corresponding vinyl-containing condensation product, which is then reduced to give the corresponding compound of Formula I.

Conversion of N-tert-butoxycarbonylamino acid or its peptidomimetic derivative to the corresponding aminomethylaldehyde is preferably carried out with N,0-dimethylhydroxylamine hydrochloride in the presence of triethylamine and dicyclohexylcarbodiimide in dichloromethane. Alternatively, the conversion is accomplished by treating the amino acid or peptidomimetic derivative with triethylamine and the coupling agent benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and then reduced with lithium aluminum hydride to give the corresponding aldehyde (see, e.g., the method of Fehrentz, J-A. and Castro, B.; Synthesis, (1983), 676-678). Conversion of the aldehyde to the corresponding vinyl-containing ester can be carried out with the sodium anion of triethylphosphonic acetate. Deprotection of the vinyl-containing ester can be carried out with hydrogen chloride in dioxane. The hydrogenation is typically carried out in the presence of palladium.

Compounds with Formula I in which X is ethylene are suitably prepared by the method shown in Scheme 5, Figure 5.

Treatment of a suitable N-tert-butoxycarbonyl-α-aminoaldehyde, prepared as described in Equation 5, with the sodium anion of the appropriate sulfonylmethane phosphate (SMP)

(eg diethyl phenylsulfonylmethane phosphonate diethyl 2-naphthylsulfonylmethane phosphonate, diethyl methylsulfonylmethane phosphonate, etc.) gives the corresponding vinyl-containing sulfone. The sulfone is deprotected with anhydrous p-toluenesulfonic acid in ether and then coupled with N-protected amino acid or peptide or a peptidomimetic derivative thereof to give the corresponding vinylic condensation product, which is then reduced to give the corresponding compound of Formula I. Suitable arylsulfonylmethanephosphonates can be prepared by treating aryl thiols with paraformaldehyde in the presence of hydrogen chloride and reacting with triethyl phosphite to give the corresponding diethylphosphonomethylaryl sulfide and then oxidizing the sulfide. Alternatively, suitable sulfides can be obtained commercially (eg, diethylphosphonomethylmethylsulfide obtained from Aldrich Chemical Co., diethylphosphonomethylphenylsulfide, etc.) and oxidized to their corresponding sulfones.

Structure 1

Synthesis of ketones is carried out using Wadswori. 2mmon reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, followed by catalytic reduction with hydrogen in the presence of palladium. In general, the aldehyde moiety is synthesized as indicated above. The phosphonate, if not commercially available, is synthesized by treating the enolate anion of methyl or substituted methyl ketones, such as acetone or acetophenone, with diethyl chlorophosphonate. The enolate anion is generated e.g. by treating the tetrahydrofuran solution of diisopropylamine with butyllithium, followed by addition of ketone to lithium diisopropylamide (LDA) solution (H.O. House, Modern Synthetic Reactions, 2nd Ed. (W. Benjamin, Inc., Menlo Park, CA, Chapter )). After the formation of the enolate, diethyl chlorophosphonate is added. Wadsworth-Emmons reagent forms as a consequence of coupling of enolate with diethyl chlorophosphate.

For the synthesis of cysteine protease inhibitors with nitriles such as EWG, Structure II is used:

Structure II

Synthesis of nitriles is carried out by means of the Wadsworth-Emmons reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst. In general, the aldehyde moiety is synthesized as indicated above. The phosphonate is commercially available.

For the synthesis of cysteine protease inhibitors with sulfoxides such as EWG, structure III is used

Structure III

Synthesis of sulfoxides was carried out by means of the Wadsworth-Emmons reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst. The aldehyde moiety is generally synthesized as indicated above. Phosphonate is synthesized by treating the anion of methyl sulfoxides with diethyl chlorophosphate. The anion is generated by the addition of BuLi to diisopropylamine, followed by the addition of methyl sulfoxide.

For the synthesis of cysteine protease inhibitors with sulfonamides such as EWG, structure IV is used:

Structure IV

Synthesis of the sulfonamides is carried out by means of the Wadsworth-Emmons reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst. The aldehyde moiety is generally synthesized as indicated above. Phosphonate is synthesized, e.g. by a method such as the following: a) diethylphosphoryl methanesulfonates, as prepared by the method of Carretero and Ghosez (Tetrahedron Lett., 28, 1104-1108 (1987)), and converted to sulfonyl chlorides by treatment with phosphorus pentachloride (M. Quaedvlieg, in "Method der Organische Chemic (Houben-Weyl)", ed. E. Muller, Thieme Verlag, Stuttgart, 4th Ed., 1955, Vol. IX, Chapter 14); or b) treating the sulfonyl chloride with an amine, such as ammonia, a primary amine (including an amino acid derivative), or a secondary amine, resulting in the formation of the sulfonamide (Quaedvilieg, supra, Chapter 19). The sulfonamide phosphonate was then reacted with Boc-α-aminoaldehydes to form the target compounds as in the Wadsworth-Emmons reaction.

For the synthesis of cysteine protease inhibitors with sulfinamides such as EWG, Structure V is used:

Structure V

Synthesis of sulfinamides is carried out by means of the Wadsworth-Emmons reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst. In general, the aldehyde moiety is synthesized as indicated above. Phosphonate can be synthesized using one of the following methods. Treatment of methyl dialkyl phosphonates such as commercially available methyl diethyl phosphonate (Aldrich) with thionyl chloride in the presence of aluminum chloride gives dialkyl phosphoryl methanesulfinyl chloride (Vennstra et al., Synthesis (1975) 519. See also Anderson, "Comprehensive Organic Chemistry (Pergamon Press)", Vol. 3, Chapter 11.18, (1979).Alternatively, treatment of dialkylphosphorylsulfinyl chloride with amines (Stirling, Internat. J. Sulfur Chem. (B) 6: 277 (1971)) gives dialkylphosphorylsulfinamide.

For the synthesis of cysteine protease inhibitors with sulfoximines such as EWG, structure VI is used:

Structure VI

Synthesis of sulfoximines is accomplished by the Wadsworth-Emmons reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst. In general, the aldehyde moiety is synthesized as indicated above. Phosphonate can be synthesized in several ways. For example, N-alkyl or N-arylphenylmethylsulfoximines are made by the method described by Johnson in "Comprehensive Organic Chemistry (Pergamon Press), supra, Chapter 11.11. Alternatively, the lithium anion of compounds such as N-alkylphenylmethylsulfoximine is prepared by treating the neutral compound with butyl lithium in THF (Cram et al., J. Amer. Chem. Soc. 92: 7369 (1970)). Reaction of this lithium anion with dialkyl chlorophosphates such as commercially available diethyl chlorophosphate (Aldrich) results in the Wadsworth-Emmons reagent required for the synthesis of the sulfoximine compounds .

For the synthesis of the cysteine protease inhibitors with sulfonates such as EWG, structure VII is used:

Structure VII

Synthesis of sulphonates is carried out by means of the Wadsworth-Emmons reaction between Boc-α-aminoaldehydes and the appropriate phosphonate, e.g. diethyl phosphoryl methanesulfonate, followed by hydrogenation in the presence of a suitable catalyst, such as Raney nickel. The phosphonate can be synthesized as follows. The anion of methyl dialkyl phosphonates such as commercially available methyl diethyl phosphonate (Aldrich) is generated by treating this phosphonate with a strong base such as LDA. The resulting anion is sulfonated with sulfur trioxide/trimethylamine complex (Carreto et al., Tetrahedron Lett., 28: 1104-1108 (1987)) to form diethyl phosphoryl methanesulfonate, which is capable of reacting in the Wadsworth-Emmons procedure with aldehydes to form a ,B-unsaturated sulfonates.

The chloride compounds containing R 5 and R 9 groups are generally made using commercially available reagents and products using techniques well known in the art. The reaction generally produces a mixture of cis and trans configurations, with the trans isomer favored. Upon reduction to the cysteine protease inhibitors in this embodiment, the cis-trans isomerism disappears by definition and a single compound is formed.

The cysteine protease inhibitors are further purified if necessary after synthesis, e.g. to remove unreacted materials. The cysteine protease inhibitors in the present invention can e.g. be crystallized, or passed through silica chromatography columns using solvent mixtures to elute pure inhibitors.

In one embodiment, the cysteine protease inhibitors in the present invention are labeled. By "labeled cysteine protease inhibitor" is meant here a cysteine protease inhibitor that has at least one element, isotope or chemical compound attached to enable detection of the cysteine protease inhibitor or the cysteine protease inhibitor bound to a cysteine protease. In general, the markers fall into three classes: a) isotopic markers, which may be radioactive or heavy isotopes; b) immune markers which may be antibodies or antigens; and c) colored or fluorescent colors. The markers can be incorporated into the cysteine protease inhibitor at any position. A marker can e.g. be attached as the "R<1>" group in Formula 1, or a radioisotope incorporated in any position. Examples of useful markers include <14>C, <3>H, biotin, and fluorescent markers well known in the art.

PHARMACOLOGY AND UTILIZATION

Once produced, the cysteine protease inhibitors of the present invention can be easily screened for their inhibitory effect. The inhibitor is first tested against the cysteine protease for which the targeting group of the inhibitor was chosen, as indicated above. Alternatively, many cysteine proteases and their corresponding chromogenic substrates are commercially available. Numerous cysteine proteases are thus routinely assayed with synthetic chromogenic substrates in the presence and absence of the cysteine protease inhibitor, to confirm the inhibitory action of the compound, using techniques well known in the art. Effective inhibitors were then subjected to kinetic analysis to calculate Ki values, and dissociation constants were determined.

If a compound inhibits at least one cysteine protease, it is a cysteine protease inhibitor in the context of the present invention. Preferred embodiments have inhibitors that exhibit correct kinetic parameters against at least one targeted cysteine protease.

In some cases, the cysteine protease is not commercially available in a purified form. The cysteine protease inhibitors in the present invention can also be analyzed for effect by using biological analyses. The inhibitors can e.g. be added to cells or tissues containing cysteine protease, and the biological effects are measured.

In one embodiment, the cysteine protease inhibitors of the present invention are synthesized or modified so that in vivo and in vitro proteolytic degradation of the inhibitors is reduced or prevented. This is generally done by incorporating synthetic amino acids, derivatives or substituents into the cysteine protease inhibitor. Only a non-naturally occurring amino acid or amino acid side chain is preferably incorporated into the cysteine protease inhibitor, so that targeting of the inhibitor to the enzyme is not significantly affected. Some embodiments using longer cysteine protease inhibitors containing a number of targeting residues may tolerate more than one synthetic derivative. In addition, non-naturally occurring amino acid substituents can be designed to mimic binding of naturally occurring side chains to the enzyme, so that more than one synthetic substituent is tolerated. Alternatively, peptide isosteres are used to reduce or prevent degradation of the inhibitor.

In this embodiment, resistance of modified cysteine protease inhibitors can be tested against a wide variety of known commercially available proteases in vitro to determine their proteolytic stability. Promising candidates can then be routinely screened in animal models, e.g. by using labeled inhibitors, to determine in vivo stability and efficacy.

Specific cysteine proteases that can be inhibited by the inhibitors of the present invention are those from the family of cysteine proteases that carry a thiol group in the active site. These proteases are found in bacteria, viruses, eukaryotic microorganisms, plants and animals. Cysteine proteases can generally be classified by belonging to one to four or more distinct superfamilies. Examples of cysteine proteases that can be inhibited by the novel cysteine protease inhibitors of the present invention include, but are not limited to, plant cysteine proteases such as papain, ficin, aleurain, oryzain and actinidain; mammalian cysteine protease such as cathepsins B, H, J, N, S, T, O and C (cathepsin C is also known as dipeptidyl peptidase I), interleukin converting enzyme (ICE), calcium-activated neutral proteases, calpain I and II; bleomycin hydrolase, viral cysteine proteases such as picornian 2A and 3C, aphthovirus endopeptidase, cardiovirus endopeptidase, comovirus endopeptidase, potyvirus endopeptidases I and II, adenovirus endopeptidase, two chestnut disease virus endopeptidases, togavirus cysteine endopeptidase as well as poliovirus and rhinovirus cysteine proteases; and cysteine protease known to be essential for parasite life cycle, such as proteases from species of Plasmodia, Entamoeba, Onchocera, Trypansoma, Leishmania, Haeonchus, Dictystelium, Therileria and schistosoma, such as those associated with malaria (P. falciparium), trypanosomes ( T. cruzi, the enzyme is also known as cruzain or cruzipain), mouth P. vinckei and C. elegans cysteine protease. For a comprehensive listing of cysteine proteases that can be inhibited by the cysteine protease inhibitors of the present invention, see Rawlings et al., Biochem. J. 290: 205-218 (1993), herein expressly incorporated by reference.

The inhibitors of cysteine proteases are thus useful in numerous applications. The inhibitors in the present invention are thus used to quantify the amount of cysteine protease present in a sample, and are thus used in analyzes and diagnostic kits for the quantification of cysteine proteases in blood, lymph, saliva or other tissue samples, in addition to bacterial, fungal, plant, yeast, viral or mammalian cell cultures.

Thus, in a preferred embodiment, the sample is analyzed to use a standard protease substrate. A known construction of cysteine protease inhibitor is added, and is given the opportunity to bind to a special cysteine protease that is present. The protease assay is then rerun, and loss of activity is checked for cysteine protease activity using techniques well known in the art.

The cysteine protease inhibitors are also useful for removing or inhibiting contaminating cysteine proteases in a sample. The cysteine protease inhibitors in the present invention are e.g. added to samples where proteolytic degradation due to contamination by cysteine proteases is undesirable. The cysteine protease inhibitors of the present invention may alternatively be bound to a chromatographic support, using techniques well known in the art, to form an affinity chromatography column. A sample containing an unwanted cysteine protease is run through the column to remove the protease.

In a preferred embodiment, the cysteine protease inhibitors are useful for inhibiting cysteine proteases implicated in a number of diseases. In particular, cathepsins B, L and S, cruzain, calpain I and II, and interleukin lii converting enzyme are inhibited. These enzymes are examples of lysosomal cysteine proteases implicated in a wide spectrum of diseases characterized by tissue breakdown. Such diseases include, but are not limited to, arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, parasite-borne infections, Alzheimer's disease, periodontal disease, and cancer metastases. Mammalian lysosomal thiol proteases play e.g. an important role in intracellular degradation of proteins and in the processing of some peptide hormones. Enzymes similar to cathepsins B and L are released from tumors and may be involved in tumor metastasis. Cathepsin L is present in diseased human synovial fluid and transformed tissue. Release of cathepsin B and other lysosomal proteases from polymorphonuclear granulocytes and macrophages is similarly observed in trauma and inflammation.

The cysteine protease inhibitors also find application in many other diseases, including but not limited to gingivitis, malaria, leishmaniasis, filariasis and other bacterial and parasite-borne infections. The compounds can also be used in viral diseases, based on the way of inhibiting proteases necessary for viral replication. Many picornoviruses including poliovirus, foot-and-mouth disease virus, and rhinovirus encode cysteine proteases essential for cleavage of viral polyproteins.

In addition, these compounds offer the possibility of application in disorders involving interleukin-lli converting enzyme (ICE), a cysteine protease responsible for processing interleukin lii; e.g. in the treatment of inflammation and immune-based disorders in the lung, respiratory tract, central nervous system and surrounding membranes, eyes, ears, joints, bones, connective tissue, cardiovascular system including pericardium, gastrointestinal and urogenital systems, skin and mucosal membranes. These conditions include infectious diseases where the active infection exists in any body site, such as meningitis and salpingitis; complications of infections including septic shock, disseminated intravascular coagulation, and/or adult respiratory distress syndrome; acute or chronic inflammation due to antigen, antibody and/or complement deposition; inflammatory conditions including arthritis, chalangitis, colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion injury and vasculitis. Immune-based diseases include, but are not limited to conditions involving T cells and/or macrophages such as acute and delayed hypersensitivity, graft rejection, and graft-versus-host disease; auto-immune diseases including Type I diabetes mellitus and multiple sclerosis. Bone and cartilage absorption as well as disease resulting in extensive deposition of extracellular matrix such as interstitial pulmonary fibrosis, cirrhosis, systemic sclerosis and keloid formation can also be treated with the inhibitors of the present invention. The inhibitors may also be useful in the treatment of certain tumors that produce IL 1 as an autocrine growth factor and in the prevention of cachexia associated with certain tumors. Apoptosis and cell death are also associated with ICE and ICE-like activities and can be treated with the inhibitors of the present invention.

Furthermore, the cysteine protease inhibitors from the present invention find use in drug potentiation applications. Therapeutic agents such as antibiotic or antitumor drugs can e.g. be inactivated through proteolysis by endogenous cysteine proteases, thus rendering the administered drugs less effective or inactive. It has e.g. has been shown by bleomycin, an antitumor drug, to be hydrolyzed by bleomycin hydrolase, a cysteine protease (see Sebti et al., Cancer Res. January 1991, pages 227-232). The cysteine protease inhibitors from the invention can thus be administered to a patient in connection with a therapeutic agent to potentiate or increase the activity of the drug. This co-administration may be by simultaneous administration, such as a mixture of the cysteine protease inhibitor and the drug, or by separate simultaneous or sequential administration.

In addition, the cysteine protease inhibitors have been shown to inhibit the growth of bacteria, particularly human pathogenic bacteria (see Bjorck et al., Nature 337: 385 (1989)). The cysteine protease inhibitors in the present invention can thus be used as antibacterial agents to retard or inhibit the growth of certain bacteria.

The cysteine protease inhibitors in the invention also find use as agents for reducing damage caused by bacterial cysteine protease to the host organism. Staphylococcus produces a very active extracellular cysteine protease that breaks down insoluble elastin, and possibly contributes to connective tissue destruction seen in bacterial infections such as septicaemia, septic arthritis and otitis media. See Potempa et al., J. Biol. Chem. 263 (6); 2664-2667 (1988). The cysteine protease inhibitors from the present invention can thus be used to treat bacterial infections such as internal tissue damage.

ADMINISTRATION AND PHARMACEUTICAL COMPOSITION

The cysteine protease inhibitors of the invention will generally be administered in therapeutically effective amounts via any of the usual and acceptable ways known in the art, either singly or in combination with other cysteine protease inhibitors from the invention or with another therapeutic agent. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound being used and other factors. Therapeutically effective amounts of the cysteine protease inhibitors in the invention can be in the range from 10 micrograms per kilogram body weight (ug/kg) per day to 1 milligram per kilogram body weight (mg/kg) typically 100 ug/kg/day to 1 mg/kg/day. A therapeutically effective amount for an 80 kg human can range from 1 mg/day to 1000 mg/day, typically 10 mg/day to 100 mg/day.

Someone with knowledge in the field of treatment of such diseases will have the ability, without unreasonable experimentation and based on personal knowledge and the description of this invention, to be able to determine a therapeutically effective amount of a cysteine protease inhibitor from the invention for a given disease.

The cysteine protease inhibitors of the invention will generally be administered as pharmaceutical compositions by one of the following routes: oral, systemic, (e.g. transdermal, intranasal, intrapulmonary or by suppository) or parenteral (e.g. intramuscular, intravenous, intrapulmonary or subcutaneous). The composition may be taken in the form of tablets, pills, capsules, semi-solids, powders, sustained release formulation, solution, suspensions, elixirs, aerosols or any other suitable composition and is generally comprised of a cysteine protease inhibitor of the invention in combination with at least one pharmaceutical acceptable excipient. Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the cysteine protease inhibitor of the invention. Such excipients can be any solid, liquid, semi-solid, or in this case with an aerosol composition, gaseous excipient generally available to those skilled in the art.

Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, lime, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skimmed milk and the like. Liquid and semi-solid excipients may be selected from water, ethanol, glycerol, propylene glycol, and various oils, including those of petroleum, animal, vegetable, or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil). Preferred liquid carriers, particularly for injectable solutions including water, saline, aqueous dextrose and glycols.

Compressed gases can be used to disperse the cysteine protease inhibitor in the invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, nitrogen oxides, etc. Other suitable pharmaceutical carriers and their formulations are described in A. R. Alfonso Reminton's Pharmaceutical Sciences 1985, 17th ed. Eason, Pa.: Mack Publishing Company, hereby expressly incorporated by reference.

The amount of the cysteine protease inhibitor in the invention in composition can vary widely depending on the type of formulation, size of unit dose, type of excipients and other factors known within the field of pharmaceutical research. The final composition will generally comprise from 0.1% v to 10% v of the cysteine protease inhibitor, preferably 1% v to 10% v, the remainder being excipient or excipients.

The pharmaceutical composition is preferably administered in a single unit dosage form for continuous treatment or in a single unit dosage form ad libitum when relief of the symptoms is specifically desired. Representative pharmaceutical formulations containing a cysteine protease inhibitor of the invention are described in Example 20, below.

The following examples serve to describe the manner in which the invention described above is used, as well as to indicate the best ways to implement the various features of the invention. It is understood that these examples are in no way intended to limit the scope of the invention, but rather are given for illustrative purposes. All references cited herein are expressly incorporated by reference.

The following abbreviation conventions have been used to simplify the examples.

Mu = morpholinurea

Xaaj = amino acid in the Pl position relative to the active site of the enzyme

Xaa2 = amino acid in P2 position relative to active site of enzyme y-C02Et = y-aminoethyl ester

Y-SO2PI1 = y-aminosulfone with a phenyl terminal

y-CO 2 H = y-aminocarboxylate

γ-PEt = γ-aminophosphonate

γ-AM = γ-aminoamide

y-Ar(sub) = y-aminoaromatic compound (substituted as appropriate)

6-SO2Ph = B-aminosulfone with phenyl substituent

ot-SC>2Ph = oc-aminosulfone with phenyl substituent

Hph = homophenylalanine

PSMP = diethylphenylsulfonylmethylenephosphonate

Np2 = 2-naphthylalanine

SO22Np = sulfone with 2-maphthyl terminus

Phac = phenylacetyl

B-Ala = J-alanine

MeOSuc = methoxysuccinyl

Mu-Phe-Hph-fl-SC^Ph where Xaa2 = Phe (phenylalanine) and Xaai = Hph (homophenylalanine), transformed to J5-aminophenylsulfone according to the procedure described in the examples.

Example 1

Synthesis of cysteine protease inhibitor containing a γ-amino ester as in EWG

Unless otherwise stated, all reactions were carried out under an inert atmosphere of argon or nitrogen at room temperature. THF was distilled from sodium benzophenone ketyl. All other solvents and commercially available reagents were used without further purification.

Synthesis of ethyl (S)-4-(4-mofrolinecarbonyl-phenylalanyl)-amino-6-phenylhexanoate, abbreviated Mu-Phe-Hph-y-CC^Et, was as follows. Unless otherwise noted, all reagents were obtained from Aldrich, Inc. 0.393 g of a 60% mineral oil dispersion (9.82 mmol) of sodium hydride was added to a solution of triethylphosphonoacetate (2.20 g, 9.82 mmol) in THF (50 ml) at -10°C. The mixture was stirred for 15 min, whereby a solution of Boc-homophenylalanine (Boc-HphH) (2.35 g, 9.82 mol), prepared by conversion of Boc-homophenylalanine (Synthetech) to its N,0-dimethylhydroxamide, at using the Fehrentz method, followed by lithium aluminum hydride reduction) in THF (20 mL) was added. The mixture was stirred for 45 minutes. 1M HCl (30 mL) was added. The product was extracted with ethyl acetate (50 mL), washed with saturated aq. HCl in dioxane (20 mL) was added. The mixture was stirred for 30 min. Solvent was removed under reduced pressure and the residue, ethyl (S)-4-amino-6-phenyl-2-hexenoate hydrochloride, was pumped dry.

4-morpholinecarbonylphenylalanine (Mu-PheOH, 2.74 g, 9.82 mmol, prepared according to the method described in Esser, R. et al., Arthritis & Rheumatism (1994), 37, 236) was dissolved in THF ( 50 ml) at -10°C. 4-Methylphorfoline (1.08 mL, 9.82 mmol) was added, followed by isobutyl chloroformate (1.27 mL, 9.82 mmol). The mixed aldehyde was stirred for 10 min, upon which a solution of ethyl (S)-4-amino-6-phenyl-2-hexenoate hydrochloride from the previous step in DMF (10 mL) was added, followed by 4-methylmorpholine (1 .08 ml, 9.82 mmol). The mixture was stirred for 1 hour. 1M HCl (50 mL) was added. The product was extracted with ethyl acetate (100 mL), washed with saturated aqueous NaHCO 3 (50 mL), dried over MgSO 4 and decolorized charcoal (DARCO), filtered and

evaporated to dryness to give 3.80 g of the intermediate (80% yield from Boc-homo-phenylalaninal).

To a solution of this intermediate (1.45 g, 3.09 mmol) in ethanol (25 mL) was added 5% palladium on activated carbon (0.5 g). The mixture was reduced on a Parr hydrogenator for 36 hours. The solution was filtered and solvent was removed under reduced pressure, yielding 1.19 g (82%) of the product.

Thin layer chromatography (TLC) was performed on each sample. Visualization was carried out using UV light at 254 nm, followed by ninhydrin, bromocresol green or p-anisaldehyde dye. The retention factor (Rf) of Mu-Phe-Hph-γ-CO 2 Et was 0.35 (5% MeOH/CH 2 Cl 2 ).

NMR spectra were recorded on a Varian Gemini 300 Mhz instrument. All <*>H NMR data from this and subsequent examples are reported as delta values in parts per million relative to internal tetramethylsilane, top acquisitions in bold type. The following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; br, broad. An asterisk (<*>) implies that a signal is indistinct or covered by other resonance.

Example 2

Synthesis of a cysteine protease inhibitor containing a γ-aminosulfone as EWG.

Synthesis of (S)-3-tert-butoxycarbonylamino-5-phenyl-l-phenylsulfonylpentane (Boc-Hph, y-SO2Ph). To a solution of PSMP (8.87 g, 30.34 mmol) in THF (150 mL) at 0°C was added sodium hydride (1.21 g of a 60% mineral oil dispersion). The mixture was stirred for 20 minutes, whereupon a solution of Boc-homophenylalaninal, synthesized by the method of Fehrentz and Castro, above, (7.99 g, 30.34 mmol) in THF (20 mL) was added. The solution was stirred for 30 minutes at 0°C. 1M HCl (100 mL) was added. The product was extracted into ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (100 mL), brine (50 mL), dried over MgSO 4 , filtered and solvent removed under reduced pressure. The residue was dissolved in ethanol (100 ml) and transferred to a Parr flask filled with 5% palladium on activated charcoal (0.92 g). The mixture was reduced in a Parr apparatus for 24 hours. The solution was filtered through Celite and solvent was removed under reduced pressure. TLC of the product showed a single product in quantitative yield, Rf = 0.29 (30% ethyl acetate/hexane) as colored white with paraanisaldehyde spray.

Example 3

Synthesis of a cysteine protease inhibitor containing a γ-aminosulfone as EWG.

Synthesis of (S)-3-amino-5-phenyl-l-phenylsulfonyl-pentane hydrochloride (HCl.Hph-y-SO2Ph). To a solution of Boc-Hph-γ-SO 2 Ph (12.24 g, 30.34 mmol) in dichloromethane (20 mL) was added hydrogen chloride in dioxane (50 mL of a 4.0 M solution). The mixture was stirred for 90 minutes. Solvent was removed under reduced pressure and the residue was dissolved in CH 2 Cl 2 (50 mL). Ether (500 mL) was carefully added to the solution with stirring. The solid was filtered, washed with ether (50 mL) and dried in vacuo.

Example 4

Synthesis of a cysteine protease inhibitor containing a γ-aminosulfone as EWG.

Synthesis of (S)-3-(4-morpholinecarbonylphenylalanyl)-amino-5-phenyl-1-phenylsulfonylpentane (Mu-Phe-Hph-y-SO2Ph). To a solution of Mu-PheOH (2.94 g, 10.56 mmol) in THF (75 mL) at -10°C was added 4-methylmorpholine (1.16 mL, 10.56 mmol) and isobutyl chloroformate (1 .37 ml, 10.56 mmol). The mixture was stirred for 5 minutes. (S)-(E)-3-amino-5-phenyl-l-phenylsulfonyl-1-pentene p-toluenesulfonate, synthesized by Wadsworth-Emmons condensation between Boc-homophenylalaninal and p-toluenesulfonic acid deprotection (5.00 g, 10.56 mmol) was added, followed by 4-methylmorpholine (1.16 mL, 10.56 mmol). The mixture was stirred for 45 minutes. The solution was diluted with ethyl acetate (100 mL), washed with 1M HCl (2x50 mL), saturated aqueous sodium bicarbonate (50 mL), brine (50 mL), dried over MgSO 4 , filtered and solvent removed under reduced pressure. The residue was crystallized from CH 2 Cl 2 /ether to give 4.27 g (72%) of the intermediate. 1.17 g of this material (2.08 mmol) was dissolved in ethanol (25 mL). The solution was transferred to a Parr bottle filled with 5% palladium on activated charcoal (0.30 g). The mixture was hydrogenated at room temperature overnight on a Parr shaker. Ethyl acetate was added to the suspension of the product, which had crystallized from the reaction mixture. The solution was filtered and concentrated in vacuo and then recrystallized from CH 2 Cl 2 /hexane. Temp. = 176

- 178°C. TLC: (50% ethyl acetate/CH 2 Cl 2 ) Rf = 0.24.

Example 5

Synthesis of cysteine protease inhibitor containing a γ-aminosulfone such as EWG.

Synthesis of (S)-3-(4-morpholinecarbonyltyrosyl)-amino-5-phenyl-1-phenylsulfonylpentane (Mu-Tyr-Hph-y-SChPh). To a solution of 4-morpholinecarbonyltyrosine (Mu-TyrOH, synthesized according to the method described in Esser, R. et al., Arthritis & Rheumatism (1994), 37.236, 0.50 g, 1.70 mmol) in THF ( 10 ml) at -10°C was added 4-methylmorpholine (0.187 ml, 1.70 mmol) and isobutyl chloroformate (0.220 ml, 1.70 mmol). After 5 min, HCl.Hph-γ-SO 2 Ph (0.577 g, 1.70 mmol, described in Example 3) added, followed by 4-methylmorpholine (0.187 mL, 1.70 mmol). The mixture was stirred for 45 minutes. Ethyl acetate (50 mL) was added. The solution was washed with 1M HCl, saturated aqueous sodium bicarbonate and brine (30 mL each), dried over MgSO 4 , filtered, and solvent was removed under reduced pressure. The residue was precipitated from C 2 C 2 /ether to give 0.58 g (59%) of Mu-Tyr-Hph-y-SO 2 Ph. Temp. 104 - 107°C. TLC: (10% MeOH/CH 2 Cl 2 ) Rf = 0.59.

Example 6

Synthesis of a cysteine protease inhibitor containing a γ-aminosulfone as EWG.

Synthesis of (S)-3-(4-morpholinecarbonyl-2-naphthyl-alanyl)amino-5-phenyl-1-(2-naphthylsulfonyl)pentane (Mu-Np2-Hph-y-SO22Np). 2-Naphthalenethiol (9.64 g, 60.16 mmol) was dissolved in toluene (75 mL). Paraformaldehyde (3.97 g, 132 mmol) and HCl/dioxane (33 mL of a 4.0 M solution) were added. The mixture was stirred for several days at room temperature. Solvent was removed under reduced pressure and the residue was suspended in hexane (200 mL), dried over MgSO 4 , filtered and evaporated to dryness. The material, crude chloromethyl 2-naphthyl sulfide, was combined with triethyl phosphite (10.93 g, 65 mmol) and heated at reflux for 4 hours. The mixture was cooled to room temperature, diluted with ether (200 mL), washed with 1M HCl, saturated aqueous sodium bicarbonate and brine (150 mL each), dried over MgSO 4 , filtered and concentrated in vacuo to give 17.35 g (93% crude yield ) of diethyl 2-naphthylthiomethylene phosphonate. This material was dissolved in CH 2 Cl 2 (300 mL) and cooled to 0°C. Peracetic acid (23.5 ml of a 32% dilute acetic acid solution) was carefully added. The mixture was stirred overnight while warming to room temperature. The solution was washed with freshly prepared saturated aqueous sodium bisulfite solution (100 mL), then with several portions of saturated aqueous sodium bicarbonate, until the aqueous phase became basic. The organic phase was dried over MgSO 4 , filtered and solvent was removed under reduced pressure. Chromatography on 60 - 200 mesh silica gel (0 -10% ethyl acetate/CH2Cl2) gave 6.5 g

(34%) of pure Wadsworth-Emmons reagent, diethyl 2-naphthylsulfonyl methylene phosphonate together with an appropriate equal amount of impure material. TLC: (20% ethyl acetate/C₂C₂) Rf = 0.37.

To a solution of diethyl 2-naphthylsulfonyl methylene phosphonate (3.91 g, 11.42 mmol) in THF (60 mL) at 0°C was added sodium hydride (0.457 g of a 60% mineral oil dispersion). The mixture was stirred for 15 min, whereupon a solution of Boc-homophenylalaninal (3.00 g, 11.42 mmol) in THF (5 mL) was added. The mixture was stirred for 30 minutes. 1M HCl (100 mL) was added. The product was extracted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (75 mL), brine (50 mL), dried over MgSO 4 , filtered and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (10 ml), to which was added HCl/dioxane (25 ml of a 4.0 M solution). The mixture was stirred at room temperature for 1 hour, poured into ether (300 mL), and filtered. The solids were washed with ether (2x50 mL) and dried in vacuo to give 3.30 g (74% from Boc-homophenylalaninal) of (S)-(E)-3-amino-5-phenyl-1-( 2-naphthylsulfonyl)-1-pentene.

To a solution of Boc-2-naphthylalanine (2.68 g, 8.51 mmol), (Synthetech, Oregon) in THF (50 mL) at -10°C was added 4-methylmorpholine (0.936 mL, 8.51 mmol) and isobutyl chloroformate (1.103 mL, 8.51 mmol). The mixture was stirred for 5 minutes, after which (S)-(E)-3-amino-5-phenyl-1-(2-naphthylsulfonyl)-1-pentene (3.30 g, 8.51 mmol) was added, followed by of 4-methylmorpholine (0.936 mL, 8.51 mmol). The mixture was stirred for 45 min, diluted with ethyl acetate (100 mL), washed with 1M HCl (50 mL), saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over MgSO 4 , filtered and solvent removed under reduced pressure . The intermediate (S)-(E)-3-(tert-butoxycarbonyl-2-naphthylalanyl)amino-5-phenyl-1-(2-naphthylsulfonyl)-1-pentene was crystallized from a suitable mixture CH2Cl2/ether/hexane in 69% yield. The resulting material (3.83 g, 5.90 mmol) was dissolved in CH 2 Cl 2 (5 mL) and treated with HCl/dioxane (15 mL of a 4.0 M solution. The mixture was stirred at room temperature for 1 h. The solution was poured with stirring into ether (500 mL) and filtered.The solids were washed with ether (2 x 50 mL) and dried in vacuo to give the intermediate, (S)-(E)-3-(2-naphthylalanyl) -amino-5-phenyl-1-(2-naphthylsulfonyl)-1-pentene, 3.41 g, 99% yield.

2.00 g of this material (3.42 mmol) was dissolved in THF (15 mL) and cooled to 0°C. 4-Methylmorpholine carbonyl chloride (0.400 mL, 3.42 mmol) and triethylamine (0.953 mmol) were added. The mixture was stirred for 1 hour at 0°C and then at room temperature for 2 hours.

Ethyl acetate (50 mL) was added. The solution was washed with 1M HCl (30 mL), saturated aqueous sodium bicarbonate (30 mL), brine (30 mL), dried over MgSO 4 , filtered and evaporated to dryness to give 1.58 g (69%) of the intermediate, ( S)-(E)-3-(4-morpholinecarbonyl-2-naphthylalanyl)amino-5-phenyl-1-(2-naphthylsulfonyl)-1-pentene. TLC: (50% ethyl acetate/hexane) Rf= 0.37.

0.73 g (1.10 mmol) of this material was dissolved in ethanol (20 mL) and transferred to a Parr flask filled with 5% palladium on carbon (0.30 g). The mixture was reduced on a Parr hydrogenator for 36 hours. The solution was filtered and the solvent removed under reduced pressure. The product was purified by column chromatography on 60-200 mesh silica gel (50% ethyl acetate/CH2Cl2 as eluent) to give 0.14 g (19%) of the crude product, Mu-Np2-Hph-y-SO22Np, along with impure material. TLC: (50% ethyl acetate/CH 2 Cl 2 ) Rf = 0.34.

Example 7

Synthesis of a cysteine protease inhibitor containing a γ-aminosulfone which

EWG.

Synthesis of 3-acetyltyrosylvaly lalanylamino-4-hydroxy-carbonyl-1-phenylsulfonylbutane (Ac-Tyr-Val-Ala-Asp-y-SO2Ph). Sodium hydride (0.489 g of a 60% mineral oil dispersion, 12.23 mmol) was added to a solution of diethylphenylsulfonyl methylene phosphonate (3.58 g, 12.23 mmol) in 50 mL of THF at 0°C. The mixture was stirred for 15 minutes. A solution of Boc-AspH(B-Ot-Bu), (3.04 gm, 11.12 mmol), prepared by conversion of Boc-Asp(B-O-t-Bu) to N,0-dimethylhydroxamide and reduced with lithium aluminum hydride), in THF (10 mL) was added. The mixture was stirred for 1 hour, after which 1M HCl (30 mL) was added. The product was extracted with ethyl acetate (100 mL), washed with saturated aqueous NaHCO 3 (30 mL), brine (30 mL), dried over MgSO 4 , filtered and evaporated to dryness to give the intermediate. Chromatography on silica gel (20 - 30% ethyl acetate/hexane, gradient elution) gave 2.07 g 45%) of the intermediate, (S)-(E)-3-tert-butoxycarbonylamino-4-tert-butoxycarbonyl-1-phenylsulfonyl-1 -buten. This material was dissolved in ether (2 mL) and treated with a solution of anhydrous p-toluenesulfonic acid (1.0 g, 5.87 mmol) in ether (2 mL). The mixture was stirred at room temperature overnight, then diluted with ether (25 mL). The white precipitate was filtered, washed with ether and dried in vacuo to give 0.80 g (95%) of the next intermediate, (S)-(E)-3-amino-4-tert-butoxycarbonyl-1-phenylsulfonyl- 1 -butene-p-toluenesulfonate.

This material was coupled using mixed anhydride chemistry to Ac-Tyr-Val-AlaOH, itself prepared by standard peptide chemistry, yielding the next intermediate, (S)-(E)-3-acetyltyrosylvalylalanylamine-4-tert-butoxycarbonyl-1 - phenylsulfonyl-1 - butene.

This material was treated with trifluoroacetic acid to remove the t-butyl ester of the aspartic acid side chain, and this gave (E)-3-acetyltyrosylallylalanylamino-4-hydroxycarbonyl-1-phenylsulfonyl-1-butene. 0.28 g (0.444 mmol) of this material was dissolved in ethanol (10 mL). The solution was transferred to a Parr bottle, filled with 5% palladium on carbon (0.1 g). The solution was reduced on a Parr hydrogenator overnight. The solution was filtered and solvent was removed under reduced pressure. The residue, dissolved in methanol (5 mL) and diluted with 40x1:1 CH 2 Cl 2 /ether, formed a gelatinous precipitate, which was collected in a Buchner funnel to give 0.18 g (64%) yield. The isomer ratio of S to R with regard to the Asp residue was estimated to be approximately 3:1 based on integration of doublets associated with the aromatic region of the NMR belonging to the Tyr residue.

Example 8

Synthesis of a cysteine protease inhibitor with a γ-aminocarboxylate which

EWG.

Synthesis of (S)-4-(4-morpholinecarbonylphenylalanyl)amino-6-phenylhexanoic acid, Mu-Phe-Hph-y-CO2H. To a solution of Mu-Phe-Hph-γ-CO 2 Et prepared according to the procedure described in Example 1 (0.5 g, 1.06 mmol) was added aqueous NaOH (1 ml of a 2 M solution). After 4 hours the reaction was complete. 1M HCl (4 mL) was added along with water (10 mL). The product was extracted with CH 2 Cl 2 (2x10 mL), THF (dried over MgSO 4 , the solvent was removed under reduced pressure and the residue, Mu-Phe-Hph-y-CO 2 H, was pumped to a solid. Yield = 0.30 g (60% ).

Example 9

Synthesis of a cysteine protease inhibitor with a γ-aminophosphonate as EWG.

Synthesis of diethyl (S)-4-(4-morpholinecarbonyl-phenylalanyl)amino-6-phenylhexanephosphonate (Mu-Phe-Hph-y-SC>2Ph) was as follows. To a solution of tetraethyl methylene diphosphonate (2.00 g, 6.94 mmol) in THF (30 mL) was added sodium hydride (0.278 g of a 60% mineral oil dispersion, 6.94 mmol). The mixture foamed rapidly and then became clear. After 5 min, a solution of Boc-HphH (1.83 g, 6.94 mmol) in THF (5 mL) was added. The mixture was stirred for 1 hour. 1M HCl (20 mL) was added. The product was extracted into ethyl acetate (50 mL), washed with saturated aqueous NaHCO 3 (20 mL), brine (10 mL), dried over MgSO 4 , filtered and evaporated to dryness to give 2.46 g (89%) of the intermediate, diethyl (S)-(E)-4-tert-butoxycarbonylamino-6-phenyl-2-hexenephosphonate. To a solution of this material in CH 2 Cl 2 (3 ml) was added 10 ml of a 4.0 M solution of HCl in dioxane. The mixture was stirred at room temperature for 1.5 hours. Solvent was removed under reduced pressure and the residue dissolved in methanol (10 mL). The solution was poured into ether (400 ml). The precipitate was collected in a Buchner funnel, washed with ether (2x20 mL), and pumped dry to give 1.25 g (60%) of the intermediate, diethyl (S)-(E)-4-amino-6- phenyl-2-hexenephosphonate hydrochloride. To a solution of Mu-PheOH (1.04 g, 3.74 mmol) in THF (15 mL) at -10 °C was added 4-methylmorpholine (0.412 mL, 3.74 mmol), followed by isobutyl chloroformate (0.486 ml, 3.74 mmol). The mixed anhydride was stirred for 5 min, after which a solution of (S)-(E)-4-amino-6-phenyl-2-hexene-phosphonate hydrochloride (1.25 g, 3.74 mmol) in DMF (5 mL ) was added, followed by 4-methylmorpholine (0.412 mL, 3.74 mmol). The mixture was stirred for 1 hour. Ethyl acetate (50 mL) was added. The solution was washed with 1M HCl (25 mL), saturated aqueous NaHCO 3 (25 mL) and brine (10 mL), dried over MgSO 4 , filtered and evaporated to dryness. The product on treatment with CfyCVeter/hexane (315 ml in a 15:200:100 ratio) gave an oil which solidified on drying in vacuo to give 1.44 g (69%) of diethyl (S)-(E)-4- (4-morpholinecarbonyl-phenlalanyl)-amino-6-phenyl-2-hexenephosphonate. 0.85 g of this material was dissolved in ethanol (10 ml) and transferred to a Parr flask filled with 5% palladium on activated charcoal. The solution was reduced on a Parr hydrogenator for 36 hours. The solution was then filtered through Celite and solvent was removed under reduced pressure to give 0.66 g (76%) of the final product as an oil. TLC: (5% MeOH/CH 2 Cl 2 ) Rf = 0.27.

Example 10

Synthesis of a cysteine protease inhibitor with a γ-aminoamide as EWG.

Synthesis of benzyl (S)-3-(4-morpholinecarbonylphenyl-alanyl)-amino-6-phenylhexanamide (Mu-Phe-Hph-y-AMBzl). Wadsworth-Emon's reagent diethyl benzylamido-carbonylmethylenephosphonate was synthesized in two steps, first by saponification of triethylphosphonoacetate to diethylphosphonoacetic acid, which was then dissolved in ethyl acetate to a 0.2 M concentration, treated with one equivalent of benzylamine, 0.1 equivalents of 4-dimethylamino -pyridine and an equivalent of dicylohexyl carbodiimide. To a solution of Wadsworth-Emmons reagent (2.59 g, 9.08 mmol) in THF (40 mL) at 0°C was added sodium hydride (0.363 g of a 60% mineral oil dispersion, 9.08 mmol). The mixture was stirred at room temperature for 15 min, after which a solution of Boc-homophenylalaninal (2.39 g, 9.08 mmol) in THF (10 mL) was added. The mixture was stirred for 1 hour. 1M HCl (30 mL) was added. The product was extracted into ethyl acetate, washed with saturated aqueous sodium bicarbonate (50 mL), brine (30 mL), dried over MgSO4, filtered, concentrated and crystallized from ether/hexane to give 1.81 g (51%) of benzyl (S )-(E)-3-tert-butoxycarbonylamino-6-phenyl-2-hexenamide. This material was dissolved in CH 2 Cl 2 (5 mL). To the solution was added HCl/dioxane (10 ml of a 4.0 M solution). The mixture was stirred for 3 hours at room temperature. Solvent was removed under reduced pressure. The residue was dissolved in methanol (5 mL) and emptied into ether (300 mL), whereupon the intermediate, benzyl (S)-(E)-3-amino-6-phenyl-2-hexenamide hydrochloride separated as an oil, in 82 % yield (1.25 g). To a solution of Mu-PheOH (1.05 g, 3.78 mmol) in THF (15 mL) at -10 °C was added 4-methylmorpholine (0.416 mL, 3.78 mmol) and isobutyl chloroformate (0.490 mL, 3 .78 mmol). The mixture was stirred for 10 min, whereby a solution of benzyl (S)-(E)-3-amino-6-phenyl-2-hexenamide hydrochloride (1.25 g, 3.78 mmol) in THF (3 mL) was added, followed by 4-methylmorpholine (0.416 mL, 3.78 mmol). The mixture was stirred for 45 minutes. Ethyl acetate (40 mL) was added. The solution was washed with 1M HCl (10 mL), saturated ether sodium bicarbonate (10 mL), brine (5 mL), dried over MgSO 4 , filtered and evaporated to dryness. The intermediate, benzyl (S)-(E)-3-(4-morpholinecarbonyl-phenylalanyl)amino-6-phenyl-2-hexenamide, was precipitated from CH2Cl2/ether in 56% yield. 0.48 g (0.865 mmol) of this material was dissolved in ethanol (10 mL) and transferred to a Parr flask filled with 5% palladium on activated carbon. The mixture was reduced on a Parr hydrogenator for 4 hours. The solution was filtered through Celite and solvent was removed under reduced pressure. The final product (Mu-Phe-Hph-γ-AMBzl) was crystallized from ethanol/hexane to give 0.25 g (52%). TLC: (50% ethyl acetate/hexane) Rf = 0.45.

Example 11

Synthesis of a cysteine protease inhibitor with a γ-aminoamide as EWG.

Synthesis of phenyl (S)-3-(4-morpholinecarbonylphenyl-alanyl)-amino-6-phenylhexanamide (Mu-Phe-Hph-y-AMPh). To a solution of Mu-Phe-Hph-γ-CC^H, (0.30 g, as prepared according to Example 8), in THF (5 mL) at -10°C was added triethylamine (90 µl, 1 eq.) followed by the addition of isobutyl chloroformate (0.083 mL, 1 eq.). After 5 minutes, aniline (0.058 mL) was added. The cooling bath was removed and the reaction stirred at room temperature for 2 hours. CH 2 Cl 2 (30 mL) was added. The solution was washed with 1M HCl and saturated aqueous sodium bicarbonate (10 mL each), dried over MgSO 4 , filtered and solvent was removed under reduced pressure. The residue was triturated with Et 2 O, filtered and dried in vacuo to give 0.29 g (85%) of the product, Mu-Phe-Hph-γ-AMPh. TLC (10% MeOH/CH2Cl2) Rf= 0.70, strongly absorbs UV (254 nm), 12.

Example 12

Synthesis of a cysteine protease inhibitor with a γ-aromatic like EWG.

Synthesis of (S)-4-aminophenyl-3-(4-morpholine-carbonylphenylalanyl)amino-5-phenylpentane hydrochloride, (Mu-Phe-hPhe-y-C6H4HN2.HCl).

Triphenylphosphine (38.17 g, 0.146 mol) and 4-nitrobenzyl chloride (25 g, 0.146 mol) were dissolved in CH 3 CN (100 mL) and heated at reflux for 2 h, then allowed

opportunity to cool to room temperature. The reaction mixture was diluted with Et 2 O (300 mL), the white solid was filtered, washed with Et 2 O (200 mL) and dried in vacuo to give 53.3 g 884%) of 4-nitrobenzyltriphenylphosphonium chloride as a single spot on TLC: ( Rf = 0.71, 4:1:1 butanol:acetic acid:water) <*>H-NMR (d6-DMSO): 5.40 - 5.50 (2H, d, CH2P, J=20Hz); 7.20 - 7.40 (2H, dd, aromatic), 7.40 - 7.80 (12H, m, aromatic); 7.90

- 8.00 (3H, m, aromatic); 8.10 - 8.20 (2H, d, aromatic).

To a stirred suspension of 4-nitrobenzyltriphenylphosphonium chloride (10.02 g, 23.1 mmol) in CH 2 Cl 2 (100 mL) was added 4-methylmorpholine (2.54 mL, 23.1 mmol). When all solid was dissolved, Boc-HphH (4.04 g, 15.4 mmol) was added. After 24 h, the reaction mixture was diluted with CH 2 Cl 2 (200 mL) and filtered. The filtrate was washed with 1M HCl (200 mL), saturated aqueous sodium bicarbonate (200 mL); dried over MgSO 4 , filtered and concentrated under reduced pressure to give 4.00 g of the crude intermediate, part of which was purified by chromatography (gradient elution: 10 - 30% ethyl acetate/hexane to enable an NMR analysis of the intermediate, ( S)-t-butoxycarbonyl-3-amino-1-(4-nitrophenyl)-5-phenyl-1-pentene. TLC: (30% EtOAc/hexane) Rf = 0.49. To a solution of this material (2 .76 g, 7.2 mmol) in Et 2 O (25 mL), was added a solution of anhydrous r>toluenesulfonic acid (2.76 g, 16.0 mmol) in Et 2 O (10 mL). The reaction was stirred for 16 hr, filtered; solid was washed with Et 2 O (25 mL), and dried in vacuo to give 2 g (61%) of (S)-3-amino-1-(4-nitrophenyl)-5-phenyl-1- pentene as a single spot on TLC: (Rf = 0.49.10% MeOH/CH2Cl2).

To a solution of Mu-PheOH (1.29 g, 4.63 mmol) in THF (20 mL) was added 4-methylmorpholine (0.51 mL, 4.63 mmol) and isobutyl chloroformate (0.61 mL, 4, 63 mmol). After 3 minutes, a solution of (S) 3-amino-1-(4-nitrophenyl)-5-phenyl-1-pentene hydrochloride, prepared by HCl/dioxane-mediated deprotection of (S) 3-tert-butoxycarbonylamino-1 -(4-nitrophenyl)-5-phenyl-1-pentene precursor, (1.34 g, 4.20 mmol) in CH 2 Cl 2 (20 mL), followed by 4-methylmorpholine (0.51 mL, 4.63 mmol) . The mixture was stirred overnight while warming to room temperature. The solution mixture was diluted with CH 2 Cl 2 (100 mL), washed with 1M HCl (200 mL), saturated aqueous sodium bicarbonate (200 mL); dried over MgSC<4, filtered and concentrated under reduced pressure to give a yellow oil. This material was crystallized from CH2Cl2/ether (2:100, 20 mL) to give 1.00 g (40%) of (S)-3-(4-morpholinecarbonylphenylalanyl)amino-1-(4-nitrophenyl)-5 -phenyl-l-pentene as an approximate 4:1 E/Z mixture. 0.27 g (0.49 mmol) of this material was dissolved in ethanol (50 mL), transferred to a Parr flask filled with 5% palladium on carbon (0.10 g) and reduced in a Parr hydrognator for 8 h. The mixture was filtered and the solvent removed under reduced pressure. The residue was dissolved in 4:1 ether/CH 2 Cl 2 (100 mL) and to this was added HCl/dioxane (0.135 mL of a 4.0 M solution). The product, Mu-Phe-Hph-Y-C6H4NH2.HCl was filtered and dried in vacuo. Yield = 0.15 g (54%). TLC: (10% methanol/CH 2 Cl 2 ) Rf=0.31.

Example 13

Synthesis of a cysteine protease inhibitor with a B-amino sulfone as EWG.

Synthesis of (S)-2-(4-morpholinecarbonylphenyl-alanyl)amino-4-phenyl-l -phenylsulfonylbutane (Mu-Phe-Hph-B-SO2Ph). Preparation of Boc-homophenylalaninol (Boc-Hph-B-OH) and (S)-2-tert-butoxycarbonylamino-1-methanesulfonyloxy-1-phenylbutane (Boc-Hph-B-OMs or Boc-homophenylalaninol mesylate) followed a similar scheme as reported by Spaltenstein, Carpino, Miyake, and Hopkins, above. To a solution of Boc-homophenylalanine (10.29 g, 36.84 mmol) in THF (100 mL) at -10°C was added 4-methylmorpholine (4.05 mL, 36.84 mmol) and isobutyl chloroformate (4, 78 ml, 36.84 mmol). The solution was stirred for 10 minutes and then filtered. The filtrate was carefully added to a stirred solution of sodium borohydride (2.77 g, 73.67 mmol) in water (100 mL) at 0°C. The mixture was stirred for 30 minutes. Saturated aqueous sodium bicarbonate (200 mL) was added. The product was extracted with CH 2 Cl 2 (2 x 100 mL), dried over MgSO 4 , filtered and solvent removed under reduced pressure to give 9.78 g (100%) of Boc-homophenyl-alaninol. TLC: (30% ethyl acetate/hexane) Rf = 0.15. 5.83 g (21.97 mmol) of this material was dissolved in CH 2 Cl 2 (150 mL), cooled to 0 °C and treated with methanesulfonyl chloride (4.15 mL, 53.71 mmol) and triethylamine (9.24 mL, 66.3 mmol). The mixture was stirred for 30 minutes. Water (100 mL) was added; the mixture was stirred vigorously. The organic phase was separated, dried over MgSO 4 , filtered and the solvent was removed under reduced pressure to give 7.31 g (97%) yield. TLC: (30% ethyl acetate/hexane) Rf = 0.21. A similar procedure was used to prepare corresponding benzenesulfonate esters of Boc-Hph-15-OH.

To a solution of thiophenol (0.653 mL, 6.36 mmol) in THF (5 mL) was added sodium hydride (0.254 g, 6.36 mmol as a 60% mineral oil dispersion. The mixture was stirred for 10 min. A solution of Boc-homophenylalaninol Benzenesulfonate (2.58 g, 6.36 mmol) in THF (5 mL) was added. The solution was stirred at room temperature for 10 min. Methanol (2 mL) was then added and the mixture was heated at reflux for 1 h. The solution was cooled, diluted with 1M NaOH (25 mL), extracted with CH 2 Cl 2 (100 mL), dried over MgSO 4 , filtered and the solvent removed under reduced pressure. The residue was dissolved in CH 2 Cl 2 (35 mL) and cooled to 0 °C. To the soln. 4-chloroperbenzoic acid (3.71 g, 13.99 mmol, estimated peracid content 65 wt%) was added. The mixture was stirred for 1 h, at which time 10% NaOH (35 mL) and saturated aqueous NaHSO 3 (35 mL) were added The mixture was extracted with CH 2 Cl 2 (3 x 50 mL portions), dried over MgSO 4 , filtered and solvent was removed under reduced pressure to give a waxy solid, (S)-2-tert-butoxycarbonylamino-4-phenyl-1-phenylsulfonylbutane. TLC: (30% ethyl acetate/hexane) Rf = 0.32. 1.25 g of this material was dissolved in CH 2 Cl 2 (5 mL) and treated with HCl/dioxane (5 mL of a 4.0 M solution). The mixture was stirred for 2 hours at room temperature. The solution was poured into ether (200 mL) and an oily residue formed. The supernatant was discarded. The residue was redissolved in CH 2 Cl 2 (10 mL) and poured into ether (200 mL). The intermediate, (S)-2-amino-4-phenyl-1-phenylsulfonylbutane hydrochloride precipitated. The solid was filtered and dried in vacuo to give 0.40 g of the material (38% yield from Boc-homophenylalaninol benzenesulfonate.

To a solution of Mu-PheOH (.342 g, 1.23 mmol) in THF (10 mL) at -10°C was added 4-methylmorpholine (0.135 mL, 1.23 mmol) and isobutyl chloroformate (0.159 mL, 1 .23 mmol). The mixture was stirred for 10 min, at which time (S)-2-amino-4-phenyl-1-phenylsulfonylbutane hydrochloride (0.40 g, 1.23 mmol) was added, followed by 4-methylmorpholine (0.135 mL, 1.23 mmol). The mixture was stirred for 45 minutes. 1M HCl (15 mL) was added. The product was extracted with ethyl acetate (30 mL), washed with saturated aqueous sodium bicarbonate (15 mL), brine (15 mL), dried over MgSO4 , filtered and the solvent was removed under reduced pressure. The final product, Mu-Phe-Hph-B-SO 2 Ph, weighed 0.68 g (100% yield).

Example 14

Synthesis of a cysteine protease inhibitor with a B-amino sulfone as EWG.

Synthesis of (S)-2-tert-butoxycarbonylamino-4-phenyl-1-(1'-trimethylsilylethyl)-sulfonylbutane (Boc-Hph-B-SO2CH2CH2TMS). To a solution of 2-trimethylsilyletanethiol (0.86 g, 6.41 mmol), the synthesis described by Anderson, Ranasinghe, Palmer, and Fuchs) in THF (10 mL) was added sodium hydride (0.256 g, 6.41 mmol as a 60% mineral oil dispersion). The mixture was stirred for 10 minutes. Boc-homophenylalaninol mesylate (2.00 g, 5.82 mmol, synthesis described in Example 13, above) was added. The solution was stirred for 2 hours. Ethyl acetate (50 mL) was added. The solution was washed with 30 mL each of 1M HCl, saturated aqueous bicarbonate and brine, dried over MgSO 4 , filtered and solvent removed under reduced pressure to give the intermediate (S)-2-tert-butoxycarbonylamino-4-phenylbutyl-trimethylsilylethylsulfide. TLC: (5% ethyl acetate/hexane) Rf = 0.22. This material was dissolved in CH 2 Cl 2 (50 mL), cooled to -10°C and treated with 4-chloroperbenzoic acid (3.24 g, 12.22 mmol, estimated 65% peracid content). The mixture was stirred overnight. The suspension was filtered and saturated aqueous NaHSO 3 (40 mL) and saturated aqueous sodium bicarbonate (50 mL) were carefully added to the filtrate. The organic phase was separated, dried over MgSO 4 , filtered and solvent was removed under reduced pressure to give the product, Boc-Hph-B-SO 2 CH 2 CH 2 TMS in quantitative mass recovery from the mesylate. TLC: (30% ethyl acetate/hexane) Rf = 0.49.

Example 15

Synthesis of a cysteine protease inhibitor with a B-amino sulfone as EWG.

Synthesis of (S)-2-(4-morpholinecarbonylphenylalanyl)-amino-1-chloromethylsulfonyl-4-phenylbutane (Mu-Phe-Hph-B-SO2CH2C1). To a solution of Boc-Hph-B-SO2CH2CH2TMS (0.90 g, 2.18 mmol as described in Example 14) in THF (2 ml) was added tetrabutylammonium fluoride (8.7 ml of a 1.0 M THF solution) and multiple molecular sieves. The mixture was stirred overnight at room temperature. Bromochloromethane (5 mL) was added. The mixture was heated at reflux for 1 hour, cooled and the volatile components were removed under reduced pressure. The residue was dissolved in ethyl acetate (75 mL), washed with 1M HCl (50 mL), dried over MgSCM, filtered and the solvent was removed under reduced pressure. The residue, crude (S)-2-tert-butoxycarbonylamino-1-chloromethylsulfonyl-4-phenylbutane, was dissolved in ether (3 mL). A solution of anhydrous 4-toluenesulfonic acid (0.80 g, 4.70 mmol) in ether (3 mL) was added. The mixture was stirred at room temperature overnight. Ether (100 mL) was added. The solid intermediate, (S)-2-amino-1-chloromethyl-sulfonyl-4-phenylbutane 4-toluenesulfonate (TsOH.Hph-1f-SO2CH2Cl), was filtered and the solids were washed with ether (2 x 20 mL) , and dried in vacuo to give 0.193 g of the material (24% from Boc-Hph-fi-SO 2 CH 2 CH 2 TMS).

To a solution of Mu-PheOH (0.109 g, 0.392 mmol) in THF (3 mL) at -10 °C was added 4-methylmorpholine (43 µl, 0.392 mmol) and isobutyl chloroformate (51 µl, 0.392 mmol). The mixture was stirred for 10 min, whereupon TsOH.Hph-15-SO 2 CH 2 Cl (0.17 g, 0.392 mmol) was added, followed by 4-methylmorpholine (43 µl, 0.392 mmol). The mixture was stirred for 45 minutes. Ethyl acetate (20 mL) was added. The solution was washed with IM HCl, saturated aqueous sodium bicarbonate and brine (2 mL each), dried over MgSO4, filtered, and solvent removed under reduced pressure to give the final product, Mu-Phe-Hph-B-SO2CH2C1 ( 90 mg, 48% yield).

Example 16

Synthesis of a cysteine protease inhibitor with an α-amino sulfone as EWG.

Synthesis of l-(tert-butoxycarbonyl)amino-2-methyl-1-phenylsulfonylpropane (Boc-Val-α-SO2Ph). To a stirred suspension of t-butyl carbamate (2.34 g, 20 mmol) and sodium benzenesulfinate (3.28 g, 20 mmol) in water (20 mL) was added a solution of isobutyraldehyde (2.00 mL, 22 mmol) in formic acid (5 ml). The mixture was stirred at room temperature overnight. The precipitate was filtered, washed with water (2 x 50 mL) and crystallized from isopropanol/water to give 4.72 g (75%) of the product.

Example 17

Synthesis of a cysteine protease inhibitor with an α-amino sulfone as EWG.

Synthesis of l-benzyloxycarbonylamino-3-phenyl-l-phenylsulfonylpropane (Z-Hph-a-SO2Ph). To a suspension of sodium benzenesulfinate (10 g, 60.9 mmol) and benzyl carbamate (9.21 g, 69.9 mmol) in water (40 mL) was added hydrocinnaldehyde (8.8 mL, 67 mmol) in formic acid (10 mL) ). The mixture was heated at 70°C for 1 hour and then allowed to cool to room temperature overnight. The product crystallized out; it was filtered and recrystallized from hot isopropanol, yielding 23 g (100%). TLC: (30% ethyl acetate/hexane) Rf = 0.37.

Example 18

Synthesis of a cysteine protease inhibitor with α-aminosulfone as EWG.

Synthesis of (R)-1-(4-morpholinecarbonylphenylalanyl)amino-3-phenyl-1-phenylsulfonylpropane and (S)-1-(4-morpholinecarbonylphenylalanyl)amino-3-phenyl-1-phenylsulfonylpropane (Mu-Phe-Hph- a-SChPh, epimers separated).

Method A: Z-Hph-α-SO 2 Ph (1.0 g, 2.44 mmol) was treated with 30% hydrogen bromide in acetic acid (5 mL). After 30 minutes the mixture was diluted with ether (300ml), filtered, washed with ether (2 x 30ml) and dried in vacuo to give 0.74g (86%) of 1-amino-3-phenyl-1-phenylsulfonylpropane hydrobromide (HBr.Hph-a-SC«2Ph). To a solution of Mu-PheOH (0.64 g, 2.3 mmol) in THF (15 mL) was added 4-methylmorpholine (0.302 mL, 2.3 mmol) and isobutyl chloroformate (0.312 mL, 2.3 mmol) . The mixture was stirred for 10 minutes. HBr.Hph-α-SC^Ph (0.74 g, 2.1 mmol) was added, followed by 4-methylmorpholine (0.302 mL, 2.3 mmol). After 45 min the mixture was diluted with ethyl acetate (30 mL), washed with 15 mL each of 1M HCl, saturated aqueous sodium bicarbonate and brine, dried over MgScu, filtered and solvent removed under reduced pressure to give 0.75 g (65% ) of the product, Mu-Phe-Hph-a-SC^Ph. Method B: To a solution of phenylalanine amide hydrochloride (10 g, 50 mmol) in DMF (50 mL) and CH 2 Cl 2 (50 mL) was added triethylamine (13.9 mL, 100 mmol) and 4-morpholinecarbonyl chloride (5.9 mL, 50 mmol). The mixture was stirred overnight. Solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and filtered. Ether was added to the filtrate until the solution became turbid. 7.2 g (80% yield) of the intermediate, 4-morpholinecarbonylphenylalanine amide (Mu-Phe-NH 2 ) crystallized from solution after 3 days. To a solution of Mu-PheNH 2 (2.24 g, 8.1 mmol) in formic acid (5 mL) was added, with stirring, hydrocinnaldehyde (1.17 mL, 8.9 mmol). The mixture was stirred for 5 h, whereupon sodium benzenesulfinate (1.33 g, 8.1 mmol) was added. The mixture was rapidly heated to reflux over a 5 minute period and allowed to cool to room temperature. The solution was then allowed to stir for 3 days. An equal volume of water was added. The product was extracted with CH 2 Cl 2 (3 x 100 mL), dried over MgSO 4 , filtered and the solvent was removed under reduced pressure. The yield of the product, diastereomer (R)- and (S)-l-(4-morpholine-carbonylphenylalanyl)amino-3-phenylsulfonyl-l-phenyl-propane was 3.9 g (90%). TLC (50% ethyl acetate/CH 2 Cl 2 ) Rf = 0.27, 0.34.

The diastereomers were separated by flash chromatography on 230 - 400 mesh silica gel (20 - 50% ethyl acetate/CH2Cl2, gradient elution).

Example 19

Inhibition of cysteine proteases with inhibitors from the invention.

Conditions for cathepsin B: 50 mM phosphate, pH 6.0, 2.5 mM EDTA, 2.5 mM DTT. Substrate: [Z-Arg-Arg-AMC] = 50 mM (Km = 190 mM). The assay at 25°C was initiated by the addition of eat B (final concentration approximately 10 nM) and an increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 minutes. A decrease in the rate of substrate hydrolysis after the addition of varying concentrations of inhibitors was noted. The analysis was linear in the observed range. Double runs were measured.

Conditions for cathepsin L: 50 mM acetate, pH 5.5, 2.5 mM EDTA, 2.5 mM DTT. Substrate: [Z-Phe-Arg-AMC] = 5 mM (Km = 2 mM). The assay at 25°C was initiated by the addition of eat L (final concentration approximately 1 nM) and the increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 minutes. Decrease in rate of substrate hydrolysis after addition of different concentrations of inhibitors was noted. The analysis was linear throughout the observed range. Double runs were measured.

Conditions for cathepsin S: 50 mM phosphate, pH 6.5, 2.5 EDTA, 2.5 mM DTT. Substrate: [Z-Val-Val-Arg-AMC] = 10 mM (Km = 18 mM). The assay at 25°C was initiated by the addition of eat S (final concentration approximately 30 pM) and the increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 minutes. A decrease in the rate of substrate hydrolysis after the addition of varying concentrations of inhibitors was noted. The analysis was linear throughout the observed area. Double runs were measured.

Conditions for cruzain were the same as for cathepsin L except that the Km for the substrate was 1 mM.

The respective Ki values were estimated by using Dixon plots as described by Irwin Segel in Enzyme Kinetics: Behavior and analysis of rapid equilibrium and steady-state enzyme systems, 1975, Wiley-Interscience Publication, John Wiley & Sons, New York.

The results are shown in Table 2.

Example 20

The following are representative pharmaceutical formulations containing a cysteine protease inhibitor of the invention.

ORAL FORMULATION

A representative solution for oral administration contains:

INTRAVENOUS FORMULATION

A representative solution for intravenous administration contains:

TABLET FORMULATION

Claims (28)

1. Compound characterized by formula I: where W is SO2R<2>, PO(OR10)2(where R<10> is independently C1C6 alkyl) phenyl (optionally substituted with amino, C14 alkoxy or C1-C4 alkyl), COOR<2> (where R<21> is H or C16 alkyl), or CONHR5 where R<5> is benzyl, phenyl or phenyl-C14 alkyl; n is 0 to 4; A-B represents -C(O)NH-; X represents -(CH2)m-, where m is 0-4: Z is -C(R<6>)(R<7>)-, where R 6 is hydrogen or C 1 -C 1 alkyl and R 7 is as defined below; Z<1> is -C(R<6>)(R<8>)-, where R6 is hydrogen or C 1 -C alkyl and R<8> is defined below; R<1> is methyl, morpholinyl, benzyloxyaminoalkyl, 0-(CH2)2COOR<22> where R<22> is H or Ci-C4 alkyl, naphthyl-OC(0)(CH2)2CO2H, phenylacetyl, benzyloxycarbonyl, Ci- C6 alkoxy, phenyl-C1-C4-alkoxy, phenyl-C1-C4-alkyl (optionally substituted with OH in the phenyl ring and with -C1-C4-alkylcarbonylamino in the alkyl chain) or naphthyl, morpholinylnaphthyl; R 7 and R 8 are independently hydrogen, (Ci.10) alkyl (optionally substituted with a radical selected from amino, carboxy, phenyl or naphthyl (optionally substituted in the aryl ring with a radical selected from hydroxy, halo, amino or a protected thereof) ; R<2> is (Ci. 10) alkyl (optionally substituted with one or more radicals selected from chlorine, bromine, fluorine, iodine, phenyl, C]-C4-alkoxyphenyl or C1-C4-alkyl-silyl-tri-Ci- C 4 -alkyl) or a group selected from phenyl or naphthyl or a protected form thereof; and pharmaceutically acceptable salts, single isomers and mixtures of isomers thereof. 2. Compound according to claim 1, characterized in that W is SO2R<2>. 3. Compound according to claim 2, characterized by atnerO to 2, where R<7> and R<8> are independent (Ci-Cio) alkyl (optionally substituted with a group selected from phenyl, 1-naphthyl, and 2-naphthyl, (which group is optionally substituted in its aryl ring with a radical selected from hydroxy, amino, chlorine, bromine and fluorine, or a protected form thereof); R<2> is Ci-Cio) alkyl (optionally substituted with one or two radicals selected from chlorine , bromine, fluorine and iodo) or a group selected from phenyl or naphthyl. 4. Compound according to claim 3, characterized in that n is 0 to 1; R is methyl, trifluoromethyl, optionally substituted phenyl, 2-naphthyl or 2-phenylethyl; wherein R 7 and R 8 are independently (C 1 -C 10 ) alkyl (optionally substituted with a group selected from phenyl, 1-naphthyl, 2-naphthyl and 2-phenyl (which group is optionally substituted in its aryl ring with a radical selected from hydroxy, amino, chlorine, bromine and fluorine, or a protected form thereof)). 5. Compound according to claim 4, characterized in that R 9 is phenyl or 2-naphthyl, and R 7 is benzyl, 1-naphthylmethyl or 2-naphthylmethyl. 6. Compound according to claim 5, characterized in that X represents a bond, R<8> is 2-phenylethyl, R<2> is phenyl and R7 is benzyl, namely A^<2->(4-morpholinylcarbonyl)-A^<; ->(3-phenyl-1-phenylsulfonylpropyl)-L-phenylalanine amide. 7. Compound according to claim 5, characterized in that X represents methylene, R<8> is 2-phenylethyl, R<2> is phenyl and R7 is benzyl, namely iV<2->(4-morpholinylcarbonyl)-V-(3-phenyl) -15'-phenylsulfonylmethylpropyl)-L-phenylalanine amide. 8. Compound according to claim 5, characterized in that X represents -CH2CH2-, R<8> is 2-phenylethyl, R<2> is 2-naphthyl and R7 is 2-naphthylmethyl, namely A^-(4-morpholinylcarbonyl)-A^ </->{3-Phenyl-15'-[2(2-natylsulfonyl)ethyl]propyl}-p-(2-naphthyl) L-alanine amide. 9. Compound according to claim 5, characterized in that X represents -CH2CH2-, R is 2-phenylethyl, R is phenyl and R is 4-hydroxybenzyl, namely A^-(4-morpholinylcarbonyl)-jN7-(3-phenyl-1S- [2-(Naphthylsulfonyl)ethyl]propyl}-L-tyrosinamide. 10. Compound according to claim 5, characterized in that X represents -CH2CH2-, R<8> is 2-phenylethyl, R<2> is phenyl and R7 is 4-benzyl, namely A^<2->(4-morpholinylcarbonyl)-A ^;-[3-phenyl-lS'-[2-(phenylsulfonylethyl)propyl]-L-phenylalanine amide. 11. Compound according to claim 2, characterized in that W is PO(OR<,0>)2, where R<10> is independent C1-6 alkyl. 12. Compound according to claim 11, characterized in that R10 is -CH2CH2-. 13. Compound according to claim 1, characterized in that W is phenyl (optionally substituted with amino C 1 -C 1 alkoxy or C 1 -C 1 alkyl). 14. Compound according to claim 13, characterized in that W is methoxyphenyl. 15. Compound according to claim 14, characterized in that methoxyphenyl is 4-methoxyphenyl, and where R<1> is 4-morpholinyl, R<8> is 2-phenylethyl, R<7> is benzyl, namely A^-(4-morpholinylcarbonyl) -N-{3-phenyl-15[2-(4-methoxyphenyl)ethyl]propyl}-L-phenylalanine amide. 16. Compound according to claim 13, characterized in that W is phenyl substituted with amino. 17. Compound according to claim 16, characterized in that W is 4-aminophenyl, R 1 is 4-morphohnyl, R R is 2-phenylethyl, R 7 is benzyl, namely N 7-(4-morpholinylcarbonyl)-Af7-{3-phenyl-15 '[2-(4-aminophenyl)ethyl]propyl}-L-phenylalanine amide. 18. Compound according to claim 1, characterized in that W is COOR<21> (where R<21> is H or C1-6 alkyl). 19. Compound according to claim 18, characterized in that R21 is CH2CH3. 20. Compound according to claim 19, characterized in that R1 is morpholinyl, R<8> is 2-phenylethyl, R7 is 2-naphthylmethyl, namely ethyl-4S-[A^-(4-morpholinylcarbonyl)-P-(2-naphthyl)- L-alanylamino]-6-phenylhexanoate. 21. Compound according to claim 19, characterized in that R<1> is morpholinyl, R<8> is 2-phenylethyl, R7 is benzyl, namely ethyl 4S-[7Vr-(4-morpholinylcarbonyl)-L-phenylalanylamino]-6-phenylhexanoate . 22. Compound according to claim 1, characterized in that W is CONHR5 where R<5> is benzyl, phenyl or phenyl-C1 alkyl. 23. Compound according to claim 22, characterized in that R<5> is benzyl. 24. Compound according to claim 23, characterized in that R<1> is morpholinyl, R<8> is 2-phenylethyl, R7 is benzyl, namely A^<2->4-(morpholinylcarbonyl)-V-[3-phenyl-1 S -(2-benzylcarbamoylethyl)propyl]-L-phenylalanine amide. 25. Compound according to claim 22, characterized in that R<5> is phenyl. 26. Compound according to claim 25, characterized in that R<1> is morpholinyl, R<8> is 2-phenylethyl, R7 is benzyl, namely A/<2->4-(morpholinylcarbonyl)-A^</->[3- phenyl-1S-(2-phenylcarbamoylethyl)propyl]-L-phenylalanine amide. 27. Compound according to claim 22, characterized in that R<5> is phenyl-C1-4 alkyl. 28. Compound according to claims 1-5 or 11-27, characterized by atmerO.