MXPA00004970A - N-[2-(5-benzyloxycarbonyl-amino-6-oxo-2-(4- fluorophenyl)-1,6-dihydro -1-pyrimidinyl)aceto- xyl]-l-aspartic acid aldehyde as an i(in vivo) inhibitor of interleukin-1b converting enzyme (ice) - Google Patents

Abstract

This invention comprises the compound (1) and the pharmaceutically acceptable salts thereof. Compound (1) is an interleukin-1b converting enzyme inhibitor and is useful for treating inflammatory diseases such as arthritis and inflammatory bowel disease, septic shock, reperfusion injury, shigellosis, and neuroinflammatory disorders such as stroke, multiple sclerosis, and Alzheimer's disease.

Description

ALDEHYDE OF ASPARTIC ACID-LN- [2- (5-BENZYLOXICARBONYL-AMINO-6-OXO-2- (4-FLUOROFENYL) -1,6-DIHYDRO-1-PYRIMIDINYL) ACETOXYL] AS AN IN VIVID INHIBITOR OF THE CONVERTIBLE ENZYME INTERLEUKIN -1ß (ICE) " BACKGROUND OF THE INVENTION Field of the Invention This invention relates to the compound of the aspartic acid aldehyde-LN- [2- (5-benzyloxycarbonyl-amino-6-oxo-2- (4-fluorophenyl) -1,6-dihydro-1-pyrimidinyl) acetoxyl] as an inhibitor of the interleukin-1 converting enzyme. This invention also relates to a method of treating fulminating attack, inflammatory diseases, septic shock, reperfusion injury, Alzheimer's disease and shigellosis and to a pharmaceutically acceptable composition containing aspartic acid aldehyde-lN- [2 - (5-benzyloxycarbonyl-amino-6-oxo-2- (4-fluorophenyl) -1,6-dihydro-1-pyrimidinyl) acetoxy].

Previous Art The converting enzyme interleukin-1β (ICE or Caspase-1) in pro-interleukin-1β (pro-IL-1ß) to produce interleukin-1β (IL-1ß), which is an inflammatory cytosine (Kostura MJ et al., Proc. Nat. Acad. Sci., 1989; 86: 5227-5231 and Black RA et al., FEBS Lett., 1989; 247: 386-391). Several diseases are associated with the activity of interleukin-1. Examples of diseases in which interleukin-1 is involved include, but are not limited to, inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease and neuroinflammatory disorders; such as fulminating attack, multiple sclerosis and Alzheimer's disease (Dinarello C.A., Eur. Cytokine Netw., 1994; 5: 517). Other diseases include septic shock, reperfusion injury and shigellosis.

It has been shown that agents that modulate the activity of IL-1ß have beneficial effects in vivo. For example, it has been shown that the compounds which are antagonists receptors of the interleukin-1 exhibit ischemic and excitotoxic damage in the brain of the rat (Relton J.K. et al., Brain Research Bulletin, 1992; 29: 243-246). Additionally, ICE inhibitors demonstrated that they reduce inflammation and pyrexia in rats (Elford P.R. et al., British Journal of Pharmacology, 1995; 115: 601-606).

ICE inhibitors can also inhibit other cysteine proteases in the ICE family. Recently, the nomenclature of these cysteine proteases has also been defined in the ICE family (also known as Caspazas with ICE, also known as Caspasa-1). The following proteases are representative members of this class of enzymes using the nomenclature described in Alnemri et al., Cell, 1996; 87: 171: Caspase-2 (also known as lch-1), Caspase-3 (also known as CPP32, Yama and apopain), Caspasa-4 (also known as TX, lch-2 and ICE rel-ll), Caspasa-5 (also known as ICE rel-III), Caspasa-6 (also known as Mch2), Caspasa-7 (also known as Mch3), Caspasa-8 (also known as FLICE and Mch5), Caspasa-9 (also known as ICE-LAP6 and Mch6), Caspasa-10 (also known as Mch4). It is known that members of this family of enzymes play key biological roles in both inflammation and apoptosis (programmed cell death) (Thornberry NA et al., Perspectives in Drug Discovery and Design, 1994; 2: 389-399). .

In addition to its effects on the production of IL-1ß, ICE has been shown to play a role in the production of interferon-? intermediary of inflammation (Ghayur et al., Nature, 1997, 386 (6625): 619-623). In the processes of ICE, the inactive pro- formula of interferon-? (IGIF; Interleukin-18) activates IGIF, a protein that induces the production of interferon-? through T-cells and natural killer cells. The interferon-? It has been implicated in the pathogenesis of diseases such as inflammatory disorders and septic shock. Therefore, it can also be expected that ICE inhibitors have beneficial effects in such disease states through the effects of interferon-α.

The majority of the ICE inhibitors described in the art are based on peptides (Dolle R. et al., J. Med. Chem., 1994; 37: 563). However, the use of peptide mimetic inhibitors based on pyridone or pyrimidone has recently been reported (Dolle R. et al, WO 9526958, 1995; Dolle R. et al, J. Med. Chem., 1996; 39: 2438; and Semple G. et al., Bioorg, Med. Chem. Lett., 1997; 7: 1337). X-ray crystallography and molecular molding have shown that pyridone-based inhibitors are suitable replacements for P2-P3 found in peptide-based inhibitors (Golee J. et al., Bioorg.Med.Chem. Lett., 1997; 7: 2181-2186).

The application WO 9526958 describes the use of ICE inhibitors based on pyrimidone, such as compound 2: As the in vitro models possess some activity, the compounds of the application WO 9526958 have an IC 50 range from 0.1 to 10 μm, reflecting the percent inhibition of the release of IL-1β. Although these values reflect some activity, research for better ICE inhibitors for the treatment of diseases such as inflammation, Alzheimer's disease, fulminating attack and septic shock is desirable.

SUMMARY OF THE INVENTION This invention comprises the compound 1 and the stereoisomers and pharmaceutically acceptable salts, the esters, the amides and the prodrugs thereof: As an ICE inhibitor, compound 1 exhibits superior and unexpected in vivo activity compared to the prior art of ICE inhibitors. In fact, this is the first ICE inhibitor that demonstrates in vivo activity in an animal model of fulminant attack after peripheral administration. Compounds of the prior art, such as compound 2, do not show such activity.

DETAILED DESCRIPTION OF THE INVENTION This invention comprises compound 1 and its use as an inhibitor of the interleukin-1β converting enzyme. This invention also comprises a method of treating inflammatory diseases such as arthritis and the disease of inflammation of the intestines, neuroinflammatory disorders; such as multiple sclerosis and Alzheimer's disease and other diseases such as fulminating attack, septic shock, reperfusion injury and shigellosis; the method comprises administering to a mammal in need of such treatment, a therapeutically effective amount of compound 1. This invention further comprises a pharmaceutically acceptable composition containing compound 1. This invention also comprises the use of compound 1 to study the biological effects of the inhibition of ICE in in vitro and in vivo models.

Compound 1, notwithstanding a species of the genus described in the application WO 9526958 (application '958), has demonstrated unexpectedly good in vivo activity as an ICE inhibitor.

The side-by-side comparison of compound 1 and the compound was not performed 2 (described and claimed in the '958 application). While both compounds show similar activity in enzyme inhibition and in cell-based tests, compound 1 is active in in vivo models, whereas compound 2 is not.

Compound 1 significantly blocks the production of IL-1β, while compound 2 is inactive. Compound 1 also significantly reduces the extent of excitotoxic brain damage in mice, a property not observed in compound 2. In addition, the mice treated with compound 1 showed a reduced size of the arterial lesion in the ischemic brain damage, followed by the Medial cerebral arterial occlusion after reperfusion.

Compound 1 can be administered to a patient alone or as part of a pharmaceutically acceptable composition. The compositions can be administered to patients such as humans and animals, either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a oral or nasal spray.

Compositions suitable for parenteral injection may comprise sterile aqueous or non-aqueous physiologically acceptable solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles, include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures thereof, vegetable oils (such as olive oil). and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. The prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like . Prolonged absorption of the injectable pharmaceutical form can be achieved by the use of absorption-dilating agents, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dose forms, the active compound is mixed with at least one ordinary inert excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) filling or extension agents, such as, for example, starches, lactose , sucrose, glucose, mannitol and silicic acid; (b) binders, such as, for example, carboxymethyl cellulose, alignates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, such as, for example, glycerol; (d) disintegrating agents, such as, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (e) solution retarders, such as, for example, paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (h) adsorbents, such as, for example, kaolin and bentonite and (i) lubricants, such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. same. In the case of capsules, tablets and pills, the dosage forms may also comprise stabilizing agents.

Solid compositions of similar type can also be used as filling agents in hard and soft gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms such as tablets, "chochos", capsules, pills and granules can be prepared with coatings such as the enteric layer and other coatings well known in the art. They may contain opacifying agents and may also be of such composition that they may release the active compound in a certain part of the intestinal tract in a delayed manner. Examples of encapsulation compositions that can be used are polymeric substances and waxes. The active compound may also be in micro-encapsulated form, if appropriate, with one or more of the aforementioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate , benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, peanut oil, corn germinate oil, olive, castor oil and sesame oil, glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances and the like.

In addition to such inert diluents, the composition may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

The suspensions, in addition to the active compound, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, chickenpoxethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances and the like.

The compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compound 1 with suitable non-irritating carriers or carriers such as cocoa butter, polyethylene glycol or a suppository wax, all of which are solid at ordinary temperature, but liquid at room temperature. body temperature and therefore, they melt in the rectum or in the vaginal cavity and release the active component.

Dosage forms for topical administration of compound 1 include ointments, powders, sprays and inhalants. The active compound is mixed under sterile conditions with a physiologically acceptable carrier and some preservatives, stabilizing agents or propellants may be required. Ophthalmic formulations, ophthalmic ointments, powders and solutions are also contemplated within the scope of this invention.

Compound 1 can be administered to a patient in the range of about 0.1 to about 1000 mg per day. For a normal human adult who has a body weight of about 70 kg, a dose in the range of about 0.01 to about 100 mg per kilogram of body weight per day is preferable. However, the specific dose used may vary. For example, the dose may depend on several factors including the needs of the patient, the severity of the condition to be treated and the pharmacological activity of the compound used. The determination of optimal doses for a particular patient is well known to those skilled in the art.

In this document as the term "pharmaceutically acceptable salts, esters, amides and prodrugs" is used, it refers to those carboxylated salts, addition salts of amino acids, esters, amides and prodrugs of the compound of the present invention which are within the scope of the invention. medical judgment, suitable for coming into contact with the tissues of patients without any undue toxicity, irritation, allergic response and the like, commensurate with a reasonable risk / benefit ratio and effective to be used in their amphoteric and tautomeric forms when possible, with the compounds of the invention. The term "salts" refers to the relatively non-toxic organic and inorganic acid addition salts of compound 1. These salts can be prepared in situ during the final isolation and purification of the compound or by separate reaction of the purified compound in its free base with a suitable organic or inorganic acid and the isolation of the salt formed. Representative salts include the salts of hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laureate, borate, benzoate, lactate, phosphate, tosylate, citrate, mealate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate and lauryl sulfonate and the like. These may include cations based on alkaline metals and alkaline steams, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to ammonium, tetramethylammonium. , tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like (see, for example, Berge SM et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference). reference).

Examples of pharmaceutically acceptable non-toxic esters of compound 1 include C 1 -C 6 alkyl esters wherein the alkyl group is a long or branched chain. Acceptable esters also include C5-C7 cycloalkyl esters as well as arylalkyl esters such as, but not limited to benzyl, C1-C4 alkyl esters are preferred. The esters of compound 1 can be prepared to conventional methods.

Examples of pharmaceutically acceptable non-toxic amides of compound 1 include amides derived from ammonium, primary C 1 β alkyl amines and secondary Ci-C β dialkyl amines, wherein the alkyl groups are long or branched chains. In the case of secondary amides, the amine may also be in the form of a 5- to 6-membered heterocycle containing a nitrogen atom. Amides derived from ammonium, C1-C3 primary alkyl amines and C1-C2 secondary dialkyl amines are preferred. The amides of compound 1 can be prepared according to conventional methods.

The term "prodrug" refers to compounds that are rapidly transformed in vivo to compounds related to compound 1, for example, by hydrolysis in blood. An additional description is provided in Higuchi T. and Stella V., "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series and in Bioreversible Carriers n Drug Design. ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both incorporated herein by reference.

In addition, compound 1 can exist in solvated and non-solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. In general, solvated forms are considered unsolvated equivalents for the purposes of the present invention.

Compound 1 is administered to a patient in need of inhibition of ICE. In general, patients with such a need are those who have a disease or condition in which ICE plays a role. Examples of such diseases include, but are not limited to, inflammatory diseases such as rheumatoid arthritis and the disease of inflammation of the intestines and neuroinflammatory disorders such as fulminating attack. Other diseases include reperfusion injury, Alzheimer's disease and shigellosis.

A "therapeutically effective amount" is an amount of compound 1 that, when administered to a patient having a disease that can be treated with compound 1, improves the symptomatology of such a disease. A therapeutically effective amount of compound 1 is readily determined by one skilled in the art by administering compound 1 to a patient, observing the results that can be achieved routinely using techniques recognized in the art.

An illustration of the preparation of compound 1 is shown in Scheme 1. SCHEME 1 ÍAIH4 in EljO The starting materials and the various intermediates can be obtained from commercial sources, can be prepared from commercially available organic compounds, or routinely prepared using well-known synthetic methods.

The descriptions in this application of all articles and references, including patents, are incorporated herein by reference.

The invention is further illustrated by the following examples which are not intended to limit the scope of the invention or the spirit of the specific procedures described therein.

EXAMPLE 1 Preparation of Compound 1 To a solution of acid 3 (50.0 g, 154.6 mmol) in dry dichloromethane (200 mL) and N-methylmorpholine (42.5 mL, 386.5 mmol) at -75 ° C is added dropwise isobutylchloroformate (24.1 mL, 185.5 mmol) followed by stirring at -75 ° C for 30 minutes. A solution of N, 0 -dimethylhydroxylamine hydrochloride (18.10 g, 185.5 mmol) in dry dichloromethane (50 mL) and N-methylpiperidine (22.55 mL) is added.185.5 mmol). The solution is left at room temperature and stirred for 1 hour, quenched by the addition of 10% H_SO4 and extracted into ethyl acetate. The organic layer is washed sequentially with H2SO4, 10% H2O and brine, dried and concentrated in vacuo. The resulting oil is crystallized with the addition of 20% ether / hexane and the solids are collected and dried to give 45.0 g (80%) of the corresponding amide 4. 1 H NMR (400 MHz, CDCl 3) d 7.30 (5H, s). 5.62 (1H, d), 5.05 (2H, s), 4.95 (1H, m), 3.78 (3H, s), 3.20 (3H, s), 2.65 (I H.dd), 2.55 (1 H, dd ), 1.39 (9H, s); MS (APCI) 367 (M + H); room temperature = 18.2 minutes, elution gradient: 0 to 100 in 20 minutes, 0.10% TFA in CH3CN / water. 2.

To a suspension of lithium aluminum hydride (2.80 g, 74.0 mmol) in dry ether (250 mL) at -75 ° C is added dropwise compound 4 (22.60 g, 61.70 mmol) in Et2?: THF (120 mL , 10: 1). The resulting solution is stirred at -75 ° C for 3 hours. The reaction is quenched by the dropwise addition of potassium hydrogen sulfate (16.80 g, 123.40 mmol) in water (15 mL) and extracted into ethyl acetate. The organic layer is sequentially washed with H2O and brine. The solution is dried and the solvent is removed to give 18.0 g (99%) of the product 5 as an oil. 1 H NMR (400 MHZ, CDCl 3) d 9.60 (1 H, s), 7.28 (5 H, bs), 5.12 (2 H, s), 5.03 (1 H, s), 2.78-2.58 (2 H, m), 1.38 ( 9H, s); room temperature = 16.9 minutes, elution gradient: 0 to 100 in 20 minutes, TFA to 0.10% in CH3CN / water. 3.

To a solution of aldehyde 5 (21.50 g, 61.40 mmol) in ethanol (200 mL) is added semicarbazide hydrochloride (6.85 g, 61.40 mmol) and sodium acetate (5.10 g, 61.40 mmol) in water (25 mL). The resulting mixture is stirred at room temperature for 5 hours. The ethanol is removed, the crude product is extracted into ethyl acetate (300 mL) and the organic layer is subsequently washed with water (150 mL) three times, dried and concentrated to give 21.77 g (97%) of the product. like a foam. 1 H NMR (400 MHz, DMSO) d 9.97 (1H, s), 7.59 (1 H, d), 7.23 (5H, m), 7.05 (1H, s), 6.21 (2H, s), 4.98 (2H, s) ), 4.42 (1 H, m), 2.58 (1 H, dd), 2.41 (1 H, dd), 1.28 (9H, s), MS (APCI) 365.6 (M + H); room temperature = 15.9 minutes, elution gradient: 0 to 100 in 20 minutes, TFA to 0.10% in CH3CN / water. 4.

To a solution of compound 6 (2.10 g, 5.80 mmol) in MeOH (20 mL) is added 20% Pd / C (150 mg) and the mixture is stirred for 2 hours under an atmosphere of hydrogen at atmospheric pressure. The catalyst is filtered and the solvent is removed. The resulting oil solidifies after the addition of ether. The solid is dried to give 1.15 g, (86%) of compound 7 as a grayish solid. The solid is used immediately in the next stage. It is important to solidify this intermediary before continuing to the next stage. 1 H NMR (400 MHz, DMSO) d 9.84 (1 H, s), 7.02 (1 H, d), 6.07 (2 H, s), 3.57 (1 H, m), 3.38 (2 H, m), 2.38 (1 H, dd), 2.25 (1 H, dd), 1.37 (9H, s), room temperature = 6.4 minutes, elution gradient: 0 to 100 in 20 minutes, 0.10% TFA in CH3CN / water.

. To a solution of acid 7 (6.10 g, 15.30 mmol) and N-methylmorpholine (1.70 mL, 15.30 mmol) in dry dichloromethane (300 mL) at -25 ° C is added freshly distilled isobutyl chloroformate (2.0 mL, 15.30 mmol) and the resulting solution is stirred for 30 minutes. A second portion of N-methylmorpholine (1.70 mL, 15.30 mmol) is added followed for amine 8 (3.50 g, 15.30 mmol). The solution is kept at -25 ° C for 30 minutes and left at room temperature and stirred for 1 hour. The solution is filtered and the solvent is removed. The resulting residue is dissolved in ethyl acetate (200 mL), washed with water, saturated sodium bicarbonate, dried and concentrated. The resulting oil is chromatographed (20% THF in dichloromethane) to give 4.60 g (49%) of compound 9 as a white solid. 1 H NMR (400 MHz, DMSO) d 10.2 (1 H, s), 8.87 (1 H, s), 8.39 (2 H, s), 7.54 (2 H, m), 7.34 (8 H, m), 7.0 (1 H, s), 6.25 (2H, s), 5.13 (2H, s), 4.62 (1H, m), 4.43 (1H, d), 4.36 (1H, d), 2.56 (1H, dd), 2.38 (1H , dd), 1.24 (9H, s); MS (APCI) 610.3 (M + H); room temperature = 16.2 minutes, elution gradient: 0 to 100 in 20 minutes, 0.10% TFA in CH3CN / water.

To a solution of ester 9 (1.0 g, 1.60 mmol) in dichloromethane (50 mL) at 0 ° C is added 25% TFA in dichloromethane (20 mL). The resulting solution is stirred at 0 ° C for 8 hours before being diluted with toluene (100 mL) and evaporated to provide an oil. The resulting oil is chromatographed (elution gradient: 2.5% MeOH in dichloromethane to 10% MeOH in dichloromethane) to give 0.70 g (79%) of compound 10 as a white solid. 1 H NMR (400 MHz, DMSO) d 10.03 (1H, s), 8.88 (1H, s), 8.39 (2H, s), 7.50 (2H, m), 7.32 (8H, m), 7.03 (1H, s). , 6.20 (2H, bs), 5.12 (2H, s), 6.64 (1H, m), 4.42 (2H, dd), 2.58 81 H, m), 2.42 (1 H, m), MS (APCI) 554.4 ( M + H); room temperature = 12.6 minutes, elution gradient: 0 to 100 in 20 minutes, TFA to 0.10% in CH3CN / water. compound 1 To the semicarbazone 10 (6.0 g, 10.80 mmol) is added a solution of 37% aqueous paraformaldehyde and acetic acid (100 mL, 1: 1). The reaction mixture is stirred for 2 hours before the solvent is removed and the resulting oil is diluted with CH3CN (100 mL) and evaporated. The resulting residue is chromatographed using mega bond elut C18 S¡O2 (elution gradient: 25% acetonitrile in water to 55% acetonitrile in water) to give 4.20 g (78%) of final product 1 as a white powder. 1 H NMR (400 MHz, CD 3 OD) d 8.59 (1 H, s), 7.58 (2 H, m), 7.35 (7 H, m), 5.19 (2 H, s), 4.68-4.42 (3 H, m), 4.28 (1 H, m), 2.62 (1 H, m), 2.37 (1 H, m); MS (APCI) 497.4 (M + H); room temperature = 13.4 minutes, elution gradient: 0 to 100 in 20 minutes, 0.10% TFA in CH3CN / water.

EXAMPLE 2 In Vitro Tests Compounds 1 and 2 were tested for the inhibition of ICE as demonstrated by the measurement of K i (μM) using the protocol described herein. 1. Inhibition Studies The ICE (2.24 nM, final concentration) is added to 400 μL of HGDE stabilizer (100 mM HEPES, 20% glycerol, 5mM DTT, 0.5mM EDTA) containing 15 μM of substrate (Ac-Tyr-Val- Ala-Asp-AMC; KM = 15 μM) plus vehicle (DMSO) or inhibitor in the concentrations in square brackets in the formula of K¡. Hydrolysis of the substrate was monitored for 300 seconds by observing the fluorescence of the AMC released using excitation at 380 nm and emission at 400 nm. Mean hydrolysis rates were evaluated by regression-linear analysis of fluorescence vs. time traces. To evaluate K, the points of percent inhibition vs. inhibitor concentration are adjusted by non-linear regression to a reversible competitive model: % inhibition = 100 * [I] [l] + K, (l + [S] / KM) where the competition factor (1+ [S] / K) = 2 2. Colorimetric dose of the ICE - Response Test (IC50) Reserve materials of the diluted inhibitor are prepared by a series of double dilutions from a primary pool whose concentration is selected (based on analysis tests or sequences prior to the IC50 evaluation) to achieve an inhibition of approximately 25% in the majority of the concentrate. The aliquots of each dilution were transferred to a microtiter plate in triplicate.

The ICE enzyme was diluted to approximately 24 nM in HGE stabilizer (100 mM Hepes pH 7.5, 0.5 mM EDTA 20% glycerol, 0.1% Bovine Serum Albumin (BSA) and activated by the addition of dithiothreitol (DTT) to a final concentration of 5 mM The activated enzyme is subsequently aliquoted in the sources containing the inhibitor or the vehicle and the plate was pre-incubated for 60 minutes at room temperature The substrate (Ac-Tyr-Val-Ala-Asp- pNA) was added to each source at a final concentration of 50 μM and the plates were placed in the microtiter plate reader heated at 25 ° C. 5 minutes after the addition of the substrate, the absorbance (405 nm) was monitored by 1 hour and the activity was calculated as the average rate of change in absorbance during this interval. 3. Determinations of the Cellular PBMC Test (IC50) In addition to evidencing that compound 1 is a potent inhibitor of ICE, its ability to inhibit the production of IL-1β in human mono nuclear peripheral blood cells (PBMCs) was demonstrated as described herein. The PBMCs were isolated from heparinized blood by centrifugation in a ficoll centrifuge, then washed three times with phosphate stabilizing saline. The PBMCs were suspended in a medium containing RPMI 1640 with glutamine, penicillin, streptomycin and 2% human AB serum, then placed in 106-cell sources in ninety-six flat-bottomed source plates. The PBMCs were stimulated overnight with 10 ng / mL of lipopolysaccharide (LPS, E. Coli strain 0111: B4; Calbiochem) in the presence or absence of compound 1 or 2. The medium is harvested and the maturity level of IL-1β was determined using an ELISA test kit from R &D Systems. Inhibition of the compound was analyzed by determining the concentration of the agent that reduces the levels of IL-1β by 50%. Cells were cultured for an additional 4 hours in the presence of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT). To determine the viability. The toxicity of the compound can, therefore, be analyzed by determining the concentration of the agent that kills 50% of the cells (IC50). 4. Colorimetric dose of the lch-2-Response Test (IC50) Inhibition of the lch-2 enzyme (Caspase 4) was analyzed as described above for ICE, except that the enzyme was used at 64 nM and 60 μM of the specific substrate-lch-2-specific substrate Ac-Leu-Glu Val-Asp-pNA, instead of the ICE substrate Ac-Tyr-Val-Ala-Asp- pNA. The results of said tests are shown in table 1 below.

Table 1 Compound Weight Mode of 1.ICE 2.ICE 3.PBMC 4.lch-2 Molecular Inhibition K, (μm) IC 0 (μm) IC50 (μm) IC50 (μm) 1 496 reversible 0.060 0.485 1.8 9.800 2 737 irreversible no 0.004 2.2 0.005 determined EXAMPLE 3 1. In Vivo Models 1. Mouse Model Female C57BL / 6 mice were injected intravenously with 5 mg / kg of lipopolysaccharide (LPS) (£ Coli 0111: B4) to TO; this induced the production of interleukin-1ß cytosine. After 3.75 hours from the administration of the LPS, the mice were injected intravenously with a vehicle or a test compound. The mice bled at T4h and the plasma was analyzed for IL.-1β by the ELISA test. The vehicle is 5% DMSO / 20% Trappsol / 3% Dextrose / NMDG. Compound 1 showed an inhibition of IL-1β production of 48% (p <0.03, student's t-test) when compared to animals treated with the vehicle. Compound 2 showed a reduction of IL-1β of 11% (not significant). 2. Cerebral Excitotoxic Damage in Newborn Rats Compound 1 and compound 2 were tested in an excitotoxic brain injury model in newborn rats. 7-day-old mice were anesthetized with ether, placed in a head-holding apparatus and injected with 15 nMoles of MDA into the right caudal nucleus. 0.25 hours later, the mice were treated via intraperitoneal injection with drug (30 mg / kg) or vehicle (2% methocel in water) and returned to their cages. 5 days after this excitotoxic change, the mice were sacrificed and their brains were removed and the cerebral hemispheres weighed. The weight loss of the hemisphere injected with respect to the contralateral hemisphere is an estimate of the degree of cerebral necrosis. Table 2 summarizes the results that demonstrate that compound 1 significantly reduces the extent of excitotoxic brain damage, expressed as percent loss of hemisphere weight. Importantly, such protection is not observed with the same dose of compound 2.

Table 2 Treatment% of loss SD Number of p-value mouse cells Vehicle 23.1 5.3 8 Compound 1 16.1 3.9 8 < 0.009 Vehicle 24.6 7.2 9 Compound 2 20.8 3.7 8 < 0.26 3. Brain Ischemic Damage Model Compound 1 was tested in the permanent (MCAOp) and transient (MCAOt) models of focal cerebral ischemia in the mouse. The middle cerebral artery was occluded by an intraluminal suture in CD-1 mice. In the transient model, the suture was removed after 1 hour in both studies, the mice were sacrificed in 24 hours and the brain lesion size was determined from sections marked H and E. The drug the vehicle was administered IP in -5.60 and 180 minutes from the moment of occlusion of the blood vessel at doses of 25 and 50 mg / kg x 3 (MCAOp) or 50 mg / kg x 3 (MCAOt). In the most severe MCAOp, there is a small reduction in the size of the lesion (13%, p <0.04, a t-test of the tail) at high doses, in the less severe MCAOt, the size of the lesion is 60% smaller in mice treated with drug (p <0.002). The invention and the manner and process for using it have been described in complete, clear, concise and accurate terms to enable any person skilled in the art to manufacture and make use of the rhisma. It should be understood that the preferred embodiments of the present invention described above may be modified without departing from the spirit or scope thereof. To clarify the subject matter claimed in the present invention, the following claims conclude this description.

Claims (3)

1. The compound, and the stereoisomers and pharmaceutically acceptable salts, the esters, the amides and prodrugs thereof.

2. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier.

3. A method of treatment of the interleukin-1β converting enzyme that mediates diseases, comprising administering to a mammal in need of such treatment a therapeutically effective amount of the compound according to claim 1. A method of inhibition of the converting enzyme interleukin-1β by contacting the converting enzyme interleukin-1β according to claim 1. A method of treatment or prevention of fulminating attack, the method comprises administering to a mammal in need of such treatment a therapeutically effective amount of the compound according to claim 1.