HK1032045A - New derivatives from piperidine-keto acid, their preparation and use - Google Patents

Description

Novel piperidinecarboxylic acid derivatives, their preparation and use

The present invention relates to novel ketoesters and ketoamides which are enzyme inhibitors, especially cysteine proteases, such as Calpain (a calcium dependent cysteine protease) and its isoenzymes and cathepsins, such as: b and L forms.

Calpain is an intracellular proteolytic enzyme from the cysteine protease group that is present in many cells. The Calpain enzyme is activated by calcium after increasing concentration, Calpain I or u-Calpain and Calpain II or m-Calpain are different, the former is activated by mu-mol concentration of calcium ions, and the latter is activated by m-mol concentration of calcium ions. (p.johnson, int.j.biochem.1990, 22(8), 811-22.) currently, a further callain catabolic enzyme has been devised (k.suzuki and others, biol.chem.hoppe-Seyler, 1995, 376(9), 523-9).

Calpain is believed to play a very important role in a variety of physiological activities, including the cleavage of regular proteins, such as protein kinase C, cytoskeletal proteins, such as MAP2 and intraerythrocytic proteins, muscle proteins, protein degradation in rheumatoid arthritis, proteins that activate platelet and peptide metabolism, and proteins that act during mitosis and other physiological processes, as mentioned in M.J. Barrett et al, Life Sci.1991, 48, 1659-69, and K.K. Wang et al, trends in Pharmacol.Sci.1994, 15, 412-9.

Increased levels of Calpain can be measured in various pathological processes, for example, cardiac ischemia (e.g., myocardial infarction), renal ischemia or central nervous system ischemia (e.g., stroke), inflammation, muscular dystrophy, cataracts, central nerve injury (e.g., trauma), alzheimer's disease, etc. (see k.k.wang, supra).

It is estimated that these diseases are associated with a continuously increasing calcium content in the cells, and therefore the calcium demanding physiological activities are over-activated and no longer subject to physiological regulation. Accordingly, hyperactivation of Calpain also causes pathological activity.

Inhibitors of the Calpain enzyme are believed to be effective in these diseases. Various studies have confirmed this. Thus, Seung-chyl Hong and other authors, Stroke, 1994, 25(3), 663-9 and R.T. Bartus et al, Neurological Res.1995, 17, 249-58 disclose that Calpain inhibitors have a neuroprotective effect in the treatment of acute neurodegeneration such as that produced following stroke. Likewise, in experimental brain trauma cases, Calpain inhibitors promote the recovery of impaired memory and avoid the development of neuromuscular motor disturbances (k.e. saatmane et al proc. natl. acad. sci. usa, 1996, 93, 3428-. Calpain inhibitors were found to play a protective role in the treatment of renal injury due to hypoxia, c.l. edelstein et al, proc.natl.acad.sci USA, 1995, 92, 7662-6. Yoshida, Ken Ischi et al, jap. circ. j.1995, 59(1), 40-8, also demonstrated that Calpain inhibitors have significant therapeutic effects on myocardial injury caused by ischemia or reperfusion. Calpain inhibitors also have potential for the treatment of Alzheimer's disease because they are resistant to the release of β -AP4 protein (J.Higaki et al, Neuron, 1995, 14, 651-59). Similarly, the release of cross-linked leukocidin-1 α can be inhibited by Calpain inhibitors (n.watanabe et al, Cytokine 1994.6(6). 597-. Further, it was found that Calpain inhibitors have cytotoxic effects on tumor cells (E.Shiba et al.20)^th Meeting Int.Ass.Breast Cancer Res.Sendai.JP.1994；25.-28.Sept.，Int.J.Oncol.5(Suppl.)，1994，381)。

Other possible uses of Calpain inhibitors are described in k.k.wang Trends in pharmacol.sci.1994, 15, 412-9.

Calpain inhibitors have been described in the literature. But these are mainly irreversible or peptide inhibitors. Generally, irreversible inhibitors are alkylating substances and undergo non-selective reactions or are unstable in the human body. These inhibitors are therefore often of limited or no use due to side effects, such as toxicity. Among the irreversible inhibitors are: epoxy derivatives E64(E.B.McGowan et al. biochem.Biophys.Res. Commun.1989, 158, 432-5), alpha-haloketones (H.Angliker et al.J.Med.chem.1992, 35, 216-20) or disulfides (R.Matsueda et al.chem.Lett.1990, 191-.

Many known reversible inhibitors of cysteine proteases, such as Calpain, are compounds of peptide aldehydes, especially di-and tripeptide aldehydes, such as Z-Val-Phe-H (MDL 28170) (S.Mehdi, Tendsin biol.Sci.1991, 16, 150-3) and EP 520336. Under physiological conditions, peptide aldehydes are unstable due to strong reactivity, are easily metabolized rapidly, and are prone to non-specific reactions, which may be responsible for toxic side effects (J.A. Fehrentz and B.Castro Synthesis 1983, 676-78). Thus, peptide aldehydes are often limited or not functioning at all when used to treat diseases. The surprising result of this is: only a few aldehydes can be used as active compounds, i.e. in particular when the aldehyde groups are stabilized, for example by formation of hemiacetals

One advance was the discovery that certain peptidone derivatives are also cysteine protease inhibitors, particularly Calpain inhibitors. Thus, for example in the case of serine proteases, ketone derivatives are known as inhibitors, the ketone group being activated by an electron-withdrawing group such as CF 3. In the case of cysteine proteases, there is little or no activity with ketone derivatives activated by CF3 or similar groups (m.r.angelastro et al, j.med.chem.1990, 33, 11-13).

Surprisingly, in the case of Calpain, only ketone derivatives in which, on the one hand, the leaving group in the alpha position leads to irreversible inhibition, and, on the other hand, a carboxylic acid derivative activates the ketone group, have been found to be active inhibitors (cf. m.r. angelastro et al, supra; WO 92/11850; WO92, 12140; WO94/00095 and WO 95/00535). However, of these ketoamides and ketoesters, only peptide derivatives have been considered active (Zhao Zhao Li et al, J.Med.Chem.1993, 36, 3472-80; S.L.Harbenson et al, J.Med.Chem.1994, 37,2918-29 and see M.R.Angelastro et al, supra).

One of the objectives of the present invention is to make available non-peptide inhibitors derived from more stable ketones, which do not have the common complaints shared by peptides (metabolic stability, difficulty in passing through cell membranes, etc.).

The present invention relates to piperidinecarboxylic acid derivatives of formula I:and their tautomeric and isomeric forms, and the physiologically acceptable salts thereof, where the variable groups have the following meanings: r¹is-CO-R⁴，-SO₂-R⁴，-CONH-R⁴，COOR⁴，-C(＝N)-R⁴，-C(＝O)-NHR⁴and-C (═ S) -NHR⁴R²is-C₁-C₆Alkyl, in branched or unbranched form, which may additionally bear a phenyl, pyridine or naphthalene ring, which may then be substituted by up to two R⁵Radical substitution, R⁵C which may be branched or unbranched₁-C₄-alkyl, -O-C₁-C₄Alkyl, OH, Cl, F, Br, I, CF₃、NO₂、NH₂、CN、COOH、-COO-C₁-C₄-alkyl, -NHCO-C₁-C₄-alkyl, -NHCOPh, -NHSO₂-C₁-C₄Alkyl, NHSO₂-Ph、-SO₂-C₁-C₄-alkyl and-SO₂Ph；R³is-OR₆or-NHR₆；R⁴Is branched or unbranched-C₁-C₆Alkyl, the chain of two or more carbon atoms may also contain double or triple bonds, or be substituted by one or two rings, for example phenyl, naphthalene, quinoxaline, quinoline, isoquinoline, pyridine, thiophene, benzothiophene, benzofuran, pyrimidine, thiazole, isothiazole, triazole, imidazole, cyclohexyl, cyclopentyl, fluorene, indole, benzimidazole, oxazole, isoxazole, and furan, each ring itself possibly carrying up to two further R' s⁵A group.

R⁶Is a hydrogen atom, one of which may additionally carry one to two R⁵The phenyl ring, branched or unbranched C of the radical which may contain a double or triple bond and a ring such as phenyl, naphthalene, pyridine, pyrimidine, piperidine, pyrrolidine, morpholine, thiophene, quinoline, and isoquinoline₁-C₆Alkyl, aromatic rings may additionally bearUp to two radicals-NR⁷R⁸Or R⁵，R⁷And R⁸May be independently of one another a hydrogen atom or a branched or unbranched C₁-C₆-an alkyl group.

Preferred piperidinecarboxylic acid derivatives of the general formula I are those claimed in claim 2 for which: r¹is-C (═ O) R⁴，-SO₂R⁴R²Is branched or unbranched C₁-C₆-alkyl, -CH₂-Ph or-CH₂-pyridinyl R³is-OR⁶or-NHR⁶And R⁴，R⁵，R⁶The meaning of (a) is indicated in claim 2.

Particularly preferred piperidinecarboxylic acid derivatives of the general formula I are those indicated in claim 3 for which: r¹is-C (═ O) R⁴，-SO₂R⁴R²is-C₁-C₄-alkyl or-CH₂-PhR³is-NHR⁶R⁴is-CH ═ CH-R⁹，R⁹Possibly phenyl, naphthalene or quinoline, and R⁶Is a hydrogen atom; c substitutable by phenyl, pyridine or morpholine₁-C₉-alkyl radical

The compounds of formula I may be used as their racemates or enantiomerically pure compounds, or as diastereomers. When enantiomerically pure compounds are desired, they can be obtained by resolution of the compounds of formula I or their intermediates using optically active acids or bases.

The invention also relates to meso compounds or tautomers of the compounds of formula I, e.g. compounds in which the keto group of formula I is present as enolic substructures.

The invention further relates to physiologically acceptable salts of the compounds of formula I, which are obtainable by converting the compounds of formula I using a suitable acid or base.

The piperidinecarboxylic acid derivatives I according to the invention can be prepared by different methods, as is briefly indicated in the schemes 1 and 2.

Starting from piperidinecarboxylic acid II, derivative III is obtained by conversion under customary conditions with an activated acid derivative R1-L, L being a leaving group such as Cl, imidazole or N-hydroxybenzotriazole. The reaction is carried out in an anhydrous inert solvent, such as dichloromethane, tetrahydrofuran or dimethylformamide, at from-20 to +25 ℃ and generally under conventional conditions [ outlined in Houben-Weyl, methods of organic chemistry, 4 th edition, E5, chapter V ].

The piperidine carboxylate ester III is converted to the acid IV in an aqueous medium or a mixture of water and an organic solvent such as an alcohol or tetrahydrofuran with an acid or a base such as lithium hydroxide, sodium hydroxide or potassium hydroxide at room temperature or at an elevated temperature such as 25 to 100 ℃.

These acids IV are linked to an alpha-amino acid derivative using the conventional conditions as described above and listed in Houben-Weyl. Reaction scheme 1

The derivative V, generally an ester, is converted to the ketocarboxylic acid VI, analogously to the hydrolysis process described above. Ketoesters VII were prepared by a reaction analogous to the Dakin-West reaction, which was carried out according to the method of Zhao ZhaoLi et al, J.Med.chem., 1993, 36, 3472-80. In this process, a carboxylic acid such as V is reacted with oxalic acid monoester acid chloride at elevated temperature (50-100 ℃ C.) in a solvent such as tetrahydrofuran, and the resulting product is then reacted with a base such as sodium ethoxide in ethanol at 25-80 ℃ to give the ketoester I' of the invention. As mentioned above, the ketoester I' can be hydrolyzed to give, for example, the ketocarboxylic acids of the invention.

The reaction to give ketoamide I' can also be carried out analogously to ZhaoZhao Li et al (see above). The keto group in I' is protected at room temperature by addition of 1, 2-ethanedithiol in an inert solvent such as dichloromethane with Lewis acid catalysis, e.g. boron trifluoride etherate, to obtain a dithiane. These derivatives are reacted with amines R in polar solvents, for example alcohols, at 0 to 80 DEG C³-H generationReaction to give the ketoamide I'. Reaction formula 2

Another method is illustrated in equation 2. Piperidinecarboxylic acid IV was reacted with aminohydroxycarboxylic acid derivative VII (see s.l.harbenson et al, j.med.chem.1994, 37,2918-29) in the conventional peptide coupling manner (see above, Houben-Weyl) to give amide VIII. These alcohol derivatives VIII can be oxidized to the ketocarboxylic acid derivatives I' of the invention. For this purpose, various conventional oxidation reactions (c.r. larock, commercial organic transformations, VCH publishers, 1989, page 604 and below) such as the Swern oxidation and oxidation similar to the Swern class (t.t. tidwell, Synthesis1990, 857-70) or sodium hypochlorite/TEMPO (s.l. harbenson et al, supra) can be employed.

When VIII is an α -hydroxy ester (X ═ alkoxy), these can be hydrolyzed to carboxylic acid IX, and this reaction can be carried out in a similar manner to the above method, but is preferably carried out at room temperature using lithium hydroxide in a water/tetrahydrofuran mixed solvent. Other esters or amides X may be prepared by reaction with alcohols or amines under the coupling conditions described above. The alcohol derivative X can be oxidized again to produce the ketocarboxylic acid derivative I of the present invention.

Derivatives of ketones I comprised in the present invention are inhibitors of cysteine proteases, in particular cysteine proteases such as calpain I and II and cathepsins B and L.

The inhibition of ketone derivatives I is determined according to conventional enzyme assays in the literature, wherein, as an activity scale, the concentration of inhibitor (═ IC) is determined when 50% of the enzyme activity is inhibited₅₀). The inhibition of Calpain I, Calpain II and cathepsin B by ketone derivative I was determined in this manner. Cathepsin B assay

The method of determining the inhibition of cathepsin B is similar to S.Hasnain et al, J.biol.chem.1993, 268, 235-40.

mu.L of inhibitor solution prepared from inhibitor and DMSO (final concentration: 100. mu.M to 0.01. mu.M) was added to 88. mu.L of cathepsin B (cathepsin B from human liver (Calbiochem) diluted to 5 units in 500mM buffer). This mixture was preincubated at room temperature (25 ℃) for 60 minutes, and then 10. mu.L of 10mM Z-Arg-Arg-pNA (in 10% DMSO-containing buffer) was added to start the reaction. The reaction was monitored at 405nM for 30 minutes using a microtiter plate reader. Then, IC was determined from the maximum increment₅₀Test of the values of Calpain I and II

The inhibitory properties of the Calpain inhibitor were determined in a buffer containing 50mM tris-HClpH 7.5; 0.1M NaCl, 1mM dithiothreitol; 0.11mM CaCl₂And the fluorogenic Calpain matrix Suc-Leu-Tyr-AMC (dissolved in DMSO at 25mM, Bachem/Switzerland) (Sasaki et al J. biol. chem.1984, Vol.259, 12489-. Mu-calpain in humans follows Croall and Demartino (BBA1984, Vol.788, 348-.&Med.Lett.1995, Vol.5, 387-392) from erythrocytes. After several chromatographic steps (DEAE-Sepharose, phenyl-Sepharose, Superdex 200 and blue Sepharose), the enzyme obtained was < 95% pure according to SDS-PAGE, Western blot analysis and N-terminal sequencing. The fluorescence intensity of the cleavage product 7-amino-4-methylcoumarin (AMC) was monitored at λ ex-380 nm and λ em-460 nm in a Spex fluorologue fluorometer. The cleavage of the matrix was linear over the measurement range of 60min, the autocatalytic activity of Calpain was low when the experiment was performed at 12 ℃ (see Chatterjee et al, 1996, Bioorg.&Med. chem lett., vol.6, 1619-. The inhibitor and Calpain base were added to the experimental mixture as a DMSO solution, where DMSO should not exceed 2% in the final concentration.

In a typical experimental mixture, 10. mu.l of matrix (last 250 μm) and then 10. mu.l of μ -Calpain (2. mu.g/ml, last, i.e.18 nM) were added to a 1ml cuvette containing buffer. Calpain-mediated lysis of the matrix was measured for 15 to 20 minutes. Followed by addition of 10. mu.l of inhibitor (C)50 to 100 μ M DMSO solution) and further measure the inhibition of lysis for 40 minutes. The Ki value is determined according to the conventional reversible inhibition equation, which is: k: i (v)₀V) -1; wherein I is the inhibitor concentration, v₀Initial rate before inhibitor addition; v. of_iThe reaction rate in equilibrium. Platelet assay for determining the cellular activity of Calpain inhibitors

The progression of Calpain-mediated degradation of proteins in platelets is described in Zhao Liet et al, j.med.chem., 1993, 36, 3472-. Human platelets were isolated from fresh sodium citrate blood from donors and adjusted to 10 in buffer⁷Cells/ml (5mM Hepes, 140mM NaCl and 1mg/ml BSA, pH 7.3).

Platelets (0.1ml) were preincubated with 1 μ l of various concentrations of inhibitor (dissolved in DMSO) for 5 minutes. Calcium ionophore A23187 (1. mu.M in this assay) and calcium (5mM in this assay) were then added and incubated at 37 ℃ for an additional 5 minutes. After the centrifugation step, platelets were collected in SDS-Page sample buffer and heated at 95 ℃ for 5 minutes before separating the proteins into 8% concentration gels. The degradation of actin-binding protein (ABP) and talin, two proteins, which disappeared since the addition of calcium and ionophore, was monitored by quantitative densitometry, and a new color band appeared in the 200Kd molecular weight region. The half-maximum of the enzyme activity was thus determined. Glutamate-induced cell death of cortical neurons

This test followed glutamate neurotoxicity in cultures of cortical cells in accordance with Choi d.w., Maulucci-Gedde m.a., and Kriegstein A.R (1987). Neurosci.7, 357-368.

Individual cells were obtained by cutting two halves of the lower cortex from 15-day-old mouse embryos and then using an enzymatic (trypsin) method. These cells (glia und cortical neurons) were seeded into 24-well plates. Mitotic treatment with FDU (5-fluoro-2-desoxyuridine) was performed after three days (laminin coated plates) or seven days (ornithine coated plates). After 15 days of cell preparation, glutamate was added to induce cell death (15 min). After glutamate removal, Calpain inhibitor was added. After 24 hours, cell damage was determined by measuring Lactate Dehydrogenase (LDH) in the cell culture supernatant.

Calpain is also thought to play a role in apoptosis (M.K.T.Squier et al.J.cell.Physiol.1994, 159, 229-59237; T.Patel et al.Fasseb Journal 1996, 590, 587-597). Thus in another model in human cell lines, cell death is induced by calcium in the presence of calcium ionophores. Calpain inhibitors must enter the cell and inhibit Calpain there in order to prevent induced cell death. Calcium-mediated cell death in NT2 cells

Calcium in the presence of ionophore a23187 induced cell death in the human cell line NT 2. 10 hours before the experiment⁵Cells/well were placed into microtiter plates. After this interval, cells were incubated with various concentrations of inhibitor in the presence of 2.5. mu.M vehicle and 5mM calcium. 0.05ml of XTT (cell proliferation kit II, Boehringer Mannheim) was added to the reaction mixture after 5 hours. After about 17 hours, the optical density was determined in Easy Reader EAR400 of SLT according to the manufacturer's instructions. From two control samples without inhibitor, which were incubated in the absence and presence of ionophore, respectively, the optical density at which half of the cells died was calculated.

Ketone derivatives I are inhibitors of cysteine proteases, such as calpain I or II, and cathepsins B or L, and are therefore useful in the control of diseases associated with increased enzymatic activity of the calpain enzyme or cathepsins. The ketone derivatives I herein can accordingly be used for the treatment of neurodegenerative diseases occurring after ischemia, trauma and massive cerebral haemorrhage and neurodegenerative diseases such as multi-infarct dementia, alzheimer's disease and Huntington's disease, and further for the treatment of cardiac injury after cardiac ischemia, renal injury after renal ischemia, skeletal muscle injury, muscular dystrophy, injury caused by smooth muscle cell proliferation, coronary spasm, cerebral vasospasm, cataracts, and vascular restenosis after angioplasty.

Furthermore, the ketone derivatives I can be used for chemotherapy of tumors and their metastases, and for the treatment of diseases in which increased levels of cross-linked leukocidin-1 occur, such as inflammation and rheumatism-induced diseases.

In addition to conventional pharmaceutical adjuvants, the pharmaceutical preparations of the present invention comprise a therapeutically effective amount of compound I.

For topical application, such as a powder, ointment or spray, the active compound may be included in conventional concentrations. Generally, from 0.0001 to 1% by weight, preferably from 0.001 to 0.1% by weight, of the active compound is included.

In the case of internal administration, the preparation is administered in an individual dose. In a single dose, 0.1 to 100mg per kg body weight is administered. One or more doses per day are administered depending on the nature and severity of the disease.

The pharmaceutical preparations of the present invention contain, in addition to the effective compounds, conventional pharmaceutical excipients and auxiliaries, depending on the desired method of administration. For topical application, it is possible to use pharmaceutical auxiliaries, such as ethanol, isopropanol, ethoxylated castor oil, ethoxylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol stearate, ethoxylated fatty alcohols, liquid paraffin, petroleum jelly and lanolin. In the case of oral administration, for example, lactose, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone are suitable.

Antioxidants such as vitamin E and butylated hydroxyanisole and butylated hydroxytoluene, flavour enhancing additives, stabilisers, emulsifiers and lubricants may further be included.

The substances contained in the preparation in addition to the active compounds and used in the production of the pharmaceutical preparations should be toxicologically harmless and compatible with the respective active compounds. Pharmaceutical formulations are prepared in a conventional manner, for example by mixing the active compound with other conventional excipients and diluents.

Pharmaceutical formulations are prepared by methods familiar to those skilled in the art (see, e.g., h.sucker et al, Pharmazeutische technology, Thieme Verlag, Stuttgart, 1991).

The pharmaceutical preparation can be administered by various types of administration methods, for example, orally, parenterally such as intravenous infusion, subcutaneous injection, intraperitoneal injection, local injection. Formulations such as tablets, emulsions, infusion and injection solutions, pastes, salves, gels, ointments, lotions, powders and sprays are possible.

EXAMPLES example 14-methyl-2-oxo-3- (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) -amidopentanoic acid ethyl estera)1- (E-phenyl-1-acryloyl) piperidine-4-carboxylic acid

32.0 g (0.248 mol) of piperidine-4-carboxylic acid are dissolved in 500 ml of pyridine and reacted batchwise with 43.3 g (0.26 mol) of cinnamoyl chloride. The whole was left to stir at room temperature for 16 hours. The reaction mixture was then concentrated under reduced pressure and the resulting residue was partitioned between 2M hydrochloric acid and ethyl acetate. The organic phase was separated, dried and concentrated in vacuo to yield 47.0 g (76%) of product. M.p.: 178 ℃ and 179 ℃.

b) 4-methyl-2 (1- (E-3-phenyl-1-acryloyl) -piperidin-4-yl) amido-butyric acid methyl ester

20.0 g (77.1mmol) of the product in 1a and 12.5 g (77.1mmol) of L-valine methyl ester hydrochloride were added to 350 ml of methylene chloride, and 25.6 ml (185.1mmol) of triethylamine was added dropwise under cooling on ice. After stirring for one hour, 3.1 g (23.1mmol) of 1-hydroxy-1H-benzotriazole (HOBT) were added. The reaction mixture was cooled to 0 ℃ and then 14.8 g (77.1mmol) of N' - (3-dimethylaminopropyl) -N-Ethylcarbodiimide (EDC) were added in portions for reaction. The whole was left to stir at room temperature for 16 hours. The organic phase is then washed with water, aqueous sodium bicarbonate solution, 5% strength citric acid solution, then with water, dried and concentrated under reduced pressure. 27.3 g (96%) of product are obtained.

1H-NMR(CDCl₃): δ is 0.9(6H), 1.6-2.0(3H), 2.2(1H), 2.5(1H), 2.8(1H), 3.2(1H), 3.8(3H), 4.2(1H), 4.6(1H), 4.7(1H), 6.0(1H), 6.9(1H), 7.3-7.6(5H) and 7.6(1H) ppm.

c) 4-methyl-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amido-butyric acid

27.0 g (72.5mmol) of the product of 1c are dissolved in 200 ml of tetrahydrofuran and 3.5 g (145mmol) of lithium hydroxide dissolved in 250 ml of water are reacted, the whole being stirred at room temperature for 1 hour. The tetrahydrofuran is then removed under reduced pressure and the remaining aqueous solution is extracted with ethyl acetate, neutralized with 1M hydrochloric acid and extracted again with ethyl acetate. The latter organic phase is dried and concentrated under reduced pressure to yield 26 g (100%) of product.

1H-NMR (CDCl 3): δ is 1.0(6H), 1.6-2.2(6H), 2.5(1H), 2.9(1H), 3.2(1H), 4.6(2H), 6.4(1H), 6.9(1H), 7.3-7.6(5H) and 7.7(1H) ppm.

d) 4-methyl-2-oxo-3- (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidopentanoic acid ethyl ester

26.0 g (72.5mmol) of the product from 1c, 0.9 g (7.25mmol) of 4-Dimethylaminopyridine (DMAP) and 23.4 ml (0.29 mol) of pyridine are dissolved in 150 ml of anhydrous tetrahydrofuran, and then 16.2 ml (0.15 mol) of ethyloxalyl chloride are rapidly dropped to raise the temperature to 50 ℃ and the whole is refluxed for 3 hours. After the reaction mixture was stirred at room temperature for 16 hours, 100 ml of water was carefully added and the mixture was stirred for another 30 minutes, allowing it to partition between water and ethyl acetate. The organic phase is washed several times with water, dried and concentrated under reduced pressure.

The enol ester obtained is dissolved in 200 ml of ethanol, treated with 0.47 g (5.6mmol) of potassium ethoxide and stirred at room temperature for 16 hours, the whole is concentrated under reduced pressure, and the residue is purified by chromatography (effluent: dichloromethane/methanol ═ 20/1) to yield 10.8 g (36%) of product.

MS(FAB)：m/e＝414(M⁺). Example 2:

4-methyl-2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amido-pentanamide

a)2, 2-Ethylenedimercapto) -4-methyl-E-3- (1- (3-phenyl) -1-acryloyl) piperidin-4-yl) -amidopentanoic acid ethyl ester

6.0 g (14.6mmol) of the material obtained in 1d are dissolved with 1.5 ml (17.5mmol) of 1, 2-ethanedithiol in 20 ml of dry dichloromethane, and the mixture is reacted with 4 ml of boron trifluoride etherate. The whole was stirred at room temperature for 16 hours, diluted with 10 ml of dichloromethane and washed 3 times with saturated sodium chloride solution. The organic phase was dried and concentrated under reduced pressure to give 7.3 g of crude product, which was reacted further in an unpurified state.

b) 4-methyl-2-oxo-3- (E-1 (3-phenyl) -1-acryloyl) piperidin-4-yl) amidovaleramide

1.7 g (3.6mmol) of the product from 2a are added to 20 ml of a 2M ethanolaminum solution, the mixture is stirred at room temperature for 16 hours and the whole is concentrated under reduced pressure. The residue was purified by chromatography (effluent: dichloromethane/methanol-40/3) to yield 0.22 g of product.

MS(FAB)：m/e＝385(M⁺) Example 3

N-ethyl-4-methyl-2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidovaleramide

1.7 g (3.6mmol) of the product from 2a were reacted in ethanolaminoethane solution in a procedure similar to 2 b. 0.15 g of product can be obtained.

MS(FAB)：m/e＝413(M⁺) Example 4

4-methyl-N- (3- (morpholin-1-yl) propyl) -2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidovaleramide.

1.3 g (3.6mmol) of the product formed in 2a were reacted with 0.8 g (5.4mmol) of 3- (morpholin-1 yl) propylamine in a similar manner to 2b to give 1.1 g of the product.

MS(FAB)：m/e＝512(M⁺) Example 5:

4-methyl-2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) -amido-N- (2- (pyridin-2-yl) ethyl) pentanamide

Reaction of 1.3 g (2.8mmol) of the product from 2a with 0.7 g (5.5mol) of 2- (2-aminoethyl) pyridine was carried out in a similar manner to 2b to give 0.85 g of product.

MS(FAB)：m/e＝490(M⁺) Example 6

2-oxo-4-phenyl-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidobutyric acid ethyl ester.

a) Methyl 3-phenyl-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidopropionate.

The product was prepared from intermediate 1a and phenylalanine methyl ester in a similar procedure to 1 b.

1H-NMR(CDCl₃): δ is 1.6-2.0(3H), 2.35(1H), 2.9(1H), 3.0-3.3(4H), 3.7(3H), 4.1(1H), 4.6(1H), 4.9(1H), 5.9(1H), 6.9(1H), 7.1(2H) and 7.2-7.7(9H) ppm.

b) 3-phenyl-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amido-propionic acid.

The product was prepared from intermediate 6a in a similar procedure to 1 c.

1H-NMR(CDCl₃): δ is 1.4-2.0(4H), 2.3(1H), 2.8(1H), 3.0-3.4(3H), 4.0(1H), 4.6(1H), 4.9(1H), 6.2(1H), 6.8(1H), 7.0-7.8(11H) and about 8.2 (wide) ppm.

c) 2-oxo-4-phenyl-3 (1- (E-3 phenyl-1-acryloyl) piperidin-4-yl) amidobutyric acid ethyl ester.

The product was prepared from intermediate 6b in a similar procedure as 1 d.

MS(FAB)：m/e＝462(M⁺) Example 7

N- (3- (morpholin-1-yl) propyl) -2-oxo-4-phenyl-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidobutanamide.

The product was prepared from example 6 with 1- (3-aminopropyl) -morpholine in analogy to 2 b.

1H-NMR(CDCl₃): δ is 1.4-1.9(6H), 2.3-2.6(6H), 2.8(1H), 2.2(2H), 2.3-2.5(3H), 2.6-2.8(4H), 4.1(1H), 4.6(1H), 5.5(1H), 6.1(1H), 6.9(1H), 7.1(1H), 7.2-7.7(10H) and 8.9(1H) ppm. Example 8

2-oxo-4-phenyl-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidobutanamide

This product was prepared from example 6 and ethanolamino solution in a procedure similar to 2 b.

1H-NMR(D₆-DMSO): 1.2-1.9(4H), 2.4(1H), 2.7-2.9(2H), 3.0-3.2(2H), 4.1-4.3(3H), 5.1(1H) and 7.0-8.2(14H) ppm. Example 9

4-methyl-N (2- (morpholin-1-yl) ethyl) -2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidovaleramide

This product was prepared from intermediate 2a and 1- (2-aminoethyl) morpholine in analogy to 2 b. MS: 498 (M/e)⁺) Example 10

2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidopentanoic acid ethyl ester

a)3- (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidobutyric acid ethyl ester

This product was prepared from intermediate 1a and methyl 2-aminobutyrate in a similar procedure as 1 b.

1H-NMR(CDCl₃): δ is 0.9(3H), 1.6-2.0(6H), 2.5(1H), 2.9(1H), 3.2(1H), 3.8(3H), 4.2(1H), 4.5-4.7(2H), 6.3(1H), 6.9(1H), 7.4(3H), 7.6(2H) and 7.7(1H) ppm.

b)3- (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidobutyric acid

This product was prepared from intermediate l0a in analogy to 1 c.

1H-NMR(D₆-DMSO): δ is 0.9(3H), 1.3-1.9(6H), 2.6(1H), 2.7(1H), 3.1(1H), 4.1(1H), 4.3(1H), 4.5(1H), 7.2-7.6(5H), 7.7(2H), 8.0(1H) and 12.5 (width) ppm.

c) 2-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidopentanoic acid ethyl ester

This product was prepared from intermediate 10b in a similar procedure to 1 d.

1H-NMR(CDCl₃): δ is 0.9(3H), 1.4(3H), 1.8-2.2(6H), 2.5(1H), 2.8(1H), 3.2(1H), 4.2(1H), 4.4(2H), 4.6(1H), 5.1(1H), 6.7(1H), 6.9(1H), 7.4(3H), 7.5(2H) and 7.7(1H) ppm. Example 112-oxo-3 (1- (E-3-phenyl-1-acryloyl) piperidin-4-yl) amidovaleramide

This product was prepared from product 10 and ethanolamino solution in a similar procedure as 2a and b.

MS：m/e＝371(M⁺) Example 12

3- (1- (2-Naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenylbutanoic acid ethyl ester

a)1- (2-naphthylsulfonyl) piperidine-4-carboxylic acid

26.0 g (0.2 mol) of piperidine-4-carboxylic acid are dissolved in 250 ml of pyridine and the solution is treated with 47.6 g (0.2 mol) of 2-naphthylsulfonyl chloride in portions at room temperature. The whole was then stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was partitioned between ethyl acetate and 2M hydrochloric acid. This organic phase is dried and concentrated under reduced pressure. 48.5g (75%) of product are obtained.

b)3- (1- (2-Naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenylpropionic acid ethyl ester

This product was prepared from intermediate 12a in a similar procedure to 1 b.

1H-NMR(D₆-DMSO): δ ═ 1.1(3H), 1.4 to 1.8(5H), 2.3 to 2.6(2H), 2.7 to 3.2(3H), 3.5 to 3.8(2H), 4.0(2H), 4.5(1H), 7.2(4H), 7.7(3H), 8.1 to 8.3(3H), and 8.5(1H) ppm.

c)3(1- (2-naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenyl-propionic acid

This product was prepared from intermediate 12b in a similar procedure to 1 c.

1H-NMR(D₆-DMSO): δ ═ 1.3-1.8(5H), 2.3-2.6(3H), 2.8-3.2(2H), 3.4-3.8(2H), 4.4(1H), 7.2(4H), 7.7(3H), 8.0-8.3(4H) and 8.4(1H) ppm.

d) 3- (1- (2-Naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenylbutanoic acid ethyl ester

This product was prepared from intermediate 12c in a similar procedure to 1 d.

1H-NMR(D₆-DMSO): δ ═ 1.2(3H), 1.3-1.9(4H), 2.2(1H), 2.3-2.5(2H), 2.8(1H), 3.1(1H), 3.6(2H), 4.2(2H), 4.4(1H), 7.0-7.3(5H), 7.7(3H), 8.0-8.3(3H), and 8.4(2H) ppm. Example 13

3- (1- (2-naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenylbutanamide

This product was prepared from example 12 in analogy to 2a and b

MS(FAB)：m/e＝493(M⁺) Example 14

N- (3- (morpholin-1-yl) pop-1-yl) -3(1- (2-naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenylbutanamideThis product was prepared from product 12 and 1- (3-amino-propan-1-yl) morpholine, in analogy to 2a and b

MS(FAB)：m/e＝620(M⁺) Example 15

3- (1- (2-naphthylsulfonyl) piperidin-4-yl) amido-2-oxo-4-phenyl-N (2- (2-pyridyl) ethyl) butanamide

This product was prepared from example 12 and 2- (2-aminoethyl) -pyridine in analogy to 2a and b.

MS(FAB)：m/e＝598(M⁺) Example 16

3(S) - (1- (2-naphthoyl) piperidin-4-yl) amido-2-oxo-4-phenylbutanamide

a)3(S) - (N-tert-Boc-amino) -2(R, S) -hydroxy-4-phenylbutanamide

17.7 g (60mmol) of 3(S) - (N-tert-Boc-amino) -2(R, S) -hydroxy-4-phenylbutyric acid (S.L. Harenson et al, J.Med.chem.1994, 37,2918-29) and 8.1 g (60mmol) of 1-hydroxybenzo-triazole are dissolved in 150 ml of anhydrous dimethylformamide. 12.6 g (66mmol) of N' - (3-dimethylamino-propyl) -N-ethylcarbodiimide hydrochloride and 48 ml (about 2molar) of ethanolamino solution are added successively at-5 ℃ and the mixture is stirred at this temperature for 1 hour. Then stirred at room temperature for another 16 hours. After that, 500 ml of water was added, and the whole was extracted with ethyl acetate. The organic phase is washed with dilute sodium hydroxide solution and water, dried and concentrated under reduced pressure. The residue was further treated with n-heptane and the resulting precipitate was filtered off with a suction device. 13.5 g (76%) of product are finally obtained. 1H-NMR (D)₆-DMSO): δ is 1.3(9H), 2.6-2.9(2H), 3.7(1H), 5.7(1H), 6.2(1H) and 7.3(5H) ppm.

b)3(S) -amino-2 (R, S) -hydroxy-4-phenylbutanamide

13.4 g (46mmol) of the 17a compound are dissolved in 300 ml of dichloromethane and treated with 100 ml of trifluoroacetic acid. The whole was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was partitioned between water and diethyl ether, and the aqueous phase was concentrated under reduced pressure. 12.3 g (88%) of the product are finally obtained as trifluoroacetate.

c)2- (R, S) -hydroxy-3 (S) - (1- (2-naphthoyl) piperidin-4-yl) -amido-4-phenylbutanamide.

1.1 g (3.6mmol) of compound 17b are reacted with 1.0 g (3.6mmol) of 1- (2-naphthoyl) piperidine-4-carboxylic acid in analogy to 17 a. 1.0 g (61%) of product was obtained. 1H-NMR (D)₆-DMSO): δ ═ 1.2-1.9(6H), 2.6-3.2(4H), 3.6(1H), 3.7-4.0(1H), 4.2-4.6(2H), 5.8(1H) and 7.0-8.2(14H) ppm.

d)3(S) - (1- (2-naphthoyl) piperidin-4-yl) amido-2-oxo-4-phenyl-butyramide.

0.46 g (1mmol) of compound 17c and 0.4 g (4mmol) of triethylamine are dissolved in 10 ml of dimethyl sulfoxide and the solution is treated at room temperature with 0.48 g (3mmol) of sulfur trioxide-pyridine complex dissolved in 5ml of dimethyl sulfoxide. The whole was stirred for 16 hours. Then 150 liters of water were added and the precipitate was filtered with a suction device. 0.33 g (72%) of product was finally obtained. MS: m/e 457 (M)⁺)

The following examples of formula I can be prepared by the method mentioned in example 16.

Claims (16)

1. Piperidinone carboxylic acid derivatives of formula I:

or their tautomeric and isomeric forms, or physiologically acceptable salts, where the variable groups have the following meanings: r¹is-CO-R⁴，-SO₂-R⁴，-CONH-R⁴，COOR⁴，-C(＝N)-R⁴，-C(＝O)-NHR⁴and-C (═ S) -NHR⁴；

R²is-C₁-C₆Alkyl, in branched or unbranched form, which may additionally bear a phenyl, pyridine or naphthalene ring, which may then be substituted by up to two R⁵Radical substitution, R⁵C which may be branched or unbranched₁-C₄-alkyl, -O-C₁-C₄Alkyl, OH, Cl, F, Br, I, CF₃、NO₂、NH₂、CN、COOH、-COO-C₁-C₄-alkyl, -NHCO-C₁-C₄-alkyl, -NHCOPh, -NHSO₂-C₁-C₄Alkyl, NHSO₂-Ph、-SO₂-C₁-C₄-alkyl and-SO₂Ph；R³is-OR⁶or-NHR⁶；R⁴Is branched or unbranched-C₁-C₆Alkyl, the chain of two or more carbon atoms may also contain double or triple bonds, or be substituted by one or two rings, for example phenyl, naphthalene, quinoxaline, quinoline, isoquinoline, pyridine, thiophene, benzothiophene, benzofuran, pyrimidine, thiazole, isothiazole, triazole, imidazole, cyclohexyl, cyclopentyl, fluorene, indole, benzimidazole, oxazole, isoxazole and furan, each ring itself may additionally carry up to two R⁵A group; r⁶Is a hydrogen atom, one of which may additionally carry one to two R⁵The phenyl ring of the group, branched or unbranched, C which may contain a double or triple bond and a ring such as phenyl, naphthalene, pyridine, pyrimidine, piperidine, pyrrolidine, morpholine, thiophene, quinoline and isoquinoline₁-C₆Alkyl, the aromatic ring may additionally carry up to two radicals-NR⁷R⁸Or R⁵，R⁷And R⁸May be independently of one another a hydrogen atom or a branched or unbranched C₁-C₆-an alkyl group.

2. Piperidinone carboxylic acid derivatives of formula I in claim 1, wherein R¹is-C (═ O) R⁴，-SO₂R⁴，R²Is branched or unbranched C₁-C₆-alkyl, -CH₂-Ph or-CH₂-pyridinyl R³is-OR⁶or-NHR⁶，R⁴，R⁵，R⁶The meaning of (a) is indicated in claim 1.

3. Piperidinone carboxylic acid derivatives of formula I in claim 1, wherein R¹is-C (═ O) R⁴，-SO₂R⁴，R²is-C₁-C₄-alkyl or-CH₂-Ph，R³is-NHR⁶，R⁴is-CH ═ CH-R⁹，R⁹Possibly phenyl, naphthalene or quinoline, R⁶Is a hydrogen atom or C which may be substituted by phenyl, pyridine or morpholine₁-C₄-an alkyl group.

4. Use of piperidinecarboxylic acid derivatives of the formula I as claimed in claims 1 to 3 for controlling diseases.

5. Use of a piperidinone carboxylic acid derivative of the formula I in claims 1 to 3 as a cysteine protease inhibitor.

6. Use according to claim 5 as inhibitor of the cysteine proteases Calpain, cathepsin B and cathepsin L.

7. Use of a piperidinecarboxylic acid derivative of formula I as claimed in claims 1 to 3 for the manufacture of a medicament for the treatment of diseases in which the activity of Calpain is increased.

8. Use of a piperidinecarboxylic acid derivative of formula I as in claims 1-3 for the manufacture of a medicament for the treatment of neurodegenerative diseases and nerve injury.

9. The use according to claim 8 for the treatment of neurodegenerative diseases and neurological damage caused by ischemia, trauma and massive cerebral haemorrhage.

10. Use according to claim 9 for the treatment of stroke and craniocerebral trauma.

11. The use according to claim 8 for the treatment of alzheimer's disease and Huntington's disease.

12. Use of a piperidinecarboxylic acid derivative of formula I in claims 1-3 for the manufacture of a medicament for the treatment of cardiac injury after cardiac ischemia, renal injury after renal ischemia, skeletal muscle injury, muscular dystrophy, injury due to smooth muscle cell proliferation, coronary artery spasm, cerebral artery spasm, cataracts, and vascular restenosis after angioplasty.

13. Use of a piperidinecarboxylic acid derivative of formula I as claimed in claims 1 to 3 for the manufacture of a medicament for the treatment of tumors and their metastases.

14. Use of a piperidinecarboxylic acid derivative of formula I in claim 1 for the manufacture of a medicament for the treatment of diseases in which increased levels of cross-linked leukocidin-1 occur.

15. The use according to claim 14 for the treatment of inflammatory and rheumatic diseases.

16. A pharmaceutical preparation for oral, parenteral or intraperitoneal injection, which comprises, in addition to conventional pharmaceutical auxiliaries, per unit dose at least one piperidinecarboxylic acid derivative of the formula I as claimed in claims 1 to 3.