SK122199A3 - Methods and compositions for modulating responsiveness to corticosteroids - Google Patents

Abstract

Method for modulating responsiveness to corticosteroids in a subjet are provided. In the method of the invention, an agent which antagonizes a target that regulates production of IFN- gamma in the subject is administered to the subject in combination with a corticosteroid such that responsiveness of the subject to the corticosteroid is modulated as compared to when a corticosteroid alone is administered to the subject. In one embodiment, the agent is an IL-18 antagonist. In another embodiment, the agent is an interleukin-12 (IL-12) antagonist. In yet another embodiment, the agent is an NK cell antagonist. In a preferred embodiment, the agent is an inhibitor of a caspase family protease, preferably an ICE inhibitor. In another preferred embodiment, the agent is an anti-IL-12 monoclonal antibody. In yet another preferred embodiment, the agent is an anti-asialo-GM1 antibody or an NK1.1 antibody. Other preferred agents include phosphodiesterase IV inhibitors and beta-2 agonists. The methods of the invention can be used in the treatment of a variety of inflammatory and immunological diseases and disorders. Pharmaceutical compositions comprising an agent which antagonizes a target that regulates production of IFN- gamma in a subject, a corticosteroid and a pharmaceutically acceptable carrier are also provided. A preferred composition comprises an ICE inhibitor, a corticosteroid and a pharmaceutically acceptable carrier.

Description

Preparations for modulating the response to corticosteroids

BACKGROUND OF THE INVENTION

Standard therapy for various immune disorders and inflammatory diseases involves the administration of corticosteroids that have the ability to suppress immunological and inflammatory reactions. Corticosteroids are used in the treatment of diseases such as asthma, autoimmune disorders (e.g. rheumatoid arthritis, systemic lupus erythematosus) and transplant rejection (as reviewed by corticosteroids reported, for example, Truhan, AP et al. (1989) Annals of Allergy 62: 375-391 Baxter, JD (1992) Hospital Practice 27: 111134, Kimberly, RP (1992) Curr Opin Rheumatol 4: 325-331, Weisman, M.

H. (1995) Curr. Opin. Rheumatol. 7: 183-190. Corticosteroids are also currently used in the treatment of various dermatological diseases such as contact dermatitis, general psoriasis, flat lichen, keloids and urticaria pigmentosa (reviewed in Sterry, W. (1992) Ar. Dermatol. Res. 284 (suppl.): S27 -S29).

While corticosteroids are therapeutically beneficial, their use is associated with a number of side effects ranging from mild to potentially life-threatening. Complications associated with prolonged and / or high use of steroid doses include musculoskeletal effects (e.g. osteoporosis, myopathy, aseptic bone necrosis,), ophthalmic effects (e.g. back subcapsular cataracts), gastrointestinal effects (e.g. ulcers, pancreatitis, nausea, nausea) effects (e.g. hypertension, atherosclerosis), central nervous system effects (e.g. pseudotumor cerebri, psychiatric reactions), dermatological effects (e.g. hirsutism, redistribution of subcutaneous fat, impaired wound healing, skin thinning) and suppression of the hypothalamic pituitary adrenal (e.g., adrenal) Truhan, AP et al (1989) Annals of Allergy 62: 375-391). Several dose-dependent side effects may occur with corticosteroids (Kimberly, R. P.

(1992) Curr. Opin. Rheumatol. 4: 325-331).

Thus, methods and compositions that allow the use of lower effective dosages of corticosteroids (referred to as "steroid limited effects") may be desirable to avoid unwanted side effects.

Another problem that limits the usefulness of corticosteroids is the phenomenon of steroid resistance. Certain inflammatory or immunological diseases show resistance to steroid treatment. For example, attempts to use corticosteroid therapy to treat septic shock in humans have met with disappointing results, and so corticosteroids are generally not recommended as adjunctive therapy for severe sepsis or septic shock (see, for example, Putterman, C. (1989, Israel J. Med. Sci.). 25: 332-338; Bone, RC and Brown, RC (1990) in Vincent, JL (ed.) "Update in Intensive Line and Emergency Medicine 10," Heidelberg.Springer Verlag, p. 121). exhibiting resistance to corticosteroid therapy include inflammatory bowel disease (see, for example, Hepi, T. et al. (1995) J. Gastroenterol. 30: 121-123) and graft-versus-host ("graft-versus-host") disease ( Antin, JH et al (1994) Blood 84: 1342-1348, Racadot, E. et al (1995) Bone Marrow Transplantation 15: 669-677) Therefore, methods and compositions that can be used to overcome or alteration of corticosteroid resistance in z Paľová and immunological diseases are still needed.

Yet another drawback of corticosteroid therapy is the occurrence of a "spontaneous steroid rebound effect" when corticosteroid administration is discontinued. The spontaneous response to steroids is characterized by worsening inflammatory conditions (s) that are treated after discontinuation of steroid therapy. Methods and compositions that can be used to improve the spontaneous response to steroids are still needed.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for modulating a corticosteroid response in a subject. For example, the methods and compositions of the invention can be used to reverse steroid resistance ("reversal of steroid resistance") in a subject, to allow treatment of the subject with corticosteroids. The methods and compositions of the invention can be used to increase steroid sensitivity in a subject and thereby achieve therapeutic efficacy of corticosteroid treatment at lower doses (e.g., to avoid the harmful side effects of high doses of corticosteroids or to enable treatment of steroid-dependent diseases at lower doses). ). Further, the methods and compositions of the invention can be used to improve the spontaneous response to steroids when the subject undergoes treatment with corticosteroids removed.

In modulatory methods of the invention, a composition that antagonizes an object that regulates IFN-γ production in a subject is administered to the subject in combination with a corticosteroid such that the subject's response to the corticosteroid is modulated compared to when the corticosteroid is administered to the subject. For example, the object that is antagonized may be a cytokine or an enzyme that regulates IFN-γ production or a cell that regulates IFN-γ production. The composition is administered at a dosage and route of administration sufficient to inhibit IFN-γ production in the subject. In various embodiments, the compositions and the corticosteroid are administered at the same time, the composition being applied first and then the corticosteroid or vice versa. The methods may be applied to a prophylactic and to a therapeutic regimen of corticosteroid treatment.

In an embodiment, the method comprises administering a composition that is an IL-18 antagonist. The IL-18 antagonist is administered at a dosage and route of administration sufficient to inhibit IL-18 activity in the subject. The IL-18 antagonist may act at the level of IL-18 synthesis, IL-18 cytokine activity, or IL-18 interaction with the IL-18 receptor. In a preferred embodiment, the IL-18 antagonist is a caspase family protease inhibitor, preferably an inhibitor

Interleukin-β converting enzyme (ICE) ("Interleukin-β Converting Enzyme (ICE) inhibitor"). In another embodiment, the IL-18 antagonist is an antibody, antibody fragment, or exposed binding protein that binds to IL-18 or an IL-18 receptor.

In another embodiment, the method comprises administering a composition that is an Interleukin-12 (IL-12) antagonist. For example, an IL-12 antagonist may act on the level of IL-12 synthesis, IL-12 cytokine activity, or IL-12 interaction with the IL-12 receptor. In a preferred embodiment, the IL12 antagonist is an antibody, antibody fragment, or displayed binding protein that binds to IL-12 or an IL-12 receptor. In another embodiment, the IL-12 antagonist is a composition that stimulates production of cyclic AMP (cAMP) in cells that produces IL-12. Examples of a composition that can be used to stimulate cAMP include phosphodiesterase IV inhibitors and beta-2 antagonists. In yet another embodiment, the IL-12 antagonist is a STAT4 inhibitor.

In yet another embodiment, the method comprises administering a composition that deprives or eliminates NK cells and NK-like cells (referred to herein as "NK cell antagonists") from the subject. The NK cell antagonist is administered at a dosage and route of administration sufficient to inhibit IFN-γ production in the subject. Preferred NK cell antagonists are NK / NK-like cell-specific antibodies that deplete these cells in vivo. Examples of preferred antibodies for use as NK cell antagonists are asialo-GM1 antibodies and NK1.1 antibodies.

Another aspect of the invention relates to a method for modulating a corticosteroid response in a subject, wherein a caspase family protease inhibitor, preferably ICE, is administered to the subject together with a corticosteroid such that the subject's response to the corticosteroid is modulated compared to when the subject is administered alone. corticosteroid.

Yet another aspect of the invention relates to a method for modulating a corticosteroid response in a subject, wherein the IL-12 antagonist is administered to the subject together with a corticosteroid such that the subject's response to the corticosteroid is modulated compared to when the corticosteroid is administered to the subject.

Yet another aspect of the invention relates to a method for modulating a corticosteroid response in a subject, wherein an NK cell antagonist (e.g., an antibody against NK / NK-like cells) is administered to the subject together with a corticosteroid such that the subject's response to the corticosteroid is modulated when the subject is administered corticosteroid alone.

Yet another aspect of the invention relates to a method for modulating a corticosteroid response in a subject, wherein the subject is selected for the need to modulate a corticosteroid response, and a composition that antagonizes an object that regulates IFN-γ production in the subject the corticosteroid is compared to when the subject is administered the corticosteroid alone. The composition is administered at a dosage and route of administration sufficient to inhibit IFN-γ production in the subject. For example, the selected subject may be a steroid-resistant subject prior to treatment, a steroid susceptible subject whose steroid sensitivity will grow, or a steroid recipient subject whose spontaneous steroid response is improved.

The invention also provides pharmaceutical compositions for modulating the response to corticosteroids in a subject. In an embodiment, the composition of the invention comprises a composition that antagonizes an object that regulates IFN-γ production in a subject, a corticosteroid, and a pharmaceutically acceptable carrier. In another embodiment, the formulation of the invention comprises an IL-18 antagonist (such as a caspase family protease inhibitor, preferably an ICE or anti-IL-18 inhibitor or an anti-IL-12 monoclonal antibody), a corticosteroid and a pharmaceutically acceptable carrier In yet another embodiment, the composition of the invention comprises an IL-12 antagonist (e.g., anti-IL-12 or anti-IL-12 monoclonal antibody, phosphodiesterase IV inhibitor, beta-2 agonist, STAT4 inhibitor), a corticosteroid and a pharmaceutically acceptable carrier. In another embodiment, the composition of the invention comprises an NK cell antagonist (for example, an antibody against NK cells / NK-like cells), a corticosteroid, and a pharmaceutically acceptable carrier The pharmaceutical compositions of the invention may be formulated for administration by a preferred route of administration to achieve the desired therapeutic effect. In another preferred embodiment, the pharmaceutical composition is formulated for administration by inhalation. Other preferred routes of administration include oral and intravenous administration.

The methods and compositions of the invention can be used in the treatment of any disease or disorder in which it is desired to modulate the response to steroids. In preferred embodiments, the methods and compositions of the invention are used to treat a subject suffering from septic shock. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from Crohn's disease. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from asthma. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from an autoimmune disease or disorder. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from graft disease against a host or transplant rejection. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from an acute inflammatory disorder. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from a chronic inflammatory disorder.

BRIEF DESCRIPTION OF THE DRAWINGS FIG

Figure 1 is a bar graph showing serum TNFα levels (in ng / ml) in wild type and ICE-poor mice (ICE KO) treated with vehicle alone or dexamethasone (4 mg / kg) 30 minutes after LPS at LPS / P. acnes septic shock model demonstrating that ICE-poor but not wild-type mice exhibit suppression of TNFα production and are therefore steroid sensitive.

Figure 2 is a bar graph showing serum TNFα levels (in ng / ml) in wild-type (solid bars) and ICE-poor mice (shaded bars) pretreated with vehicle alone or a decreasing amount of dexamethasone (0.05, 0.005 or 0.0005 mg / kg) in LPS / P. acnes of a septic shock model demonstrating that ICE-poor mice maintain the steroid response to reduce steroid doses compared to wild type mice.

Figure 3 is a bar graph showing LPS-induced serum IL-12 (in pg / ml) in B6 mice treated with vehicle or a phosphodiesterase IV inhibitor, rolipram, demonstrating that phosphodiesterase IV inhibitor treatment inhibits IL-12 production.

Figure 4 is a bar graph showing serum TNFα levels (in ng / ml) in B6 mice treated with vehicle alone (saline) or ICE inhibitor (Ac-YVAD-CHO) in combination with dexamethasone treatment in an LPS / P.acnes septic shock model.

Figure 5 is a bar graph showing serum levels of IL-6 (in ng / ml) in B6 mice treated with vehicle alone (saline) or ICE inhibitor (Ac-YVAD-CHO) in combination with dexamethasone treatment in the LPS / P.acnes septic model shock.

Figure 6 is a bar graph showing serum levels of IL-Iβ (in ng / ml) in B6 mice treated with vehicle alone (saline) or ICE inhibitor (Ac-YVAD-CHO) in combination with dexamethasone treatment in an LPS / P acnes septic shock model .

The present invention is based, at least in part, on the discovery that ICE-poor mice as compared to standard-treated mice are sensitive to corticosteroids upon eliciting an LPS response in a septic shock model (see Example 1). In addition, ICE-poor mice exhibit increased sensitivity to low-dose corticosteroids compared to standard treated mice when corticosteroid treatment is performed prior to inducing an LPS response in a septic shock model (see Example 2). The invention is further based, at least in part, on the discovery that depletion of NK / NK-like cells upon eliciting an LPS response in wild-type mice results in a substantially increased survival rate.

It has previously been described that the administration of interferon-γ (IFN-γ) can overcome corticosteroid suppression of TNFα biosynthesis of mouse macrophages (Leudke, C. E. and Cerami, A. (1990) J. Clin. Invest. 86, 1234-1240). Moreover, ICE and other proteases from the caspase family can cleave the precursor forms of IL-18 into their mature, active forms (see Example 4). Although not intended to limit the mechanism, the ability to deliver a corticosteroid response by inhibiting ICE activity in a subject, in accordance with the present invention, is intended to result in inhibition of IL-18 processing by ICE such that mature IL-18 production is inhibited resulting in a decrease in IFN-γ production in the subject. Thus, it is intended that NK / NK-like cells form positive feedback in the production of IFN-γ, which can be down-modulated by depletion or elimination of NK / NK-like cells.

In view of the foregoing, the invention broadly provides methods and compositions for modulating the response to corticosteroids, wherein the object that regulates IFN-γ production is antagonized in a subject. This object that regulates IFN-γ production and which is antagonized may be IL-18 (which can be antagonized, for example, indirectly by inhibiting ICE activity or directly using an anti-IL-18 antibody). Alternatively, another factor that regulates IFN-γ production such as IL-12 may be antagonized to modulate the corticosteroid response in a subject. Further, a composition that deprives or eliminates NK / NK-like cells by inhibiting IFN-γ production can be used to modulate the corticosteroid response in a subject.

The invention will be better understood by reference to the following description. Terms are defined first.

The term "corticosteroid," as used herein, refers to a class of therapeutic agents useful in the treatment of inflammatory conditions, including those resulting from infection, transplant rejection, and autoimmune disorders. Corticosteroids include those that occur naturally, are synthetic or semi-synthetic in origin, and are characterized by the presence of steroid nuclei having four-membered fused rings such as found in the cholesterol, dihydroxycholesterol, stigmasterol and lanosterol structures. Corticosteroid drugs include cortisone, cortisol, hydrocortisone (11β17dihydroxy-21- (phosphonooxy) -pregn-4-ene3,20-disodium), dihydroxycortisone, dexymetazone (21- (acetyloxy) -9-fluoro-11β, 17-dihydroxy-, 17-dihydroxy- 16a-methylpregna-1,4diene-3,20-dione) and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-11β, 17,21, βπβ-16β-methylpregna-1,4-diene) 3,20-dione 17,21-dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone.

The term "object that regulates IFN-γ production" is intended to include chemical factors (e.g., cytokines, enzymes, etc.) and cells that directly or indirectly direct IFN-γ synthesis in a subject. Examples of factors that regulate IFN-γ production include IL-18 (see Okamura, H. et al. (1995) Nature 378.88-91; Ushio, S. et al. (1996) J. Immunol. 156: 42744279) and interleukin-12 (IL-12) (see Schoenhaut, D. et al. (1992, J. Immunol. 148: 3433; PCT Patent Application No. WO 951059; European Patent Application No. EP 433 827 A2). Examples of cells that regulate IFN-γ production includes NK cells and NK-like cells.

Compositions as used herein that "antagonize" a factor are intended to include agents that inhibit factor activity and a agent that downregulates (namely inhibits) factor synthesis or production.

The term "IL-18" refers to a cytokine having the amino acid sequence as set forth in Okamura, H. et al. (1995) Nature 378: 88-91 (mouse) or Ushio, S. et al (1996) J. Immunol. 156: 4274-4279 (human) and other mammalian homologues thereof.

Cytokine IL-18 has also been referred to in the art as Interferon γ Inducing Factor (IGIF) induction (IL-γ).

The term "IL-18 antagonist" is intended to include agents that inhibit the synthesis or production of IL-18, agents that inhibit IL-18 activity once synthesized, agents that inhibit IL-18 interaction with the IL-18 receptor, which inhibit IL18 receptor activity. Examples of the IL-18 antagonist include protease inhibitors of the caspase family (e.g., ICE inhibitors) and antibodies, antibody fragments, or displayed binding protein that binds to IL-18 or the IL-18 receptor.

The term "interleukin-12 (IL-12)" refers to a cytokine having an amino acid sequence as set forth in Schoenhaut, D. et al. (1992J J. Immunol. 148: 3433; PCT Patent Application No. WO 951059; European Patent Application No. EP 433 827 A2 and other mammalian homologues thereof.

The term "IL-12 antagonist" is intended to include agents that inhibit IL-12 synthesis or production, agents that inhibit IL-12 activity once synthesized, agents that inhibit IL-12 interaction with the IL-12 receptor, and agents that inhibit IL12 receptor activity. Examples of the IL-12 antagonist include antibodies, antibody fragments, or displayed binding proteins that bind to either IL-12 or the IL12 receptor, agents that stimulate intracellular cAMP production in cells that produce IL-12 (such as phosphodiesterase IV or phosphodiesterase IV inhibitors). beta-2 agonists) and agents that inhibit STAT4.

The term "caspase family proteases" is intended to include members of the caspase family proteases as described in Alnemri, E. et al. (1996) Cell 87: 171 including caspase-1 (ICE), caspase-2 (ICH-1), caspase-3 (CPP32, Yama, apopain), caspase-4 (TX, ICH-2, ICE _re i-II) ), Caspases-5 (ICE _re i-III, TY), Caspases-6 (Mch2), Caspases-7 (Mch3, ICE-LAP3, CMH-1), Caspases-8 (MACH, FLICE, Mch5), Caspases- 9 (ICE-LAP6, Mch6) and Caspase-10 (Mch4). In addition, the term "caspase family proteases" is intended to include any protein that participates in the amino acid sequence identity of more than 20% with ICE in the active domains of the protease (namely the active domains of the p10 and p20 ICE subunits) that contains the glutamine peptide sequence. -alanine cysteine-X-glycine (QACXG), in which cysteine (C) is a catalytically active cysteine residue and X is any amino acid, and which contains the serine-histidine-glycine (SHG) sequence, located N-terminus to the QACXG motif in which histidine (H) is a catalytic essential residue of histidine. Proteases from the caspase family typically demonstrate a significant preference for hydrolysis of the peptide bond immediately followed by an acidic amino acid (namely aspartic acid or glutamic acid).

Proteases from the caspase family are known in humans and other organisms, including mice and Caenorhabditis elegans. Examples of proteases from the caspase family include, for example, ICH-1 (Wang, L. et al. (1994) Cell 78: 739-750); ICH-2 (Kamens, J. et al. (1995) J. Biol. Chem. 270: 15250-15256); Mch 2 (Fernandes-Alnemri, T. et al. (1995) Cancer Res. 55: 2737-2742); CPP32 (Fernandes-Alnemri, T. et al. (1994) J. Biol. Chem. 269: 3076130764); Yama / CPP32p (Tewari, M. et al. (1995) Cell 81: 801-809); the mouse Nedd2 gene product (Kumar, S. et al. (1992) Biochem. Biophys. Res. Commun. 185: 1155-1161; Kumar, S. et al. (1994) Genes Dev. 8: 1613-1626); the product of the C. elegans gene, ced-3 (Yuan, J. et al. (1993) Cell 75: 641-652); human TX protein (Faucheu, C., et al., (1995) EMBO J. 14: 1914-1922); ICE _re , II and ICE _re , III (Munday, NA et al. (1995) J. Biol. Chem. 270: 15870-15876).

The term "interleukin-β-converting enzyme (ICE)" is intended to refer to a protease having an amino acid sequence as described in Cerretti, D.P. et al. (1992) Science 256 97-100 (human) or Nett, M.A. et al. (1992) J. Immunol. 149: 3254-3259 (mouse), and other mammalian homologues thereof.

The term "ICE inhibitor" is intended to include chemical agents that inhibit the proteolithic activity of ICE. Examples of ICE inhibitors are known in the art, including, for example, those disclosed in U.S. patent application Ser. No. 5,585,357 (pyrazolyl derivatives); U.S. patent application Ser. No. 5,677,283 (pyrazolyl derivatives); U.S. patent application Ser. No. 5,656,627 (inhibitors containing a hydrogen bonding group, a hydrophobic group, and an electronegative group); U.S. patent application Ser. No. 5,411,985 (gamma-pyrone-3-acetic acid compounds); U.S. patent application Ser. No. 5,430,128 (tripeptidyl derivatives); U.S. patent application Ser. No. 5,434,248 (tripeptidyl compounds); U.S. patent application Ser. No. 5,565,430 (Ν, Ν'-diacylhydrazinoacetic acid compounds); U.S. Patent Application No. 5,416,013 (peptidyl derivatives); PCT Patent Application No. WO 94/21673 (alpha-ketoamide derivatives); PCT Patent Application No. WO 97/22619 (N-acylamino compounds); PCT Patent Application No. WO 97/22618 (amino acids or di- or tripeptidamide derivatives); PCT Patent Application No. WO 95/3 5308 (inhibitors comprising a hydrogen bonding group, a hydrophobic group, and an electronegative group); PCT Patent Application No. WO 93/14777 (peptidyl derivatives); PCT Patent Application No. WO 93/16710 (peptidyl derivatives); PCT Patent Application No. WO 95/05152 (substituted ketone derivatives); PCT Patent Application No. WO 94/03480 (4-amino-2,2-difluoro-3-oxo-1,6-hexanedioic acid peptidyl derivatives); PCT Patent Application No. WO 94/00154 (peptidyl derivatives); PCT Patent Application No. WO 93/05071 (peptidyl derivatives); European Patent Application EP No. 519,748 (peptidyl derivatives); European Patent Application EP number EP 590 650 (cyclopropene derivatives); European Patent Application No. 628,550 (pyridazines); European Patent Application No. 644,198 (alpha-heteroaryloxymethyl ketones); European Patent Application No. 644,197 (peptide phosphinyloxymethyl ketones); European Patent Application No. 547,699 (peptidyl derivatives); GB Patent Application No. 2,278,276 (gamma-pyrone-3-acetic compound); and Canada Patent Application No. 2,109,646 (paranitroanilide peptides). The present invention encompasses the use of the ICE inhibitors disclosed in any prior disclosure in the methods described herein

Additional preferred inhibitors for use in the methods of the invention include ICE substituted aspartic sulfonamide inhibitors having the general formula I:

OR 'm

Ο Ο (%)) * //

I x

wherein R ¹ is hydrogen, C 1 -C 6 alkyl or benzyl;

R ² is -CHO, COR ^a or -CN;

each R * is independently hydrogen or C 1 -C 6 alkyl;

common name X is a bond, CH _2, CHR ^5, NH, NR ⁵ or O;

R ³ is aryl, substituted-aryl, heteroaryl, substituted-heteroaryl, cycloalkyl, substituted-cycloalkyl, heterocycle or substituted heterocycle;

the general designation Y is absent, NR ⁵ , CO, S, O, SO 2, -O (CHR ⁵ ) -, CHR ⁵ , NR ⁵ CO, NCR ⁵ , CONR ⁵ , OCHR ⁵ , CHR ⁵ O, SCHR ⁵ , CHR ^with S, SO 2 NR ⁵ , C 1 -C ⁶ alkyl, NR ⁵ SO 2. CH ₂ CHR ⁵ , CHR ⁵ CH ₂ , COCH ₂ or CH ₂ CO;

R ⁴ is absent, aryl, substituted-aryl, C 1 -C 6 alkyl, heteroaryl, substituted-heteroaryl, cycloalkyl, C 1 -C ₆ alkyl, substituted-cycloalkyl, heterocycloalkyl or substituted-heterocycloalkyl;

each R ⁵ is independently C 1 -C 6 alkyl, aryl, - (CH ₂ ) n aryl or (CH ₂ ) n cycloalkyl;

each n is independently 0 to 5, m is 1 or 2, and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

In an embodiment of the invention, R ² is CHO.

In another embodiment of the invention, R ¹ is hydrogen.

In another embodiment of the invention, R ^a is hydrogen.

In another embodiment, X is a bond.

In another embodiment of the invention, R ³ is phenyl or substituted phenyl.

In another embodiment of the invention, the general designation Y is a bond

In another embodiment of the invention, the general designation Y is O.

In another embodiment of the invention, the general designation Y is CH 2.

In another embodiment of the invention, R ⁴ is phenyl or substituted phenyl.

In another embodiment, R ² is CHO, R 1 is H, R ¹ is hydrogen, X is a bond, R ³ and R ⁴ are phenyl or substituted phenyl, and Y is a bond, CH 2 or O.

In another embodiment of the invention, m is 1 and R ⁵ is hydrogen.

Other preferred sulfoamide substituted ICE inhibitors have the general formula II

O O TOm W //

X ^{'S N} J> ^R2

R

II wherein R ¹ is hydrogen, C 1 -C 6 alkyl or benzyl;

R ² is -CHO, COR 'or -CN;

each R c is independently hydrogen or C 1 -C 6 alkyl;

X is a bond, CH 2, CHR ⁵ , NH, NR ⁵ or O;

the general designation Y is absent NR ⁵ , CO, S, O, SO 2, CHR ⁵ , NR ^S CO, CONR ⁵ , OCHR ⁵ , CHR ⁵ O, -O (CHR ⁵ ) n -, SCHR ⁵ , CHR ^with S, SO 2 NR ⁵ , NR ⁵ SO ² , CH ₂ CHR ⁵ , CHR ⁵ CH 2, COCH ₂ or CH ₂ CO;

each R ⁵ is independently C 1 -C 6 alkyl, aryl or - (CH ₂ ) _n aryl; each n is independently 0 to 5; m is 1 or 2, each Z independently is hydrogen or aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic group that is fused to a phenyl group containing Z as a substituent;

R ^b , R ^c , R ^d and R ^e are each hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, -OH, C 1 -C 6 thioalkoxy, halogen, trifluoromethyl, dialkylamino, -NO 2, -CN, -CF 3, CO 2 alkyl, -SO 3 H , -CHO, -COalkyl, -CONH-alkyl, -CONHR ', -CON (alkyl) 2, (CH2) n-NH2, - (CH2) n-NH-alkyl, -NHR ^q , -NHCOR ^q , - ( CH 2) OH, - (CH ₂ ) _n CONH ₂ or - (CH ₂ ) _n CO ₂ H; and R ^q is hydrogen or C 1 -C 6 alkyl, and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

Within other embodiments with regard on the compound of formula n. R < ¹ & gt ^; is hydrogen. Within other embodiments with regard on the compound of formula n. R ² is CHO. Within other embodiments with regard on the compound of formula II.

R * is hydrogen.

In another embodiment, with respect to compounds of Formula II, the general designation X is a bond.

In another embodiment, with respect to compounds of Formula II, the general designation Y is a bond, O or CH ₂ .

In another embodiment, with respect to compounds of Formula II, R ^b and R ^c are hydrogen.

In another embodiment, with respect to compounds of formula II, wherein R ^b , R ^c and R ^d are hydrogen and R ^e is C 1 -C 6 alkyl.

In another preferred embodiment with respect to compounds of formula II, R ^b and R ^c are located at the para position of the phenyl ring with respect to the general designation X and R ^b or R ^c is -OCH ₃ .

In another embodiment, with respect to compounds of formula II, m is 1 and R ⁵ is hydrogen.

Preferred compounds include:

3- (Biphenyl-2-sulfoamino) -4-oxo-butanoic acid;

3- (2-Benzyl-benzenesulfonylamino) -4-oxo-butanoic acid;

4-Oxo-3- (2-phenoxy-benzenesulfonylamino) -butanoic acid;

4-Oxo-3- (2-p-tolyloxy-benzenesulfonylamino) -butanoic acid;

3- [2- (4-Isopropyl-phenoxy) -benzenesulfonylamino] -4-oxo-butanoic acid;

4-Oxo-3- (2-m-tolyloxy-benzenesulfonylamino) -butanoic acid;

3- [2- (3-Isopropyl-phenoxy) -benzenesulfonylamino] -4-oxo-butanoic acid; and 3- (4'-Methyl-biphenyl-2-sulfonylamino) -4-oxo-butanoic acid.

Other ICE inhibitors include compounds of Formula III.

III wherein R ¹ is hydrogen, C 1 -C 6 alkyl or benzyl;

R ² is -CHO, COR ^a or -CN;

each R * is independently hydrogen or C 1 -C 6 alkyl;

X is a bond, CH ₂ , CHR ⁵ , NH, NR ^S or O;

R ⁵ is hydrogen, C 1 -C 6 alkyl, aryl or - (CH ₂ ) n -aryl;

each n is independently 0 to 5;

m is 1 or 2;

Z is absent or is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, which is fused to a phenyl group containing Z as a substituent;

R ^f , R ⁸ are independently hydrogen, C 1 -C 6 alkyl, hydroxy, halogen, trifluoromethyl, dialkylamino, -NO ₂ , -CN, -CO ₂ H, -CO ₂ alkyl, -SO ₃ H, -CHO, COalkyl, - CONH ₂₎ -CONH (CH ₂ ) n -aryl, -CONH (CH ₂ ) _and -substituted-aryl, 17

CONH-alkyl, -CONHR ^q , -CON (alkyl) 2, - (CH 2) n -NH 2, - (CH 2) n -N-alkyl, NHR ^q , -NHCOR ^q , -OR ^q , -SR ^q or - ( CH 2) _n aryl; and R ^q is hydrogen or C 1 -C 8 -alkyl and acceptable salts, esters, amides, and prodrugs thereof.

In a preferred embodiment of the compounds of formula III, R ^f is ortho to X on the phenyl ring and R ⁸ is hydrogen.

In a preferred embodiment of the compounds of formula III, the general designation Z is hydrogen, m is 1, R ⁵ is hydrogen and R 8 is hydrogen.

In a preferred embodiment of the compounds of formula III, the compound is 3-benzenesulfonylamino-4-oxo-butanoic acid;

The above-described sulfonamide substituted aspartic acids of ICE inhibitors may generally be prepared as follows:

Other sulfonamide ICE inhibitors that can be used in the invention are compounds of formula IV:

wherein R ¹ is

ABOUT

II .s

R 4 ^Z 4 H

ABOUT

R ⁵

R 6x

R <S ^ _Ν φ (Αλ <

⁶ R ^

R ³ is hydrogen,

C, -C «alkyl,

- (CH ₂ ) _n aryl or - (CH 2) _n heteroaryl;

R ⁴ is C 1 -C 6 alkyl,

- (CH ₂ ) _n aryl or - (CH ₂ ) _n heteroaryl;

R ⁵ and R ⁶ are each independently hydrogen,

C 1 -C ₆ alkyl, - (CH ₂ ) _n aryl or - (CH ₂ ) _n heteroaryl;

R ⁷ is C 1 -C 6 alkyl, - (CH ₂ ) _n aryl or - (CH ₂ ) _n heteroaryl;

each n is 0 to 6;

each m is 0, 1, 2, or 3;

general designation A is alanine, leucine, isoleucine, proline, phenylalanine, glycine, tyrosine, serine, threonine, tryptophan, cysteine, methionine, valine, asparagine, glutamine, aspartic acid, lysine, glutamic acid, arginine or histidine; ^R2 is - _{(CH2) n-Z;} and the general designation Z is aryl, heteroaryl, cycloalkyl, C 1 -C ₆ alkyl,

fluorenyl, substituted fluorenyl, substituted aryl, substituted heteroaryl or substituted cycloalkyl and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

In a preferred embodiment of the compounds of formula IV, R ¹ is

R ⁷ O

(A)

m

In a preferred embodiment of the compounds of formula IV, R is

R ⁷ O

m is 0 and R ⁷ is - (CH ₂ ) _n aryl.

In a preferred embodiment of the compounds of formula IV, R ¹ is

R ⁷ O

m is 0 and R ⁷ is -CHiaryl.

In a preferred embodiment of the compounds of formula IV, R ² is (CH ₂ ) n -aryl.

In a preferred embodiment of the compounds of formula IV, aryl is phenyl or naphthyl.

In a preferred embodiment of the compounds of formula IV, R ² is (CH ₂ ) _n cycloalkyl.

In a preferred embodiment of the compounds of formula IV, R ¹ is

O ^ _{ζ Α κ θ ζ χ} (ΟΗ _{2) η} phenyl or SO ₂ -phenyl.

In another preferred embodiment of the compounds of formula IV, the substituent

R ² is

ABOUT

In a preferred embodiment of the compounds of formula IV, R ² is

Other sulfonamide ICE inhibitors containing compounds of formula V

wherein ^R2 is -CH2CH2-aryl, -CH2-cycloalkyl, -CKhCKh cycloalkyl or CH ₂ CH 2 -heteroaryl;

R ¹ is

R 'is - (CH ₂ ) _n -aryl or - (CH ₂ ) _n heteroaryl; R ^b is aryl or heteroaryl; R ^c is -CH ₂ aryl or aryl;

R ^d is hydrogen or C 1 -C 6 alkyl;

R ^e is -CH ₂ aryl or is -CH ₂ heteroaryl; and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

In a preferred embodiment of the compounds of formula V, R ¹ is

R ^e O

In another preferred embodiment of the compounds of formula V, R ¹ is S.

B ^ ll ^

K Q

In another preferred embodiment of the compounds of formula V, R ^e is - (CH ₂ ) n -aryl.

In another preferred embodiment of the compounds of formula V, aryl is phenyl or naphthyl.

In another preferred embodiment of the compounds of formula V, R ^b is aryl.

Preferred compounds include:

3-Benzyloxycarbonylamino-4-oxo-5- (2-phenoxy-ethanesulfonylamino) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-propane-1-sulfonylamino) -pentanoic acid;

3-Benzenesulfonylamino-4-oxo-5- (2-phenylethane-1-sulfonylamino) -pentanoic acid;

5-Benzenesulfonylamino-3-benzyloxycarbonylamino-4-oxo-pentanoic acid, 3-Benzyloxycarbonylamino-5-methanesulfonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (naphthalene-1-sulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-cyclohexyl-ethanesulfonylamino) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (2-naphthalen-1-yl-ethanesulfonylamino) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1- (R) ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (indan-1-ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (9-fluoro-9H-fluoren-9-ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1- (S) ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino) -4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

3- {2- [4-Carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methyl-butyrylamino} -4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

3- (2- {2- [2-Acetylamino-3- (4-hydroxyphenyl) propionylamino] -4-carboxybutyrylamino} -3-methyl-butyrylamino) -4-oxo-5- (2-phenyl-ethanesulfonylamino pentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -5- (7,7-dimethyl-2-oxobicyclo [2.2.1] hept-1- (S) -ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- (2-Acetylamino-propylamino) -5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1 (S) -ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-biityrylamino) -propionylamino] -5 (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1-ylmethanesulfonylamino) -4-oxopentanoic acid;

3- {2- [4-Carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methyl-butyrylmino} -5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept -1-ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- (2- {2- [2-Acetylamino-3- (4-hydroxyphenyl) propionylamino] -4-carboxybutyrylamino} -3-methyl-butyrylamino) -5- (7,7-dimethyl-2-oxo- -bicyclo [2.2.1] hept-1-ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1-ylmethanesulfonylamino) - 4-pentopanoic acid;

3- (1,2,3,4-tetrahydro-1-oxo-isoquinolin-2-yl-acetamino-5-benzenesulfonyl-amino-4-hydroxy-pentanoic acid;

(S) -5- (Bicyclo [2.2.1] hept-1-ylmethanesulfonylamino) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] - pentanoic acid;

(S) -4-Oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -5- (2-phenyl-ethanesulfonylamino) -pentanoic acid; and

4-Oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -5-phenylethanesulfonylamino-pentanoic acid.

Other sulfonamides of ICE inhibitors contain compounds of formula VI.

wherein R * is

Ο

Ο

R ^e O

Ο

R R ^a is - (CH 2) n - aryl or - (CH ₂ ) n heteroaryl;

R ^b is aryl or heteroaryl;

R ^c is -CH ₂ aryl or aryl;

R ^d is hydrogen or C 1 -C 6 alkyl;

R ^e is -CH ₂ aryl or is -CH ₂ heteroaryl and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

In a preferred embodiment of the compounds of formula VI, R ¹ is

R ^e O

In a preferred embodiment of the compounds of formula VI, R ¹ is

ABOUT

II

In another preferred embodiment of the compounds of formula VI, R ^e is - (CH ₂ ) n -aryl.

In another preferred embodiment of the compounds of formula VI, aryl is phenyl or naphthyl.

In another preferred embodiment of the compounds of formula VI, R ^b is aryl.

The compounds of formulas IV, V or VI described above can generally be prepared by converting the corresponding starting sulfonamide 1 to Boc-sulfonamide 2 using a reagent such as di- tert -butyl dicarbonate. Bocsulfonamide 2 can then be reacted with an appropriately substituted bromoethyl ketone-β- tert -butyl ester of aspartic acid 3 in the presence of a base, followed by treatment with an acid to give the desired product 4.

Scheme 1

ABOUT

II

HJM — S — R ^{2 2} II o

diphenyl / f-butyl dicarbonate

Boe-NS-R ² II H

ABOUT

1. Boo - NS - R ² H II

O base

2. HCl or CF ₃ COOH

Alternatively, compounds of formulas IV, V or VI may generally be prepared by reacting a suitably substituted aspartic acid aldehyde 1 with nitromethane in the presence of a base such as potassium ZerZ-butoxide to give the nitroalcohol 2. Reduction of 2 to amine 3. followed by reaction with a suitable sulfonyl chloride yields 4, which can be oxidized to the ketone 5 with a reagent such as "Dess Martin periodinane" or Swern oxidation. Acid removal of the t-butyl ester protecting group with HCl or trifluoroacetic acid affords the desired product 6.

Scheme 2

H

R ¹ = hL = COOMe-COOt-Bu _t H

R ¹ = N = COOH

COOt-Bu

R ¹ = N 2, CH ₂ OH - COO-Bu _t H

R ¹ —N. XHO

H

R ¹ —N

OH

NO.

H

R ¹ —N

OH

NH, —COOt-Bu —COOt-Bu —COOt-Bu

R ² -SO, Cl

H

R ¹ —N

OH

H

N-SO ₂ -R ² -COO-Bu

H

R ¹ —N

ABOUT

H

N-SO ^ - R ²

R ¹

H II H ^—N x ^ \ / ^N ” ^SO2 — ^r2 —COOt-Bu — CO ₂ H

6 v

Further other ICE inhibitor compounds that may be used in the invention include hydroxamate compounds including compounds of formula

wherein R ¹ is or

R @ ⁷ CT @ (A) @

O O

JL ¹¹

R '-' (NA), /, R ^/ n ^x - (A) / '

OO ^R5 \

ABOUT

R <s /

^ ^Υ R 6, each R is independently hydrogen or Ci-Cealkyl;

R ³ is hydrogen, C 1 -C 6 alkyl, - (CH ₂ ) _n aryl, - (CH ₂ ) n heteroaryl, - (CH ₂ ) _p X -aryl or - (CH ₂ ) _p -X-heteroaryl;

R ⁴ is C ₆ alkyl, - (CH ₂₎ "aryl, - _(CH2)" heteroaryl, - _(CH2) jX-aryl or - _(CH2) jX-heteroaryl;

R ⁵ and R ⁶ are each independently hydrogen, C 1 -C 6 alkyl, - (CH 2) n aryl, (CH 2) n heteroaryl, - (CH 2) n 1 -aryl or - (CH 2) n 1 -heteroaryl; R ⁷ is C 1 -C 6 alkyl, - (CH 2) _p aryl, - (CH ₂ ) _p heteroaryl, - (CH ₂ ) 1 x -aryl or - (CH ₂ ) 1 x -heteroaryl;

each n is independently 0 to 6;

each p is independently 1 to 6;

each j is independently 2 to 6;

each m is 0 to 2;

the general designation A is alanine, valine, serine, threonine, glutamic acid, lysine, arginine, histidine, glutamine or α-aminobutanoic acid; R 'is hydrogen, C 1 -C 6 alkyl or - (CH ₂ ) _n phenyl; X is O or S; and the general designation Q is C 1 -C 6 alkyl, - (CH ₂ ) n aryl, - (CH ₂ ) n heteroaryl, and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

In an embodiment of the compounds of Formula VII, each R is hydrogen.

R ⁷ O

In another embodiment of the compounds of Formula II, R ¹ is m is 0.

In another embodiment of the compounds of Formula VII, R ¹ is

ABOUT

m is 0 and R ⁷ is - (CH 2) n -aryl.

In another embodiment of the compounds of Formula VII, the general designation Q is - (CH ₂ ) _n phenyl or - (CH ₂ ) _n naphthyl.

In another embodiment of the compounds of Formula VII, R 1 is hydrogen or methyl.

In another embodiment of the compounds of Formula VII, the general designation Q is -CH ₂ -phenyl, -CH ₂ -naphthyl, is -CH ₂ CH ₂ -phenyl, -CH ₂ CH ₂ -naphthyl.

Other ICE inhibitor hydroxamate compounds include compounds having

Formula VIII wherein Z is

VIII

each g is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, - (CH ₂ ) n CO ₂ R (CH ₂ ) naryl, aryl, - (CH ₂ ) n heteroaryl or heteroaryl; The common name is O or CH _2;

R ¹ is

R ³ (A) of

II _R A (A) _m

R ⁵ rsA

R ⁵ (A)

R ⁷ O (A) or

R 5 ^/ each R 5 is independently hydrogen or C 1 -C 6 alkyl;

R ³ is hydrogen, C 1 -C 6 alkyl, - (CH 2) _n aryl, - (CH 2) _n beteroaryl, - (CH ₂ ) _p X -aryl or - (CH ₂ ) _p -X-heteroaryl;

R ⁴ is C ₆ alkyl, - (CH _{2) n} -aryl, - _(CH2) nheteroaryI, - _(CH2) jX-aryl or - _(CH2) jX-heteroaryl;

R ⁵ and R ⁶ are each independently hydrogen, C 1 -C 6 alkyl, - (CH ₂ ) n aryl, (CH ₂ ) _n heteroaryl, - (CH ₂ ) n 1 -aryl or - (CH ₂ ) n 1 -heteroaryl; each n is independently 0 to 6;

each p is independently 1 to 6;

each j is independently 2 to 6;

each m is 0 to 2;

the general designation A is alanine, valine, serine, threonine, glutamic acid, lysine, arginine, histidine, glutamine or α-aminobutanoic acid; and the general designation X is O or S and pharmaceutically acceptable salts, esters, amides and prodrugs thereof.

In an embodiment of the compounds of formula VIII, the general designation Z is

and each g is hydrogen.

In an embodiment of the compounds of Formula VIII, R ¹ is

ABOUT

and m is 0.

In an embodiment of the compounds of Formula VIII, R ¹ is

ABOUT

m is 0 and R ⁷ is - (CH ₂ ) n -aryl.

In an embodiment of compounds of Formula VIII, R ⁷ is (CH ₂ ) n -phenyl.

In a preferred embodiment of the compounds of formula VIII, the general designation Z is

O g

g g g and each g is hydrogen.

Preferred ICE inhibitor hydroxamate compounds include:

3-Benzyloxycarbonyl-amino-4-oxo-5-phenylacetylaminooxy-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5- (3,5-dioxo-10-oxa-4-aza-tricyclo [5.2.1.0 ^2,6 ] dec8-en-4-yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indol-yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yloxy) -pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -4-oxo-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5- (2,5-dioxo-pyrrolidin-1-yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (2,2,3-trimethyl-5-oxo-pyrrolidin-1-yloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5- (1,3-dioxo-octahydro-isoindol-2-yloxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (1,3-dioxo-1,3-dihydro-isoindol-2-yloxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [3- (4-bromo-phenyl) -2,5-dioxo-2,5-dihydro-pyrrol-yloxy] -4-oxo-pentanoic acid;

3-benzyloxycarbonylamino-5- (3,5-dioxo-4-aza-tricyclo [5.2.1.0 ^{2 '6]} dec-8-en-4-yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2,4-dioxo-3-aza-spiro [5.5] undeca-3-yloxy) -4-oxo-pentanoic acid;

5- (2-Biphenyl-4-yl-5-oxo-pyrrolidin-1-yloxy) -4-oxo-3- (2-propenyl-penta-2,4-dienyloxycarbonylamino) -pentanoic acid;

5-Benzoylaminooxy-3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-propionyl-aminooxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-naphthalen-1-yl-acetylamino-amino) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (3-naphthalen-1-yl-propionylaminooxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [methyl- (3-phenyl-propionyl) -aminooxy] -4-oxopentanoic acid;

5- (Benzoyl-methyl-aminooxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- [methyl- (3-naphthalen-1-yl-propionyl) -aminooxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- [methyl- (naphthalen-1-yl-acetyl) -aminooxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [benzyl- (3-phenyl-propionyl) -aminooxy] -4-oxopentanoic acid;

5- [Benzyl- (3-naphthalen-1-yl-propionyl) -aminooxy] -3-benzyloxycarbonylamino-4- 21-pentanoic acid;

5- (3-Benzyl-2-oxo-pyrrolidin-1-yloxy) -4-oxo-3- (2-propenyl-penta-2,4-dienyloxycarbonylamino) -pentanoic acid;

5- (3-Methyl-2-oxo-pyrrolidin-1-yloxy) -4-oxo-3- (2-propenyl-penta-2,4-dienyloxycarbonylamino) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- [methyl- (phenylacetyl) -aminooxy] -pentanoic acid or

3-Benzyloxycarbonylamino-4-oxo-5- (1-oxo-1,3-dihydro-isoindol-2-yloxy) -pentanoic acid.

Hydroxamate ICE inhibitors as described above can be prepared, for example, as in Example 12.

Still other types of ICE inhibitors that may be used in the invention include aspartate ester inhibitors including compounds having the general Formula IX:

ABOUT

ABOUT

R 2 wherein R ¹ is R ³ OC (O) -,

R ³ CO-, r ³ so ₂ ,

R ⁵ N (R 1) CHR ⁶ CO-,

IX

each R ^a is independently hydrogen, C 1 -C 6 alkyl or - (CH ₂ ) n -aryl;

R ² is - (CRR) n - aryl,

- (CRR) '- X-aryl,

- (CRR) "- heteroaryl,

- (CRR) n-X-heteroaryl,

- (CRR) n (substituted heteroaryl);

- (CRR) '- (substituted aryl);

- (CRR) n-X- (substituted-aryl).

- (CRR) n-aryl-aryl,

- (CRR) n-aryl-heteroaryl,

- (CRR) _n -aryl- (CH ₂ ) _n aryl,

- (CRR) _n -CH (aryl) ₂ ,

- (CRR) n-cycloalkyl,

- (CRR) '- X-cycloalkyl,

- (CRR) n-heterocycle,

- (CRR) n-X-heterocycle,

- (CRR) n-substituted heterocycle

- (CRR-CH ₂ CH ₂ ) -aryl, substituted aryl

- (CRR-CH 2 -C ₂ ) -aryl.

O- (CRRJ — N /

- (CRR) -CH, (CH 2) 4 -heteroaryl ( ^CH 2 ⁾ n ^-aryl

ABOUT

ABOUT

N-S- [aryl »substituted aryl.]

II ’

O o

N-CO (CH2) JJJ [aryl. ^or substituted aryl

ABOUT

Λ,

N aryl.,

AJ

Ο

N, N = O (CH ₂ ) _n aryl

T ⁴ f ^N (CHR) _n - _or 00 ^{N> (CHR} ).

each R is independently hydrogen, C 1 -C 6 alkyl, halogen or hydroxy;

X is O or S;

R ³ is C 1 -C ₆ alkyl, aryl, heteroaryl,

- (CHR) "- aryl,

- (CHR) "- heteroaryl,

- (CHR) _n -substituted heteroaryl, - (CHR) _n -substituted aryl,

- (CRR) "C (O) OR a,

- (CRR) n - S (CH ₂ ) n - aryl, cycloalkyl, substituted cycloalkyl, heterocycle, substituted heterocycle,

- (CRR) _n -C (O) NR a R a,

- (CRR) _n -SO ₂ - (CH ₂ ) _n -aryl,

- (CRR) _n -SO ₂ -C 1 -C ₆ alkyl,

J-CH ₂ C (R) H,

-CHR ⁶ C (O) -heteroaryl,

- (CRR) _n -S (CH ₂ ) n C (O) OR \

- (CRR) n - SO ₂ - (CH ₂ ) n C (O) OR a,

- (CRR) n - S (CH ₂ ) _n -aryl,

- (CRR) _n -SO ₂ - (CH ₂ ) _n -aryl,

- (CRR) n -SC (O) C 1 -C ₆ alkyl,

- (CRR) n -S (O) (CH ₂ ) n -aryl,

- (CRR) n - S (O) (CH ₂ ) _{n and} CO ₂ R ^a ,

- (CRR) -N

_R NHC ₆ alkyl

-CH ₂ NHC (O) -C 1 -C ₆ alkyl,

Ο

- (CRR) H-N

(CH ₂ ) _n aryl,

CNR ^b R ^b

I aryl with v

I — Ν ^{ζ Ή ζΧχ} \ / <CH ₂ ) _n θ R 'R'

(\ / (CH ₂ ) _n

ABOUT

II aryl — S

II o

• N, w //

CH ₂ C (O) NR ^b R ^b ,

Ο R 'R'

R 'R'

X) o

II

C 1 -C 6 alkyl N,

ABOUT

II

CC ^ -Cgalkyl

OCH ₂ -aryl

R (CRR) _n or each R 'is independently C 1 -C ₆ alkyl, C 1 -C 6 alkylaryl, aryl or hydrogen;

each generic designation J is independently

-CO ₂ R ^b

-SO ₂ NR ^b R ^b or -SO ₂ R ^b ;

-CONR ^b R ^b

aryl o 4 = o, N ¹¹ tr

C- (CRR) _n - each R ^b is independently hydrogen, C 1 -C ₆ alkyl, aryl, substituted aryl; arylalkyl, heteroarylalkyl, substituted arylalkyl or substituted heteroarylalkyl;

R ⁴ is C 1 -C 6 alkyl,

CH ₃ OC (O) -, phenyl or C 1 -C ₆ alkyl C (O) -;

R ⁵ is C 1 -C 6 alkyl-CO-,

- (CH ₂ ) n -aryl,

C 1 -C ₆ alkylOC (O) -,

C 1 -C 6 alkyl-X- (CH ₂ ) n CO,

C 1 -C ₆ alkyl-X- (CH ₂ ) _n OC (O) -,

C (O) (CRR) '- aryl,

-C (O) NR ^and R 1

-SO ₂ -C 1 -C 6 alkyl,

-C (O) (CH ₂ ) _n C (O) NR'R ',

-C (O) O (CH ₂ ) _n aryl,

-C (O) O (CH ₂₎ "substituted aryl,

-C (O) (CRR) n NHC (O) O (CH ₂ ) n - aryl,

ABOUT

II —c—

- (CH ₂ ) n X (CH ₂ ) _n -aryl,

-C 1 -C 6 alkyl-C 1 -C 6 alkyl aryl or OOO

-C-CH-NHC-CH-NHC 1 -C ₆ alkyl (CH 2) _n CH 2 -heteroaryl

CO ₂ R ^and R ⁵ 'is C (O) C 1 -C ₆ alkyl,

-C (O) _OC-C6 alkyl,

-C-CH-NHC 1 -C 6 alkyl,

I (CH ₂ ) _n aryl substituted aryl

-C (O) O (CH ₂ ) n aryl, -C (O) (CH ₂ ) _n aryl or

OO « ¹¹

CCHNHC 1 -C 6 alkyl;

I

CH ₂ heteroaryl substituent R ⁶ is hydrogen, C 1 -C 6 alkyl, - (CH ₂ ) _n aryl, - (CH ₂ ) _B CO ₂ R *, hydroxyl substituted with C 1 -C ₆ alkyl or imidazole substituted with C 1 -C ₆ alkyl;

each n is independently 0 to 3, and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

In an embodiment of the compounds of Formula IX, R ¹ is phenyl-CH ₂ OC (O) -.

Within further embodiments compound of formula ix. substituent R ¹ is a phenyl-SO ₂ -. Within further embodiments compound of formula ix. substituent R ¹ is a CH ₃ -OC (O) -. Within further embodiments compound of formula IX. substituent R ¹ is a

phenyl-CH ₂ CH ₂ -CO-.

In another embodiment of the compounds of Formula IX, R ¹ is

H ₃ C

In another embodiment of the compounds of Formula IX, R 1 is:

In another embodiment of the compounds of Formula IX, R ¹ is phenyl-CH ₂ -CO-.

In another embodiment of the compounds of Formula IX, R ¹ is

WHAT- .

In another embodiment of the compounds of Formula IX, each R is hydrogen.

In another embodiment of the compounds of Formula IX, R ² is (CH ₂ ) n -phenyl.

In another embodiment of the compounds of Formula IX, R ² is (CH ₂ ) n -naphthyl.

In another embodiment of the compounds of Formula IX, R ² is (CH ₂ ) n -O-phenyl.

In another embodiment of the compounds of Formula IX, R ² is (CH ₂ ) n -naphthyl.

In another embodiment of the compounds of Formula IX, R ² is (CH ₂ ) _n -S-phenyl.

In another embodiment of the compounds of Formula IX, R ² is (CH ₂ ) n -CH (phenyl) ₂ .

In another embodiment of the compounds of Formula IX, each R ^a is hydrogen; R ¹ is benzyloxycarbonyl; ^R2 is aryl-X (CRR) "-, aryl- (CRR)" -, heteroaryl- (CRR) "-, or cycloalkyl- (CRR)"-; n is 1, 2 or 3; X is O or S; and R is hydrogen, methyl or benzyl.

In another embodiment of the compounds of Formula IX, each R 8 is hydrogen;

R ¹ is benzyloxycarbonyl; and R ² is - (CH ₂ ) n - naphthyl,

- (CH ₂ ) n -phenyl,

- (CH _{2) n} -cycloalkyl,

- (CH ₂ ) "O (CH ₂ )" - naphthyl,

- (CH ₂ ) _n O (CH ₂ ) n -phenyl or - (CH ₂ ) n S (CH ₂ ) n -phenyl.

In another embodiment of the compounds of Formula IX, each R ^a is hydrogen;

R ¹ is benzyloxycarbonyl; and R ² is -CH ₂ -naphthyl.

In another embodiment of the compounds of Formula IX, each R 8 is hydrogen; R ² is benzyloxycarbonyl, ο

II ο

II ο

ιι ο

ιι

C-CH-NHC-CHNHC-CHNHCCpCg alkyl, ₃

CH ₂ CH ₂ CO ₂ H ^CH 2 ^heteroaryl

Ο Ο

II II

_-C-CH-CH3 NHCch ^

-CHNHC-CHNHC 1 -C 8 alkyl,

CH ₂ CH ₂ CO ₂ H ^CH 2 ^aryl »

-C-CH-CH ₂ -S-aryl, <I, _CH3 oo

II H

-C-CHCH ₂ -S-aryl.

I II ch ₃ o

Other ICE inhibitor aspartate ester compounds that can be used in the invention include compounds of formula X:

X wherein R ¹ is -C (O) OCH ₂ phenyl, -SO ₂ -phenyl, -C (O) OCH ₃ , C (O) CH ₂ CH ₂ phenyl,

O O

II II

C-CHNHCCH3, _CH3

C (O) _CH2 thienyl, -C (O) _Ci-6 alkyl,

ABOUT

II ch ₃ c

NH-CH = CH,

ABOUT

II

-COCH ₂ phenyl,

- (CH ₂ ) ₃ phenyl,

O O

H II

-CCH-NHS-CH3,

IB ch ₃ o

000

II II N

-c-ch-nh-c-ch-nhcch ₃

I I

CH ₃ (CH ₂ ) 2

Phenyl o o n 11

-c-ch-nhcch ₃ , ch ₂

I ch ₃ ο ο σ

II II II

CCHNHC (CH2) 3CNH?

I ch ₃ ο ο

II II

C-CHCH ₂ S-phenyl

I II ch ₃ ο

-C-CH-NHCNH2,

CH ₃

II. .

-CCH-C- thienyl,

C ιι c-ch ₃ ch ₃

Ο Ο

II c-chch ₂ -s-ch ₃

I II ch ₃ ο ο ο

II II

C (CH ₂ ) ₂ CNH ₂ , ο ο

II II

C (CH ₂ ) ₃ CNH ₂ , ο ο

II Ηchch ₂ cnh ₂ ,

-S (CH ₂ ) _n SCH ₃ ;

Ο ο

Ο ο

II II

-ch ₂ sch _3>

II ch ₃ ο

CI

CH

CH

-CCHNH

H ·

ABOUT

II '—chnhcoch ₂ phenyl o

II o

II

C-CH-NHCOCH-, phenyl

₃ h ₃ c ch.

phenyl o

II

O = S = O i Ň ch —ch ₂ ch ₂ c — C>

0 0

II II II

C-CH-NHC-CHNHCCH3,

I I

CH 3 (CH ₂₎ 2

I Co ₂ H o

II —C-CH — N N- phenyl

I v_y ^y CH,

ABOUT

II

CCH-SCH ·? phenyl.

I ch ₃ o

s? Λ

-C-CH-N N- phenyl ¹ kJ

CH,

Ο Ο Ο

-C-CH-NHC-CH-NHCOCH ₂ phenyl

Λ.

h ₃ c 'ch ₃ (ch ₂ ) ₂ co ₂ ho

II o

II

ABOUT

II

ΌΓ1

Λ.

H ₃ Cl ₃

about

II

C-NH-CH-CH-NHC _{(CH2) 2} -phenyl

A.

h ₃ c

CH, (ch ₂ ) ₂ co ₂ ho

II

-C-CH-NHC (CH ₂ ) 2 phenyl I ch ₃ o

II

II c-CH-NHC-CH / CH ₃

CH, _{CH2 CH3} o

II

ABOUT

II

ABOUT

II - CH - NHC - CH - NHCCHNHCCH ₃ (CH ₂ ) ₂ ch ₂ co ₂ hh ₃ c

CH, imidazoyl

H ιι h ₃ c

Ο

II

CH-NHCCH,

ABOUT

OH

-CGHCH ₂ CNH (CH 2) 2 phenyl, CH ₃

ABOUT

II

ABOUT

II

ABOUT

II h ₃ c

CH,

ABOUT

II

ABOUT

II

-CH 2 -CH 2 -S-phenyl

I

CH ₃

O O

II. II

-CCH ₂ CH ₂ -S- phenyl

I II ch ₃ oo

II

-CH ₂ CH ₂ -S- (CH ₂ ) 2 phenyl, CH ₃

O o

II u

C-CH ₂ -S-CH (CH _{2) 2} phenyl.

I II ch ₃ oo

-C-CHCH ₂ SCH ₂ phenyl

I ch ₃

Ο Ο

B- II

C-CH-CH2-S (CH2) 2 phenyl

I ιι ch ₃ ο ο ο

Β «

C-CH-SCH 2 phenyl

I II ch ₃ ο

-C-CH-NHCCHnCHn phenyl

₃ h ₃ c \; h ₃

o o

II II

S-CHCH2SCH _3,

II I II o ch ₃ ooo

II II

-CCHCH2SCCH ₃ ,

I

CH ₃ o

II

-CCHCH2CO2H.

I

CH ₃

ABOUT

-CCHCH 2 S- (CH 2) 3 -phenyl

C-CH-CH ₂ S- (CH ₂ ) 3 phenyl,

-c-ch-ch ₂ s (ch ₂ ) ₂ e ₂ H, ch ₃ ooo

-CCHCH ₂ S (CH2) 2CO2H;

R ¹¹ is CH ₃

-CH ₂ CH ₂ phenyl,

-CH ₂ naphthyl,

-CH ₂ CH ₂ cyclohexyl,

-CH ₂ O naphthyl,

-CH ₂ O phenyl,

-CH ₂ S-phenyl,

-CH ₂ -substituted naphthyl,

-CH ₂ CH (phenyl) ₂ ,

-CH ₂ -imidazole,

- (CH _{2) 3} phenyl,

-C _(CH3) H-naphthyl,

O - CH

_CH2 NF _EN yl

-CH [CH ₂ phenyl] ₂ , -C (OH) H-naphthyl, -CH ₂ -NH phenyl,

CH ₂ phenyl substituted naphthyl

-CH ₂ -naphthyl-phenyl,

-CH ₂ -fluorenyl,

-CH ₂ naphthylthienyl,

ABOUT

Λ 2

N - S - substituted phenyl δ

-CH ₂ -benzofuranyl,

-CH ₂ -benzothienyl,

-CH ₂ -naphthyl-CH ₂ -phenyl, -CH ₂ -substituted phenyl,

CH ₂ -substituted indolyl, ο

ιι

C ^- phenyl

Zľ) o

C OCH-, phenyl I ² N, phenyl-CH ₂ -CH

_CH2 phenyl / phenyl

/ pyridyl - _CH2 --CH phenyl ¹

ABOUT

- ch ₂ -n

- (CH ₂ ) ₂ pyridyl, or

each n is independently 0 to 3, and pharmaceutically acceptable salts, esters, amides, and prodrugs thereof.

Preferred ICE inhibitor aspartate ester compounds include:

3-Benzyloxycarbonylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5 - (- cyclohexyl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(naphthalen-1-yloxy) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5-phenoxyacetoxypentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5-phenylsulfanylacetoxy-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(6-methoxy-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (naphthalen-2-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (3-naphthalen-2-yl-propionyloxy) -4-oxo-pentanoic acid,

3-Benzyloxycarbonylamino-5- (3,3-diphenyl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(1H-indol-3-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (indol-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-naphthalen-1-yl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(2-oxo-pyrrolidin-1-yl) -acetoxy] -pentanoic acid;

5 - [(Acetyl-phenyl-amino) -acetoxy] -3-benzyloxycarbonyl-amino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (hydroxy-naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(phenyl-amino) -acetoxy] -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(6-hydroxy-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [3- (4-hydroxy-phenyl) -2-naphthalen-1-yl-propionyloxy) -4-oxo-pentanoic acid;

(S) -3-Benzyloxycarbonylamino-4-oxo-5-phenylacetoxy-pentanoic acid;

(S) -3-Benzyloxycarbonylamino-4-oxo-5- (4-phenyl-butyryloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(4-phenyl-naphthalen-1-yl) -acetoxy] pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(4-methyl-naphthalen-1-yl) -acetoxy] 4-oxopentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(4-thiophen-2-yl-naphthalen-1-yl) -acetoxy] pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(4-fluoro-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2-methyl-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2-fluoro-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

5- (Benzofuran-4-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzo [b] thiophen-7-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzo [b] thiophen-4-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5 - [(4-Benzyl-naphthalen-1-yl) -acetoxy] -3-benzyloxycarbonylamino-4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5 - [(3,4-dihydro-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-bromo-1H-indol-3-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (3,4-diphenyl-butyryloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-phenylamino-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(1,2,3,4-tetrahydro-naphthalen-2-yl) -acetoxy] -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(1-methanesulfonyl-piperidin-4-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(2,3,5,6-tetramethylphenyl) acetoxy] pentanoic acid;

5- (Benzothiazol-4-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzofuran-3-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzo [b] thiophen-3-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-pyridin-2-yl-propionyloxy) pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2,3-dichloro-phenyl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-methyl-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2-iodo-phenyl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-pyridin-3-yl-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-methoxy-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5 - [(8-methyl-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5 - [(9H-fluoren-9-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5-yl) -acetoxy] -4-oxo-pentanoic acid;

5-Oxo-1- (toluene-4-sulfonyl) -pyrrolidine-2-carboxylic acid 3-benzyloxycarbonylamino-4-carboxy-2-oxo-butyl ester;

2- (3-Benzyloxycarbonylamino-4-carboxy-2-oxo-butyl) 5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester;

1-Benzoyl-pyrrolidine-2-carboxylic acid 3-benzyloxycarbonylamino-4-carboxy-2-oxo-butyl ester

Pyrrolidine-1,2-dicarboxylic acid 2- (3-benzyloxycarbonyl amino-4-carboxy-2-oxo-butyl) ester 1-benzyl ester;

3-Benzyloxycarbonylamino-5- (2-benzyl-3-phenyl-propionyloxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-cyano-naphthalen-1-yl) -acetoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-pyridin-3-yl-propionyloxy) pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-pyridin-4-yl-propionyloxy) pentanoic acid; and

3-Benzyloxycarbonylamino-4-oxo-5 - [(1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) acetoxy] pentanoic acid.

3-Benzenesulfonylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Methoxycarbonylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (3-phenyl-propionylamino) -pentanoic acid;

3-Methoxycarbonylamino-4-oxo-5-phenoxyacetoxypentanoic acid; and

3- (2-Methanesulfonyl-1-methyl-ethylsulfanylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid.

[S- (R *, R *)] - 3- (2-Acetylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3 - [(thiophene-3-carbornyl) -amino] -pentanoic acid;

3 - [(Furan-3-carbonyl) amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (4-phenyl-butyrylamino) -propylamino] -pentanoic acid;

3- (2-Methanesulfonylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- [2- (2-Acetylamino-4-phenyl-butyrylamino) -propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- [2- (4-Carbamoyl-butyrylamino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-Benzyloxycarbonylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-ureido-propionylamino) -pentanoic acid;

3- (2-Acetylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3 - [(1-Acetyl-pyrrolidine-2-carbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Methyl-3-oxo-3-thiophen-2-yl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-acetylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-propionylamino) -5- (3,3-diphenyl-propionyloxy) -4-oxopentanoic acid;

3- [2- (2-Acetylamino-4-carboxy-butyrylamino) -propionylamino] -5- (naphthalenyl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (3-phenyl-propionylamino) -propionylamino] -pentanoic acid;

3- [2- (3-Methyl-butyrylamino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3 - [(1-Acetyl-4-benzyloxy-pyrrolidine-2-carbonyl) -amino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- (4-Carbamoyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and

3- [2- (1-Methyl-1H-imidazol-4-yl) -acetylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid.

(S) -5- (Naphthalen-1-yl-acetoxy) -4-oxo-3-phenylacetylamino-pentanoic acid;

(S) -5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-thiophen-2-yl-acetylamino) -pentanoic acid;

3 - [(2-Carbamoyl-cyclopentanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3 - [(3-Carbamoyl-bicyclo [2.2.1] heptane-2-carbonyl) -amino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- (3-Methanesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Benzenesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Butyrylamino-5- (naphthalen-2-yl-acetoxy) -4-oxo-pentanoic acid;

3-Acetylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Methanesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Carbamoyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

[S- (R *, R *)] - 3- (3-Acetylsulfanyl-2-methyl-propionylamino) -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid; and trans-3 - [(3-Carbamoyl-cyclopentanecarbonyl) -amino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid.

3- (1,2,3,4-tetrahydro-1-oxo-isoquinolin-2-yl) -acetamino-5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- (2-Methyl-3-phenethylcarbamoyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-2-yl-acetoxy) -4-oxo-3- [2- (2-oxo-6-phenyl-piperidin-1-yl) -acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-6-phenyl-piperidin-1-yl) -acetylamino] -pentanoic acid;

3- [3-methyl-2- (3-phenyl-propionylamino) -butyrylamino] -4-oxo-5 - [(l-oxo-2,3,4-tetrahydro-naphthalen-2-yl) acetoxy] - pentanoic acid;

5- (Naphthalen-2-yl-acetoxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (2-oxo-6-phenyl-piperidin-1-yl) -acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl) -acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -propionylamino] -pentanoic acid;

5- (Naphthalen-2-yl-acetoxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -propionylamino] -pentanoic acid;

3- [4- (1-Benzenesulfonyl-1H-pyrrol-2-yl) -4-oxo-butyrylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl) -acetylamino] -pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -propionylamino] -pentanoic acid;

4-oxo-3- [2- (l-oxo-3,4-dihydro-lH-isoquinolin-2-yl) propionylamino] -5 - [(l-oxo-l, 2,3,4-tetrahydro-naphthalene 2-yl) -acetoxy] -pentanoic acid;

3- [4- (1-Benzenesulfonyl-1H-pyrrol-2-yl) -4-oxo-butyrylamino] -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

4-Oxo-5 - [(1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl) -acetoxy] -3- [2- (1-oxole, 2,3,4-tetrahydro- naphthalen-2-yl) -acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-3-phenyl-imidazolidin-1-yl) -propionylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-3-phenyl-tetrahydro-pyrimidin-1-yl) -propionylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-3-phenyl-tetrahydro-pyrimidin-1-yl) -acetylamino] -pentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -5- (3-benzyl-4-phenyl-butyryloxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -5- (4-benzyl-5-phenyl-pentanoyloxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -4-oxo-5 - [(1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl) -acetoxy] -pentanoic acid,

5- (3-Benzyl-4-phenyl-butyryloxy) -3- [3-methyl-2- (3-phenyl-propionylamino) butyrylamino] -4-oxo-pentanoic acid;

3- [2- (3-Acetylamino-2-oxo-2H-pyridin-1-yl) -acetylamino] -5- (3,3-diphenylpropionyloxy) -4-oxo-pentanoic acid; and

3- [2- (3-Acetylamino-2-oxo-2H-pyridin-1-yl) -acetylamino] -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid.

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5 (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-3-methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5 (3,3-diphenyl-propionyloxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5 (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- {2- [4-carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methyl-butyrylamino} -4-oxo-pentanoic acid ;

3- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -5- (3,3-diphenylpropionyloxy) -4-oxo-pentanoic acid;

3- (2-Acetylamino-3-hydroxy-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Acetylamino-3-hydroxy-butyrylamino) -5- (3,3-diphenyl-propionyloxy) -4-oxo-pentanoic acid;

3- (2- {2- [2-Acetylamino-3- (lH-indol-3-yl) propionylamino] -4-carboxybutyrylamino} -3-methyl-butyrylamino) -5- (2-benzyl-3-phenyl (propionyloxy) -4-oxo-pentanoic acid; and

5- (3,3-Diphenyl-propionyloxy) -4-oxo-3- [2- (4-phenyl-butyrylamino) -propionylamino] -pentanoic acid.

3- (2- {2- [2-Acetylamino-3- (lH-indol-3-yl) propionylamino] -4-karboxybutyryIamino} -3-methyl-butyrylamino) -5- (naphthalen-l-yl-acetoxy -4-oxo-pentanoic acid; and

3- (2- {2- [2-Acetylamino-3- (4-hydroxyphenyl) propionylamino] -4-carboxybutyrylamino} -3-methyl-butyrylamino) -5- (naphthalen-l-yl-acetoxy) - 4-Oxopentanoic acid.

3 - [(2-Carboxy-cyclohexanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3 - [(2-Methoxycarbonyl-cyclohexanecarbonyl) -amino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and

3 - [(2-Carbamoyl-cyclohexanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid.

3- (3-Benzylsulfanyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Methyl-3-phenylmethanesulfonyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- [3- (2-Carboxy-ethanesulfanyl) -2-methyl-propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (2-carboxy-ethanesulfonyl) -2-methyl-propionylamino] -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (3-carboxy-propane-1-sulfinyl) -2-methyl-propionylamino] -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-phenylmethanesulfanyl-propionylamino) -pentanoic acid;

3- (2-Methyl-3-phenylsulfanyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-methyl-3-phenylsulfanyl-propionylamino) -4-oxo-pentanoic acid;

3- (2-Methyl-3-phenethylsulfanyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-methyl-3-phenethylsulfanyl-propionylamino) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (3-benzylsulfanyl-2-methyl-propionylamino) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-benzylsulfanyl-propionylamino) -4-oxo-pentanoic acid;

3- [2-Methyl-3- (3-phenyl-propylsulfanyl) -propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- (3-Benzenesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Benzenesulfonyl-2-methyl-propionylamino) -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [2-methyl-3- (2-phenyl-ethanesulfonyl) -propionylamino] -4-oxo-pentanoic acid;

- [2-Methyl-3- (2-phenyl-ethanesulfonyl) -propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-phenylmethanesulfonyl-propionylamino) -pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-methyl-3-phenylmethanesulfonyl-propionylamino) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- (2-phenylmethanesulfonyl-propionylamino) -pentanoic acid;

3- [2-Methyl-3- (3-phenyl-propane-1-sulfonyl) -propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [2-methyl-3- (3-phenyl-propane-1-sulfonyl) -propionylamino] -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (2-carboxyethylsulfanyl) -2-methylpropionylamino] -4-oxo-pentanoic acid;

3- [3- (3-Carboxy-propylsulfanyl) -2-methyl-propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (3-carboxy-propylsulfanyl) -2-methyl-propionylamino] -4-oxo-pentanoic acid;

3- (3-Carboxymethylsulfanyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy)

4-Oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (3-carboxymethylsulfanyl-2-methylpropionylamino) -4-oxo-pentanoic acid;

3- [3- (2-Carboxy-ethanesulfonyl) -2-methyl-propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- [3- (3-Carboxy-propane-1-sulfonyl) -2-methyl-propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Carboxymethanesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (3-carboxy-propane-1-sulfonyl) -2-methyl-propionylamino] -4-oxo-pentanoic acid;

- (2-Benzyl-3-phenyl-propionyloxy) -3- (3-carboxymethanesulfonyl-2-methylpropionylamino) -4-oxo-pentanoic acid;

3- [3- (3-Carboxy-propane-1-sulfinyl) -2-methyl-propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- [2-Methyl-3- (3-phenyl-propane-1-sulfinyl) -propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid; and

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [2-methyl-3- (3-phenyl-propane-1-sulfinyl) -propionylamino] -4-oxo-pentanoic acid.

3- [3-Methyl-2- (phenethylcarbamoyl-methyl) -butyrylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid; and

3- (3-Carboxy-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid.

3- (2-Methyl-3-sulfamoyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid.

3- (3-Carbamoyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Benzyloxycarbonylamino-3-methyl-naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3 - [(2-Carbamoyl-cyclopentanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3 - [(1-Carbamoyl-pyrrolidine-2-carbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2- {2- [2-Acetylamino-3- (4-hydroxyphenyl) propionylamino] -4-carboxybutyrylamino} -3-methyl-butyrylamino) -5- (2-benzyl-3-phenyl-propionyloxy -4-oxo-pentanoic acid;

3- (3-Carbamoyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Carbamoylmethyl-3-methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Benzyloxy-2-ureido-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

3- {2- [4-Carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methyl-butyrylamino} -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and

3- [2- (2-Acetylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -5 (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid. .

The above ICE aspartate ester inhibitors can be made according to the following schemes 1-11:

Scheme 1

3-Benzyloxycarbonylamino-5-bromo-4-oxopentozoic acid tert -butyl ester, also known as Z-asp (OtBu) -bromomethylketone, is commercially available or can be prepared according to the following published procedure: Dolle, et al., J. Med. Chem., 1994; 37: 563-564. This methyl bromoketone is reacted with a suitably substituted carboxylic acid and a base such as potassium fluoride. In another method, bases such as cesium carbonate or potassium t-butoxide may be used. The reagents should be mixed in dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), acetonitrile, or other suitable solvent, and stirred at room temperature for 8 to 24 hours. The t-butyl ester protecting group may be removed in an acidic medium, preferably trifluoroacetic acid, to give the carboxyaspartyl esters shown in Scheme 1.

Scheme 2

or ROCOCI

The mixture of the appropriately substituted acyloxymethyl ketone t-butyl carbobenzoxyaspartyl ester is hydrogenated with an equivalent hydrochloric acid or other acid in the presence of a catalyst such as palladium on charcoal to give the amine salt. The salt may be acylated with a suitably substituted isocyanate, sulfonyl chloride, chloroformate or phenylpropionyl chloride to give N-substituted derivatives. These isocyanates, sulfonyl chlorides or chloroformates are commercially available or can be synthesized using methods described in the chemical literature. The t-butyl ester protecting group can be removed in the final step using an acidic medium, preferably trifluoroacetic acid, to give the acyloxymethyl ketone derivatives shown in Scheme 2.

Scheme 3

Step A

R 2 R ¹ CO ₂ H

Spojovacie prost. HOBT, Base

Step B CF ₃ CO ₂ H

The amine salt of the acyloxymethyl ketone Z-asp (Ot-Bu) OH is synthesized and reacted with an appropriately substituted carboxylic acid and coupling agent. The coupling agent may be, but is not limited to, such as 1,3-dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1,1'-carbonyldiimidazole (CDI), 1,1'- carbonylbis (3-methylimidazolium) triflate (CBMIT), isobutyl chloroformate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), 2- (3,4-dihydro-4-oxo-1,2,3-benzotriazin-3yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TDBTU) and 2- (1H-benzotriazoll-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). 1-Hydroxybenzotriazole hydrate should be added to the reaction to improve yield, prevent isomerization, increase basicity, preferably an amine such as trimethylamine or methyl morpholine should be added as an acid scavenger. The resulting amide product is reacted with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester to give the end products as described in Scheme 3.

Scheme 4 hci.h ₂ n

CO ₂ 'Bu

Step A

R 2 R ¹ COX (X = Cl, F)

Step B

CF ₃ CO ₂ H ^{R 1}

ABOUT

O to O

R 2

CO ₂ H

The amine salt of the acyloxymethyl ketone Cbz-Asp (Ot-Bu) OH is synthesized and reacted with an appropriately substituted acid chloride or acid fluoride to generate the amide product. Acid chlorides are commercially available or can be prepared by reacting carboxylic acids with agents such as thionyl chloride, phosphorus tribromide or oxalyl chloride / DMF. The acid fluorides are prepared by reacting the carboxylic acid with cyanuric acid. The penultimate amide product is reacted with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester to give the final products as described in Scheme 4.

Scheme 5

2. NaOH

Step B

Step A

1. R ¹ C0 ₉ H, Coupler

Step C

Formation of Bromomethylketone ¹

RCO ₂ H, KF DMF

Step D CF ₃ CO ₂ H 0 R ² 0 <0 every ₂ h

The H-Asp (OtBu) OMe hydrochloride salt is reacted with a suitably substituted carboxylic acid and coupling agent. 1-Hydroxybenzotriazole hydrate should be added to the reaction to improve yield, prevent isomerization, increase basicity, preferably an amine such as trimethylamine or methyl morpholine should be added as an acid scavenger. The resulting amide product is reacted with an alkaline reagent such as sodium hydroxide to hydrolyze the methyl ester to give the carboxylic acid. The resulting carboxylic acid is reacted with a chloroformate such as isobutyl chloroformate, followed by reaction with diazomethane and then hydrobromic acid to give methyl bromoketone. Reaction of methyl bromoketone with an appropriately substituted carboxylic acid and a base such as potassium fluoride affords the desired ketones which are deprotected with trifluoroacetic acid to give final compounds as described in Scheme 5.

Scheme 6

Step E

Formation of bromomethylketone jjj. Cbz-AA binding iv. atcf

The salt of H-Asp (OctBu) OMe hydrochloride is reacted with a suitably protected amino acid and coupling agent. 1-Hydroxybenzotriazole hydrate should be added to the reaction to improve yield, avoid isomerization and increase basicity, preferably an amine such as trimethylamine or methyl morpholine should be added as an acid scavenger. The resulting amide product is reacted with an alkaline reagent such as sodium hydroxide to hydrolyze the methyl ester to give the carboxylic acid. The Cbz-amine protecting group is removed using standard catalytic hydrogenation conditions, and the coupling of the additional protected amino acid can take place as described above. This procedure is repeated until the peptide has the desired length. The resulting peptide product is reacted with an alkaline reagent such as sodium hydroxide to hydrolyze the methyl ester to give the carboxylic acid. The resulting acid is then reacted with a chloroformate such as isobutyl chloroformate, followed by diazomethane and then hydrobromic acid to give methyl bromoketone. Reaction of methyl bromoketone with a preferably substituted carboxyl group and a base such as potassium fluoride affords the desired acyloxymethyl ketones which are deprotected with an acid to give the final compounds as described in the scheme.

6th

Scheme 7

CBZ-AA

ABOUT

Step A

H _{2 liters} of 20% Pd / C

Suitably substituted acylmethylketones of the protected amino acid are synthesized. The Cbz-amine protecting group is removed using standard catalytic hydrogenation conditions and the amine product is reacted with a suitably substituted carboxyl group and coupling agent. 1-Hydroxybenzotriazole hydrate should be added to the reaction to improve yield, avoid isomerization and increase basicity, preferably an amine such as trimethylamine or methyl morpholine should be added as an acid scavenger. The penultimate amide product is reacted with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester and obtain the end products as described in Scheme 7.

Scheme 8

pyridine, DMAP

1. Formation of an amide or ester

2. CF ₃ CO ₂ H

The trans-1,2-cyclobexanedicarboxylic acid anhydride is reacted with an amine salt suitably substituted with the acyloxymethyl ketone of t-butyl aspartyl ester in the presence of pyridine and 4-dimethylaminopyridine (DMAP) to afford the amide product. The carboxylic acid can be functionalized with suitably substituted amines or alcohols and standard coupling reagents to provide ester products. The penultimate product is reacted with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester and yield the end products as described in Scheme 8.

Scheme 9

1.R ¹ SH, NaH

Me

CO ₂ R

2. NaOH

1. fasteners .

2. NaOH

1. Oxidation

2. CF ₃ CO ₂ H

The methyl methacrylate is reacted with an anion of a suitably substituted sulfide to provide a Michael adduct which is hydrolyzed in a basic medium such as sodium hydroxide to give the carboxylic acid. This acid is combined with the amine salt of the acyloxymethyl ketone of t-butyl aspartyl ester and the coupling agent to give the amide product. If the desired product is a sulfide (n = 0), the oxidation step is not used and the amide t-butyl ester is removed (protecting group) in an acidic medium. Alternatively: When the sulfoxide (n = 1) or sulfone (n = 2) is the final product, the amide intermediate is reacted with an oxidizing agent, which may be, but is not limited to, chloroperbenzoic acid, potassium monoperoxysulfate or sodium perborate, thereby to give the desired oxidized product. The t-butyl ester protecting group of the penultimate intermediate is removed in an acidic medium, preferably trifluoroacetic acid, to give the final compounds as described in Scheme 9.

Ο χ

Ο NH

Η

R

Ο Ο

i. n-BuLi, -78 ° C, X., A / R 'NaNfSiMeJ; -78 ° C, ON

H ii. R HjCOCI

LiOH, H ₂ O ₂₁ BuO, C

THF, water

ABOUT

Spojovacie prost.

ii. BrCH ₂ CO ₂ ^t Bu R ¹

R ³ N

1. CF ₃ CO ₂ H

u.

⁰ Yr °

2. R ₃ R ₂ NH or PhCH ₂ ONH ₂ coupling

PhSiH ₃ , Pd (PPh 3) ₄

2. Formation of bromomethylketone

3. R ₂ CO ₂ H, KF, DMF

Ph

1. PhSiH _3, Pd _{(PPh3) 4}

2. Formation of bromomethylketone

3. R ₂ CO ₂ H, KF, DMF

R ¹

R ³ N

ABOUT

Ύ

R ₂ 'BuO ₂ C

R ¹

H,

.ph

Auxiliary chiral 4-substituted-2-oxazolidine as described in Evans, et al.,

Chem., 1985; 50: 1830 is mixed with a base such as n-butyllithium, but is not limited thereto, followed by reaction with an appropriately substituted acid chloride or other activated carboxylic acid to give the Nacylated product. This product is then reacted with a base such as, but not limited to, sodium bis (trimethylsilyl) amide and t-butyl bromoacetate, resulting in the formation of an alkylated chiral product. Then, the chiral auxiliary is removed using lithium hydroxide and hydrogen peroxide to give the chiral acid. Treatment of the acid with an amine salt of H-Asp (OBz) O-allyl and a coupling agent affords the succinylamide product.

At this stage of the process, the product can be developed in one of two ways. The first in which the t-butyl ester is removed in an acidic medium, preferably trifluoroacetic acid, and the resulting acid is combined with a suitably substituted amine in the presence of a coupling agent, resulting in the formation of a new amide product. The allyl ester is removed with phenylsilane and tetrakis (triphenylphosphine) palladium or other Pd (O) catalyst to give the carboxylic acid which is converted to methyl bromoketone and then to acyloxymethyl ketone. The penultimate intermediate is subjected to catalytic hydrogenation to remove the benzyl ester to give the final amide products as described in Scheme 10.

Alternatively, in a second way to obtain the end products, the allyl ester is removed using phenylsilane and tetrakistetrakis (triphenylphosphine) palladium or other Pd (0) catalyst to give the carboxylic acid. This acid is converted to methyl bromoketone and then to acyloxymethylketone. Removal of the t-butyl acyloxymethyl ketone with trifluoroacid and subsequent conversion of the resulting carboxylic acid to the ester results in a new ester product. Esterification can be accomplished using a variety of techniques reported in the relevant literature, including, but not limited to, reacting a carboxylic acid with a suitably substituted alcohol in the presence of a coupling agent. The penultimate intermediate is subjected to catalytic hydrogenation to remove the benzyl ester and provide the final ester products as described in Scheme 10.

Scheme 11

1. PhCH ₂ Br, DBU

2- Cl ₂ (g) EtOH: CCl ₄ (1: 9)

R ¹

JL cK ^{x /} XO ₂ CH ₂ Ph

1. (R) ₂ NH, Et 3 N, CH ₂ Cl ₂

2. _H2, 20% Pd / C

2. H-Asp (O'Bu) -OMe.HCl NMM, CH ₂ Cl ₂

3. NaOH, H ₂ , EtOH

1. SOCl ₂ or Cyanofluoride

The appropriately substituted S-acetyl mercaptocarboxylic acid is reacted with benzyl bromide and 1,8-diazobicyclo [5.4.0] undec-7-ene (DBU) to give the benzyl ester, which is then reacted with chlorine gas to give the sulfonyl chloride. The sulfonyl chloride is reacted with N, N-bis (pmethoxybenzyl) amine to provide the sulfonamide, which is subjected to catalytic hydrogenation to provide the carboxylic acid intermediate. The acid is activated using cyanuric fluoride, which is then mixed with the amine salt of H-Asp (Ot-Bu) OMe to afford the amide product. The methyl ester is hydrolyzed with sodium hydroxide and the carboxylic acid is treated to an acyloxymethyl ketone. The p-methoxybenzyl sulfonamide protecting groups are removed under oxidizing conditions, preferably, but not limited to, ammonium cerium nitrate, and the t-butyl ester protecting group is removed in an acidic medium, preferably trifluoroacetic acid to provide the desired sulfonamide products as described in the scheme. 11th

The term "alkyl" as used herein means a branched or unbranched hydrocarbon chain. Typical examples of alkyl groups are methyl, ethyl, propyl, isopropyl, isobutyl, butyl, tert-butyl, sec-butyl, pentyl and hexyl.

The term "alkoxy" means an alkyl group attached to an oxygen atom.

Typical examples of alkoxy groups include methoxy, ethoxy, tert-butoxy, propoxy and isobutoxy.

The term "halogen" includes chlorine, fluorine, bromine and iodine.

The term "aryl" means an aromatic hydrocarbon. Typical examples of aryl groups include phenyl and naphthyl.

The term "heteroatom" includes an oxygen, nitrogen, sulfur and phosphorus atom.

The term "heteroaryl" means an aryl group in which one or more carbon atoms of the aromatic hydrocarbon have been replaced by a heteroatom. Examples of heteroaryl groups include furan, thiophene, pyrrole, thiazole, pyridine, pyrimidine, pyrazine, benzofuran, indole, coumarin, quinoline, isoquinoline and naphthyridine.

The term "cycloalkyl" means a cyclic alkyl group. Examples of cycloalkyl groups include cyclopropane, cyclobutane, cyclopentane and cyclohexane.

The term "heterocycle" means a cycloalkyl group in which one or more carbon atoms have been replaced by a heteroatom. Examples of heterocycles include piperazine, morpholine and piperidine.

Aryl, heteroaryl or cycloalkyl groups may be substituted by one or more substituents, which may be the same or different. Examples of suitable substituents include alkyl, alkoxy, thioalkoxy, hydroxy, halogen, trifluoromethyl, amino, alkylamino, dialkylamino, NO ₂ , -CN, -CO ₂ H, -CO ₂ alkyl, -SO ₃ H, -CHO, -COalkyl, - CONH ₂ , -CONHalkyl, -CONHR ^q , -CON (alkyl) ₂ , - (CH ₂ ) n -NH ₂ , -OH, -CF ₃ , -OC 1 -C 6 alkyl, (CH ₂ ) -NH-alkyl, -NHR q , -NHCORq, phenyl, - (CH ₂ ) _n -OH, - (CH ₂ ) _n C (O) NH _2, or - (CH ₂ ) n CO ₂ H, wherein n is 1 to 5 and R ^q is hydrogen or alkyl.

Symbol means bond.

Examples of other caspase family inhibitors contemplated for use in the invention include ICH-1 inhibitors such as those described in PCT Patent Application Publication No. 97/27220.

The term "phosphodiesterase IV inhibitor" is intended to refer to compositions that inhibit the activity of enzyme phosphodiesterase IV. Examples of phosphodiesterase IV inhibitors are known in the art and include 4arylpyrrolidinones such as rolipram (see, for example, Sekut, L. et al. (1995) Clin. Exp. Immunol. 100: 126-132), nitraquazone (see, for example, Van Wauwe). , J. et al. (1995) Inflamm. Res. 44: 400-405), denbufylline, tibenelast (see, for example, Banner, KH et al. (1996) Br. J. Pharmacol. 119: 1255-1261) , CP-80633 (see, for example, Cohan, VL et al. (1996) J.Pharmacol. Exp.Therap. 278: 1356-1361), quinazolinone dione such as CP-77059 (see, for example, Sekut, L. et al. (1995) Clin. Exp. Immunol. 100: 126-132).

The term "beta-2 agonist" is intended to refer to agents that stimulate the adrenergic beta-2 receptor. Examples of beta-2 angonists are known in the art and include salmeterol (see, for example, in Sekut, L. et al. (1995) Clin. Exp. Immunol. 99: 461-466), phenoterol and isoproterenol (see, for example, in Severn). , A. et al (1992) J. Immunol. 148: 3441-3445).

The term "STAT4" is intended to refer to the transcription factor involved in IL-12 responses (see, for example, Thierfelder, WE et al. (1996) Nature 382: 171-174; Kaplan, MH et al. (\ 996) Nature 382). : 174177). The term "STAT4 inhibitor" refers to a composition that inhibits STAT4 transcription factor activity such that responses to IL-12 are inhibited.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, namely, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.

The term "antibody" is further intended to include bispecific and chimeric molecules having at least one antigen binding determinant in the antibody.

derived from an antibody molecule. Furthermore, although the H and L chains of the Fv fragment are encoded by individual genes, synthetic binding can be made to allow them to be made by recombinant methods as a single protein chain (known as a single chain antibody, sAb; Bird et al. 1988) Science 242: 423-426 and Huston et al., 1988 PNAS 85: 5879-5883). Such single chain antibodies are also included in the term "antibody" and can be used as binding determinants in the pattern and technique of a multispecific binding molecule.

The term "antibody fragment" as used herein refers to an active antibody fragment that retains the ability to bind (immunoreact with) an antigen. Examples of antibody fragments include: a Fab fragment consisting of the V _L , V _H , C _L and C _H domains; a Fd fragment consisting of the V _H and C m domains; a F _v fragment consisting of the VL and VH domains of single arm antibodies; a dAb fragment (Ward et al., 1989 Nature 341: 544-546) consisting of a domain

vh; isolated complementarity determining region (CDR) and F '(ab') 2 fragment, a bivalent fragment consisting of two Fab 'fragments linked by a disulfide bridge in the ginglyme region. These antibody fragments are obtained using conventional techniques well known in the art to those of skill in the art, and the fragments are screened for usefulness in the same manner as intact antibodies.

The term "displayed binding protein" as used herein is intended to include molecules derived from an antibody or other binding molecule (e.g., a receptor or ligand) that retains the desired binding specificity but which has been displayed by recombinant DNA techniques and / or are expressed using recombinant DNA techniques. Examples of displayed binding proteins include soluble and restricted forms of receptors, receptor dimers (e.g., IL-12 p40 receptor dimers) and altered or mutated forms of antibodies, ligands, or receptors selected using combinatorial libraries (e.g., a phage display library method).

The term "NK cell antagonist" as used herein is intended to include antibodies, antibody fragments, and protein binding techniques that are capable of emptied NK / NK-like cells when administered to a subject. Examples of NK cell antagonists include antibodies against asialo-GM1 and NK1.1 antibodies.

The terms "steroid resistance disease" and "steroid resistant subject" as used herein are intended to include diseases and subjects that do not significantly respond to corticosteroid therapy prior to treatment, according to the methods of the invention. Steroid resistance is also referred to as steroid resistance.

The term "immunoinflammatory disease or disorder" is intended to include inflammatory diseases or disorders in which immune cells and / or cytokines are involved in the pathophysiology of the disease or disorder. The term "acute inflammatory disorder" is intended to include disorders and episodes of disorders characterized by the rapid onset of symptoms associated with an inflammatory reaction and a relatively short duration of symptoms, while the term "chronic inflammatory disorder" is intended to include disorders characterized by the continued presence of inflammatory symptoms reaction and the lasting duration of symptoms.

I, Examples

In an embodiment, the invention provides a method for modulating a corticosteroid response in a subject, comprising administering to the subject:

a composition that antagonizes an object that regulates interferon-γ (IFN-γ) production in a subject, the composition is administered at a dose and route of administration sufficient to inhibit the IFN-γ production in the subject and a corticosteroid such that the subject's response to corticosteroid is modulated in compared to when a corticosteroid alone is administered to a subject.

In an embodiment, the method relates to the administration of a composition that is an IL-18 antagonist. The IL-18 antagonist is administered to the subject at a dose and route of administration sufficient to inhibit IL-18 activity in the subject. For example, an IL-18 antagonist may act by inhibiting IL-18 synthesis in a subject, inhibiting IL-18 cytokine activity in a subject, inhibiting IL-18 interaction with an IL-18 receptor, or inhibiting IL-18 receptor activity.

In a preferred embodiment, the IL-18 antagonist is a caspase family protease inhibitor. Proteases from the caspase family, and in particular ICE, convert the forms of the precursor IL-18 into a mature (namely active) form (see, e.g., Example 4). Accordingly, although not intended to be limited by the mechanism, it is contemplated that a protease inhibitor of the caspase family antagonizes IL-18 activity by inhibiting the processing of IL-18 from its precursor form to its mature (namely active) form. A preferred caspase family protease inhibitor for use in the methods of the invention is an ICE inhibitor. Additionally or alternatively, other caspase family proteases that are capable of cleavage of precursor IL-18 to mature IL-18 (such as Ich-2 (caspase-4) and _re iiii ICE (caspase-5)), they may be inhibited. Chemical agents that can inhibit the activity of ICE and other proteases from the caspase family are known in the art, including peptidyl derivatives, azaaspartic acid analog and gamma-pyrone-3-ethanoic acid (see USPatent No. 5,411,985, USPatent No. 5,430,128, USPatent No. 5,434,248, USPatent No. 5,565,430, USPatent No. 05,416,013, PCT patent application published under WO 94/00154, PCT patent application published under WO 93/16710, PCT patent application published under WO 93/14777, PCT patent application published under WO 93 / No. 05071, PCT patent application published under WO 95/35308, European patent application EP 547 699 and European patent application EP 519 748). Other suitable ICE inhibitors and other inhibitors of the caspase family are disclosed in US Application Ser. 08 / 700,716 and US Provisional Applications Serial Nos. 60 / 028,322,60 / 028,324,60 / 028,313 and 60 / 028,323). The precise dose and regimen for administration of an ICE inhibitor or an ICE family protease will necessarily depend on the treatment needs of the subject, the type of treatment, the efficacy of the compound, and the severity of the disease in the subject. A more or less non-limiting example of a dose range for an ICE inhibitor and other caspase family proteases is from about 0.05 to about 150 mg / kg body weight / day.

In other embodiments, the IL-18 antagonist is an antibody, antibody fragment, or displayed binding protein that binds IL-18 or an IL-18 receptor. Such binding agents may be prepared by standard methods known in the art for preparing polyclonal and monoclonal antibodies and recombinant binding proteins and are further described, for example, in European Patent Application 692,536, European Patent Application 712,931, PCT Patent Application published under WO 97/24441 and PCT Publication No. WO 97/44468.

In another embodiment, the method of the invention comprises administering an agent that is an IL-12 antagonist. The IL-12 antagonist is administered to the subject at dosages and by a route of administration sufficient to inhibit IL-12 activity in the subject. For example, an IL-12 antagonist may cause inhibition of IL-12 synthesis in a subject, inhibition of IL-12 cytokine activity in a subject, inhibition of IL-12 interaction with an IL-12 receptor, or inhibition of IL-12 receptor activity.

In an embodiment, the IL-12 antagonist is an antibody, antibody fragment, or displayed binding protein that binds IL-12 or an IL-12 receptor. A preferred IL-12 antagonist is a monoclonal antibody against IL-12. Such antibodies have been described in the art (see, for example, Chizzonite, R., et al., (1991) J. Immunol. 147: 1548-1556). The ability of anti-IL-12 monoclonal antibodies to inhibit disease responses has also been described in the art (see, for example, Leonard, J.P., et al., (1995) J. Exp. Med. 18Γ. 381386); Neurath, M. F., et al., (1995) J. Exp. Med. 182: 1281-1290. Another type of IL-12 antagonist is the p40 homodimer (see, for example, Gillessen, S., et al., (1995) Eur. J. Immunol. 25: 200-206; Gately, MK, et al. (1996) NY Acad. Sci. 795: 1-12; Ling, P., et al. (1995) J. Immunol. 154: 116-127). Yet another type of IL-12 antagonist is a low IL-12 receptor activity form as described in European Patent Application EP 638 644 and U.S. Pat. No. 5,536,657.

Non-limiting examples of IL-12 antagonists for use in embodiments of the invention include monoclonal and polyclonal antibodies and their, chimeric antibodies and chimeric antibody fragments, soluble IL-12 receptors and fragments thereof, reactive peptides or fragments thereof, chemically or genetically modified IL-12 peptides. , IL-12 subunits and fragments thereof, IL-12 subunit homopolymers and fragments thereof, and small organic molecules designed to inhibit the bioactivity of IL-12 or IL-12 receptors. The preparation of IL-12 antagonists, including: (i) a species that binds IL-12 or biologically active fragments thereof, and (ii) a species that interferes with IL-12 binding on receptors or other binding proteins, has been described in the art (for reference). in PCT Patent Application Publication No. WO 95/24918 to Leonard, et al., the contents of which are expressly incorporated by reference; see also in Presky, DH et al., (1995) Res. Immunol. 146: 439- 445).

In another embodiment, the IL-12 antagonist is used in the method of the invention as a means to stimulate the production of cyclic AMP (cAMP) in cells that produce IL-12. IL-12 production has been shown to be inhibited by an increase in intracellular cAMP production (see, for example, van der Pouw Kraan et al. (1995) J. Exp. Med. 181: 775-779). Examples of compositions that can be used to stimulate intracellular cAMP production include phosphodiesterase IV inhibitors and a beta-2 agonist. As demonstrated in Example 3, administration of a phosphodiesterase IV inhibitor in septic shock models is inhibited by LPS-induced IL-12 production. Examples of suitable phosphodiesterase IV inhibitors for use in the methods of the invention include rolipram, denbufylline, tibenelast, nitroquazone and CP-80633. Examples of beta-2 agonists for use in the methods of the invention include salmeterol, phenoterol and isoproterenol. The precise dose and regimen for administration of the phosphodiesterase IV inhibitor or beta-2 agonist will necessarily depend on the needs of the subject being treated, the type of treatment, the efficacy of the compound, and the degree of disease severity in the subject. A more or less non-limiting example of a dose range for phosphodiesterase IV inhibitors or beta-2 agonists is from about 0.05 to about 150 mg / kg per body weight / day. In a preferred embodiment, a composition that stimulates the production of cyclic AMP (cAMP) (e.g., a phosphodiesterase IV inhibitor or beta-2 agonist) is administered consistently (e.g., orally or intravenously) to inhibit IL-12 production by monocytes and macrophages.

In another embodiment, the IL-12 antagonist used in the methods of the invention is a STAT4 inhibitor. STAT4 is a transcription factor that has been shown to be involved in IL-12 responses. (See, for example, Thierfelder, WE et al. (1996) Nature 382: 171-174; Kaplan, MH et al. (1996) Nature 382: 174-177). Therefore, IL-12 responses in a subject can be inhibited by administration of an inhibitor. STAT4.

Other inhibitors of IL-12 activity known in the art may also be used in the methods of the invention. For example, PCT Patent Application No. WO 96/40093 discloses biphenyl derivatives for antagonizing the induced immune responses of IL-12. Such biphenyl derivatives may be used as IL-12 antagonists in the methods of the invention.

In another embodiment, the method of the invention relates to the administration of a composition that is an NK cell antagonist. The NK cell antagonist is administered to the subject at dosages and by a method of administering a medicament sufficient to inhibit IFN-γ activity in the subject. Preferably, the NK cell antagonist is an antibody, antibody fragment, or displayed binding protein that specifically binds NK / NK-like cells so that the subject is deprived or eliminated of the cells. Therefore, preferred NK cell antagonists bind to specific surface indicators present on NK / NK-like cells. Individual preferred NK cell antagonists are anti-asialo-GM1 antibodies and NK1.1 antibodies, which have been shown to be effective in depriving NK / NK-like cell activity from a subject (see Example 10; Axelsson, LG. Et al. ( 1996) Inflamm Res 45: 181-191, Heremans, H. et al (1994) Eur J Immunol 24: 1155-1160).

Other antibodies that designate surface indicators that identify NK / NK-like cells include antibodies reactive with Fc-IgG receptors B73.1 ("Fc-IgG receptors B73") and Leu 11 (CD16) (Lancer, LL et al. (1983) J. Immunol. 131: 1789-1796; Perussia, B. et al. (1983) J. Immunol. 130: 21332141), Leu 7 (against HNK1, which identifies NK cells by 40-60%; Abo, T. and Balch, CM (1981) J. Immunol. 127: 1024-1029) and OKT11 (CD2, which identifies 50% or more of NK cells; Lancer, LL et al. supra; Perussia,

B. et al., Supra). Other specific surface antigens of NK cells and the antigens described above include DX1 antigen (see PCT Patent Application No. WO 95/02611), PEN5-alpha and PEN5-beta glycoprotein pair (PCT Patent Application No. WO 95/1993). And NKB1 antigen (see PCT Patent Application No. WO 95/20604 for reference). The precise dosage and regimen for administration of the NK cell antagonist will necessarily depend on the needs of the subject being treated, the type of disease, the efficacy of the compound, and the degree of potency in the subject. A more or less non-limiting example of a dosage range for antibodies against NK / NK-like cells is from 0.01 to about 150 mg / kg per body weight / day. A single dose of the antibody may be sufficient to deprive or eliminate the activity of NK / NK-like cells or, alternatively, multiple doses may be administered as needed to deprive or eliminate the activity of NK / NK-like cells. Preferably, the NK cell antagonist is administered by the intravenous or intraperitoneal route of administration.

In the methods of the invention, a composition that antagonizes an object that regulates the production of interferon-γ (IFN-γ) is administered to a subject in combination with one or more corticosteroids. The term "in combination with" a corticosteroid is intended to include the simultaneous administration of the composition and the corticosteroid, the administration of the composition being the first followed by the corticosteroid or vice versa. Any therapeutically useful corticosteroids known in the art can be used in the methods of the invention. Corticosteroids are typically classified by the duration of their tissue action: short acting compounds (beclomethasone, flunisolide, hydrocortisone, cortisone), moderate compounds (e.g. prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort) and long acting compounds (eg dexamethasone). One or more corticosteroids may be administered to the subject by the route of administration and at effective doses to achieve the desired therapeutic result. Examples of suitable modes of administration include intravenous administration, oral administration, topical administration, administration by inhalation (e.g. bronchial administration) and local injection (e.g. intra-joint). The precise dosage and regimen for administering to the subject a corticosteroid will necessarily depend on the needs of the subject being treated, the type of disease, the efficacy of the compound, and the degree of disease severity in the subject. A more or less non-limiting example of a dose range for corticosteroids is from about 0.05 mg / day to about 1 mg / day, wherein the dose range depends on the corticosteroid use itself. Certain preferred dosage regimens utilize alternative daily administration (e.g., high dose intravenous pulse therapy).

Corticosteroid formulations suitable for administration are well known in the art and commercially available. For example, dexamethasone acetate, a 16 mg / ml aqueous suspension, is suitable for intramuscular injection in the treatment of rheumatic, dermatological, ophthalmic, gastrointestinal, haematological, neoplastic, allergic conditions and collagen disorders. Non-limiting examples of dosage ranges include 0.8 mg, 1.5 mg, 4 mg and 16 mg dexamethasone by injection.

Hydrocortisone is suitable as a sterile aqueous solution for intravenous, intramuscular and subcutaneous injections and is a potent anti-inflammatory agent for conditions such as osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, acute chronic bursitis. Preferred starting doses may be from 15 mg to 250 mg per human subject per day. Preferred dosages are oral or parenteral and may be administered in half-daily doses twice or more. The hydrocortisone injection may be added to the sodium chloride injection or to the dextrose injection and injected intravenously. Hydrocortisone valerate, 0.2% by weight, is formulated as a topical cream under the name Westcort. Preferred dosing involves the application of a thin thin coating to the affected areas several times a day.

Beconase (beclometasone) is suitable for inflammation of the nasal passages and cavities, for example as 8.4 mg for 200 measured spray doses in 0.042% aqueous suspension, supplied in 100 mg specific doses containing 42 μg per unit dose such that the daily nasal delivery consists of preferably 42 μg through the nostril, 84 μg through the nostril, 168 μg through the nostril, 336 μg through the nostril, 672 μg through the nostril or 1,344 μg through the nostril. It is preferably supplied, for example, in an aqueous medium in suspension with microcrystalline cellulose, sodium carboxymethylcellulose, dextrose, benzalkonium chloride, polysorbate 80, and 0.25% v / w phenylethyl alcohol. Additional propellants and media are included in some formulations.

In certain embodiments, wherein the composition of the invention is co-administered with a corticosteroid, the composition is administered consistently to regulate IFN-γ production, while the corticosteroid is administered either topically or continuously. For example, in certain embodiments, when a phosphodeiesterase IV inhibitor or a beta-2 agonist is administered together with a corticosteroid, the phosphodeiesterase IV inhibitor or a beta-2 agonist is administered continuously by an intravenous or oral route and the corticosteroid is administered either continuously or topically. Additionally, in certain embodiments of the methods of the invention, the use of a phosphodeiesterase IV inhibitor or a beta-2 agonist in combination with a corticosteroid for the treatment of asthma is specifically excluded from the scope of the invention.

The methods of the invention can be used in the treatment of various immune disorders and inflammatory disorders. For example, in a preferred embodiment, the subject is suffering from septic shock (namely, the methods of the invention allowing corticosteroids to be used in the treatment of septic shock). In another preferred embodiment, the subject to be treated is having Crohn's disease. In another preferred embodiment, the subject being treated is suffering from asthma. In another preferred embodiment, the subject being treated is suffering from graft-versus-host disease or transplant rejection. In another preferred embodiment, the subject to be treated is suffering from an autoimmune disease.

In another embodiment, the subject is suffering from a disease or immunoinflammatory disorder. Non-limiting examples of immuno-inflammatory diseases or disorders that can be treated according to the invention include asthma, adult respiratory distress syndrome, systemic erythematous lupus, inflammatory bowel disease (including Crohn's disease and ulcerative sclerosis), multiple sclerosis, insulin arteritis, autoimmune relapse, autoimmune relapse, arthritis, juvenile rheumatoid arthritis ("juvenile rheumatoid arthritis", psoriatic arthritis), inflammatory pulmonary syndrome, general pemphigus, idiopathic thrombocytopenic purpura, autoimmune meningitis, severe myasthenia, dermatitis, autoimmune, dermatitis Sjögren's Syndrome (including secondary dry keratoconjunctivitis to Sjögren's syndrome), general baldness, allergic reactions due to arthropod temptation reactions, aphthous ulcus, irite epidemiosis, conjunctivitis, keratoconjunctivitis, cutaneous erythematous lupus, scleroderma, vaginitis, proctitis, eruption of the drug (such as Stevens Johnson syndrome), leprosy reactions to reversal, nodular erythema , autoimmune uveitis, allergic encephalomyelitis, aplastic anemia, pure red cell anemia (id red cell anemia), idiopathic thrombocytopenia, polychondritis, Wegener's granulomatopathy, or chronic neuropathy, ), primary biliary cirrhosis, posterior uveitis and interstitial lung fibrosis.

In another embodiment, the subject is suffering from an acute inflammatory disorder. Examples of acute inflammatory disorder that can be treated include graft versus host disease, transplant rejection, septic shock, endotoxemia, Lyme arthritis ("Lyme arthritis"), infectious meningitis (eg, viral, bacterial, associated Lyme disease), acute asthma attack, and acute attack of autoimmune disease.

In another embodiment, the subject is suffering from a chronic inflammatory disorder. Non-limiting examples of chronic inflammatory disorder that can be treated include asthma, rubella arthritis, and chronic autoimmune disease such as systemic erythematous lupus, psoriasis, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, multiple sclerosis, and rheumatoid arthritis.

In certain instances, a composition that antagonizes individual objects that regulate IFN-γ in a subject may be advantageous for treating individual disorders. For example, although it is not intended to be a limiting mechanism, disorders in which IFN-γ is preferably or predominantly produced by NK cells are preferably treated using a composition that antagonizes IL-18 (such as an ICE inhibitor) or directly antagonizes NK cells. (namely, an NK cell antagonist, such as an antibody against NK / NK-like cells), in combination with a corticosteroid. Alternative disorders in which IFN-γ is preferably or predominantly produced by T cells are more preferably treated using a composition that antagonizes IL-12 (e.g., an anti-IL-12 antibody or composition that stimulates intracellular cAMP production) in combination with a corticosteroid. In other circumstances, it may be beneficial to use both. an IL-18 antagonist and an IL-12 antagonist (for example, in the treatment of disorders in which IFN-γ production is a contribution of both T cells and NK cells).

The composition and the corticosteroid are administered to a subject in need of treatment according to standard drug delivery methods well known in the art, the particular route of administration and the dosage of the composition and corticosteroid are selected based on the subject's treatment needs, treatment type, compound efficacy and severity of disease in the subject. The composition and the corticosteroid are administered at an "effective therapeutic dose," which means that the amount of therapeutic agent that, when administered to a subject, induces an improvement in the disorder compared to those subjects not receiving the drug. The ordinary skill in the art may be to determine and prescribe the desired effective therapeutic amount of the composition and the corticosteroid. The composition and corticosteroid of the invention are administered to a subject in biocompatible forms suitable for pharmaceutical administration in vivo to elicit the desired therapeutic response. "Biologically compatible forms suitable for pharmaceutical in vivo application" means a form of a medicinal product which is administered in such a way that the therapeutic effects of the preparation outweigh any toxic effects and side effects. In addition, a composition of the invention that antagonizes an object that regulates IFN-γ production in a subject is administered to the subject at a dose and route of administration sufficient to inhibit IFN-γ production in the subject. Similarly, an IL12 antagonist or IL-18 antagonist of the invention is administered, alone, to a subject at a dose and route of administration sufficient to inhibit IL-12 activity or IL-18 activity in the subject.

Animal models of inflammatory and immunological disorders that are accepted in the art as models of human disease can be used to evaluate the various therapeutic regimens of the invention. For example, the P.acwei / LPS septic shock model described in the examples can be used to evaluate the efficacy of therapeutic regimens for treatment, non-limiting examples that include experimental colitis (see, for example, Neurath, MF et al. (1995) J. Exp. Med. 182). : 1281-1290), experimental allergic encephalomyelitis (for example, Leonard,

K. J. et al. (1995 / J. Exp. Med. 181: 381-386), collagen-induced arthritis (Banerjee, S. et al. (1989) J. Immunol. 142: 2237-2243) and the human TNFα transgenic model of polyarthritis (see, for example, Keffer, J. et al. EMBO J (1991) 10: 4025-4031). For therapeutic regimens related to inhibition of ICE activity, ICE-depleted mice can be used as a model of complete inhibition of ICE activity. Such ICE - / - mice have been described in the art (see, for example, Li, P., et al. (1995) Cell 80: 401-411 and PCT Patent Application No. WO 96/12025).

The methods of the invention are useful for modulating the corticosteroid response in various clinical assays. For example, in one embodiment, the methods of the invention are used to reverse steroid resistance in a subject as compared to when the corticosteroid is administered to the subject. In another embodiment, the methods of the invention are used to increase steroid sensitivity in a subject compared to when the corticosteroid is administered to the subject. In yet another embodiment, the corticosteroid is administered to a subject on a schedule that reduces corticosteroid doses over time and a method of ameliorating a spontaneous steroid response associated with administration of reduced doses of corticosteroids. The ability of the methods of the invention to increase steroid sensitivity (namely, having "steroid limited effect") may therefore allow the use of corticosteroid therapy in clinical assays in which such therapies have previously been contraindicated. For example, the use of the methods of the invention may allow corticosteroid therapy in patients who have not previously been treated in such a way as it has not allowed the harmful side effects of corticosteroid therapy such as young children (e.g. juvenile rheumatoid arthritis), patients with uncontrolled diabetes and hypertensive patients.

A further aspect of the invention relates to a method of modulating a corticosteroid response in a subject comprising:

selecting the subject in need of modulating the corticosteroid response; and administering to the subject a composition that antagonizes an object that regulates interferon-γ (IFN-γ) production in the subject, a composition that is administered at a dose and a route of administration sufficient to inhibit IFN-γ production in the subject such that the subject's response to a corticosteroid. it is modulated as compared to when a corticosteroid alone is administered to a subject.

For example, the subject selected for treatment according to the method of the invention may be a subject that is corticosteroid resistant prior to administration of the composition. Alternatively, the subject selected for treatment may be a subject that is susceptible to corticosteroids prior to administration of the compositions, but which exhibits increased sensitivity to corticosteroids following administration of the composition. Examples of such a subject are patients suffering from a steroid dependence disorder in whom the disorder can be treated with lower doses of corticosteroids when treated according to the methods of the invention. Another example of such a subject is a patient whose steroid therapy has caused contraindications to side effects when the corticosteroid itself is administered, but who can tolerate a lower dose of the corticosteroid when the corticosteroid is administered in accordance with the methods of the invention. Further, the subject selected for treatment according to the method of the invention may be one whose administration of the composition ameliorates a spontaneous response to steroids in the subject but has already undergone corticosteroid therapy followed by cessation of the therapy. Means for antagonizing an object that regulates IFN-γ production in a subject are described above.

II. Pharmaceutical preparations

Another aspect of the invention relates to pharmaceutical compositions for modulating the response to corticosteroids. In an embodiment, the pharmaceutical composition of the invention comprises a composition that antagonizes an object that regulates the production of interferon-γ (IFN-γ) in a subject, a corticosteroid, and a pharmaceutically acceptable carrier. As discussed above, the object being antagonized may be, for example, IL-18, IL-12 or NK cells (namely, the pharmaceutical composition may comprise an IL-18 antagonist, IL-12 antagonist or NK cell antagonist, as previously described, a corticosteroid). and a pharmaceutically acceptable carrier).

In a preferred embodiment, the pharmaceutical composition of the invention comprises a caspase family protease inhibitor, a corticosteroid and a pharmaceutically acceptable carrier. Examples of caspase family protease inhibitors and non-limiting exemplary doses are previously described herein. In a preferred embodiment, the caspase family protease inhibitor is an ICE inhibitor.

In another embodiment, the pharmaceutical composition of the invention comprises an IL-12 antagonist, a corticosteroid, and a pharmaceutically acceptable carrier. Examples of such IL-12 antagonists are previously described herein. In a preferred embodiment, the IL-12 antagonist is a monoclonal antibody against IL-12. In another preferred embodiment, the IL-12 antagonist is a phosphodiesterase IV inhibitor. In yet another preferred embodiment, the IL-12 antagonist is a beta-2 agonist.

In another embodiment, the pharmaceutical composition of the invention comprises an NK cell antagonist, a corticosteroid, and a pharmaceutically acceptable carrier. Examples of such NK cell antagonists are previously described herein. In a preferred embodiment, the NK cell antagonist is an antibody against NK / NK-like cells, preferably an anti-asialo-GM1 antibody or an NK1.1 antibody.

The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retention agents, and the like, compatible with pharmaceutical application. The use of such media and compositions for pharmaceutical active substances is well known in the art. Without use in formulations of any conventional media or compositions that are incompatible with the active compound. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical composition of the invention is formulated to be compatible with its intended mode of administration. For example, solutions or suspensions used for parenteral, intradermal or subcutaneous administration may include the following components; sterile diluent such as water for injection, saline solution, solid oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and a tonicity agent such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as HCl or sodium hydroxide. The patenteral preparation may be provided in ampoules, disposable syringes or multiple dose divided vials made of glass or plastic.

Pharmaceutical preparations suitable for injectable use include sterile aqueous solutions (water-soluble) or dispersions and sterile dusts for a preparation prepared as necessary for a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (Cremophor EL ™) (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the formulation must be sterile and should be a fluid that has the ability to be easily in the syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the effects of contamination with micro-organisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like) and suitable mixtures thereof. The intrinsic fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the formulations. Prolonged absorption of injectable preparations may be brought about by the addition of such formulations to preparations which delay absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile carrier that contains a basic dispersion medium and the desired other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, which provide a dusting of the active ingredient plus any additionally desired ingredient from the previously sterile filtered solution.

Oral formulations generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be attached to excipients and used in the form of tablets, lozenges or capsules. Oral formulations may also be prepared using a liquid carrier for use as a gargle, wherein the compound in the liquid carrier is administered orally and swallowed and expectorated or swallowed. The pharmaceutical compatible binding agent and / or adjuvant materials may be included as part of the formulation. Tablets, pills, capsules, lozenges, etc. they may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, acacia or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silica; a soothing agent such

100 such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or flavoring orange.

In an embodiment, the active compounds are formulated with carriers that will protect the compound against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated dose systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acids, collagen, plyortoesters and polyactic acids. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and New Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. patent application Ser. No. 4,522,811.

The pharmaceutical compositions of the invention may be formulated for administration by a single administration route, such as oral administration, intravenous administration, ophthalmic administration, and the like.

In a preferred embodiment, the pharmaceutical composition of the invention is formulated for topical application. Therefore, a composition that antagonizes an object that regulates the production of interferon-γ (IFN-γ) in a subject, a corticosteroid, and a pharmaceutically acceptable carrier may be formulated into a cream, ointment, ointment (wound) and the like suitable for use on the skin.

In a preferred embodiment, the pharmaceutical composition of the invention is formulated for administration by inhalation. Therefore, a composition that antagonizes an object that regulates the production of interferon-γ (IFN-γ) in a subject, a corticosteroid, and a pharmaceutically acceptable carrier can be formulated into a nasal spray or inhalation composition to facilitate delivery.

101 therapeutic agents into the nasal or cavity or lung (e.g., bronchial) routes by inhalation.

The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, published patents, and patent applications cited throughout the application are hereby incorporated by reference.

Example 1: Inhibition of ICE activity in septic shock models resulting in a steroid response

In this example, the effect of inhibiting ICE activity on steroid response in a septic shock model was examined. A septic shock model was developed in ICE-depleted mice (ICE - / -) and wild-type (ICE + / +) mice, followed by corticosteroid treatment. ICE - / - mice served as a model for complete inhibition of ICE activity (see Li, P., et al. (1995) Cell 80: 401-411 for further description of ICE deficient mice). The response of animals to corticosteroid treatment was determined by monitoring the levels of inflammatory cytokine TNFα in the serum of mice.

ICE deficient and wild-type mice were first sensitized with propionibacterium acnes cell wall material (1 mg per mouse) to induce low-grade inflammation and then six days later a lipopolysaccharide (LPS) response (1 gg per mouse at 0.1 ml saline) was induced. intravenously). 30 minutes after LPS application, mice were treated with corticosteroid dexamethasone (4 mg / kg per mouse at 0.5 ml 95% saline / 0.5% ethanol, i.p.). Controled mice were treated with vehicle alone. All mice were bled 90 minutes after LPS administration and serum samples were analyzed for the presence of TNF-α by standard ELISA.

The results are shown in Figure 1. Wild type and ICE-poor mice treated with vehicle alone had similar levels of TNF-α serum.

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Treatment of wild type mice with dexamethasone did not significantly affect serum TNF-α levels, demonstrating their resistance to steroid treatment in this septic shock model. Serum suppressed serum TNF-α levels by 74% (p <0.002) compared to treatment of mice with ICE deficiency with dexamethasone. These data show that inhibition of ICE activity reverses resistance to steroid treatment in septic shock models.

Example 2: Inhibition of ICE activity in septic shock models increases steroid sensitivity

In this example, the effect of inhibiting ICE activity on steroid sensitivity in a septic shock model was examined. The same LPS / P.acnes septic shock model described in Example 1 was used, without ICE deficient mice and wild-type mice were pretreated with vehicle or corticosteroid prior to eliciting a response with LPS. The response of animals to corticosteroid treatment was re-determined by monitoring the serum levels of the inflammatory cytokine TNFα in mice.

ICE deficient and wild-type mice were first sensitized with propionibacterium acnes cell wall material (1 mg per mouse) to induce low-grade inflammation and then six days later a lipopolysaccharide (LPS) response (1 µg per mouse at 0.1 ml saline) was induced. iv). 15 minutes prior to challenge, animals were treated with decreasing amounts of dexamethasone corticosteroid (0.05, 0.005 or 0.0005 mg / kg per mouse in 0.5 ml 95% saline / 0.5% ethanol, i.p.). Controled mice were treated with vehicle alone. All mice were bled 90 minutes after LPS administration and serum samples were analyzed for the presence of TNF-α by standard ELISA.

The results are shown in Figure 2. Both wild type and ICE deficient mice showed a pre-treatment response with 0.05 mg / kg dexamethasone. Pretreatment of ICE-poor mice with only 0.005 or 0.005 mg / kg dexamethasone showed 76% and 78% (p <0.005), respectively.

The levels of serum TNF-α, respectively, compared to the lack of TNF-α suppression in wild-type animals similarly treated. These data indicate that inhibition of ICE activity results in an increase in steroid sensitivity in septic shock models, from 10-100 lower doses of dexamethasone phalds were therapeutically effective in animals with ICE deficiency compared to wild type animals.

Example 3: A phosphodiesterase IV inhibitor reduces IL-12 production

In this example, the effect of a phosphodiesterase IV inhibitor, Rolipram, LPS-induced IL-12 production was examined. B6 mice were pretreated with vehicle or Rolipram (30 mg / kg at 0.5 ml 0.1% methylcellulose, intraperitoneally). 15 minutes before LPS response (10 µg / mouse, intravenous). Mice were bled 90 minutes after LPS administration and serum samples were analyzed for IL-12 by standard ELISA.

The results are shown in Figure 3. Rolipram-pretreated mice had a 70% lower IL-12 serum level than mice pretreated with vehicle alone. These data show that phosphodiesterase IV inhibitors are effective in inhibiting LPS-induced IL-12 production.

Example 4: Cleavage of IL-18 by Caspase Family Proteases

The ability of various recombinant (namely, E.coli expressed) proteases from the caspase family to cleave the precursor IL-18 (for IL-18) into valid IL-18 was tested in the in vitro proteolysis assay. The cleavage of poly (ADP-ribose) polymerase (PARP) was used as a positive control. The results are summarized in Table 1 below.

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Table 1 Proteolysis of proIL-18 by caspase recombinants

U fission% caspase * Concentration (pg / ml) proIL-18 PARP ICE (ABOUT 1.25 100 99 Ich-2 (4) 5:00 82 93 ICE _re , III (5) 20:00 55 90 CPP32 (3) 5:00 0 ^a 100 MCH2 (6) 10:00 2 96 mch3 (7) 5:00 32 ^p 97 ICH-1 (2) 75.00 5 98

* Caspas are numbered in brackets as recommended by Alnemri et al. (1996) Cell 87: 171 ^and CPP32 (5 µg / ml) digested with proIL-18, but generated by the 12kDa and 10kDa fragment instead of the expected 18 kDa fragment.

^b Unlike other caspases, the precursor Mch3 obtained from E. coli does not undergo autocatalysis to produce an effective protease. The addition of ICE was required to initiate the autocatalysis of Mch3 and to produce an effective protease of Mch3. Partial cleavage of proIL-18 by Mch3 is mediated by the presence of ICE in the preparation of Mch3.

Example 5. Treatment of septic shock

Patients participating in septic shock clinical trials (for example, in conjunction with infected abrasion, protruding wounds, or systemic bacteremia from other sources) are given the agent selected

105 from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g. rolipram, 30 mg / kg) and monoclonal antibodies against IL-12, together with a corticosteroid (e.g. high dose methylprednisolone, 1 gm / day, i.v.). The corticosteroid and the composition may be administered together or separately, the composition may be administered before or after administration of the corticosteroid. Patients are also treated with appropriate antibiotic therapy.

Example 6: Treatment of transplant rejection

Patients receiving kidney transplant are administered a composition selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g. rolipram, 30 mg / kg) and a monoclonal anti-IL-12 antibody, together with a corticosteroid (e.g. oral prednisolone, 25-75 mg / day) . Preferred treatment is prior to receiving the donor kidney (for example, administration of the drug may begin 24 hours before receiving the donor kidney), with doses that may be repeated as needed (e.g., every 24 hours). The corticosteroid and the composition may be administered together or separately, the composition may be administered before or after administration of the corticosteroid. Patients are also treated with complementary immunosuppressive therapy (such as cyclosporin A or OKT3 antibody treatment) such that immune rejection and inflammatory response are simultaneously suppressed.

Example 7: Improvement of spontaneous response to steroids

Patients with asthma, allergic rhinitis or rheumatoid arthritis who are receiving a corticosteroid or systemic corticosteroid treatment with an inhaled agent and who have started discontinuing their scheduled corticosteroid therapy are given an agent selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g. and anti-IL-12 monoclonal antibodies. Patients are preferably treated before narrowing or discontinuing corticosteroid therapy to improve spontaneous response to

This may result from discontinuation of steroid therapy. If desired, patients may be treated with supplementary non-steroidal anti-inflammatory agents.

Example 8: Treatment of an acute attack of autoimmune disease

A patient suffering from an acute relapse of an autoimmune disease such as an inflammatory bowel disease (e.g. ulcerative colitis or Crohn's disease) is administered a composition selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g. rolipram, 30 mg / kg) and monoclonal anti-IL-12 antibodies together with corticosteroids. (e.g. oral prednisone, 2575 mg / day). The corticosteroid and the composition may be administered together or separately, the composition may be administered before or after administration of the corticosteroid. Patients may also be treated with complementary immunosuppressive therapy to suppress the acute relapse of autoimmune disease.

Example 9: Treatment of Chronic Autoimmune Disease

Patients suffering from chronic autoimmune disease such as Crohn's disease are administered a composition selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g. rolipram, 30 mg / kg) and a monoclonal anti-IL-12 antibody, together with a corticosteroid (e.g. oral prednisone, 25-75 mg / kg). daily). The corticosteroid and the composition may be administered together or separately, the composition may be administered before or after administration of the corticosteroid. Patients may also be treated with complementary immunosuppressive therapy to suppress autoimmune disease.

Example 10: Inhibition of IFN-γ production by NK cell elimination

In this example, shock was induced in mice by a high dose of LPS (40 mg / kg LPS administered intravenously). The effect was investigated

107 cell depletion to produce various cytokines in mice and to mortality by administering intravenous antibody (anti-ASGM1) against asialo-GM1 10 minutes prior to LPS administration. Monitored animals received rabbit IgG ("Rabbit IgG"). The effect of ASGM1 action on IL-β, TNFα, and IFN-γ production, as well as on mortality, is summarized in Table 2 below.

Table 2

effect # Mice production cytokine u (pg / ml) mortality h) (30 IL-Ιβ TNFa IFN-γ Survival LPS + rabbit IgG (controls a) 6 1946 483 ± 5857 1071 + 1663 811 ± 0 (n = 10) LPS + antibody ASGM1 6 1647 482 ± 5453 1103 ± 363 108 ± 90 (n = 11)

* All animals died within 15 hours.

These results show that elimination of NK cells by anti-asialo-GM1 antibody reduces IFN-γ production and prolongs survival after LPS administration at high dose of LPS shock.

Example 11: Effect of ICE inhibitor and corticosteroid in septic shock models

In this example, the LPS / P.acnes septic shock model described in Examples 1 and 2 was used to investigate the effect of an ICE inhibitor in combination with a corticosteroid. B6 mice were implanted with a 24 hour osmotic pump containing acetyl-tyrosine-valine-alanine-aspartic acid-CHO (Ac-YVAD-CHO) ICE inhibitor (100 mg / kg) or vehicle control, subcutaneously 18 hours prior to LPS injection. LPS was injected intravenously (0.01 gg / mouse or 10 gg / mouse) at time zero. All mice were injected with 5 mg / kg dexamethasone intraperitoneally 30 minutes after LPS injection.

108

The response of animals to corticosteroid treatment was determined by monitoring the levels of inflammatory TNFα cytokine as well as interleukin-6 (IL-6) and interleukin-1β (IL-β) in mouse serum. 90 minutes after LPS administration, all mice were bled and serum samples were analyzed for the presence of TNFα, IL-6 and IL-β by standard methods.

The results are shown in Figures 4, 5 and 6, for TNF-α, IL-6 and IL-β, respectively. The data demonstrate that mice treated with both ICE inhibitor AcYVAD-CHO and thus dexamethasone had significantly lower serum levels of TNF-α, IL-6 and IL-1b when either 0.01 gg or 10 gg of LPS was used to induce septic shock. As previously shown in Example 1, treatment of mice with dexamethasone alone did not significantly affect serum levels of TNF-α, demonstrating the resistance of mice to steroid treatment alone in this septic shock model. Treatment of mice with both dexamethasone and ICE inhibitor suppressed serum TNF-α levels by 96% (p <0.005), in mice treated with 0.01 gg LPS and by 86% (p <0.005), in mice treated with 10 gg LPS. In addition, IL-6 serum levels were reduced by 95% (p <0.00005) and 91% (p <0.00005), respectively, and IL-ββ serum levels were reduced by 94% (p <0.001) and 92% (p <0.0002) ), individually. These data show that inhibition of ICE activity using an ICE inhibitor reverses resistance to steroid treatment in septic shock models.

Example 12; Synthesis of hydroxamate of ICE inhibitors

3-Benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid

Step A N- (Phenylmethoxy) -benzeneacetamide [(0.760g, 3.15 mmol), prepared according to the method of Hearn MTW and Ward AD (Aust. J. Chem. 1969; 22: 1731)] was taken up in 10 mL of CH ₃ CN and treated with dimethylamino-pyridine (DMAP) (50 mg) and di-tert-butyl dicarbonate (0.824 g, 3.78 mmol). The reaction can be stirred under argon for 12 hours, then diluted with ethyl acetate (EtOAc) and washed with 3M K 2 SO 5 (1 x 10 mL), NaHCO ₃

109 (1 x 10 mL). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. Purification by chromatography (SiO ₂ , 9: 1Hexane-EtOAc) afforded 0.910 g (84%) of 1,1-dimethylethyl (phenylacetyl) phenylmethoxy) carbamate as a clear, viscous oil.

1 H NMR (400 MHz, DMSO-d ₆ ):? 41 [m, 5H] 7. 32 [m, 2H] .7. 24 [m, 3H] 4. 90 [s, 2H]. 09 [s, 2H], 1.48 [s, 9H] .IR ("infrared spectroscopy") 3063, 3032, 2979, 2935, 2886, 1777, 1736, 1497, 1455, 1370, 1302 cm ' ¹ . MS ( "mass spectroscopy ,,) (CI [Cl] - _NH3) 342 (M ⁺ H +).

Elemental analysis:

Calculated for C ₂ oH ₂₃ N0 ₄ .0.051 CH ₂ C1 _2: C, 69.66; H, 6.74; N, 4.05.

Found: C; 69.66; H, 6.83; N, 3.99.

Step B 1,1-Dimethylethyl (phenylacetyl) (phenylmethoxy) carbamate (810 mg, 2.37 mmol) was dissolved in 75 mL dry THF and 90 mg of 5% Pd / BaSO _{4 was added} . The reaction was allowed to react with H ₂ (20 psi per squre inch) for 20 hours. The reaction was filtered through celite and concentrated to give 588 mg (99%) of 1,1-dimethyl-ethyl-hydroxy- (phenylacetyl) carbamate as an oil. No further purification was performed.

1 H NMR (400 MHz, CDCl ₃ ):? 8. 22 [s, 1 H], 7. 31 [m, 5H]. 24 [s, 2 H], 1.55 [s, 9 H],

Step C (S) -5-Bromo-4-oxo-3 - [[(phenylmethoxy) carbonyl] amino] -pentanoic acid, 1,1-dimethylethyl ester [(297 mg, 0.742 mmol), prepared according to the procedure of Dolle RE, et. al., (J. Med. Chem., 1994; 37: 563-4)], 1,1-dimethylethyl hydroxy (phenylacetyl) carbamate (187 mg, 0.742 mmol) and KF (104 mg, 1.85 mmol) were combined in 5 ml dimethylformamide (DMF) and can be stirred under Ar for 12 hours. The reaction was diluted with EtOAc (15 mL) and washed with water (3 x 15 mL) and brine

110 (1 x 15 mL). The organic layer was dried over Na ₂ SO ₃ and concentrated. Purification by chromatography (SiO ₂ , 4: 1 Hexane-EtOAc) afforded 168 mg (40%) of [[[(1,1-dimethylethoxy) carbonyl] (phenylacetyl) amino] oxy] -4oxo-3- [ [((phenylmethoxy) carbonyl] amino] pentanoic acid as a clear oil.

1 H NMR (300 MHz, CDCl ₃ ): δ 7. 35 (m, 5H), 5.84 [d, J = 9.0 Hz, 1H], 4.79, [A of AB, J = 15.3 Hz, 1H ], 4.70 [m, 1H], 4.57 (B of AB, J = 15.3 Hz, 1H], 4.10 [s, 2H], 3.09 [dd, J = 16.8.6 Hz, 1H ], 79.7 [dd, J = 16.8,

4.9 Hz, 1H], 1.52 [s, 9H], 1.39 [s, 9H], IR (thin layer) 3374, 2980, 2935, 1726 (br), 1499, 1370, 1298, 1150 cm &^lt; -1 & gt ^; . MS (APCI, Methanol (MeOH)) 571.5 (M ⁺ H +).

Elemental analysis:

Calcd. For C 30 H 38 NO 9: C, 63.15; H, 6.71; N, 4.91.

Found: C, 62.76; H, 6.70; N, 4.69.

Step D 3-Benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid 1,1-dimethylethyl ester (208 mg, 0.365 mmol) was taken up in 3 mL of 1: 1 trifluoroacetic acid (TFA) / CH ₂ Cl ₂ and stirred for 2 hours. The reaction was diluted with acetonitrile (MeCN) (10 mL) and concentrated. The residue was eluted five times from MeCN. Purification by chromatography (SiO ₂ , 90: 9: CH ₂ Cl ₂ -acetone-methanoic acid) gave 3-benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid (51 mg, 34%) as a white foam.

1 H NMR (300 MHz, CDCl 3): δ 8.63 [s, 1H], 7.34 [broad (br) s, 10H], 5.48 [br d, J = 4 Hz, 1H], 5.08 [br dd, J = 16] 12 Hz, 2H], 4.23 [m, 1 H], 3.97 [m, 2 H], 3.58 [br s, 2 H], 2.80 [m, 1 H], 2.64 [m, 1 H],

IR (KBr) 3305 (br), 2928, 1791, 1772, 1717, 1699, 1685, 1674, 1654, 1521, 1455 cm &^lt; -1 & gt ^; . MS (APCI, MeOH) 415 (M ⁺ H +).

Elemental analysis:

Analysis calculated for C ₂ H ₂₂ N ₂ O ₇ * 06 0.1 _{CF3 CO2} H: C, 59.73; H, 5.22; N, 6.57.

111

Found: C, 59.73; H, 5.46; N, 6.28.

They were further prepared from (S) -5-bromo-4-oxo-3 [[(phenylmethoxy) carbonyl) amino] -pentanoic acid 1,1-dimethylethyl ester as described above, step C and step D.

3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) -pentanoic acid

Step A Prepared from 1-hydroxy-2-pyrrolidinone [Biswas A. and Miller MJ (Heterocycles, 1987; 26: 2849) as described above, Step C, to obtain 3-benzyloxycarbonylamino-4-oxo-1,1-dimethylethyl ester 5- (2-Oxo-pyrrolidin-1-yloxy) -pentanoic acid (74%).

1 H NMR (400 MHz, CDCl ₃ ):? 7. 37 [m, 5H]; 5.88 [br d, J = 8.9 Hz); 5.16 [A of AB, J = 12.2 Hz, 1H]; 5.11 [B of AB, J = 12.2 Hz, 1 H]; 4.95 [A of AB, J = 17.1Hz, 1H], 4.81 [B of AB, J = 17.1 Hz, 1H], 4.60 [m, 1H], 3. 62 [m, 2H], 3.01 [dd, J = 17]. 1, 4.6 Hz, 1H], 2.75 [dd, J = 17.1, 4.8 Hz, 1H], 2.30 [t, J = 7.95 Hz, 2H], 1.99 [quintet, J = 7.5 Hz, 2H], 1.41 [s, 9H], IR (KBr) 3328 (br), 2976, 2932, 1717, 1701, 1522, 1256 cm ^-1 .

MS (APCI, -MeOH) 421 (M ⁺ H +).

Elemental analysis:

Calcd for C2iH ₂ 8N2O * 0096 ₇ DMF: C, 59.81; H, 6.76; N, 6.87.

Found: C, 59.56; H, 7.00; N, 6.52.

Step B: Prepared from 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) -pentanoic acid 1,1-dimethylethyl ester as described above, Step D, to give 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidine-lyloxy) -pentanoic acid (72%).

112 1 H NMR (400 MHz, CDCl ₃ ): δ 8.55 [br s, 1H], 7.36 [m, 5H], 5.46 [br d, J =

9.4 Ηζ, ΙΗ], 5.14 [A of AB, J = 5.2 Hz, 1H], 5.11 [B of AB, J = 5.2 Hz, 1H], 4. 23 [m, 1H], 4.19 [A of AB, J = 13.3 Hz, 1H], 3.96 [B of AB, J = 13.3 Hz, 1H], 3.67 [m, 1H], 3.52 [dd, J = 15.1, 7.9 Hz, 1H], 2.84 [dd, J = 16.9, 8.2 Hz, 1 H], 2.61 [dd, J = 16.9, 10.9 Hz, 1 H], 2.42 [m, 2H], 2.11 [m, 2H], IR ( KBr) 3408 (br), 2926, 1791, 1717, 1700, 1540, 1268, 1054 cm ^{@ -1} . The MS (APCI, MeOH) 365 (M ⁺ H +).

Analysis calculated for Ci7H2oN ₂ 0 ₇ 0:32 C3H7OC3H7 *: C, 57.27; H, 6.24; N, 7.04.

Found: C, 57.27; H, 6.24; N, 6.74.

3-Benzyloxycarboylamino-5- (3,5-dioxo-10-oxa-4-aza-tricyclo [5.2.1.0 ^2,6 ] dec-8en-4-yloxy) -4-oxo-pentanoic acid

Step A Prepared from 3α, 4,7,7α-tetrahydro-2-hydroxy-4,7-epoxy-1H-isoindole-1,3 (2H) -dione [Narita M., Teramoto T, Okawara M (Bull. Chem. Soc.) Jap., 1971; 44: 1084)] as described above, Step C, to obtain 3-benzyloxycarbonylamino-5- (3,5-dioxo-10-oxa-4-azatricyclo [1,1.1.0] dimethyl-ethyl ester [5.2: 1.0 ^2.6 dec-8-en-4-yloxy) -4-oxo-pentanoic acid (64%).

1 H NMR (400 MHz, DMSO-d ₆ ):? 7. 84 [d, J = 8]. 2 Hz, 1 H], 7. 34 [m, 5H] 6. 54 [s, 2H]. 16 [s, 2 H]; 5 .07 [A of AB, J = 12.5 Hz, 1H], 5.03 [B of AB, J = 12.5 Hz, 1H], 4.93 [A of AB, J = 16.2 Hz, 1H], 4.87 [B of AB, J = 16.2 Hz, 1 H], 4.52 [m, 1 H], 2.87 [s, 2 H], 2.73 [dd, J = 16.2, 5.8 Hz, 1 H], 2.50 [Shaded Dimethylsulfoxide Resonance (DMSO)], 1.37 [ s, 9H]. IR (KBr) 3421, 2979, 2930, 1790, 1726, 1520, 1368 cm ^-1 . MS (APCI, MeOH) 445 (M ⁺ C ₄ H ₈ ).

Calcd for C25H28N2O9: C, 59.65; H, 5.70; N, 5.35.

Found: C, 59.99; H, 5.64; N, 5.60.

Step B Prepared from 3-Benzyloxycarbonylamino-5- (3,5-dioxo-10-oxa-4-aza-tricyclo [5.2.1.0 ² ' ⁶ ] dec-8-en-4-yloxy) 1,1-dimethylethyl ester -4 oxo-pentanoic acid

113 of the acid as described above, Step C, to obtain 3-benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid (78%). IR (thin layer) 3360, 1789, 1723, 1530, 1220 cm ^-1 . MS (APCI, MeOH) 445 (M ⁺ H +).

Elemental analysis:

Calcd for C ₂₁ H 20 N ₂ O 9 · 0.194 CF ₃ CO ₂ H: C, 55.06; H, 4.36; N, 5.96. Found: C, 55.06; H, 4.58; N, 5.99.

3-Benzyloxycarbonylamino-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -4-oxopentanoic acid.

Prepared from 1-hydroxyoxindole [Kende A.S. and Thurston J. (Synthetic Communications, 1990; 20: 2133-8)) to obtain 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -pentanoic acid (24% ), 1.1. 5870 ° C (decomposition).

Elemental analysis:

Calcd. For C 21 H 20 N 2 O 7: C, 61.16; H, 4.89; N, 6.79.

Found: C, 60.84; H, 4.72; N, 6.46.

3-Benzyloxycarbonylamino-5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid

Step A Hydroxylamine hydrochloride (200 mmol, 13.8 g) was dissolved in pyridine (200 mmol, 16 mL) and methanol (10 mL) and this solution was added to a mixture of cis-2-oxo-1,3-cyclohexanediacetic acid dimethyl ester, [(35 mmol,

8.5 g) prepared according to the procedure of Grieco PA, Noguez JA, Masaki Y, Hiroi K., Nishizawa M., Rosowsky A., Oppenheim S., Lazarus HJ Med. Chem., 1977; 20: 71] in 200 mL MeOH. To this solution was added portionwise NaCNBH ₄ (30 mmol, 1.9 g) over about one hour, and the resulting solution was stirred at room temperature for 4 days. The reaction mixture is then concentrated to dryness,

114 was diluted again in 500 mL ethyl acetate and washed with 3 x 50 mL saturated NaCl, dried over Na ₂ SO 4, filtered and concentrated to give the crude solid, which was mostly the desired product and pyridine. The crude octahydro-1-hydroxy-2-oxo-1H-indole-7-acetic acid methyl ester was recrystallized from EtOAc to give 4.05 g (51%) of a white solid.

1 HNMR: 9.26 [1 H, s], 3.64 [1 H, dd], 3.59 [3 H, s], 2.65 [1 H, dd], 2.49 [1 H, dd], 2.34 [1 H, dd], 2.18 [1 H, m ], 2.04 [1 H, m], 1.79, [1 H, d], 1.62 [1 H, m], 1.60 [1 H, sbr], 1.42 [1 H, m], 1.25 [2 H, m], 1.06 [1 H] , m], MS (Cl, _NH3) 228 (m ⁺ H +).

Step B Prepared from octahydro-1-hydroxy-2-oxo-1H-indole-7-acetic acid methyl ester as described above, step C, to give 3-benzyloxycarbonylamino-5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indole-, 1,1-trimethylethyl ester) 1-yloxy) -4-oxo-pentanoic acid as a glassy oil (45%).

¹ H NMR (400 MHz, DMSO-d 6, 1: 1 mixture of diastereoisomers: δ 7.85 [d, J =

5.8 Hz, 0.5 H], 7.83 [d, J = 5.8 Hz, 0.5 H], 7.35 [m, 5 H], 5. 06 [s, 2H], 4. 94 [A of AB, J = 16.9 Hz, 0.25H], 4. 87 [A of AB, J = 17.6 Hz, 0.25H], 4.82 [B of AB, J = 17.6 Hz, 0.25H], 4.74 [B of AB =, J = 16.9 Hz, 0.25H], 4.23 [m, 1 H], 3.82 [m, 0.5 H], 3.79 [m, 0.5 H], 3.57 [s, 1.5 H]; 3.57 [s, 1.5H]; 2.72 [m, 0.5H]; 2.70 [m, 0.5H]; 2.52 [m, shielded DMSO]; 2.39 [m, 2H]; 2.22 [br m, 1H]; 2.10 [br m, 1H], 1.88 [br s, 0.5H], 1.84 [br s, 0.5H], 1.61 [m, 2H], 1.42 [m, 1H], 1.36 [s, 9H], 1.25 [m, 2H], 1.06 [m, 1H], IR 3418, 3344, 3017, 2979, 2934, 2860, 1725, 1506 cm &^lt; -1 & gt ^; . MS (APCI, MeOH) 547.6 (M ⁺ H +).

Step C Prepared from 3-benzyloxycarbonylamino-5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid 1,1-trimethylethyl ester as described above, Step D to give 3-benzyloxycarbonylamino- 5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid (45%), m.p. 55-58 ° C.

115 1 H NMR (400 MHz, DMSO-d ₆ , 1: 1 mixture of diastereomers): δ 12. 4 [s, 1 H], 7.84 [m, 1 H], 7.35 [m, 5 H], 5.05 [s, 2 H], 4.86 [m, 2 H], 4.45 [m, 1 H], 3. 83 [m, 0.5H], 2.79 [m, 0.5H], 3.59 [s, 1.5H], 3.58 [s, 0.5H], 2.57 [m, shielded DMSO], 2.41 (m complex, 4H], 2.20 [m, 2H], 1.88 [m, 1H], 1.62 [m, 2H], 1.43 [m, 2H], 1.23 [m, 2H], 1.05 [m, 1H], IR (KBr) 3337, 2931, 1790, 1726, 15384 cm ^first MS (ES, NH ₄ OH) 489.5 (M ⁺ H +).

Elemental analysis:

Calcd. For C 24 H 30 N 2 O 9: C, 58.77; H, 6.16; N, 5.71.

Found: C, 59.19; H, 6.40; N, 5.34.

3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yloxy) pentanoic acid

Step A Ethyl 2-cyclohexanone acetate (4.28 g, 23.3 mmol) and Oenzylhydroxylamine hydrochloride were combined in 100 mL ethanol (EtOH) and 2.59 g (25.6 mmol, 3.55 mL) of triethylamine (Et ₃ N) was added. The reaction mixture was stirred at room temperature for 12 hours, at which point it was concentrated in vacuo. The residue was taken up in EtOAc and washed with 1N HCl (2 x 20 mL), saturated NaHCO ₃ (1 x 20 mL), dried over Na ₂ SO ₄ , filtered and concentrated. Purification by chromatography (SiO ₂ , 90: 1 Hexanes-EtOAc) gave (2-benzyloxyimino-cyclohexyl) -acetic acid ethyl ester (4.76 g, 72%) as a mixture of oxime isomers.

1 H NMR (400 MHz, CDCl 3 7: 1 mixture of oxime isomers): δ 7. 32 [m complex, 5H], 5.05 [s, 0.25 H], 5.02 [s, 1H], 4.05 [q, J = 7.2 Hz, 2H], 3.20 [m, 1H], 2.73 [m complex, 2H ], 2.46 [d, J = 8.0Hz, 0.125H], 2.21 [dd, J = 15.4, 6.3Hz, 0.875H], 1.92 [m, 1H], 1.79 [m complex, 3H], 1.43 [m] 1 H, 1.38 [complex m, 2H], 1.22 [t, J = 7.2 Hz, 3 H]. IR (thin layer) 2931, 1735, 1638, 1451 cm ^-1 . MS (Cl, _NH3) 290 (M ⁺ H +).

Calcd. For C ₁₇ H ₂₃ NiO ₃ : C, 70.56; H, 8.01; N, 4.84.

Found: C, 70.47; H, 7.92; N, 4.78.

116

Step B (2-Benzyloxyimino-cyclohexyl) -acetic acid ethyl ester (4.66 g, 16.1 mmol) was taken up in 15 mL of acetic acid (AcOH) and NaBEhCN and stirred for 72 hours. Pour the reaction into NaHCO 3 and extract into EtOAc (3 x 30 mL). The combined organic layers were washed once with brine, dried over Na ₂ SO ₄ , filtered and concentrated. The clear oil was dissolved in 50 mL of MeOH and K 2 CO 3 (5.55 g, 40.2 mmol) was added and the reaction was stirred for 12 hours. Concentrate the reaction, take up the residue in CHCl 3, filter, and concentrate. Purification by chromatography (SiO 2, 4: 1 Hexanes / EtOAc) gave 1.72 g (43%) of cis- (2-benzyloxyamino-cyclohexyl) -acetic acid ethyl ester and 0.441 g (11%) of trans- (2-benzyloxyamino-cyclohexyl) -ethyl ester. -acetic acid.

Data for cis isomer:

1 H NMR (400 MHz, CDCl ₃ ): δ 7.44 [complex m, 2H], 7.37 [complex m, 3H], 5. 05 [A of AB, J = 10.4 Hz, 1H], 4.94 [B of AB, J = 10.4 Hz, 1H], 3.47 [dd, J = 10.6, 5.3 Hz, 1H], 2.33 [dd, J = 16.4 Hz, 1 H], 2.20 [m, 1H], 2.08 [dd, J = 16.4, 4.6 Hz, 1H], 1.74 [complex m, 2H], 1.60 [m, 1H], 1.32 [complex m, 5H]. IR (solution, CHCl 3) 3031, 2932, 2856, 1717, 1453 cm ^-1 . MS (Cl, _NH3) 246 (M ⁺ H +).

Data for the trans isomer: 1.1. 79-82 ° C.

Elemental analysis:

Calcd for C 11 H 15 N 1 O 1: C, 73.44 H, 7.81; N, 5.71.

Found: C, 73.38; H, 7.89; N, 5.63.

Step C Prepared from cis- (2-benzyloxyamino-cyclohexyl) -acetic acid ethyl ester as described above, Step B, to afford cis-1-hydroxyoctahydro-indol-2-one (85%), m.p. 85-86 ° C.

1 H NMR (400 MHz, CDCl 3): δ 9.86 [br s, 1H], 3.75 [dd, J = 10.4, 4.8 Hz, 1H], 2.41 [dd, J = 16.1, 7.7 Hz, 1H], 2.33 [m, 1H], 1.97 [m, 1H], 1.71 [m, 2H], 1.54 [m, 1H], 1.44 [m, 2H], 1.31 [m, 2H].

117

IR (KBr) 3037, 2936, 2856, 2710, 1690, 1659, 1548 cm &^lt; -1 & gt ^; . MS (Cl, _NH3) 156 (M ⁺ H +).

Elemental analysis:

Calcd. For C ₈ H ₃ NiO ₂ : C, 61.91; H, 8.44; N, 9.03.

Found: C, 61.94; H, 8.49; N, 8.96.

Step D Prepared from cis-1-hydroxy-octahydro-indol-2-one by the method described above, Step C, to give 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yl) 1,1-dimethylethyl ester. yloxy) -pentanoic acid (41%). IR (thin layer) 2933, 1723, 1516, 1367 cm ^-1 . Elemental analysis:

Analysis calculated for C _{34 2} 5Hi _N2 O7: C, 63.28; H, 7.22; N, 5.90.

Found: C, 63.03; H, 7.36; N, 5.65.

Step E Prepared from 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yloxy) -pentanoic acid 1,1-dimethylethyl ester as described above, Step D, to give 3-benzyloxycarbonylamino-4-oxo- 5- (2-oxo-octahydro-indol-1-yloxy) -pentanoic acid (72%). IR (KBr) 3352 (br), 2935, 2869, 1789, 1704, 1535 cm &^lt; -1 & gt ^; . MS (APCI, MeOH) 419.5 (M ⁺ H +).

Elemental analysis:

Analysis calculated for C ₂ H ₂₆ N ₂ O ₇ * 0th 12 H ₂ O * 0.322 CH ₂ Cl ₂ : C, 57.17; H, 6.05; N, 6.26.

Found: C, 57.17; H, 6.05; N, 5.89.

The following were prepared from 5-bromo-3- [2- (2-benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -1,4-oxopentanoic acid 1,1-dimethyl ester [Dolle R.E., et al. (J. Med. Chem., 1994; 37: 563-4)] as described above, Step C and Step D.

118

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid.

Prepared from octahydro-1-hydroxy-2-oxo-1H-indole-7-acetic acid methyl ester (65%), m.p. 162-167 ° C dec.

Elemental analysis:

Analysis calculated for C 9 H ₃₄ N ₂ O _{4 9} * 0.75 _H2O (596127): C, 58.43; H, 6.00; N, 9.40. Found: C, 58.40; H, 5.68; N, 9.19.

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -proionylamino] -4-oxo-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -pentanoic acid

Prepared from 1-hydroxyoxindole [Kende A.S. and Thurston J. (Synthetic Communications, 1990; 20: 2133-8)] to give 3- (2- (2-benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -4-oxo-5- (2-oxo-2,3- dihydro-indol-1-yloxy) -pentanoic acid (67%), mp 162-167 ° C, dec.

Elemental analysis:

Calcd. For C ₂ H ₃₄ N ₄ O 9 * 0.75 H ₂ O (596: 127):

C, 58.43; H, 6.00; N, 9.40.

Found: C, 58.40; H, 5.68; N, 9.19.

Other compounds were prepared using automated parallel synthesis as follows: To a 7-ml glass screw cap vial containing 17 mg (0.3 mmol, 3 eq KF) was added a 500 μΐ (0.1 mmol, 1 eq) 0.2 M solution of the appropriate hydroxamate in DMF. Shake for several minutes until the KF is completely dissolved At this point, 500 μΐ (0.1 mmol, 1 eq) of a 0.2 M solution of (S) -5-bromo-4-oxo-3 [[(phenylmethoxy) 1,1-dimethylethyl ester] carbonyl] amino] -pentanoic acid in DMF

119 cover the lid and the stand of 30 to 40 vials is placed on top of the ring mixer for 12 hours.

The reaction mixtures were diluted with 2 mL of ethyl acetate followed by 2 mL of deionized water. Two milliliters of liquid are withdrawn from the center of the vial and quickly injected back twice. The vials were allowed to settle for 30 minutes and the organic layer was removed from the top half of the vial. Add twice more, 2 mL of ethyl acetate, mix and separate. The combined organic layers were evaporated under constant nitrogen vapor overnight.

The crude residue from the reactions was dissolved in 3-4 mL of 40% TFA in methylene chloride. The vials are shaken without caps to ensure complete dissolution in the vaporizer hood. After 2 hours, the vials were placed again under nitrogen atmosphere overnight

The crude reaction mixture was taken up in 1 mL of chloroform (MeOH was sometimes added to dissolve completely). The solution was applied to a 500 micron preparative silica gel TLC layer and then eluted with 20% acetone in methylene chloride with 1% to 2% acetic acid. The product bands are visualized by UV absorption, pooled, and the silica gel is washed with methanol into a molded vial. The vials were placed under nitrogen vapor overnight. The weighed purified products are then analyzed and biologically evaluated. The solutions can be evaporated in a vaporizer for 72 hours.

equivalents

Those skilled in the art will recognize or be able to recognize that more than one conventional experiment procedure may be used, there are many equivalent procedures for the specific embodiments of the invention described herein. Such equivalents are also intended to be within the scope of the following claims.

Claims (25)

A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject, comprising: an agent that antagonizes an object that regulates the production of interferon-γ (IFN-γ) in a subject; and a corticosteroid. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 1, wherein the agent is an IL-18 antagonist. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 2, wherein the agent is a caspase family protease inhibitor, for example an ICE inhibitor. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 2 or 3, wherein the agent is an antibody, antibody fragment or exposed binding protein that binds IL-18 or IL-18 receptor. . The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 1, wherein the agent is an interleukin-12 (IL-12) antagonist. 121 The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 5, wherein the agent is an antibody, antibody fragment or exposed binding protein that binds IL-12 or IL-12 receptor. A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 5 or 6, wherein the agent stimulates the production of cyclic AMP in cells that produce IL-12. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 7, wherein the agent is a phosphodiesterase IV inhibitor, for example selected from the group consisting of 4-arylpyrrolidinones, rolipram, denbufylline, tibenelast, nitraquazone, CP-80633, quinazolinediones, and CP-77059. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 7, wherein the agent is a beta-2 agonist, for example selected from the group consisting of salmeterol, phenoterol and isoproterenol. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 5, wherein the agent is a STAT4 inhibitor. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 1, wherein the agent is an NK cell antagonist. 122 The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to claim 11, wherein the agent is an antibody against NK / NK-like cells, for example an anti-asialo-GM1 antibody or NK1.1 antibody. A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of the preceding claims, wherein the corticosteroid is selected from the group consisting of cortisone, hydrocortisone, beclomethasone, flunizolide, prednisone, prednisolone, methylprednisolone. , triamcinolone, deflazacort, betamethasone and dexamethasone. The combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of the preceding claims, wherein the subject is suffering from septic shock, Crohn's disease, asthma, graft versus host disease or transplant rejection or autoimmune disease, or disorder. Combined preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of claims 1 to 13, wherein the subject is suffering from an immunoinflammatory disease or disorder selected from the group consisting of asthma, adult respiratory distress syndrome, systemic erythematous lupus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, insulin-dependent diabetes mellitus, autoimmune arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, pulmonary inflammation, psoriasis, psoriasis, psoriasis , severe myasthenia, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczema dermatitis, psoriasis, Sjögren's syndrome, keratoconjunctivitis sicca secondary to Sjögren's syndrome, allergic baldness, arthritis reactions, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, cutaneous erythematous lupus, scleroderma, vaginitis, proctitis, eruptions from drugs, Stevens-Johnson syndrome, leprosy reverse erythema, leprosy reverse erythema anemia, pure red blood cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Graves ophthalmopathy, primary biliary cirrhosis, posterior uveitis and interstitial lung fibrosis. A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of claims 1 to 13, wherein the subject is suffering from an acute inflammatory disorder. A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of claims 1 to 13, wherein the steroid resistance in the subject is reversed or increased compared to when the subject is administered alone. corticosteroid. A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of claims 1 to 13, wherein the corticosteroid is administered to the subject according to a regimen that reduces the corticosteroid dose over time. 124 and this method improves spontaneous reactions to steroids associated with administration of reduced doses of corticosteroid. An agent for modulating a corticosteroid response in a subject, wherein the agent antagonizes an object that regulates the production of interferon-γ (IFN-γ) in the subject. A corticosteroid response modulating agent in a subject according to claim 19, wherein: (a) the subject is corticosteroid resistant prior to administration of the agent; (b) the subject responds to the corticosteroid prior to administration of the agent, but exhibits increased sensitivity to the corticosteroid upon administration of the agent; or c) treating the subject with a corticosteroid should be stopped and the administration of the agent ameliorates spontaneous responses to steroids in the subject. A corticosteroid response modulating agent in a subject according to claim 19, wherein the agent is an IL-18 antagonist or an interleukin-12 (IL12) antagonist. A pharmaceutical composition comprising an agent according to any one of claims 1 to 12 or 19 to 21, a corticosteroid and a pharmaceutically acceptable carrier. A pharmaceutical composition according to claim 22, characterized in that it is formulated for topical application or for administration by inhalation. 125 Use of an agent according to any one of claims 1 to 12 or 19 to 21 (optionally in combination with a corticosteroid) in the manufacture of a medicament for modulating a corticosteroid response in a subject. Use of an agent according to any one of claims 1 to 12 or 19 to 21 (optionally in combination with a corticosteroid) in the manufacture of a medicament for the treatment of one or more of the disorders according to any one of claims 14 to