EP1073631A1

EP1073631A1 - New substituted amides, their production and their use

Info

Publication number: EP1073631A1
Application number: EP99917996A
Authority: EP
Inventors: Wilfried Lubisch; Achim Möller; Hans-Jörg Treiber; Monika Knopp
Original assignee: BASF SE
Current assignee: Abbott GmbH and Co KG
Priority date: 1998-04-20
Filing date: 1999-04-19
Publication date: 2001-02-07
Also published as: KR20010042836A; HUP0101624A3; NO20005266L; CN1297432A; NO20005266D0; ID26402A; US6448254B1; JP2002512221A; IL138412A0; HRP20000776A2; BR9909775A; AU3607199A; TR200003028T2; HUP0101624A2; DE19818614A1; BG104855A; AU753402B2; CA2328435A1; WO1999054294A1; ZA200006711B

Abstract

The invention relates to amides of general formula (I) and their tautomeric and isomeric forms, their possible enantiomeric and diastereomeric forms and possible physiologically compatible salts, where the variables have the following meanings: R1 is C1-C6 alkyl, phenyl, naphthyl, quinolyl, pyridyl, pyrimidyl, pyridazyl, quinazolyl and quinoxalyl, whereby the rings can still be substituted with up to 2 R4 rests; R2 is -(CH2)m-R8, where R8 is phenyl, cyclohexyl or indolyl and m is 1 to 6; X is a bond, -CH2-, -CH2CH2-, -CH=CH-, -C C-, -CONH-, -, -SO2NH-, and R1-X together can also be formula (a), R3 is hydrogen and CO-NR6R7; R4 is hydrogen, branched or unbranched C1-C4 alkyl and O-C1-C4-alkyl; R5 is hydrogen, branched or unbranched C1-C4 alkyl and O-C1-C4-alkyl; R6 is hydrogen, branched or unbranched C1-C6 alkyl; R7 is hydrogen, branched or unbranched C1-C6 alkyl; and n is 0, 1 or 2. The amides of formula (I) are inhibitors of enzymes, especially cysteine proteases such as calpains (calcium dependent cysteine proteases) and their isoenzymes and cathepsins, such as B and L.

Description

New substituted amides, their production and application

description

The present invention relates to novel amides, which are inhibitors of enzymes, especially cysteine proteases such as Cal ^¬ pain (= calcium dependent cysteine proteases) and its isoenzymes and cathepsins, for example B and L represent.

Calpains are intracellular, proteolytic enzymes from the group of so-called cysteine proteases and are found in many cells. Calpains are activated by increased calcium concentration, with a distinction between Calpain I or μ-calpain, which is activated by μ-molar concentrations of calcium ions, and Calpain II or m-calpain, which is activated by m-molar concentrations of calcium ions , (P.Johnson, Int. J.Biochem. 1990, 22 (8), 811-22). Today, further calpain isoenzymes are postulated (K.Suzuki et al., Biol. Chem. Hoppe-Seyler, 1995, 376 (9), 523-9).

It is believed that calpains play an important role in various physiological processes. These include cleavages of regulatory proteins such as protein kinase C, cytoskeleton proteins such as MAP 2 and spectrin, muscle proteins, protein degradation in rheumatoid arthritis, proteins when platelets are activated, neuropeptide metabolism, proteins in mitosis and others, the in. MJBarrett et al. , Life Be. 1991, 48, 1659-69 and K.K. ang et al. , Trends in Pharmacol. Be . , 1994, 15, 412-9.

Elevated calpain levels were measured in various pathophysiological processes, for example: ischemia of the heart (e.g. heart attack), the kidney or the central nervous system (e.g. "stroke"), inflammation, muscular dystrophies, eye cataracts, injuries to the central nervous system (e.g. rauma), Alzheimer's disease, etc. (see KK ang, above). It is suspected that these diseases are associated with increased and persistent intracellular calcium levels. As a result, calcium-dependent processes are overactivated and are no longer subject to physiological regulation. Accordingly, overactivation of calpains can also trigger pathophysiological processes.

Therefore, it has been postulated that inhibitors of calpain enzymes can be useful in the treatment of these diseases. Various studies confirm this. For example, Seung-Chyul Hong et al., Stroke 1994, 25 (3), 663-9 and RTBartus et al. , Neurologist - 2 cal res. 1995, 17, 249-58 demonstrated a neuroprotective effect of calpain inhibitors in acute neurodegenerative disorders or ischemia, such as occur after a stroke. Calpain inhibitors also improved the recovery of the memory deficits and neuromotor disorders that occurred after experimental brain trauma (KESaatman et al. Proc. Na tl. A - cad. Sci. USA, 1996, 53,3428-3433). C. Edelstein et al. , Proc. Natl. Acad. Sci. USA, 1995, 92, 7662-6, found a protective effect of calpain inhibitors on kidneys damaged by hypoxia. Yoshida, Ken Ischi et al. , Jap. Circ. J. 1995, 59 (1), 40-8, were able to show beneficial effects of calpain inhibitors after cardiac damage caused by ischemia or reperfusion. Since calpain inhibitors inhibit the release of the β-AP4 protein, a potential use as a therapeutic agent for Alzheimer's disease has been proposed (J.Higaki et al., Neuron, 1995, 14, 651-59). The release of interleukin-lα is also inhibited by calpain inhibitors (N. Watanabe et al., Cytokine 1994, 6 (6), 597-601). Furthermore, it has been found that calpain inhibitors show cytotoxic effects on tumor cells (0 E. Shhiba et al. 20th Meeting Int. Ass. Ereast Cancer Res., Sendai Jp, 1994, September 25-28, Int. J. Oncol 5 (Suppl.), 1994, 381).

Further possible uses of calpain inhibitors are in K.K. Wang, Trends in Pharmacol. Be . , 1994, 15, 412-8. 5

Calpain inhibitors have already been described in the literature. However, these are predominantly either irreversible or peptide inhibitors. Irreversible inhibitors are usually alkylating substances and have the disadvantage that they are in the

30 organism react unselectively or are unstable. So these inhibitors often show undesirable side effects, such as toxicity, and are then restricted in use or unusable. The irreversible inhibitors include, for example, the epoxides E 64 (E.B. McGowan et al., Bio formerly Biophys. Res. Commun.

35 1989, 158, 432-5), α-haloketones (H. Angliker et al.,

J.Med. Chem. 1992, 35, 216-20) or disulfides (R. Matsueda et al., Chem. Let t. 1990, 191-194).

Many known reversible inhibitors of cysteine proteases such as 40 calpain are peptidic aldehydes, in particular dipeptide and tripepidic aldehydes such as, for example, Z-Val-Phe-H (MDL 28170) (S.Mehdi, Trends in Biol. Sei. 1991, 16, 150-3). Under physiological conditions, peptidic aldehydes have the disadvantage that they are often unstable due to their high reactivity, can be metabolized quickly and lead to unspecific 3 see reactions that can be the cause of toxic effects (JA-Fehrentz and B. Castro, Synthesis 1983, 676-78.

JP 08183771 (CA 1996, 605307) and EP 520336 have described aldehydes which are derived from 4-piperidinoylamides and 1-carbonyl-piperidino-4-yl amides as calpain inhibitors. However, the aldehydes claimed here, which are derived from heteroaromatically substituted amides of the general structure I, have so far been described. Other aldehyde derivatives are in Chatterjee ez al. Bioorganic & Medicinal Chemistry Letters 1997, 7, 287-290, Bioorganic & Medicinal Chemistry Letters 1996, 6, 1619-1622, WO 97/10231 and WO 97/21690.

Peptidic ketone derivatives are also inhibitors of cysteine proteases, especially calpains. For example, in the case of serine proteases, ketone derivatives are known as inhibitors, the keto group being activated by an electron-withdrawing group such as CF3. In the case of cysteine proteases, derivatives with ketones activated by CF3 or similar groups have little or no activity (MRAngelas ro et al., J.Med. Chem. 1990, 33, 11-13). Surprisingly ^¬ so far, only ketone derivatives in which α-leaving groups cause irreversible inhibition and on the other hand a carboxylic acid derivative activates the keto group have been found as effective inhibitors in Calpain (see MRAngelastro et al., See above; WO 92/11850; WO 92,12140; WO 94/00095 and WO 95/00535). However, of these ketoamides and keto esters, almost only peptide derivatives have been described as effective (Zhaozhao Li et al., J.Med. Chem. 1993, 36, 3472-80; SLHarbenson et al., J. Med. Chem. 1994, 37 , 2918-29 and see above MRAngelastro et al.). Only in Chatterjee et al.

(see above) a xanthene derivative of a ketobenzamide has been described as a cal pain inhibitor.

Ketobenzamides are already known in the literature. The ketoester PhCO-Abu-COOCH ₂ CH _{3 has been described} in WO 91/09801, WO 94/00095 and 92/11850. The analogous phenyl derivative Ph-CONH-CH (CH ₂ Ph) -CO-COCOOCH ₃ was described in MRAngelastro et al. , J. Med. Chem. 1990, 33, 11-13 as a weak calpain inhibitor, however. This derivative is also in JPBurkhardt, Tetra - hedron Let t. , 1988, 3433-36. However, the importance of substituted benzamides has never been investigated.

In a number of therapies such as stroke, the active ingredients are administered intravenously as a solution for infusion. For this purpose it is necessary to have available substances, here calpain inhibitors, which have sufficient water solubility so that an infusion solution can be prepared. Many of the described 4

However, calpain inhibitors have the disadvantage that they wrestle only ge ^¬ or show water solubility and thus is ineligible for intravenous application in question. Such active substances can only be applied with auxiliaries which are intended to impart water solubility (cf. RT Bartus et al. J Cereb. Blood Flow Metab. 1994, 14, 537-544). However, these additives, for example polyethylene glycol, often have side effects or are even incompatible. A non-peptide calpain inhibitor, which is therefore water-soluble without auxiliaries and therefore probably more tolerable, can be used to great advantage. Well-effective non-peptide calpain inhibitors with sufficient water solubility have not been described so far and would therefore be new.

Non-peptidic aldehydes, ketocarboxylic acid esters and ketoamide derivatives have been described in the present invention. These compounds are novel and surprisingly show the possible ^¬ ness on, by incorporation of rigid structural fragments po ^¬ tent non-peptide inhibitors of cysteine proteases such as calpain to. Furthermore, salt bonds with acids are possible in the present compounds of the general formula I, which all carry at least one aliphatic amine radical. This leads to an improved solubility in water and thus the compounds show the desired profile for intravenous application, as is required, for example, in stroke therapy.

The present invention relates to substituted amides of the general formula I

R ²

HOOC _χ f I

R ⁱ -X - ^^ ^H °

and their tautomeric and isomeric forms, possible enantiomeric and diastereomeric forms, as well as possible physiologically acceptable salts, in which the variables have the following meaning:

Rl can be -C ₆ alkyl, phenyl, naphthyl, quinolyl, pyridyl, pyrimidyl, pyridazyl, quinazolyl and quinoxalyl, where the rings can also be substituted with up to 2 radicals R4, and

R2 - (CH ₂ ) _m - R8, where R8 can be phenyl, cyclohexyl or indolyl and m = 1 to 6, and X is a bond, -CH ₂ -, -CH ₂ CH ₂ -, -CH = CH ~, -C≡C-, -CONH-, S0 ₂ NH- and mean and

Rl-X together too

and mean and

R3 means hydrogen and CO-NR6R7,

R4 is hydrogen, Cl-C4-alkyl, branched or unbranched and 0-Cl-C4-alkyl;

R5 is hydrogen, C1-C4-alkyl, branched or unbranched and - 0-C1-C4-alkyl;

R6 is hydrogen, -CC ₆ alkyl, branched and unbranched, means, and

R7 is hydrogen, Ci-C _δ -Al yl, branched or unbranched, means, and

n means a number 0, 1 or 2.

The compounds of the formula I can be used as racemates, as enantiomerically pure compounds or as diastereomers. If enantiomerically pure compounds are desired, these can be obtained, for example, by carrying out a classical resolution with the compounds of the formula I or their intermediates using a suitable optically active base or acid. On the other hand, the enantiomeric compounds can also be produced by using commercially available compounds, for example optically active amino acids such as phenylalanine, tryptophan and tyrosine. The invention also relates to compounds of the formula I which are mesomeric or tautomeric, for example those in which the aldehyde or keto group of the formula I is present as an enol tautomer.

The invention further relates to the physiologically tolerable salts of the compounds I, which can be obtained by reacting compounds I with a suitable acid or base. sen. Suitable acids and bases are listed, for example, in Progress in Pharmaceutical Research, 1966, Birkhäuser Verlag, Vol. 10, pp. 224-285. These include, for example, hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid etc. or sodium hydroxide, lithium hydroxide. Potassium hydroxide and tris.

The amides I according to the invention which carry an aldehyde group can be prepared in various ways, which was outlined in synthesis scheme 1.

Synthesis scheme 1

R2 R2 'OOC \

R'OOC

-COOH + OH OH

/ H ₂ N

R '- X """/ _H , _A ^C0NH

IV

1. Hydrolysis

2. Oxidation

2 2

R'OOC

P-HN 'COOH V V-CONH "' ^{^} CHO

R '- X

1. NH (CH ₃ ) OH 2. Deprotect

1. Hydrolysis

2. Reduction

R ²

R'OOC

H ₂ N CON (CH ₃ ) OH CON (CH ₃ ) OH

R ¹ - X-

VI Reduction

Vll

2 2 RΌOC \

-► <Λ \ V- CONH- "^' CO-Y

CO-Y

VIII IX

Carboxylic acids II are linked with suitable amino alcohols III to give the corresponding amides IV. Common peptide coupling methods are used, either in CRLarock, 7

Comprehensive Organic Transformat: ons, VCH Publisher, 1989, page 972f. or in Houben-Weyl, Methods of Organic Chemistry, 4th ed., E5, Kap.V. It is preferred to work with ^• "activated" acid derivatives of II, the acid group COOH being converted into a group COL. L represents a leaving group such as Cl, imidazole and N-hydroxybenzotriazole. This activated acid is then reacted with amines to give the amides IV. The reaction takes place in anhydrous, inert Lö ^¬ sungsmitteln such as methylene chloride, tetrahydrofuran and dimethylformamide at temperatures from -20 to + 250C.

This Carbonsäureester IV (R '= O-alkyl) are reacted with acids, we ren trifluoroacetic acid or hydrochloric acid or bases such as lithium hydroxide, sodium hydroxide or potassium hydroxide in aqueous medium or in mixtures of water and organic solvents such as alcohols or tetrahydrofuran at room temperature or elevated tempera ^¬ as 25-100 ° C, converted into acids IV (R '= H).

These derivatives IV (R '= H) obtained can be oxidized to the aldehyde derivatives I according to the invention. For this you can ver ^¬ various customary oxidation reactions (see CRLarock, Comprehensive Organic Transformations, VCH Publishers, 1989, page 604 f.), Such as Swern and Swern-analogous oxidations (TT Tidwell, Synthesis 1990, 857-70), sodium hypochlorite / TEMPO (S.L. Harenson et al., See above) or Dess-Martin (J.Org.Chem. 1983, 48, 4155). It is preferred to work here in inert aprotic solvents such as dimethylformamide, tetrahydrofuran or methylene chloride with oxidizing agents such as DMSO / py x S0 ₃ or DMSO / oxalyl chloride at temperatures from -50 to + 250C, depending on the method (see literature above).

Alternatively, the carboxylic acid II can be reacted with aminohydroxamic acid derivatives VI to give benzamides VII. The same reaction procedure is used as for the preparation of IV. The hydroxam derivatives VI can be obtained from the protected amino acids V by conversion with a hydroxylamine. An amide production process already described is also used here. The Abspal ^¬ processing of the protecting group P, for example Boc, takes place in a customary manner, for example with trifluoroacetic acid. The amide hydroxamic acids VII thus obtained can be converted into the aldehydes I according to the invention by reduction. For example, lithium aluminum hydride is used as a reducing agent at temperatures from -60 to 00C in inert solvents such as tetrahydrofuran or ether. 8th

Analogously to the last process can also prepare carboxylic acids or säu ^¬ re-derivatives such as esters IX (Y = COOR ', COSR'), which can likewise be converted by reduction into the aldehydes I according to the invention •. These methods are listed in RCLarock, 5 Comprehensive Organic Transformations, VCH Publisher, 1989, pages 619-26.

The substituted amides I according to the invention, a Ketoamid- or keto group carry, can on Various - done 0 nen paths that in the synthesis scheme 2 WUR outlined ^¬.

Synthesis scheme 2

5

R'OOC

^• CONH cox

R '-

XI 0

(X = R?) 5 1. Hydrolysis

2. Oxidation

30

R ²

R ² 1. Hydrolysis R'OOC

R'OOC 2. Oxidation CONH

R ¹ - R ³ O

R ¹ - X OH

35 XIII

The carboxylic acids II are mixed with aminohydroxycarboxylic acid derivatives X (preparation of XI see SLHarbenson et al., J.Med. Chem. 1994, 37, 2918-29 or JP Burkhardt et al. Tetrahedron Let t. 40 1988, 29, 3433- 3436) under customary peptide coupling methods (see above, Houben-Weyl), whereby amides XIII are obtained.

These carboxylic acid esters XIII (R '= O-alkyl) with acids such as trifluoroacetic acid or hydrochloric acid or bases such as lithium hydroxide, 45 sodium hydroxide or potassium hydroxide in an aqueous medium or in mixtures of water and organic solvents such as alcohols 9 or tetrahydrofuran at room temperature or elevated temperatures, such as 25-100 ° C, converted into the acids XIII (R '= H).

The derivatives XIII obtained can be oxidized to the ketocarboxylic acid derivatives I 'according to the invention. Various customary oxidation reactions (see CRLarock, Comprehensive Organi c Transformations, VCH Publisher, 1989, page 604 f.) Can be used for this, such as Swern- and Swern-analogous oxidations, preferably dimethyl sulfoxide / pyridine-sulfur trioxide complex in solvents such as Methylene chloride or tetrahydrofuran, optionally with the addition of dimethyl sulfoxide, at room temperature or temperatures from -50 to 25 ° C, (TTTidwell, Synthesis 1990, 857-70) or sodium hypochlorite / TEMPO (S. L. Harbenson et al., See above) , to use.

If XI are α-hydroxy esters (X = O-alkyl), these can be hydrolyzed to carboxylic acids XII, the procedure being analogous to the above methods, but preferably with lithium hydroxide in water / tetrahydrofuran mixtures at room temperature, but note that that in this case a radical is selected for the protective group R ', for example tert-butyl-O, which allows the selective cleavage of one of the two ester groups. Other amides XIII are prepared by reaction with amines under coupling conditions already described. The alcohol derivative XIII can be oxidized again to ketocarboxylic acid derivatives I 'according to the invention.

The preparation of the carboxylic acid esters II have in some cases already been described or are carried out in accordance with customary chemical methods.

Compounds in which X is a bond are produced by conventional aromatic coupling, for example Suzuki coupling with boric acid derivatives and halides with palladium catalysis or copper-catalytic coupling of aromatic halides. The alkyl-bridged residues (X = - (CH ₂ ) _m -) can be prepared by reducing the analog ketones or by alkylating the organolithium, for example ortho-phenyloxazolidines, or other organometallic compounds (cf. IMDordor, et al., J.Chem Soc. Perkin Trans. I, 1984, 1247-52).

Alkene-bridged and alkyne-bridged compounds are prepared, for example, by Heck reaction from aromatic halides and corresponding alkenes and alkynes (cf. I. Sakamoto et al., Chem. Pharm.Bull., 1986, 34, 2754-59). 10

Amides and sulfonamides are prepared analogously to the methods described above from the amines and acid derivatives.

Alternatively, compounds of general formula I can also be synthesized by changing or swapping the reaction sequences listed in Schemes 1 and 2. For example, a sulfonamide I (R1X = RS0 ₂ NH) can be produced from a derivative IV (R1X = N0 ₂ ) by the nitro group being usually catalytically with hydrogen on a catalyst, such as palladium / carbon, to the amine reduced and then the amine obtained is reacted with a sulfonic acid chloride to give a derivative IV (R1X = RS0 ₂ NH). The further reaction to I takes place, as shown in the scheme, by ester hydrolysis and oxidation. Analogously, the intermediates IV and XI (R1X = chemical groups such as nitro, amino, halogen, etc.) can be converted into derivatives in which R1X corresponds to further radicals listed in the general claim. The reactions are carried out analogously to the methods described above or analogously to general or customary methods.

The heterocyclically substituted amides I contained in the present invention are inhibitors of cysteine proteases, in particular cysteine proteases such as calpains I and II and cathepsins B and L.

The inhibitory effect of the heterocyclically substituted amides I was determined using enzyme tests customary in the literature, a concentration of the inhibitor at which 50% of the enzyme activity is inhibited being determined as the yardstick (= IC50). The amides I were measured in this way for the inhibitory action of calpain I, calpain II and cathepsin B.

Cathepsin B test

Cathepsin B inhibition was carried out analogously to a method by S.Hasnain et al., J. Biol. Chem. 1993, 268, 235-40.

2μL of an inhibitor solution, prepared from inhibitor and DMSO (final concentrations: 100μM to 0.01μM), are added to 88μL cathepsin B (cathepsin B from human liver (Calbiochem), diluted to 5 units in 500μM buffer). This mixture is preincubated for 60 minutes at room temperature (25 ° C.) and the reaction is then started by adding 10 μl 10 mm Z-Arg-Arg-pNA (in buffer with 10% DMSO). The reaction is monitored for 30 minutes at 405nM in a microplate reader. The IC50's are then determined from the maximum gradients. 11

Calpain I and II test

The inhibitory properties of calpain inhibitors are tested in buffer with 50 mM Tris-HCl, pH 7.5; 0.1 M NaCl; 1mM dithiotreithol; 0.11 mM Ca Cl, the fluorogenic calpain substrate Suc-Leu-Tyr-AMC (25 mM dissolved in DMSO, Bachem / Switzerland) being used. Human μ-calpain is isolated from erythrocytes and after several chromatographic steps (DEAE-Sepharose, Phenyl-Sepharose, Superdex 200 and Blue-Sepharose), enzyme with a purity> 95% is obtained, assessed according to SDS-PAGE, Western blot analysis and N-terminal sequencing. The fluorescence of the cleavage product 7-amino-4-methylcoumarin (AMC) is monitored in a Spex-Fluorolog fluorimeter at λ _ex = 380 nm and λem = 460 nm. In a measuring range of 60 min. the cleavage of the substrate is linear and the autocatalytic activity of calpain is low if the tests are carried out at temperatures of 12 ° C. The inhibitors and the calpain substrate are added to the test batch as DMSO solutions, the final concentration of DMSO not exceeding 2%.

In a test batch, 10 μl substrate (250μM final) and then 10 μl of μ-calpain (2μg / ml final, i.e. 18 nM) are added to a 1 ml cuvette containing buffer. The calpain-mediated cleavage of the substrate is carried out for 15-20 min. measured. Then add 10 μl inhibitor (50 - 100 μM solution in DMSO) and measure the inhibition of the cleavage for a further 40 min.

Ki values are determined using the classic equation for reversible inhibition:

Ki = I / (vo / vj _. ) - 1; where 1 = inhibitor concentration, v ₀ = initial rate before adding the inhibitor; Vi = reaction speed in equilibrium.

The speed is calculated from v = AMC release / time i.e. Altitude / time.

Calpain is an intracellular cysteine protease. Calpain inhibitors must pass through the cell membrane to prevent the breakdown of intracellular proteins by calpain. Some known calpain inhibitors, such as E 64 and leupeptin, only poorly cross the cell membranes and accordingly, although they are good calpain inhibitors, show only poor activity on cells. The aim is to find compounds with better membrane compatibility. We use human platelets as evidence of the passage of calpain inhibitors into the membrane. 12

Calpain-mediated degradation of the tyrosine kinase ppδOsrc in platelets

After platelet activation, the tyrosine kinase ppδOsrc is cleaved by calpain. This was done by Oda et al. in J. Biol. Chem., 1993, 268, 12603-12608. It was shown that the cleavage of ppδOsrc by calpeptin, an inhibitor of calpain, can be prevented. Based on this publication, the cellular effectiveness of our substances was tested. Fresh human blood with citrate was mixed for 15 min. Centrifuged at 200g. The platelet-rich plasma was pooled and diluted 1: 1 with platelet buffer (platelet buffer: 68 mM NaCl, 2.7 mM KC1, 0.5 M MgCl ₂ × 6 H ₂ O, 0.24 mM NaH ₂ PO ₄ × H ₂ 0, 12 mM NaHC0 ₃ , 5, 6 mM glucose, 1 mM EDTA, pH 7.4). After a centrifugation and washing step with platelet buffer, the platelets were adjusted to 107 cells / ml. The human platelets were isolated at RT.

In the test mixture, isolated platelets (2 x 106) with different concentrations of inhibitors (dissolved in DMSO) for 5 min. pre-incubated at 37 ° C. The platelets were then activated with 1 μM Ionophore A23187 and 5 mM CaCl ₂ . After 5 min. Incubation, the platelets were yaws briefly at 13,000 rpm zentrifu ^¬ and the pellet in SDS sample buffer was added (SDS sample buffer: 20 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0. 5 mM PMSF, 5 μg / ml leupeptin, 10 μg / ml pepstatin, 10% glycerin and 1% SDS). The proteins were separated in a 12% gel and pp60src and its 52-kDa and 47-kDa cleavage products were identified by Western blotting. The polyclonal rabbit antibody Anti-Cys-src (pp60src) used was purchased from Biomol Fein-Chemicals (Hamburg). This primary antibody was detected using an HRP-coupled second goat antibody (Boehringer Mannheim, FRG). The Western blotting was carried out according to known methods. The cleavage of pp60src was quantified densitometrically, using non-activated controls (control 1: no cleavage) and plates treated with ionophore and calcium (control 2: corresponds to 100% cleavage). The ED ₅₀ value corresponds to the concentration of inhibitor at which the intensity of the color reaction is reduced by 50%.

Glutamate-induced cell death on cortical neurons

As with Choi DW, Maulucci-Gedde MA and Kriegstein AR, the test was "Glutamate neurotoxicity in cortical cell eulture". J. Neurosci. 1989, 7, 357-368. 13

The cortex halves were prepared from 15-day-old mouse embryos and the individual cells were obtained enzymatically (trypsin). These cells (glia and cortical neurons) are sown in 24 well plates. After three days (laminin coated plates) or 5 seven days (ornithine coated plates) with FDU

(5-fluoro-2-deoxyuridine) carried out the mitosis treatment. 15 days after the cell preparation is done by adding glutamate

(15 minutes) cell death triggered. After the glutamate removal, the calpain inhibitors are added. 24 hours later, 10 is determined by determining the lactate dehydrogenase (LDH) in the cell culture - the cell damage survived.

It is postulated that calpain also plays a role in apoptotic cell death (M.K.T.Squier et al. J. Cell. Physiol. 1994, 159,

15 229-237; T. Patel et al. Faseb Journal 1996, 590, 587-597). Therefore, in another model, cell death was triggered in a human cell line with calcium in the presence of a calcium ionophore. Calpain inhibitors must enter the cell and inhibit calpain there to prevent cell death.

20th

Calcium-mediated cell death in NT2 cells

In the human NT2 cell line, cell death can be triggered by calcium in the presence of the ionophore A 23187. 105 cells / well

25 den plated in microtiter plates 20 hours before the experiment. After this period, the cells were incubated with various concentrations of inhibitors in the presence of 2.5 μM ionophore and 5 mM calcium. After 5 hours, the reaction mixture was mixed with 0.05 ml of XTT (Cell Proliferation Kit II, Boehringer Mannnheim)

30 added. The optical density is determined approximately 17 hours later, according to the manufacturer's instructions, in the Easy Reader EAR 400 from SLT. The optical density at which half of the cells have died is calculated from the two controls with cells without inhibitors that are absent and

35 presence of ionophore were incubated.

A number of neurological diseases or mental disorders result in increased glutamate activity, which leads to states of overexcitation or toxic effects in the central nervous system.

40 lead system (CNS). Glutamate mediates its effects via various receptors. Two of these receptors are classified according to the specific agonists NMDA receptor and AMPA receptor. Antagonists against these effects mediated by glutamate can thus be used to treat these diseases,

45 in particular for therapeutic use against neurodegenerative diseases such as Huntington's and Parkinson's disease, neurotoxic disorders after hypoxia, anoxia, ischemia and 14 after lesions as they occur after stroke and trauma, or also as anti-epileptics (see Medicinal Research 1990, 40, 511-514; TIPS, 1990, 11, 334-338; Drugs of the Fu ture 1989, 14-, 1059-1071).

Protection against cerebral overexcitation by excitatory amino acids (NMDA or AMPA antagonism in the mouse)

The intracerebral application of excitatory amino acids EAA (Excitatory Amino Acids) induces such a massive overexcitation that it leads to cramps and the death of the animals (mouse) in a short time. These symptoms can be inhibited by systemic, for example intraperitoneal, administration of centrally active substances (EAA antagonists). Since the excessive activation of EAA receptors of the central nervous system plays an important role in the pathogenesis of various neurological diseases, it can be concluded from the proven EAA antagonism in vivo that the substances can be used therapeutically against such CNS diseases. An ED ₅₀ value was determined as a measure of the effectiveness of the substances, in which 50% of the animals become symptom-free by a fixed dose of either NMDA or AMPA by the previous ip administration of the measuring substance.

The heterocyclically substituted amides I are inhibitors of cysteine derivatives such as calpain I or II and cathepsin B or L and can thus be used to combat diseases which are associated with an increased enzyme activity of the calpain enzymes or cathepsin enzymes. The present amides I can thereafter be used for the treatment of neurodegenerative diseases which occur after ischemia, damage by reperfusion after vascular occlusion, trauma, subarachnoid bleeding and stroke, and of neurodegenerative diseases such as multiple infarct dementia, Alzheimer's disease, Huntington's disease and epilepsy and furthermore for the treatment of damage to the heart after cardiac ischemia, damage to the kidneys after renal ischemia, skeletal muscle damage, muscular dystrophies, damage caused by proliferation of the smooth muscle cells, coronary vasospasm, cerebral vasospasm, cataracts of the eyes, re-stenosis of the bloodstream Serve angioplasty. In addition, the amides I can be useful in the chemotherapy of tumors and their metastasis and for the treatment of diseases in which an increased level of interleukin-1 occurs, such as inflammation and rheumatic diseases. 15

In addition to the usual pharmaceutical auxiliary substances, the pharmaceutical preparations according to the invention contain a therapeutically effective amount of the compounds I.

For local external use, for example in powders, ointments or sprays, the active compounds can be present in the usual concentrations. As a rule, the active substances are contained in an amount of 0.001 to 1% by weight, preferably 0.001 to 0.1% by weight.

For internal use, the preparations are administered in single doses. 0.1 to 100 mg per kg body weight are given in a single dose. The preparation can be administered daily in one or more doses depending on the type and severity of the diseases.

In accordance with the desired type of application, the pharmaceutical preparations according to the invention contain, in addition to the active ingredient, the customary excipients and diluents. For the local external application, pharmaceutical-technical auxiliaries, such as ethanol, isopropanol, ethoxylated castor oil, ethoxylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol, polyethylene glycolostearate, ethoxylated fatty alcohols, paraffin oil, vase ^¬ line and wool fat, can be used. Milk sugar, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone are suitable for internal use.

Antioxidants such as tocopherol and butylated hydroxyanisole and butylated hydroxytoluene, taste-improving additives, stabilizers, emulsifiers and lubricants can also be present.

The substances contained in the preparation in addition to the active substance and the substances used in the manufacture of the pharmaceutical preparations are toxicologically harmless and compatible with the respective active substance. The pharmaceutical preparations are produced in a customary manner, for example by mixing the active ingredient with other customary excipients and diluents.

The pharmaceutical preparations can be administered in various modes of administration, for example orally, parenterally and intravenously by infusion, subcutaneously, intraperitoneally and topically. Forms of preparation such as tablets, emulsions, infusion and injection solutions, pastes, ointments, gels, creams, lotions, powders and sprays are possible. 16

Examples

example 1

COOH

(S) -3-carboxy-5 (2-naphthylsulfonamido) -N (3-phenyl-propan-l-al-2-yl) -benzamide a) (S) -3-ethoxycarbonyl-5-nitro-N (3-phenyl-propan-l-ol-2-yl) benzamide

10g (41.8mMol) 5-nitro-isophthalic acid monoethyl ester, 6.3g (41.8mMol) (S) -phenylalaninol and 10.6g (104.5mMol) triethylamine were dissolved in 200ml methylene chloride at room temperature and stirred for 30 minutes. Thereafter was added under ice cooling 1.9g (13.9mMol) of 1-hydroxybenzotriazole and subsequently ^¬ ßend portions 8g (41.6mMol) (N-Dimethylaminopro- pyl) -Ne hylcarbodiimid to. Everything was stirred for 16 hours at room temperature. The reaction mixture was diluted to twice the volume with methylene chloride and washed successively with 2M hydrochloric acid, water, 2M sodium hydroxide solution and again water. The organic phase was separated, dried ge ^¬ and concentrated in vacuo. This gave 9.1g of the interim rule ^¬ product

b) (S) -5-Amino-3-ethoxycarbonyl-N (3-phenyl-propan-1-ol-2-yl) benzamide

9 g (24.3 mmol) of the intermediate la were dissolved in 300 ml of ethanol and hydrogenated after the addition of 1 g of palladium / carbon (10%). The reaction mixture was then filtered and the filtrate was concentrated in vacuo. 8.1 g of the intermediate product were obtained.

c) (S) -3-Ethoxycarbonyl-5 (2-naphthylsulfonamido) -N (3-phenyl-propan-1-ol-2-yl) -benzamide

2 g (5.84 mmol) of the intermediate lb and 2.4 ml (17.5 mmol) of triethylamine were dissolved in 50 ml of tetrahydrofuran. A solution of 1.32 g (5.82 mmol) of 2-naphthalenesulfonic acid chloride in 30 ml of tetrahydrofuran was then added dropwise at 0 ° C. and the reaction mixture was then stirred at 40 ° C. for 8 h. The reaction mixture was then concentrated in vacuo and the 17

Residue distributed between water and ethyl acetate. The acetic ester phase was washed with 2M hydrochloric acid and water and then dried and concentrated in vacuo. The residue thus obtained was purified by chromatography on silica gel (eluent: methylene chloride / ethanol = 20/1), where ^{¬ were obtained} at 0.65 g of the intermediate.

d) (S) -3-carboxy-5 (2-naphthylsulfonamido) - (3-phenyl-propan-l-ol-2-yl) benzamide

0.65 g (1.2 mmol) of the intermediate lc were dissolved in 30 ml of tetrahydrofuran and 0.15 g (6.3 mmol) of lithium hydroxide, dissolved in 15 ml of water, was added. Everything was stirred for 26 hours at room temperature. The organic solvent was then removed in vacuo and the aqueous

Acidified residue with 2M hydrochloric acid. The resulting low ^¬ impact was suction filtered and dried. 0.46 g of the intermediate product was obtained.

e) (S) -3-Carboxy-5 (2-naphthylsulfonamido) -N (3-phenyl-propan-l-al-2-yl) -benzamide

0.46 g (0.91 mmol) of the intermediate ld and 0.37 g (3.65 mmol) of triethylamine were dissolved in 10 ml of dry dimethyl sulfoxide and 0.44 g (2.76 mmol) of pyridine-sulfur-trioxide complex was added. Everything was stirred for 16h at room temperature. The reaction mixture was then poured into ice water, acidified with 1M hydrochloric acid and the precipitate was filtered off with suction. 0.36 g of the product was obtained.

1H-NMR (D ₆ -DMSO): 5 = 2.9 (1H), 3.3 (1H), 4.5 (1H), 7.2 (5H), 7.6-7.9 (5H), 8.0-8.2 (4H), 8.5 (2H) , 9.K1H), 9.6 (1H) and 10.9 (1H) ppm.

Example 2

N (l-carbamoyl-2-oxo-4-phenyl-propan-2-yl) -3-carboxy-5 (2-naphthylsulfonamido) benzamide

COOH 18 a) N (l-carbamoyl-2-hydroxy-4-phenyl-propan-2-yl) -3-ethoxycarbonyl-5-nitro-benzamide

5.2g (21.7mmol) 5-nitro-isophthalic acid monoethyl ester, 5g (21.7mmol) 3-amino-2-hydroxy-3-phenylbutyric acid amide and

11.2 g (110.5 mol) of triethylamine were dissolved in 200 ml of methylene chloride at room temperature and stirred for 30 minutes. Since ^¬ according were added under ice-cooling 2.9g (21.6mMol) of 1-triazol Hydroxybenzo- and then added in portions 4.6g (22.8mMol) (N-di- methylaminopropyl) -N-ethylcarbodiimide to. Everything was stirred for 16 hours at room temperature. The reaction mixture was diluted with methylene chloride to twice the volume and successively washed with 2M he ^¬ neut water hydrochloric acid, water, 2M sodium hydroxide solution and. The organic phase was separated, dried and concentrated in vacuo. This gave 2.6g of the interim rule ^¬ product

b) 5-Amino-N (l-carbamoyl-2-hydroxy-4-phenyl-propan-2-yl) -3-ethoxycarbonyl-benzamide

2.6 g (6.25 mmol) of the intermediate 2a were dissolved in 50 ml of dimethyl formamide, diluted with 200 ml of ethanol and hydrogenated after adding 1 g of palladium / carbon (10%). The reaction mixture was then filtered and the filtrate was concentrated in vacuo. 1.8 g of the intermediate product were obtained.

c) (-l-Carbamoyl-2-hydroxy-4-phenyl-propan-2-yl) -3-ethoxycarbonyl-5 (2-naphthylsulfone-amido) -benzamide

1.8 g (4.7 mmol) of the intermediate 2b and a spatula tip of 4-dimethylaminopyridine were dissolved in 30 ml of pyridine. 1.2 g (5.1 mmol) of naphthalenesulfonic acid chloride were added dropwise at room temperature and everything was stirred for a further 16 h. The reaction mixture was then poured onto ice water and acidified with 2M hydrochloric acid. This aqueous phase was extracted several times with ^¬ It sigester. The combined organic phase was dried and concentrated in vacuo. The residue thus obtained was treated in succession with ether and a little ethyl acetate, giving 1.3 g of the intermediate.

d) N (1-Carbamoy1-2-hydroxy-4-phenyl-propan-2-yl) -3-carboxy-5 (2-naphthylsulfonamido) benzamide

1.25 g (2.2 mmol) of the intermediate 3c were dissolved in 10 ml of tetrahydrofuran and mixed with 0.21 g (8.7 mmol) of lithium hydroxide, dissolved in 50 ml of water. Everything was stirred for 1 hour at room temperature. After that it was 19 organic solvents removed in vacuo and the aqueous residue acidified with 2M hydrochloric acid. The resulting low ^¬ impact was suction filtered and dried. 1.0 g of the intermediate product was obtained.

e) N (l-carbamoyl-2-oxo-4-phenyl-propan-2-yl) -3-carboxy-5 (2-naphthylsulfonamido) benz amide

0.9 g (l. Mol) of the intermediate compound 2d and 1.4 ml (9.9 mmol) of triethylamine were dissolved in 25 ml of dry dimethyl sulfoxide and 0.78 g (4.9 mmol) of pyridine-sulfur trioxide complex, dissolved in 13 ml of dimethyl sulfoxide, were added at room temperature. Everything was stirred for lh at room temperature. The reaction mixture was then poured onto ice water, acidified with 1M hydrochloric acid and the precipitate was filtered off with suction, 0.59 g of the product being obtained.

ⁱ K-NMR (CF ₃ COOD): δ = 2.9 (1H), 3.1 (1H), 5.3 (1H), 7.0-8.3 (17H), 8.4 (1H) and 9.1 (1H) ppm.

The following examples can be similarly provides the above rules Herge ^¬:

R ²

Example R 'R ² R ³ R ¹ *

2 3-COOH (CH ₂ ) ₃ CH ₃ CO H ₂ 5-naphth-2-yl-S0 ₂ NH

3 3-COOH CH, Ph H 5-plιenyl-S0 ₂ NH

4 3-COOH CH, Ph CONH, 5-phenyl-SO ₂ NH

5 3-COOH CH ₂ Ph H 5-n-butyl SO 2 NH

6 3-COOH CK, Ph CONH, 5-n-butyl-S0 ₂ NH

7 4-COOH CH ₂ Ph H 3-p enyl-Sθ2NH

8 4-COOH CH, Ph H 5-naphth-2-yl-SO ₂ NH

9 4-COOH CH, Ph CONH, 5-aphth-2-yl-SO ₂ NH

10 4-COOH CH, Ph H 3- CH = CH-phenyl

11 4-COOH CH ^ Ph H 3- (pyrid-2-yl)

12 3-COOH CH, Ph H 4- CH = CH-J ^> henyl

If R ³ = H, then the configuration is C2 (S), in the case of CONH, it is (R, S). ; o Öi

BeiR ¹ R ² R ^ R ^x xv © 4-. game

13 3 -COOH CH ₂ PH CONH ₂ 0

N

14 3 -COOH CH ₂ Ph H 0

N σ

15 3 -COOH CH ₂ Ph H

0

N

16 3 -COOH CH ₂ Ph CONH ₂ 0

O

H N

SO SO

0 \

With R ^l R ² R ³ R ^l x SO SO game

17 3 - COOH CHoPh so

18 3 - COOH CH ₂ Ph CONH ₂

19 3 - COOH (CH ₂ ) ₃ CH ₃ t

20 3 - COOH (CH ₂ ) ₃ CH ₃ CONH ₂

21 3 - COOH CH ₂ Ph O

CONH ₂ H

N so s ^ oo _^ o \

At Ri R ² R ³ Rlχ SO SO play Tue

4-. κ>

^~ 22 3 - COOH CH ₂ Ph so

4-.

23 3 - COOH

CH ₂ SO? NH

24 3 - COOH CONH 2 S0 ₂ NH

CH ₂

25 3 - COOH CH ₂ Ph H SO9NH 10 10

26 3 - COOH CH ₂ Ph CONH; SO7NH

27 3 - COOH CH ₂ Ph H CONH

O

H so

SO o

so

BeiRl R ² R ^J R ^x x so ti game 4-. »

SO

^~ 28 3 - COOH CH ₂ Ph CONH, * -

CONH

29 4 - COOH CH ₂ Ph CONH ₂

'30 4 - COOH CH Ph CONH;

31 4 - COOH CH ₂ Ph CONH ₂

32 4 - COOH CH ₂ Ph H

2-

33 4 - COOH CH ₂ Ph CONH-

34 3 - COOH CH ₂ Ph CONH; O

SO O o

24

X a

«O o

tf u

oi ι φ -H -H

L a Lf)

Claims

25 patent claims

Amides of the general formula I

R ²

HOOC O R ³ HO

Rl -CC ₆ alkyl, phenyl, naphthyl, quinolyl, pyridyl,

Pyrimidyl, pyridazyl, quinazolyl and quinoxalyl can ^¬ signified th, which rings may be substituted with up to 2 radicals R4 to, and

R2 - (CH ₂ ) _m - R8, where R8 can be phenyl, cyclohexyl or indolyl and m = 1 to 6, and

X is a bond, -CH ₂ -, -CH ₂ CH ₂ -, -CH = CH-, -C≡C-, -CONH- S0 ₂ NH- and

Rl-X together too

(R ⁵ l and mean and

R3 means hydrogen and CO-NR6R7,

R4 represents hydrogen, Cl-C4-alkyl, branched or unbranched and -0-Cl-C4-alkyl;

R5 represents hydrogen, C1-C4-alkyl, branched or unbranched and -0-C1-C4-alkyl;

R6 hydrogen f, -CC ₆ alkyl, branched and unbranched, means, and 26

R7 is hydrogen, Ci-C _δ alkyl, branched or unbranched, means, and

n means a number 0, 1 or 2.

2. Amides of formula I according to claim 1, wherein

Rl phenyl, naphthyl, butyl and quinolyl

R2 benzyl and

R3 is hydrogen and

X S0 ₂ NH and

R4 means hydrogen.

3. Amides of formula I according to claim 1, wherein

Rl phenyl, naphthyl, butyl and quinolyl

R2 benzyl and

R3 CONH ₂ and

X S0 ₂ NH and

R4 means hydrogen.

4. Use of amides of formula I according to claims 1-3 for the treatment of diseases.

5. Use of amides of the formula I according to claims 1-3 as inhibitors of cysteine proteases.

6. Use according to claim 5 as inhibitors of cysteine proteases such as calpaine and cathepsine, in particular calpaine I and II and cathepsine B and L.

7. Use of amides of formula I according to claims 1-3 for the manufacture as a medicament for the treatment of diseases in which increased calpain activities occur.

8. Use of the amides of the formula I according to claims 1-3 for the production of medicaments for the treatment of neurodegenerative diseases and neuronal damage. 27

9. Use according to claim 8 for the treatment of such neurodegenerative diseases and neuronal damage caused by ischemia, trauma or nasal bleeding.

5 10. Use according to claim 9 for the treatment of stroke and traumatic brain injury.

11. Use according to claim 9 for the treatment of Alzheimer's disease and Huntington's disease. 0

12. Use according to claim 9 for the treatment of epilepsy.

13. Use of the compounds of the formula I according to claims 1-3 for the production of medicaments and treatment of 5 damage to the heart after cardiac ischemia, damage to the kidneys after renal ischemia, damage by re-perfusion after vascular occlusion, skeletal muscle damage, Muscular dystrophy, damage caused by smooth muscle cell proliferation, coronary vasospasm, cerebral vasospasm, cataracts of the eyes and restenosis of the bloodstream after angioplasty.

14. Use of the amides of formula I according to claims 1-3 for the manufacture of medicaments for the treatment of tumors and their metastasis.

15. Use of the amides of formula I according to claims 1-3 for the manufacture of medicaments for the treatment of diseases in which increased interleukin-1 levels occur.

30

16. Use of the amides according to claims 1-3 for the treatment of immunological diseases such as inflammation and rheumatic diseases.

35 17. Pharmaceutical preparations for oral, parenteral and intraperitoneal use, containing per single dose, in addition to the usual pharmaceutical excipients, at least one amide I according to claims 1-3.

40

45