CHIRAL 2,5-DISUBSTITUTED CYCLOPENTANECARBOXYLIC ACID DERIVATIVES AND USE THEREOF

Abstract

The present application relates to novel, chiral 2,5-disubstituted cyclopentanecarboxylic acid derivatives, to a method for their preparation, to their use on their own or in combinations for the treatment and/or prevention of diseases, and to their use for producing medicaments for the treatment and/or prevention of diseases, in particular for the treatment and/or prevention of diseases of the respiratory tracts, the lung and the cardiovascular system.

Claims

1. (1S,2S,5R)-2-[4-(Benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid of the formula (I-A) or (1R,2R,5S)-2-[4-(benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid of the formula (I-B) ##STR00026## or a mixture of these compounds or a salt, solvate or solvate of a salt of these compounds or their mixture.

2. Mixture of the compounds of the formula (I-A) and (I-B) according claim 1, wherein the compounds of the formula (I-A) and (I-B) are present as racemic mixture, or a salt, solvate or solvate of a salt of this racemic mixture.

3. Compounds according to claim 1 of the formula (I-A) ##STR00027## in enantiomerically pure form or a salt, solvate or solvate of a salt thereof.

4. Process for preparing a compound or a mixture of compounds, as defined in claim 1, wherein exo-2-(trimethylsilyl)ethyl 2-oxobicyclo[2.2.1]heptane-7-carboxylate of the formula (II) ##STR00028## is reacted with a phenyl Grignard compound of the formula (III) ##STR00029## in which X is chlorine, bromine or iodine, to give the adduct of the formula (IV) ##STR00030## then the hydroxy group is eliminated via the mesylate produced in situ of the formula (V) ##STR00031## to give the olefin of the formula (VI) ##STR00032## then oxidation is carried out with N-methylmorpholine N-oxide together with osmium tetroxide as catalyst to give the cis-1,2-diol of the formula (VII) ##STR00033## then this bicyclic diol is cleaved with the help of lead tetraacetate or sodium periodate to give the racemic mixture of the 2-benzoyl-5-formylcyclopentanecarboxylic acid esters (VIII-A) and (VIII-B) ##STR00034## this mixture is reduced with sodium borohydride to give the racemic mixture of the hydroxymethyl compounds (IX-A) and (IX-B) ##STR00035## then reaction is carried out with 1,2,3-benzotriazin-4(3H)-one of the formula (X) ##STR00036## in the presence of an alkyl- or arylphosphane and an azodicarboxylate to give the racemic mixture of the benzotriazinone derivatives (XI-A) and (XI-B) ##STR00037## and finally the 2-(trimethylsilyl)ethyl ester group is cleaved off with the help of an acid or of a fluoride reagent to give the racemic mixture of the cyclopentanecarboxylic acids according to the invention (I-A) and (I-B) ##STR00038## and optionally the resulting mixture of the compounds (I-A) and (I-B) is separated into the enantiomerically pure compounds and/or converted with the corresponding (i) solvents and/or (ii) bases to the solvates, salts and/or solvates of the salts.

5. Compound or mixture of compounds, as defined in claim 1, for the treatment and/or prevention of diseases.

6. Compound or mixture of compounds, as defined in claim 1, for use in a method for the treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in the COPD (PH-COPD), bronchiectasis, asthma, interstitial pulmonary disorders, idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis, of arteriosclerosis, carotid arteriosclerosis, viral myocarditis, cardiomyopathy and aneurysms, including their consequential diseases such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome.

7. Use of a compound or mixture of compounds, as defined in claim 1, for producing a medicament for the treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in the COPD (PH-COPD), bronchiectasis, asthma, interstitial pulmonary disorders, idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis, of arteriosclerosis, carotid arteriosclerosis, viral myocarditis, cardiomyopathy and aneurysms, including their consequential diseases such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome.

8. Medicament comprising a compound or a mixture of compounds, as defined in claim 1, in combination with one or more inert non-toxic, pharmaceutically suitable auxiliaries.

9. Medicament comprising a compound or a mixture of compounds, as defined in claim 1, in combination with one or more further active ingredients selected from the group consisting of corticosteroids, beta-adrenergic receptor agonists, antimuscarinic substances, PDE 4 inhibitors, PDE 5 inhibitors, sGC activators, sGC stimulators, HNE inhibitors, prostacyclin analogues, endothelin antagonists, statins, antifibrotic agents, antiinflammatory agents, immunomodulating agents, immunosuppressive agents and cytotoxic agents.

10. Medicament according to claim 8, for the treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in the COPD (PH-COPD), bronchiectasis, asthma, interstitial pulmonary disorders, idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis, of arteriosclerosis, carotid arteriosclerosis, viral myocarditis, cardiomyopathy and aneurysms, including their consequential diseases such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome.

11. Method for the treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in the COPD (PH-COPD), bronchiectasis, asthma, interstitial pulmonary disorders, idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis, of arteriosclerosis, carotid arteriosclerosis, viral myocarditis, cardiomyopathy and aneurysms, including their consequential diseases such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome in humans and animals by administering an effective amount of a compound or of a mixture of compounds, as defined in claim 1, or of a medicament comprising the compound or the mixture auxiliaries.

Description

EMBODIMENT EXAMPLES

Example 1

(+/)-(1RS,2RS,5SR)-2-[4-(Benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid (racemate)

[0261] ##STR00021##

[0262] A solution of 213 mg (0.365 mmol) of the compound from Example 6A in 2 ml of dichloromethane was admixed at 0 C. with 1 ml (12.98 mmol) of trifluoroacetic acid. The mixture was stirred for 1 h at 0 C. and then stored at 5 C. for ca. 18 h. The mixture was then concentrated, the residue was taken up in dichloromethane and the solution was concentrated again. This procedure was repeated several times. Finally, the residue was taken up in acetonitrile/THF and purified by preparative HPLC (method 3). This thus gave 125 mg (71% of theory) of the title compound.

[0263] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.15 (s, 1H), 8.26 (d, 1H), 8.20 (d, 1H), 8.11-8.05 (m, 1H), 8.01-7.89 (m, 3H), 7.52-7.28 (m, 5H), 7.13 (d, 2H), 5.21 (s, 2H), 4.53 (dd, 2H), 4.15-4.06 (m, 1H), 3.24 (t, 1H), 2.93-2.80 (m, 1H), 2.17-2.04 (m, 1H), 1.94-1.83 (m, 1H), 1.72-1.60 (m, 1H), 1.57-1.44 (m, 1H).

[0264] LC/MS (Method 1, ESIpos): R.sub.t=1.16 min; m/z=484 [M+H].sup.+.

Separation of the Enantiomers:

Method A:

[0265] 645 mg of the racemic compound from Example 1 were dissolved in 20 ml of dioxane and separated into the enantiomers by preparative HPLC on a chiral phase (see Examples 2 and 3) [column: Daicel Chiralpak IC, 5 m 250 mm20 mm; flow rate: 15 ml/min; detection: 220 nm; injection volume: 0.2 ml; temperature: 25 C.; mobile phase: t=0-5 min 80% methanol/20% acetonitrile].

Method B:

[0266] 510 mg of the racemic compound from Example 1 were dissolved in 10 ml of THF at elevated temperature and separated into the enantiomers by preparative SFC on a chiral phase (see Example 2 and 3) [column: Daicel Chiralpak AS-H, 5 m, 250 mm20 mm; flow rate: 100 ml/min; detection: 210 nm; injection volume: 0.25 ml; temperature: 40 C.; mobile phase: t=0-8 min 60% carbon dioxide/40% ethanol].

Example 2

(+)-(1S,2S,5R)-2-[4-(Benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid

[0267] ##STR00022##

[0268] Yield (according to Method A): 209 mg; ee-value=99%

[0269] [].sub.D.sup.20=+67.2, 589 nm, c=0.32 g/100 ml, chloroform

[0270] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.15 (s, 1H), 8.26 (d, 1H), 8.20 (d, 1H), 8.11-8.05 (m, 1H), 8.01-7.90 (m, 3H), 7.49-7.31 (m, 5H), 7.13 (d, 2H), 5.21 (s, 2H), 4.53 (dd, 2H), 4.15-4.06 (m, 1H), 3.24 (t, 1H), 2.94-2.80 (m, 1H), 2.17-2.03 (m, 1H), 1.94-1.82 (m, 1H), 1.72-1.60 (m, 1H), 1.57-1.44 (m, 1H).

[0271] LC/MS (Method 2, ESIpos): R.sub.t=2.59 min; m/z=484 [M+H].sup.+.

[0272] A single-crystal X-ray structural analysis produced a (1S,2S,5R)-absolute configuration for this enantiomer. The resulting crystal data are shown in the table below (for the description of the method see introductory paragraph of the experimental section).

Crystal Data from X-Ray Structural Analysis for Example 2:

TABLE-US-00001 Space group P21 Cell structure a () 5.7051(3) b () 31.9892(14) c () 6.3511(3) () 90 () 94.405(3) () 90 Volume (.sup.3) 1155.66(10) molecules per unit cell 2 calculated density (Mg/m.sup.3) 1.389

Example 3

()-(1R,2R,5S)-2-[4-(Benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid

[0273] ##STR00023##

[0274] Yield (according to method A): 228 mg; ee value=99%

[0275] [].sub.D.sup.20=68.3, 589 nm, c=0.35 g/100 ml, chloroform

[0276] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.15 (s, 1H), 8.26 (d, 1H), 8.20 (d, 1H), 8.11-8.05 (m, 1H), 8.01-7.89 (m, 3H), 7.49-7.31 (m, 5H), 7.13 (d, 2H), 5.21 (s, 2H), 4.53 (dd, 2H), 4.14-4.05 (m, 1H), 3.24 (t, 1H), 2.94-2.80 (m, 1H), 2.17-2.04 (m, 1H), 1.95-1.83 (m, 1H), 1.72-1.60 (m, 1H), 1.57-1.44 (m, 1H).

[0277] LC/MS (Method 2, ESIpos): R.sub.t=2.59 min; m/z=484 [M+H].sup.+.

COMPARATIVE EXAMPLES

Comparative Example A-1

(1RS,2RS,5SR)-2-[(4-chlorobiphenyl-4-yl)carbonyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid (racemate)

[0278] ##STR00024##

[0279] The racemic compound and its preparation is described in WO 97/43239-A1 as Example 1.

Separation of the Enantiomers:

[0280] 1.450 g (2.97 mmol) of (1RS,2RS,5SR)-2-[(4-chlorobiphenyl-4-yl)carbonyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid (racemate) were dissolved in a mixture of 80 ml of ethanol and 20 ml of acetonitrile and separated into the enantiomers by preparative HPLC on a chiral phase (see Comparative Examples A-2 and A-3) [column: Daicel Chiralpak ID 5 m 250 mm20 mm; flow rate: 12 ml/min; detection: 220 nm; injection volume: 1.8 ml; temperature: 45 C.; mobile phase: 100% ethanol isocratic; run time: 12 min]:

Comparative Example A-2

(1RS,2RS,5SR)-2-[(4-chlorobiphenyl-4-yl)carbonyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid (enantiomer 1)

[0281] This gave 637 mg (chemical purity 100%) of the title compound.

[0282] R.sub.t=5.59 min, ee value=99% [column: Daicel Chiralpak IC-H 250 mm4.6 mm, 5 m; flow rate: 1.0 ml/min; detection: 220 nm; temperature: 45 C.; mobile phase: 100% ethanol+0.2% TFA+1% water, isocratic].

Comparative Example A-3

(1RS,2RS,5SR)-2-[(4-Chlorobiphenyl-4-yl)carbonyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylic acid (enantiomer 2)

[0283] This gave 651 mg (chemical purity 100%) of the title compound.

[0284] R.sub.t=8.51 min, ee value=99% [column: Daicel Chiralpak IC-H 250 mm4.6 mm, 5 m; flow rate: 1.0 ml/min; detection: 220 nm; temperature: 45 C.; mobile phase: 100% ethanol+0.2% TFA+1% water, isocratic].

Comparative Example B-1

(+/)-4-Oxo-2-[2-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)ethyl]-4-[4-(pentyloxy)phenyl]butanoic acid (racemate)

[0285] ##STR00025##

[0286] The racemic compound and its preparation is described in WO 97/43237-A1 as Example 15.

Separation of the Enantiomers:

[0287] 250 mg (0.57 mmol) of (+/)-4-oxo-2-[2-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)ethyl]-4-[4-(pentyloxy)phenyl]butanoic acid (racemate) were dissolved in 7 ml of acetonitrile and separated into the enantiomers by preparative HPLC on a chiral phase (see Comparative Examples B-2 and B-3) [column: Daicel Chiralpak AD-H, 5 m, 250 mm20 mm; flow rate: 20 ml/min; detection: 280 nm; injection volume: 0.12 ml; temperature: 25 C.; mobile phase: 80% acetonitrile/20% ethanol+0.2% glacial acetic acid, isocratic; run time: 6 min]:

Comparative Example B-2

(+)-4-Oxo-2-[2-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)ethyl]-4-[4-(pentyloxy)phenyl]butanoic acid (enantiomer 1)

[0288] This gave 111 mg (chemical purity 100%) of the title compound.

[0289] [].sub.D.sup.20=+30.6, 589 nm, c=0.32 g/100 ml, chloroform

[0290] R.sub.t=8.21 min, ee value=100% [column: Daicel Chiralpak AD-H, 250 mm4.6 mm, 5 m; flow rate: 1.0 ml/min; detection: 280 nm; mobile phase: 80% acetonitrile+0.2% glacial acetic acid/20% ethanol+0.2% glacial acetic acid, isocratic].

Comparative Example B-3

()-4-Oxo-2-[2-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)ethyl]-4-[4-(pentyloxy)phenyl]butanoic acid (enantiomer 2)

[0291] This gave 119 mg (chemical purity 100%) of the title compound.

[0292] [].sub.D.sup.20=25.6, 589 nm, c=0.35 g/100 ml, chloroform

[0293] R.sub.t=10.34 min, ee value=99% [column: Daicel Chiralpak AD-H, 250 mm4.6 mm, 5 m; flow rate: 1.0 ml/min; detection: 280 nm; mobile phase: 80% acetonitrile+0.2% glacial acetic acid/20% ethanol+0.2% glacial acetic acid, isocratic].

B. ASSESSMENT OF PHARMACOLOGICAL EFFICACY

[0294] The pharmacological activity of the compounds according to the invention can be demonstrated by in vitro and in vivo studies, as known to the person skilled in the art. The application examples which follow describe the biological action of the compounds according to the invention, without restricting the invention to these examples.

ABBREVIATIONS AND ACRONYMS

[0295] APMA 4-aminophenyl mercury acetate [0296] Brij-35 polyoxyethylene lauryl ether [0297] BSA bovine serum albumin [0298] CYP cytochrome P450 [0299] Dap (or Dpa) L-2,3-diaminopropionic acid (-amino-1-alanine) [0300] DMSO dimethyl sulphoxide [0301] Dnp 2,4-dinitrophenyl [0302] EDTA ethylenediaminetetraacetic acid [0303] HEPES 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulphonic acid [0304] HME human macrophage elastase [0305] IC inhibition concentration [0306] i.v. intravenous [0307] Mca (7-methoxycoumarin-4-yl)acetyl [0308] MMP matrix metallopeptidase [0309] MTP microtiter plate [0310] NADP.sup.+ nicotinamide adenine dinucleotide phosphate (oxidized form) [0311] NADPH nicotinamide adenine dinucleotide phosphate (reduced form) [0312] Nval norvalin [0313] PEG polyethylene glycol [0314] p.o. peroral [0315] Tris tris(hydroxymethyl)aminomethane [0316] v/v ratio by volume (of a solution) [0317] w/w ratio by weight (of a solution)

B-1. In Vitro HME Inhibition Test

[0318] The activity of the compounds according to the invention towards HME (MMP-12) is ascertained in an in vitro inhibition test. The HME-mediated amidolytic cleavage of a suitable peptide substrate leads herein to a fluorescent light increase. The signal intensity of the fluorescent light is directly proportional to the enzyme activity. The active concentration of a test compound at which half of the enzyme is inhibited (50% signal intensity of the fluorescent light) is given as IC.sub.50 value.

Standard In Vitro HME Inhibition Test:

[0319] In a 384 hole microtiter plate, in a test volume of in total 41 l of the test buffer (0.1 M HEPES pH 7.4, 0.15 M NaCl, 0.03 M CaCl.sub.2, 0.004 mM ZnCl.sub.2, 0.02 M EDTA, 0.005% Brij), the enzyme (0.5 nM HME; R&D Systems, 917-MP, autocatalytic activation according to the manufacturer's instructions) and the intramolecularly quenched substrate [5 M Mca-Pro-Leu-Gly-Leu-Glu-Glu-Ala-Dap(Dnp)-NH.sub.2; Bachem, M-2670] are incubated in the absence and presence of the test substance (as solution in DMSO) for two hours at 37 C. The fluorescent light intensity of the test batches is measured (excitation 323 nm, emission 393 nm). The IC.sub.50 values are ascertained by plotting the fluorescent light intensity against the active ingredient concentration.

High-Sensitivity In Vitro HME Inhibition Test:

[0320] If subnanomolar IC values are produced for high potent test substances in the standard HME inhibition test described above, then a modified test is used for their more precise determination. Here, a ten-fold lower enzyme concentration is used (final concentration e.g. 0.05 nM), in order to achieve an increased sensitivity of the test. The incubation time of the test is accordingly chosen to be longer (e.g. 16 hours).

In Vitro HME Inhibition Test in the Presence of Serum Albumin in the Reaction Buffer:

[0321] This test corresponds to the standard HME inhibition test described above, but using a modified reaction buffer. This reaction buffer additionally comprises bovine serum albumin (BSA, fatty acid-free, A6003, Sigma-Aldrich) of a final concentration of 2% (w/w), which corresponds to approximately half of the physiological serum albumin content. The enzyme concentration in this modified test is slightly increased (e.g. 0.75 nM), as is the incubation time (e.g. three hours).

[0322] Table 1 below gives the IC.sub.50 values from these HME inhibition tests for the working examples of the present invention and also for two structurally related comparison compounds from the prior art (as racemate or separated enantiomers).fwdarw.(sometimes as average values from several independent individual determinations and rounded to two significant places). The IC.sub.50 values were determined for racemates and enantiomers from differently generated DMSO stock solutions. Whereas an automatically created DMSO stock solution from the internal substance logistics was used for racemates by means of a standard method, for enantiomers and for a more precise direct comparison of the enantiomers with one another, in each case a freshly produced, manually prepared DMSO stock solution was used.

TABLE-US-00002 TABLE 1 Inhibition of human macrophage elastase (HME/hMMP-12) in the absence () or presence (+) of serum albumin (BSA) Example HME IC.sub.50 [nM] HME IC.sub.50 [nM] No. (BSA) (+BSA) 1 0.059 n.d. 2 0.071 8.4 3 14 n.d. A-1 0.043 n.d. A-2 66 n.d. A-3 0.018 5.4 B-1 1.5 n.d. B-2 1.6 170 B-3 160 n.d. [n.d. = not determined].

[0323] As is evident from the data in Table 1, the compounds 1 to 3 according to the invention are significantly more potent compared to the relevant comparison compounds A-1 to A-3 or B-1 to B-3 (more than one order of magnitude: cf. Example 1 to B-1, Example 2 to B-2, Example 3 to B-3) or are comparably potent (same order of magnitude: cf. Example 1 to A-1, Example 2 to A-3, Example 3 to A-2). A similar picture also arises under the test conditions of a potentially competing nonspecific protein binding of the compounds according to the invention and the comparison compounds, such as for example to serum albumins (IC.sub.50 values in the presence of BSA: cf. Example 2 to A-3 or B-2).

[0324] Moreover, Tables 2A/2B and 3A/3B reveal a significantly higher selectivity of the compounds according to the invention compared to the relevant comparison compounds, in particular compared to those with a comparable HME activity (see therein).

B-2. In Vitro MMP Inhibition Tests

[0325] The activity strength of the compounds according to the invention towards other MMPs (and therefore their selectivity) is likewise ascertained in in vitro inhibition tests. The MMP-mediated amidolytic cleavage of a suitable peptide substrate also leads here to a fluorescent light increase. The signal intensity of the fluorescent light is directly proportional to the enzyme activity. The active concentration of a test compound at which half of the enzyme is inhibited (50% signal intensity of the fluorescent light) is given as IC.sub.50 value.

a) Human MMPs:

In Vitro MMP-1 Inhibition Test:

[0326] Recombinant MMP-1 (R&D Systems, 901-MP) is chemically activated in accordance with the manufacturer's instructions by using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 2 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-1 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-2 Inhibition Test:

[0327] Recombinant MMP-2 (R&D Systems, 902-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 2 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-2 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-3 Inhibition Test:

[0328] Recombinant MMP-3 (R&D Systems, 513-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 2 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-002), such that a total test volume of 50 l results. The progress of the MMP-3 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-7 Inhibition Test:

[0329] Recombinant MMP-7 (R&D Systems, 907-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 0.5 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-7 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-8 Inhibition Test:

[0330] Recombinant MMP-8 (R&D Systems, 908-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 0.5 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-8 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-9 Inhibition Test:

[0331] Recombinant MMP-9 (R&D Systems, 911-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 0.1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-9 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-10 Inhibition Test:

[0332] Recombinant MMP-10 (R&D Systems, 910-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 2 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-002), such that a total test volume of 50 l results. The progress of the MMP-10 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-13 Inhibition Test:

[0333] Recombinant MMP-13 (R&D Systems, 511-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 0.1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-13 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-14 Inhibition Test:

[0334] Recombinant MMP-14 (R&D Systems, 918-MP) is enzymatically activated in accordance with the manufacturer's instructions using recombinant furin (R&D Systems, 1503-SE). 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 0.5 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-010), such that a total test volume of 50 l results. The progress of the MMP-14 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-16 Inhibition Test:

[0335] Recombinant MMP-16 (R&D Systems, 1785-MP) is enzymatically activated in accordance with the manufacturer's instructions using recombinant furin (R&D Systems, 1503-SE). 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipette into 24 l of activated enzyme (final concentration e.g. 1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-010), such that a total test volume of 50 l results. The progress of the MMP-16 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

[0336] Tables 2A and 2B below give the IC.sub.50 values from these tests relating to the inhibition of human MMPs for representative embodiment examples of the present invention, and also for two structurally related comparison compounds from the prior art (as racemate or separated enantiomer).fwdarw.(sometimes as average values from several independent individual determinations and rounded to two significant places). The IC.sub.50 values were determined for racemates and enantiomers from differently generated DMSO stock solutions. Whereas an automatically produced DMSO stock solution from the internal substance logistics was used for racemates by means of a standard method, in the case of enantiomers a freshly produced, manually prepared DMSO stock solution was used in each case for a more precise direct comparison of the enantiomers with one another.

TABLE-US-00003 TABLE 2A Inhibition of human MMPs MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 Example IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. [nM] [nM] [nM] [nM] [nM] 1 12000 18 390 500 9.2 2 5800 11.8 175 420 10.1 A-1 220 1.1 49 310 <0.61 A-3 135 0.70 22.5 145 <0.61 B-1 >40000 450 9150 >40000 706 B-2 >40000 120 6600 22500 275

TABLE-US-00004 TABLE 2B Inhibition of human MMPs MMP-9 MMP-10 MMP-13 MMP-14 MMP-16 Example IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. [nM] [nM] [nM] [nM] [nM] 1 30 21 30 27 130 2 23 17.5 14.5 15 78.5 A-1 1.9 4.6 1.8 2.9 16 A-3 1.1 <0.61 0.82 1.2 5.2 B-1 1400 5700 1800 3800 22000 B-2 625 1900 360 1300 6700

[0337] A comparison of the inhibition data given in Tables 1 and 2A/2B reveals that the compounds according to the inventionin particular the more active enantiomerhave a very high inhibitory potency (in the two-position picomolar range) towards HME and at the same time a very high selectivity (two to four orders of magnitude or even more) towards related human MMPs.

[0338] As is moreover evident from the data in the Tables 2A/2B, the compounds according to the invention have a significantly greater selectivity (as a rule more than one order of magnitude) or a comparable selectivity (as a rule same order of magnitude) compared to the relevant comparison compounds A-1/A-3 or B-1/B-2.

[0339] Viewed overall, it is evident from this data that the compounds according to the invention are significantly more selective compared to the relevant comparison compounds or, for a comparable selectivity, are significantly more potent, i.e. have a considerably improved profile as regards the combination of activity strength and selectivity.

b) MMPs of Rodents:

In Vitro MMP-2 Inhibition Test of the Mouse:

[0340] Recombinant MMP-2 of the mouse (R&D Systems, 924-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 0.1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-2 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-3 Inhibition Test of the Mouse:

[0341] Recombinant MMP-3 of the mouse (R&D Systems, 548-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 0.5 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-002), such that a total test volume of 50 l results. The progress of the MMP-3 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-7 Inhibition Test of the Mouse:

[0342] Recombinant MMP-7 of the mouse (R&D Systems, 2967-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 0.5 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-010), such that a total test volume of 50 l results. The progress of the MMP-7 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-8 Inhibition Test of the Mouse:

[0343] Recombinant MMP-8 of the mouse (R&D Systems, 2904-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 2 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-010), such that a total test volume of 50 l results. The progress of the MMP-8 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-9 Inhibition Test of the Mouse:

[0344] Recombinant MMP-9 of the mouse (R&D Systems, 909-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 0.1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-9 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-12 Inhibition Test of the Mouse:

[0345] Recombinant MMP-12 of the mouse (R&D Systems, 3467-MP) is autocatalytically activated in accordance with the manufacturer's instructions. 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-010), such that a total test volume of 50 l results. The progress of the MMP-12 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

High-Sensitivity In Vitro MMP-12 Inhibition Test of the Mouse:

[0346] If subnanomolar IC values are produced for high-potency test substances in the above-described MMP-12 inhibition test of the mouse, then a modified test is used for their more precise determination. Here, a ten-fold lower enzyme concentration is used (final concentration e.g. 0.1 nM), in order to achieve an increased sensitivity of the test. The incubation time of the test is correspondingly chosen to be longer (e.g. 16 hours).

In Vitro MMP-2 Inhibition Test of the Rat:

[0347] Recombinant MMP-2 of the rat (R&D Systems, 924-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 0.1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 10 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-2 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-8 Inhibition Test of the Rat:

[0348] Recombinant MMP-8 of the rat (R&D Systems, 3245-MP) is chemically activated in accordance with the manufacturer's instructions using APMA. 2 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-010), such that a total test volume of 50 l results. The progress of the MMP-8 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-9 Inhibition Test of the Rat:

[0349] Recombinant MMP-9 of the mouse (R&D Systems, 5427-MM) is chemically activated in accordance with the manufacturer's instructions using APMA. 0.1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration e.g. 1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-9 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

In Vitro MMP-12 Inhibition Test of the Rat:

[0350] MMP-12 of the rat (Uniprot NP_446415.1; construct L96-V277) is expressed with an additional N-terminal His-Tag and a consecutive TEV cleavage sequence by means of a pDEco7 vector in E. coli (BL21). The thus recombinantly expressed protein forms an intracellular insoluble protein compartment (so-called inclusion body). This is solubilized after separation and intensive washing under denaturing conditions. For this, the inclusion body pellet fraction from a 250 ml E. coli culture is taken up in a volume of 120 ml of buffer A (50 mM Tris pH 7.4, 100 mM NaCl, 0.03 mM ZnCl.sub.2, 10 mM CaCl.sub.2, 8 M urea). The soluble protein is renatured by in each case dialysing 60 ml of the sample several times at 4-8 C. against buffer B (50 mM Tris pH 7.4, 100 mM NaCl, 0.03 mM ZnCl.sub.2, 10 mM CaCl.sub.2). After the dialysis, the sample is centrifuged (25 000g). The folded-back protein is obtained in the supernatant with a yield of 3.7 mg per 250 ml E. coli culture. The thus obtained protein is enzymatically active without further purification operations or protease-mediated cleavage processes.

[0351] 1 l of the test compound to be analysed (as a solution in DMSO, suitable concentrations e.g. 1 nM to 30 M) is pipetted into 24 l of MMP-12 protein (final concentration e.g. 1 nM) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35) in a white 384-hole microtiter plate (MTP). The enzymatic reaction is started by adding the intramolecularly quenched substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 (final concentration e.g. 5 M; R&D Systems, ES-001), such that a total test volume of 50 l results. The progress of the MMP-12 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period (e.g. over 120 min at a temperature of 32 C.).

[0352] Tables 3A and 3B below give the IC.sub.50 values from these tests for the inhibition of rodent MMPs for representative embodiment examples of the present invention and also for two structurally related comparison compounds from the prior art (as racemate and separated enantiomer).fwdarw.(in part as average values from several independent individual determinations and rounded to two significant places). The IC.sub.50 values were determined for racemates and enantiomers from differently generated DMSO stock solutions. Whereas an automatically created DMSO stock solution from the internal substance logistics was used for racemates by means of a standard method, in the case of enantiomers, in each case a freshly produced, manually prepared DMSO stock solution was used for a more precise direct comparison of the enantiomers with one another.

TABLE-US-00005 TABLE 3A Inhibition of MMPs of the mouse MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-12 Example IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. [nM] [nM] [nM] [nM] [nM] [nM] 1 33 290 46 71 71 0.54 2 16.5 87 22 34.5 27.5 0.13 A-1 1.3 17 15 1.1 4.9 <0.61 A-3 <0.61 11.3 6.2 <0.61 1.5 0.1 B-1 610 13000 1400 2300 2700 32 B-2 145 6750 525 1080 905 18

TABLE-US-00006 TABLE 3B Inhibition of MMPs of the rat MMP-2 MMP-8 MMP-9 MMP-12 Example IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. [nM] [nM] [nM] [nM] 1 33 100 110 <1.0 2 16.5 38.5 46.5 <0.61 A-1 1.3 1.4 4.6 <0.61 A-3 <0.61 1.4 3.3 <0.61 B-1 610 3500 3200 29.3 B-2 145 1040 1350 24.5

[0353] The compounds according to the invention therefore have a very high inhibitory potency (in the sub-nanomolar range) towards MMP-12 of mouse and rat and at the same time a very high selectivity (generally two orders of magnitude) compared to other MMPs of mouse and rat.

[0354] As is evident from the data in Tables 3A/3B, the compounds according to the invention are significantly more potent compared to the relevant comparison compounds as regards MMP-12 (cf. Example 1 to B-1, Example 2 to B-2) or comparably potent (cf. Example 1 to A-1, Example 2 to A-3). Moreover, the compounds according to the invention have a significantly higher selectivity compared to the relevant comparison compounds (as a rule more than one order of magnitude) with regard to other MMPs of mouse and rat.

[0355] By virtue of this significantly higher selectivity towards the orthologous MMPs of mouse and rat in combination with the very high potency towards MMP-12, the compounds according to the inventionin contrast to the comparison compoundsare particularly well suited for preclinical investigations in disease models in rodents prior to clinical investigations with human subjects and patients.

[0356] As a summarizing assessment of the inhibition data in Tables 1, 2A/2B and 3A/3B, it can therefore be stated that the compounds according to the invention have a very high inhibitory potency both on the human and on the orthologous MMP-12 enzyme of mouse and rat, and moreover exhibit a very high selectivity towards related human or rodent MMPs. The resulting profile in each case of the compounds according to the invention of activity strength and selectivity is always significantly better than that of the listed comparison compounds from the prior art.

B-3. Animal Model of Pulmonary Emphysema

[0357] Elastase-induced pulmonary emphysems in mouse, rat or hamster is a widespread animal model for pulmonary emphysema [The Fas/Fas-ligand pathway does not mediate the apoptosis in elastase-induced emphysema in mice, Sawada et al., Exp. Lung Res. 33, 277-288 (2007)]. The animals receive an orotracheal instillation of porcine pancreas elastase. The treatment of the animals with the test substance starts at the day of the instillation of the porcine pancreas elastase and extends over a period of 3 weeks. At the end of the study, the pulmonary compliance is determined and an alveolar morphometry is carried out.

[0358] A further mouse model for pulmonary emphysema is pulmonary emphysema induced by cigarette smoke and an influenza virus infection [Role of ribonuclease L in viral pathogen-associated molecular pattern/influenza virus and cigarette smoke-induced inflammation and remodeling, Zhou et al., J. Immunol. 191, 2637-2646 (2013)]. The animals are exposed to cigarette smoke for several weeks and are then exposed to an influenza virus infection. At the end of the study, a differential cell count in the bronchio-alveolar lavage fluid (BALF) is determined and an alveolar morphometry of the lung is carried out.

B-4. Animal Model of Silica-Induced Pulmonary Inflammation

[0359] An orotracheal administration of silica in mouse, rat or hamster leads to an inflammation in the lung [Involvement of leukotrienes in the pathogenesis of silica-induced pulmonary fibrosis in mice, Shimbori et al., Exp. Lung Res. 36, 292-301 (2010)]. The animals are treated on the day of the instillation of the silica with the test substance. After 24 hours, a bronchio-alveolar lavage is carried out to determine the cell content and the biomarker.

B-5. Animal Model of Silica-Induced Pulmonary Fibrosis

[0360] Silica-induced pulmonary fibrosis in mouse, rat or hamster is a widespread animal model for pulmonary fibrosis [Involvement of leukotrienes in the pathogenesis of silica-induced pulmonary fibrosis in mice, Shimbori et al., Exp. Lung Res. 36, 292-301 (2010)]. The animals receive an orotracheal instillation of silica. The treatment of the animals with the test substance starts on the day of the instillation of the silica or therapeutically a week later and extends over a period of 6 weeks. At the end of the study, a bronchio-alveolar lavage to determine the cell content and the biomarker, and also a histological assessment of pulmonary fibrosis are carried out.

B-6. Animal Model of ATP-Induced Pulmonary Inflammation

[0361] An intratracheal administration of ATP (adenosine triphosphate) on the mouse leads to inflammation in the lung [Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: An experimental study, Matsuyama et al., Respir. Res. 979 (2008)]. The animals are treated on the day of the instillation of ATP for a period of 24 h with the test substance (by gavage, by addition to feed or drinking water, using an osmotic mini pump, by subcutaneous or intraperitoneal injection or by inhalation). At the end of the experiment, a bronchio-alveolar lavage for determining the cell content and the pro-inflammatory marker is carried out.

B-7. CYP Inhibition Test

[0362] The ability of substances to inhibit the CYP enzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in humans is investigated using pooled human liver microsomes as enzyme source in the presence of standard substrates (see below) which form CYP-specific metabolites. The inhibition effects are investigated at six different concentrations of the test compounds [2.8, 5.6, 8.3, 16.7, 20 (or 25) and 50 M), compared with the extent of the CYP-specific metabolite formation of the standard substrates in the absence of the test compounds, and the corresponding IC.sub.50 values are calculated. A standard inhibitor which specifically inhibits an individual CYP isoform is always co-incubated in order to make the results between different series comparable.

[0363] The incubation of phenacetin, diclofenac, tolbutamide, dextromethorphan or midazolam with human liver microsomes in the presence of in each case six different concentrations of a test compound (as potential inhibitor) is carried out on a workstation (Tecan, Genesis, Crailsheim, Germany) Standard incubation mixtures comprise 1.3 mM NADP.sup.+, 3.3 mM MgCl.sub.26 H.sub.2O, 3.3 mM glucose 6-phosphate, glucose 6-phosphate dehydrogenase (0.4 U/ml) and 100 mM phosphate buffer (pH 7.4) in a total volume of 200 l Test compounds are preferably dissolved in acetonitrile. 96-Well plates are incubated for a defined period of time at 37 C. with pooled human liver microsomes. The reactions are stopped by addition of 100 l of acetonitrile comprising a suitable internal standard. Precipitated proteins are removed by centrifugation, and the supernatants are combined and analysed by LC-MS/MS.

B-8. Hepatocyte Assay for Determining the Metabolic Stability

[0364] The metabolic stability of test compounds towards hepatocytes is determined by incubating the compounds at low concentrations (preferably below or around 1 M) and at low cell counts (preferably at 1*10.sup.6 cells/ml) in order to ensure the greatest possible linear kinetic conditions in the experiment. Seven samples from the incubation solution are removed within a stipulated time frame for the LC-MS analysis in order to determine the half life (i.e. the degradation) of the particular compound. This half life is used to calculate various Clearance parameters (CL) and F.sub.max values (see below).

[0365] The CL and F.sub.max values are a measure of the phase 1 and phase 2 metabolism of the compounds in the hepatocytes. In order to keep the influence of the organic solvent on the enzymes in the incubation mixtures as low as possible, its concentration is generally limited to 1% (acetonitrile) or 0.1% (DMSO).

[0366] For all species and races, a hepatocyte cell count in the liver of 1.1*10.sup.8 cells/g of liver is estimated. CL parameters, the calculation of which is based on half lives which extend considerably beyond the incubation time (usually 90 minutes), can only be regarded as rough guide values.

[0367] The calculated parameters and their meaning are: [0368] F.sub.max well-stirred [%] Maximum possible bioavailability following oral application

Calculation: (1CL.sub.blood well-stirred/QH)*100 [0369] CL.sub.blood well-stirred [L/(h*kg)] Calculated blood clearance (well stirred model)

Calculation: (QH*CL.sub.intrinsic/(QH+CL.sub.intrinsic) [0370] CL.sub.intrinsic [ml/(min*kg)] Maximum ability of the liver (of the hepatocytes) to metabolize a compound (assuming that the liver blood flow is not rate-limiting)

Calculation: CL.sub.intrinsic, apparent*species-specific hepatocyte count [1.1*10.sup.8/g liver]*species-specific liver weight [g/kg] [0371] CL.sub.intrinsic, apparent [ml/(min*mg)] Normalizes the elimination constant by dividing this by the hepatocyte cell count used x (x*10.sup.6/ml)

Calculation: k.sub.el[1/min]/(cell count [x*10.sup.6]/incubation volume [ml])

(QH=species-specific liver blood flow).

[0372] Table 4 below shows for embodiment example 2 the CL and F.sub.max values from this assay following incubation of the compound with rat hepatocytes (as average value from several independent individual determinations):

TABLE-US-00007 TABLE 4 Calculated blood clearance and bioavailability following incubation with rat hepatocytes Example No. CL.sub.blood [L/(h*kg)] F.sub.max [%] 2 0.65 84.4

[0373] The specified embodiment example of the present invention thus shows in this model a good pharmacokinetic profile in vitro with a low calculated blood clearance and a high calculated bioavailability.

B-9. Metabolic Study

[0374] To determine the metabolic profile of the inventive compounds, they are incubated with liver microsomes or with primary fresh hepatocytes from various animal species (e.g. rats, dogs), and also of human origin, in order to obtain and to compare information about a very substantially complete hepatic phase I and phase II metabolism, and about the enzymes involved in the metabolism.

[0375] The inventive compounds are incubated with a concentration of about 1-10 M. To this end, stock solutions of the compounds having a concentration of 0.1-1 mM in acetonitrile are prepared, and then pipetted with 1:100 dilution into the incubation mixture. The liver microsomes are incubated at 37 C. in 50 mM potassium phosphate buffer pH 7.4 with and without NADPH-generating system consisting of 1 mM NADP.sup.+, 10 mM glucose-6-phosphate and 1 unit glucose-6-phosphate dehydrogenase. Primary hepatocytes are incubated in suspension in William's E medium, likewise at 37 C. After an incubation time of 0-4 h, the incubation mixtures are stopped with acetonitrile (final concentration about 30%) and the protein is centrifuged off at about 15 000g. The samples thus stopped are either analysed directly or stored at 20 C. until analysis.

[0376] The analysis is carried out by high-performance liquid chromatography with ultraviolet and mass spectrometry detection (HPLC-UV-MS/MS). To this end, the supernatants of the incubation samples are chromatographed with suitable C18 reversed-phase columns and variable eluent mixtures of acetonitrile and 10 mM aqueous ammonium formate solution or 0.05% aqueous formic acid. The UV chromatograms in conjunction with mass spectrometry data serve for identification, structural elucidation and quantitative estimation of the metabolites, and for quantitative determination of the metabolic reduction of the compounds according to the invention in the incubation mixtures.

B-10. Pharmacokinetic Investigations In Vivo

[0377] The substance to be examined is administered to rats, mice or dogs intravenously as a solution (for example in corresponding plasma with a small addition of DMSO or in a PEG/ethanol/water mixture), and peroral administration is effected as a solution (for example in Solutol/ethanol/water or PEG/ethanol/water mixtures) or as a suspension (e.g. in a water/tylose mixture), in each case via a gavage. After administration of the substance, blood is taken from the animals at fixed times. The blood is heparinized, then plasma is obtained therefrom by centrifugation. The test substance is quantified analytically in the plasma via LC-MS/MS. From the plasma concentration/time plots determined in this way, using an internal standard and with the aid of a validated computer program, the pharmacokinetic parameters, such as AUC (area under the concentration/time curve), C.sub.max (maximum plasma concentration), t.sub.1/2 (half life), V.sub.SS (distribution volume) and CL (clearance), and the absolute and relative bioavailability F and F.sub.rel (i.v./p.o. comparison or comparison of suspension to solution after p.o. administration), are calculated.

[0378] Table 5 below shows the pharmacokinetic parameters in rat, mouse and dog for embodiment example 2:

TABLE-US-00008 TABLE 5 Pharmacokinetic parameters for embodiment example 2 CL.sub.Plasma CLblood AUC.sub.norm i.v. t.sub.1/2 p.o. F F.sub.rel Animal species [l/h/kg] [kg*h/L] [h] [%] [%] Rat (Wistar) 0.011 93.6 8.4 100 97 Mouse (C57BL/6) 0.022 44.6 5.0 84 n.d. Dog (Beagle) 0.094 10.6 14.4 100 n.d. [n.d. = not determined].

[0379] The specified embodiment example of the present invention thus has in vivo a very low plasma clearance (CL), a long half life (t.sub.1/2), a very high exposure (AUC) and a very high bioavailability from solution (F) and also from suspension (F.sub.rel). When viewed overall, the compound according to the invention exhibits a very good pharmacokinetic profile in vivo in the investigated species rat, mouse and dog and thus appears to be suitable to a particular extent for a once-daily, oral administration in a low dosage to humans.

C. EMBODIMENT EXAMPLES OF PHARMACEUTICAL COMPOSITIONS

[0380] The compounds according to the invention can be converted to pharmaceutical formulations as follows:

Tablet:

Composition:

[0381] 100 mg of the compound according to the invention, 50 mg of lactose (monohydrate), 50 mg of corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25).fwdarw.(BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.

[0382] Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm

Preparation:

[0383] The mixture of inventive compound, lactose and starch is granulated with a 5% solution (w/w) of the PVP in water. The granules are dried and then mixed with the magnesium stearate for 5 minutes. This mixture is compressed in a conventional tablet press (see above for format of the tablet). The guide value used for the pressing is a pressing force of 15 kN.

Suspension which can be Administered Orally:

Composition:

[0384] 1000 mg of the compound according to the invention, 1000 mg of ethanol (96%), 400 mg of Rhodigel (xanthan gum from FMC, Pennsylvania, USA) and 99 g of water.

[0385] 10 ml of oral suspension correspond to a single dose of 100 mg of the compound according to the invention.

Preparation:

[0386] The Rhodigel is suspended in ethanol; the compound according to the invention is added to the suspension. The water is added while stirring. The mixture is stirred for about 6 h before swelling of the Rhodigel is complete.

Solution for Oral Administration:

Composition:

[0387] 500 mg of the compound according to the invention, 2.5 g of polysorbate and 97 g of polyethylene glycol 400. 20 g of oral solution correspond to a single dose of 100 mg of the compound according to the invention.

Preparation:

[0388] The compound according to the invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring. The stirring operation is continued until dissolution of the compound according to the invention is complete.

i.v. Solution:

[0389] The compound according to the invention is dissolved in a concentration below the saturation solubility in a physiologically acceptable solvent (e.g. isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%). The solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection vessels.