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2,5-DISUBSTITUTED CYCLOPENTANE CARBOXYLIC ACIDS FOR THE TREATMENT OF RESPIRATORY TRACT DISEASES

20170114049 · 2017-04-27

    Inventors

    Cpc classification

    International classification

    Abstract

    The present application relates to novel 2,5-disubstituted cyclopentanecarboxylic acid derivatives, to processes for preparation thereof, to the use thereof alone or in combinations for treatment and/or prevention of diseases and to the use thereof for production of medicaments for treatment and/or prevention of diseases, especially for treatment and/or prevention of respiratory, pulmonary and cardiovascular disorders.

    Claims

    1. Compound of the formula (I) ##STR00058## in which A is O or S, n is the number 1 or 2, and R.sup.1 is hydrogen, methyl, fluoromethyl, difluoromethyl or trifluoromethyl, or a salt, solvate or solvate of a salt of this compound.

    2. Compound of the formula (I) according to claim 1 in which A is O, n is the number 1 or 2, and R.sup.1 is hydrogen, methyl or trifluoromethyl, or a salt, solvate or solvate of a salt of this compound.

    3. Compound of the formula (I) according to claim 1 in which A is O, n is the number 2, and R.sup.1 is hydrogen, methyl or trifluoromethyl, or a salt, solvate or solvate of a salt of this compound.

    4. Compound according to claim 1 having the formula (I-A) or (I-B) ##STR00059## in which the groups bonded to the central cyclopentane ring have a relative trans arrangement, or a mixture of these compounds where A, n and/or R.sup.1 are each identical in such a mixture of (I-A) and I-B), or a salt, solvate or solvate of a salt of these compounds or the mixture thereof.

    5. Compound according to claim 1 having the formula (I-A) ##STR00060## in enantiomerically pure form, with a (1S,2R,5S) configuration on the central cyclopentane ring as shown, or a salt, solvate or solvate of a salt of this compound.

    6. Process for preparing a compound as defined in claim 1, wherein a compound of the formula (II) ##STR00061## is alkylated in the presence of a base with a compound of the formula (III) ##STR00062## and X is a leaving group, for example chlorine, bromine, iodine, mesylate, triflate or tosylate, to give a compound of the formula (IV) ##STR00063## and then the 2-(trimethylsilyl)ethyl ester group is detached with the aid of an acid or a fluoride reagent to give the carboxylic acid of the formula (I) ##STR00064## and, if appropriate, the compounds of the formula (I) thus obtained are separated into their enantiomers and/or diastereomers and/or converted with the appropriate (i) solvents and/or (ii) bases to their solvates, salts and/or solvates of the salts.

    7. Compound as defined in claim 1 for treatment and/or prevention of diseases.

    8. Compound as defined in claim 1 for use in a method for treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in 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 the sequelae thereof such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome.

    9. Use of a compound as defined in claim 1 for production of a medicament for treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in 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 the sequelae thereof such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome.

    10. Medicament comprising a compound as defined in claim 1 in combination with one or more inert, nontoxic, pharmaceutically suitable excipients.

    11. Medicament comprising a compound 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.

    12. Medicament according to claim 10 for treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in 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 the sequelae thereof such as stroke, myocardial infarction and peripheral arterial occlusive disease, and also of chronic kidney diseases and Alport's syndrome.

    13. Method for treatment and/or prevention of chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, pulmonary hypertension in 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 the sequelae thereof 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 at least one compound as defined in claim 1, or of a medicament comprising the compound in combination with one or more inert, nontoxic, pharmaceutically suitable excipients.

    Description

    A. EXAMPLES

    [0224] Abbreviations and Acronyms: [0225] abs. absolute [0226] Ac acetyl [0227] aq. aqueous, aqueous solution [0228] br. broad (in NMR signal) [0229] Ex. Example [0230] Bu butyl [0231] c concentration [0232] ca. circa, about [0233] cat. catalytic [0234] CI chemical ionization (in MS) [0235] d doublet (in NMR) [0236] d day(s) [0237] (dba).sub.3Pd2 tris(dibenzylideneacetone)dipalladium(0) [0238] TLC thin-layer chromatography [0239] DCI direct chemical ionization (in MS) [0240] dd doublet of doublets (in NMR) [0241] DEAD diethyl azodicarboxylate [0242] DMF N,N-dimethylformamide [0243] DMSO dimethyl sulphoxide [0244] dt doublet of triplets (in NMR) [0245] ee enantiomeric excess [0246] EI electron impact ionization (in MS) [0247] ent enantiomerically pure, enantiomer [0248] eq. equivalent(s) [0249] ESI electrospray ionization (in MS) [0250] Et ethyl [0251] h hour(s) [0252] HPLC high-pressure, high-performance liquid chromatography [0253] iPr isopropyl [0254] conc concentrated (in the case of a solution) [0255] LC liquid chromatography [0256] LC/MS liquid chromatography-coupled mass spectrometry [0257] Lit. literature (reference) [0258] m multiplet (in NMR) [0259] Me methyl [0260] min minute(s) [0261] MPLC medium-pressure liquid chromatography (on silica gel; also referred to as flash chromatography) [0262] Ms methanesulphonyl (mesyl) [0263] MS mass spectrometry [0264] NMO N-methylmorpholine N-oxide [0265] NMR nuclear magnetic resonance spectrometry [0266] Pd/C palladium on activated carbon [0267] Pr propyl [0268] q (or quart) quartet (in NMR) [0269] qd quartet of doublets (in NMR) [0270] quant. quantitative (in chemical yield) [0271] quint quintet (in NMR) [0272] rac racemic, racemate [0273] Rf retention index (in TLC) [0274] RP reverse phase (in HPLC) [0275] RT room temperature [0276] R.sub.f retention time (in HPLC, LC/MS) [0277] s singlet (in NMR) [0278] sept septet (in NMR) [0279] SFC supercritical liquid chromatography [0280] t triplet (in NMR) [0281] tBu tent-butyl [0282] td triplet of doublets (in NMR) [0283] TFA trifluoroacetic acid [0284] THF tetrahydrofuran [0285] UV ultraviolet spectrometry [0286] v/v ratio by volume (of a solution) [0287] Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

    [0288] HPLC- and LC/MS Methods:

    [0289] Method 1 (LC/MS):

    [0290] Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T3 1.8 501 mm; eluent A: 1 1 water+0.25 ml 99% formic acid, eluent B: 1 1 acetonitrile+0.25 ml 99% formic acid; gradient: 0.0 min 90% A.fwdarw.1.2 min 5% A.fwdarw.2.0 min 5% A; oven: 50 C.; flow rate: 0.40 ml/min; UV detection: 208-400 nm.

    [0291] Method 2 (LC/MS):

    [0292] Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column: Thermo Hypersil GOLD 1.9 501 mm; eluent A: 1 1 water+0.5 ml 50% formic acid, eluent B: 1 1 acetonitrile+0.5 ml 50% formic acid; gradient: 0.0 min 97% A.fwdarw.0.5 min 97% A.fwdarw.3.2 min 5% A.fwdarw.4.0 min 5% A; oven: 50 C.; flow rate: 0.3 ml/min; UV detection: 210 nm.

    [0293] Method 3 (LC/MS):

    [0294] MS instrument: Waters Micromass QM; HPLC instrument: Agilent 1100 series; column: Agilent ZORBAX Extend-C18 3.5, 3.050 mm; eluent A: 1 1 water+0.01 mol ammonium carbonate, eluent B: 1 1 acetonitrile; gradient: 0.0 min 98% A.fwdarw.0.2 min 98% A.fwdarw.3.0 min 5% A.fwdarw.4.5 min 5% A; oven: 40 C.; flow rate: 1.75 ml/min; UV detection: 210 nm.

    [0295] Method 4 (Preparative HPLC):

    [0296] Column: Reprosil C18, 10 m, 25030 mm; eluent: acetonitrile/water with 0.1% TFA; gradient: 0-5.00 min 10:90, sample injection at 3.00 min; 5.00-23.00 min to 95:5; 23.00-30.00 min 95:5; 30.00-30.50 min to 10:90; 30.50-31.20 min 10:90.

    [0297] Method 5 (Preparative HPLC):

    [0298] Column: Reprosil C18, 10 m, 25030 mm; eluent: acetonitrile/water with 0.1% TFA; gradient: 0-5.00 min 10:90, sample injection at 3.00 min; 5.00-20.00 min to 95:5; 20.00-30.00 min 95:5; 30.00-30.50 min to 10:90; 30.50-31.20 min 10:90.

    [0299] Method 6 (Preparative HPLC):

    [0300] Column: Reprosil C18, 10 m, 12530 mm; eluent: acetonitrile/water with 0.1% TFA; gradient: 0-6.00 min 35:65, sample injection at 3.00 min; 6.00-27.00 min to 80:20; 27.00-30.00 min 95:5; 30.00-33.00 min to 35:65.

    [0301] Method 7 (Preparative HPLC):

    [0302] Column: Reprosil-Pur C18, 10 m; eluent: water/methanol; gradient: 70:30.fwdarw.50:50 (to 6 min).fwdarw.20:80 (to 22 min), to 75 min 20:80.

    [0303] Method 8 (Preparative HPLC):

    [0304] Column: Reprosil-Pur C18, 10 m; eluent: water/methanol; gradient: 70:30.fwdarw.50:50 (to 6 min).fwdarw.20:80 (to 20 min), to 115 min 20:80.

    [0305] Method 9 (Preparative HPLC):

    [0306] Column: Reprosil-Pur C18, 10 m; eluent: water/methanol; gradient: 70:30.fwdarw.50:50 (to 6 min).fwdarw.20:80 (to 21 min), to 75 min 20:80.

    [0307] Method 10 (Preparative HPLC):

    [0308] Column: Reprosil-Pur C18, 10 m; eluent: water/methanol; gradient: 70:30.fwdarw.50:50 (to 6 min).fwdarw.20:80 (to 25 min), to 75 min 20:80.

    [0309] Method 11 (Preparative HPLC):

    [0310] Column: Reprosil-Pur C18, 10 um; eluent: water/methanol; gradient: 70:30.fwdarw.50:50 (to 6 min).fwdarw.20:80 (to 20 min), to 75 min 20:80.

    [0311] Further Details:

    [0312] The percentages in the example and test descriptions which follow are, unless indicated otherwise, percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for the liquid/liquid solutions are based in each case on volume.

    [0313] Purity figures are generally based on corresponding peak integrations in the LC/MS chromatogram, but may additionally also have been determined with the aid of the .sup.1H NMR spectrum. If no purity is indicated, the purity is generally 100% according to automated peak integration in the LC/MS chromatogram, or the purity has not been determined explicitly.

    [0314] Stated yields in % of theory are generally corrected for purity if a purity of <100% is indicated. In solvent-containing or contaminated batches, the formal yield may be >100%; in these cases the yield is not corrected for solvent or purity.

    [0315] The descriptions of the coupling patterns of .sup.1H NMR signals that follow have in some cases been taken directly from the suggestions of the ACD SpecManager (ACD/Labs Release 12.00, Product version 12.5) and have not necessarily been strictly scrutinized. In some cases, the suggestions of the SpecManager were adjusted manually. Manually adjusted or assigned descriptions are generally based on the optical appearance of the signals in question and do not necessarily correspond to a strict, physically correct interpretation. In general, the stated chemical shift refers to the centre of the signal in question. In the case of broad multiplets, an interval is given. Signals obscured by solvent or water were either tentatively assigned or have not been listed.

    [0316] Melting points and melting-point ranges, if stated, are uncorrected.

    [0317] All reactants or reagents whose preparation is not described explicitly hereinafter were purchased commercially from generally accessible sources. For all other reactants or reagents whose preparation likewise is not described hereinafter and which were not commercially obtainable or were obtained from sources which are not generally accessible, a reference is given to the published literature in which their preparation is described.

    [0318] In the intermediates and working examples described hereinafter, a 1RS,2RS,5SR identifier in the IUPAC name of the example in question, in conjunction with the term racemate, means that this is a racemic mixture of the 1R,2R,5S enantiomer (.fwdarw.1st letter in each case after the position number in 1RS,2RS,5SR) with the corresponding 1S,2S,5R enantiomers (.fwdarw.2nd letter in each case after the position number). The 1RS,2RS,5SR identifier in conjunction with the statements enantiomer 1 and enantiomer 2 means that these are the two enantiomers in separate, isolated form, without having undertaken an assignment of the absolute configuration (1R,2R,5S or 1S,2S,5R) to these enantiomers. Similar identifiers such as 1RS,2SR,5RS that arise from the altered priority and/or sequence of main constituents owing to the IUPAC nomenclature rules should be interpreted in an analogous manner according to these instructions.

    [0319] For the simplified representation of the relative stereochemical configuration of chiral centres, the structural formulae of racemic example compounds hereinbelow show only the structural formula of one of the enantiomers involved; as is evident from the term racemate in the associated IUPAC name, the second enantiomer with the respective opposite absolute configuration is always included in these cases.

    [0320] Starting Compounds and Intermediates:

    Example 1A

    6-(Trifluoromethyl)-1,2,3-benzotriazin-4(3H)-one

    [0321] ##STR00028##

    [0322] To a suspension of 24.4 g (119.51 mmol) of 2-amino-5-(trifluoromethyl)benzamide in 174 ml of a 2:1 mixture of water and conc. hydrochloric acid at 0 C. was gradually added a solution of 9.08 g (131.47 mmol) of sodium nitrite in 74 ml of water, in the course of which the internal temperature was kept below 5 C. After stirring at bath temperature 0 C. for 30 minutes, while continuing to cool with an ice bath, 74 ml (0.74 mol) of 10 M sodium hydroxide solution were added, in the course of which the internal temperature rose to about 20 C. A solution formed at first, from which a suspension then arose, which was diluted with 100 ml of water for better stirrability. After stirring at RT for 1.5 h, the mixture was cautiously acidified with conc. hydrochloric acid (pH=2). The precipitate formed was filtered off and washed three times with water. After drying under air and then under reduced pressure, 24.74 g (96% of theory) of the title compound were obtained.

    [0323] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=15.31 (br. s, 1H), 8.46 (s, 1H), 8.40 (d, 2H).

    [0324] LC/MS (Method 1, ESIpos): R.sub.t=0.78 min, m/z=216 [M+H].sup.+.

    Example 2A

    6-Methyl-1,2,3-benzotriazin-4(3H)-one

    [0325] ##STR00029##

    [0326] To a suspension of 32.0 g (213.08 mmol) of 2-amino-5-methylbenzamide in 300 ml of a 2:1 mixture of water and conc. hydrochloric acid at 0 C. was gradually added a solution of 16.17 g (234.38 mmol) of sodium nitrite in 120 ml of water, in the course of which the internal temperature was kept below 5 C. After stirring at bath temperature 0 C. for 30 minutes, while continuing to cool with an ice bath, 120 ml (1.2 mol) of 10 M sodium hydroxide solution were added, in the course of which the internal temperature rose to about 20 C. and solids that were present went into solution. After stirring at RT for 1 h, the mixture was cautiously acidified with conc. hydrochloric acid (pH=2). The precipitate formed was filtered off and washed three times with water. After drying under air and under reduced pressure, 33.80 g (98% of theory) of the title compound were obtained.

    [0327] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=14.85 (br. s, 1H), 8.08 (d, 1H), 8.02 (s, 1H), 7.90 (d, 1H).

    [0328] LC/MS (Method 3, ESIpos): R.sub.t=1.40 min, m/z=162 [M+H].sup.+.

    Example 3A

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-[4-(benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylate (racemate)

    [0329] ##STR00030##

    [0330] Step 1:

    2-(Trimethylsilyl)ethyl 2-[4-(benzyloxy)phenyl]-2-hydroxybicyclo[2.2.1]heptane-7-carboxylate

    [0331] ##STR00031##

    [0332] To a solution of 24.30 g (95.52 mmol) of exo-2-(trimethylsilyl)ethyl 2-oxobicyclo[2.2.1]heptane-7-carboxylate [WO 96/15096, Example 360/Stage 1] in 60 ml of THF were gradually added, at internal temperature about 5 C. under argon, 114.62 ml (114.62 mmol) of a 1 M solution of 4-(benzyloxy)phenylmagnesium bromide in THF, in the course of which the internal temperature rose to not more than 0 C. The cold bath was then removed and the mixture was stirred for a further 1 h. The mixture was then admixed with 200 ml of 5% citric acid solution and extracted twice with dichloromethane. The combined organic phases were dried over magnesium sulphate and concentrated. The residue was purified by means of flash chromatography on 1 kg of silica gel (eluent: cyclohexane/ethyl acetate 9:1). 28.70 g (66% of theory, 97% purity) of the title compound were obtained.

    [0333] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=7.49-7.27 (m, 7H), 6.95 (d, 2H), 5.09 (s, 2H), 5.05 (s, 1H), 4.10-4.00 (m, 2H), 2.44-2.37 (m, 1H), 2.33-2.24 (m, 1H), 2.23-2.11 (m, 1H), 1.78-1.60 (m, 1H), 1.52-1.26 (m, 4H), 0.95-0.80 (m, 2H), 0.00 (s, 9H).

    [0334] LC/MS (Method 1, ESIpos): R.sub.t=3.15 min, m/z=421 [M+HH.sub.2O].sup.+.

    [0335] Step 2:

    2-(Trimethylsilyl)ethyl 2-[4-(benzyloxy)phenyl]bicyclo[2.2.1]hept-2-ene-7 -carboxylate

    [0336] ##STR00032##

    [0337] To a solution of 28.70 g (63.466 mmol) of the compound from Example 3A/Step 1 in 150 ml of dichloromethane under argon were added, at about 0 C., first 26.50 ml (190.40 mmol) of triethylamine and then, gradually, 9.82 ml (126.93 mmol) of methanesulphonyl chloride, in the course of which the internal temperature did not exceed 5 C. This was followed by stirring at 0 C. for a further 1.5 h. Thereafter, the mixture was diluted with dichloromethane and extracted with water. The organic phase was dried over magnesium sulphate and concentrated, and the residue was purified by means of flash chromatography on 1 kg of silica gel (eluent: cyclohexane/ethyl acetate 95:5). 20.06 g (75% of theory) of the title compound were obtained.

    [0338] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=7.48-7.28 (m, 7H), 6.97 (d, 2H), 6.30 (d, 1H), 5.11 (s, 2H), 4.15-4.06 (m, 2H), 3.43 (br. s, 1H), 3.06 (br. s, 1H), 1.85-1.71 (m, 2H), 1.17-1.06 (m, 1H), 1.04-0.87 (m, 3H), 0.04 (s, 9H).

    [0339] LC/MS (Method 1, ESIpos): R.sub.t=1.61 min, m/z=421 [M+H].sup.+.

    [0340] Step 3:

    2-(Trimethylsilyl)ethyl 2-[4-(benzyloxy)phenyl]-2,3-dihydroxybicyclo[2.2.1]heptane-7-carboxylate

    [0341] ##STR00033##

    [0342] To a degassed solution of 25.37 g (60.314 mmol, not corrected for purity) of the compound from Example 3A/Step 2 in 150 ml of THF under argon was added, at 0 C., a degassed solution of 15.90 g (135.71 mmol) of N-methylmorpholine N-oxide (NMO) in 42 ml of water under argon. To this mixture were then gradually added, while stirring, 116 ml (9.05 mmol) of a 2.5% solution of osmium tetroxide in tert-butanol. This was followed by stirring at 0 C. for a further 1 h. After stirring at RT for a further 16 h, the mixture was diluted with 150 ml of ethyl acetate and extracted twice with 250 ml each time of 10% citric acid solution, twice with 300 ml each time of saturated sodium hydrogencarbonate solution and twice with 300 ml each time of saturated sodium chloride solution. The organic phase was then dried over sodium sulphate and concentrated. 27.51 g (75% of theory, 75% purity) of the title compound were obtained.

    [0343] LC/MS (Method 1, ESIpos): R.sub.t=1.40 min, m/z=437 [M+HH.sub.2O].sup.+.

    [0344] Step 4:

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-[4-(benzyloxy)benzoyl]-5-formylcyclopentanecarboxylate (racemate)

    [0345] ##STR00034##

    [0346] Method A:

    [0347] To a solution of 27.42 g (60.32 mmol, not corrected for purity) of the compound from Example 3A/Step 3 in 170 ml of methanol under argon were added gradually, at bath temperature 15 C., 30.96 g (66.34 mmol, 95% purity) of lead tetraacetate. The mixture was stirred at 15 C. for 1 h. After warming to RT, the mixture was filtered through Celite and the filtration residue was washed three times with 50 ml each time of methanol. The filtrate was concentrated and the residue was taken up in 500 ml of dichloromethane and 500 ml of water without onset of a phase separation. Thereafter, the mixture was filtered through silica gel and the silica gel was washed with dichloromethane. After phase separation, the aqueous phase was extracted once again with 150 ml of dichloromethane. The combined organic phases were dried over sodium sulphate and concentrated. 27.1 g (86% of theory, 87% purity) of the title compound were obtained.

    [0348] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=9.72 (d, 1H), 8.02 (d, 2H), 7.53-7.34 (m, 5H), 7.18 (d, 2H), 5.25 (s, 2H), 4.17 (q, 1H), 4.09 (dd, 2H), 3.74 (t, 1H), 3.23-3.14 (m, 1H), 2.24-2.13 (m, 1H), 2.08-1.88 (m, 2H), 1.61-1.49 (m, 1H), 0.87-0.79 (m, 2H), 0.00 (s, 9H).

    [0349] LC/MS (Method 1, ESIpos): R.sub.t=1.45 min, m/z=425 [M+H-28].sup.+.

    [0350] Method B:

    [0351] To a solution of 69.0 g (131 mmol, about 80% purity) of the compound from Example 3A/Step 2 in a mixture of acetone/water/THF (3:1:1) were added, at 0 C. under argon, first 76.87 g (656 mmol) of N-methylmorpholine N-oxide (NMO) and then 2.09 g (8.20 mmol) of a 4% solution of osmium tetroxide in water. The mixture was stirred at RT for 3 days. Then 105.26 g (492 mmol) of sodium periodate were added and stirring of the mixture at RT continued overnight. After ethyl acetate and 10% aqueous citric acid had been added, the aqueous phase was removed and extracted once with ethyl acetate. The combined organic phases were washed once with saturated sodium hydrogencarbonate solution and then with magnesium silicate (Florisil). After filtration, the filter residue was washed with ethyl acetate. After the filtrate had been concentrated, the residue thus obtained was combined with the residues from two similarly conducted prior experiments [amounts of the compound from Example 3A used: 3.0 g (7.13 mmol) and 3.2 g (7.61 mmol)] and purified jointly by means of flash chromatography (silica gel, eluent: petroleum ether/ethyl acetate 8:2). In this way, a total of 53 g (58% of theory taking account of the prior experiments, 89% purity) of the title compound were obtained.

    [0352] Step 5:

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-[4-(benzyloxy)benzoyl]-5-(hydroxymethyl)cyclopentane-carboxylate (racemate)

    [0353] ##STR00035##

    [0354] To a solution of 27.0 g (59.65 mmol, not corrected for purity) of the compound from Example 3A/Step 4 in 135 ml of ethanol were added gradually, at RT, 677 mg (17.895 mmol) of sodium borohydride, and the mixture was stirred at RT for 30 min Subsequently, the mixture was admixed with 400 ml each of ammonium chloride solution and water and extracted twice with 300 ml each time of ethyl acetate. The combined organic phases were dried over sodium sulphate and concentrated. 21.90 g (70% of theory, 87% purity) of the title compound were obtained.

    [0355] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=7.95 (d, 2H), 7.48-7.31 (m, 5H), 7.12 (d, 2H), 5.20 (s, 2H), 4.64 (t, 1H), 4.07-3.98 (m, 3H), 3.53-3.45 (m, 1H), 3.40-3.34 (m, 1H), 2.94 (t, 1H), 2.34-2.23 (m, 1H), 2.12-2.01 (m, 1H), 1.90-1.78 (m, 1H), 1.67-1.47 (m, 2H), 0.82-0.75 (m, 2H), 0.00 (s, 9H).

    [0356] LC/MS (Method 1, ESIpos): R.sub.t=1.34 min, m/z=455 [M+H].sup.+.

    [0357] Step 6

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-[4-(benzyloxy)benzoyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylate (racemate)

    [0358] ##STR00036##

    [0359] To a solution of 500 mg (1.10 mmol, not corrected for purity) of the compound from Example 3A/Step 5 in 6 ml of THF under argon were added 243 mg (1.65 mmol) of 1,2,3-benzotriazin-4(3H)-one and 1.11 g (5.50 mmol) of tributylphosphine. Subsequently, 1.50 ml (3.30 mmol) of a 40% solution of diethyl azodicarboxylate (DEAD) in toluene were added dropwise at 0 C. The mixture was stirred at RT for about 1 h, then diluted with ethyl acetate and extracted twice with 5 ml each time of water and twice with saturated sodium chloride solution. The organic phase was dried over magnesium sulphate and concentrated. The residue was purified by means of preparative HPLC (Method 6). 334 mg (52% of theory) of the title compound were obtained.

    [0360] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.44 (dd, 1H), 8.38 (d, 1H), 8.27 (td, 1H), 8.15-8.08 (m, 3H), 7.65-7.48 (m, 5H), 7.29 (d, 2H), 5.37 (s, 2H), 4.74-4.62 (m, 2H), 4.26 (q, 1H), 3.40 (t, 1H), 3.13-3.01 (m, 1H), 2.36-2.25 (m, 1H), 2.21-2.10 (m, 1H), 1.96-1.84 (m, 1H), 1.77-1.65 (m, 1H), 0.53-0.46 (m, 2H), 0.17 (s, 9H).

    [0361] LC/MS (Method 1, ESIpos): R.sub.t=1.51 min, m/z=584 [M+H].sup.+.

    Example 4A

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-(4-hydroxybenzoyl)-5- [(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylate (racemate)

    [0362] ##STR00037##

    [0363] To a solution of 270 mg (0.46 mmol) of the compound from Example 3A in 12 ml of ethyl acetate under argon were added 25 mg (0.024 mmol) of palladium on activated carbon (10% Pd). This was followed by hydrogenation under standard pressure for 42 h. The mixture was then filtered through kieselguhr, the filter residue was washed with ethyl acetate and the filtrate was concentrated. The residue thus obtained was taken up in a little dichloromethane and purified by column chromatography (25 g of silica gel, eluent: cyclohexane/ethyl acetate 7:3). 165 mg (72% of theory, 100% purity) of the title compound were obtained.

    [0364] .sup.1H-NMR (400 MHz, CDCl.sub.3): [ppm]=8.37 (d, 1H), 8.16 (d, 1H), 7.98-7.88 (m, 3H), 7.84-7.77 (m, 1H), 6.89 (d, 2H), 6.67 (br. s, 1H), 4.77-4.70 (m, 1H), 4.68-4.60 (m, 1H), 4.20-4.10 (m, 1H), 3.88-3.81 (m, 2H), 3.46 (t, 1H), 3.08-2.94 (m, 1H), 2.19-2.04 (m, 1H), 2.01-1.86 (m, 2H), 1.72-1.64 (m, partially hidden, 1H), 0.63-0.55 (m, 2H), 0.09 (s, 9H).

    [0365] LC/MS (Method 1, ESIpos): R.sub.t=1.23 min, m/z=494 [M+H].sup.+.

    Example 5A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetra-hydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylate (racemate)

    [0366] ##STR00038##

    [0367] To a solution of 164 mg (0.33 mmol) of the compound from Example 4A in 3.7 ml of acetonitrile under argon were added 92 mg (0.66 mmol) of potassium carbonate and 71 mg (0.40 mmol) of 4-(bromomethyl)tetrahydropyran, and the mixture was stirred under reflux for 20 h. Subsequently, a further 36 mg (0.20 mmol) of 4-(bromomethyl)tetrahydropyran were added and the mixture was stirred under reflux for another 7 h. Thereafter, another 71 mg (0.40 mmol) of 4-(bromomethyl)-tetrahydropyran and 46 mg (0.33 mmol) of potassium carbonate were added and the mixture was stirred under reflux for a further 17 h. After cooling to RT, the mixture was diluted with 30 ml of water and 30 ml of ethyl acetate, and, after the phases had been separated, the aqueous phase was extracted once with 30 ml of ethyl acetate. The combined organic phases were dried over sodium sulphate, filtered and concentrated. The residue was purified by means of preparative HPLC (Method 4). The combined product-containing fractions were adjusted to pH 7-8 with saturated aqueous sodium hydrogencarbonate solution, then concentrated down to a residue of aqueous phase, and the latter was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulphate and concentrated, and the residue was dried under reduced pressure. 85 mg (42% of theory, 97% purity) of the title compound were obtained.

    [0368] .sup.1H-NMR (400 MHz, CDCl.sub.3): [ppm]=8.37 (dd, 1H), 8.15 (d, 1H), 7.99-7.91 (m, 3H), 7.83-7.76 (m, 1H), 6.91 (d, 2H), 4.77-4.68 (m, 1H), 4.67-4.59 (m, 1H), 4.23-4.13 (m, 1H), 4.03 (dd, 2H), 3.89-3.80 (m, 4H), 3.50-3.39 (m, partly concealed, 3H), 3.09-2.93 (m, 1H), 2.19-2.03 (m, 2H), 2.01-1.86 (m, 2H), 1.76 (dd, 2H), 1.71-1.62 (m, partly concealed, 1H), 1.47 (qd, 2H), 0.65-0.53 (m, 2H), 0.09 (s, 9H).

    [0369] LC/MS (Method 1, ESIpos): R.sub.t=1.43 min, m/z=592 [M+H].sup.+.

    Example 6A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylate (racemate)

    [0370] ##STR00039##

    [0371] To a solution of 3.88 g (7.47 mmol, 95% purity) of the compound from Example 4A in 41 ml of DMF under argon were added 1.01 g (8.96 mmol) of potassium tert-butoxide. After stirring at RT for 5 min, 1.73 g (8.96 mmol) of 4-(2-bromoethyl)tetrahydro-2H-pyran were added, and the mixture was stirred at bath temperature 100 C. for 2 h. After cooling to RT, water and ethyl acetate were added to the mixture. After the phases had been separated, the aqueous phase was extracted once with ethyl acetate. The combined organic phases were washed once with saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was purified by means of column chromatography (300 g of silica gel, eluent: cyclohexane/ethyl acetate 7:3). 2.91 g (64% of theory, 99% purity) of the title compound were obtained.

    [0372] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.27 (d, 1H), 8.20 (d, 1H), 8.10 (t, 1H), 7.97-7.89 (m, 3H), 7.03 (d, 2H), 4.57-4.44 (m, 2H), 4.14-4.03 (m, 3H), 3.82 (dd, 2H), 3.63-3.46 (m, 2H), 3.31-3.17 (m, partly concealed, 3H), 2.97-2.84 (m, 1H), 2.18-2.05 (m, 1H), 2.04-1.92 (m, 1H), 1.80-1.48 (m, 6H), 1.29-1.13 (m, 3H), 0.37-0.26 (m, 2H), -0.17 (s, 9H).

    [0373] LC/MS (Method 1, ESIpos): R.sub.t=1.46 min, m/z=606 [M+H].sup.+.

    Example 7A

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-[4-(benzyloxy)benzoyl]-5-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}cyclopentanecarboxylate (racemate)

    [0374] ##STR00040##

    [0375] To a solution of 13.88 g (30.53 mmol, not corrected for purity) of the compound from Example 3A/Step 5 in 200 ml of toluene under argon were added 7.88 g (36.64 mmol) of the compound from Example 1A and 9.88 g (48.85 mmol) of tributylphosphine. Subsequently, 13.90 ml (30.53 mmol) of a 40% solution of diethyl azodicarboxylate in toluene was added dropwise at 0 C. The mixture was stirred at RT for 1 day and then concentrated. The residue was purified by means of flash chromatography (1 kg of silica gel, eluent: cyclohexane/ethyl acetate 9:1). 9.06 g (44% of theory, 98% purity) of the title compound were obtained.

    [0376] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.52 (s, 1H), 8.47-8.43 (m, 2H), 7.95 (d, 2H), 7.48-7.30 (m, 5H), 7.12 (d, 2H), 5.20 (s, 2H), 4.60-4.50 (m, 2H), 4.10 (q, 1H), 3.65-3.49 (m, 2H), 3.25 (t, 1H), 2.96-2.83 (m, 1H), 2.19-2.07 (m, 1H), 2.07-1.95 (m, 1H), 1.80-1.68 (m, 1H), 1.63-1.50 (m, 1H), 0.39-0.22 (m, 2H), 0.18 (s, 9H).

    [0377] LC/MS (Method 1, ESIpos): R.sub.t=1.57 min, m/z=652 [M+H].sup.+.

    Example 8A

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-(4-hydroxybenzoyl)-5-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}cyclopentanecarboxylate (racemate)

    [0378] ##STR00041##

    [0379] To a solution of 9.05 g (13.89 mmol) of the compound from Example 7A in a mixture of 100 ml of ethyl acetate and 100 ml of ethanol under argon were added 1.05 g (16.66 mmol) of ammonium formate and 369 mg (0.35 mmol) of palladium on activated carbon (10% Pd). The mixture was then stirred at 75 C. for 1 h. Thereafter, a further 105 mg (1.67 mmol) of ammonium formate were added, and the mixture was stirred once again at 75 C. for 30 min After cooling to RT, the mixture was filtered through kieselguhr, the filter residue was washed with ethyl acetate and ethanol, and the filtrate was concentrated. 7.86 g (100% of theory) of the title compound were obtained.

    [0380] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=10.40 (br. s, 1H), 8.52 (s, 1H), 8.47-8.42 (m, 2H), 7.85 (d, 2H), 6.84 (d, 2H), 4.60-4.51 (m, 2H), 4.05 (q, 1H), 3.64-3.48 (m, 2H), 3.23 (t, 1H), 2.96-2.82 (m, 1H), 2.17-2.05 (m, 1H), 2.05-1.94 (m, 1H), 1.79-1.67 (m, 1H), 1.62-1.49 (m, 1H), 0.38-0.22 (m, 2H), 0.18 (s, 9H).

    [0381] LC/MS (Method 1, ESIpos): R.sub.t=1.31 min, m/z=562 [M+H].sup.+.

    Example 9A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylate (racemate)

    [0382] ##STR00042##

    [0383] To a solution of 1.07 g (1.90 mmol) of the compound from Example 8A in 20 ml of DMF under argon were added 256 mg (2.28 mmol) of potassium tert-butoxide. After stirring at RT for 5 min, 408 mg (2.28 mmol) of 4-(bromomethyl)tetrahydropyran were added, and the mixture was stirred at bath temperature 100 C. for 2 h. Subsequently, a further 136 mg (0.76 mmol) of 4-(bromomethyl)-tetrahydropyran were added and the mixture was stirred at bath temperature 100 C. for another 2 h. After cooling to RT, the mixture was combined with the reaction mixtures from two similarly conducted prior experiments (batch size in each case 47 mg (0.08 mmol) of the compound from Example 8A). After removing the DMF, 60 ml of water and 60 ml of ethyl acetate were added to this combined mixture. After the phases had been separated, the aqueous phase was extracted once with 30 ml of ethyl acetate. The combined organic phases were dried over sodium sulphate, filtered and concentrated. The residue was taken up in a mixture of cyclohexane and ethyl acetate (9:1) and purified by means of column chromatography (120 g of silica gel, eluent: cyclohexane/ethyl acetate 9:1). 590 mg (47% of theory, purity 100%) of the title compound were obtained.

    [0384] .sup.1H NMR (400 MHz, CDCl.sub.3): [ppm]=8.66 (s, 1H), 8.28 (d, 1H), 8.14 (dd, 1H), 7.95 (d, 2H), 6.92 (d, 2H), 4.78-4.62 (m, 2H), 4.21-4.13 (m, 1H), 4.03 (dd, 2H), 3.89-3.81 (m, 4H), 3.50-3.40 (m, 3H), 3.07-2.93 (m, 1H), 2.19-2.03 (m, 2H), 2.03-1.87 (m, 2H), 1.76 (dd, 2H), 1.72-1.61 (m, 1H), 1.47 (qd, 2H), 0.63-0.53 (m, 2H), -0.09 (s, 9H).

    [0385] LC/MS (Methode 1, ESIpos): R.sub.t=1.51 min, m/z=560 [M+H].sup.+.

    Example 10A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylate (racemate)

    [0386] ##STR00043##

    [0387] To a solution of 250 mg (0.45 mmol) of the compound from Example 8A in 4.5 ml DMF under argon were added 60 mg (0.53 mmol) of potassium tert-butoxide. After stirring at RT for 5 min, 103 mg (0.53 mmol) of 4-(2-bromoethyl)tetrahydro-2H-pyran were added, and the mixture was stirred at bath temperature 100 C. for 1 h. After cooling to RT, 60 ml of water and 60 ml of tert-butyl methyl ether were added to the reaction mixture. After the phases had been separated, the aqueous phase was extracted once with 30 ml of tert-butyl methyl ether and twice with 50 ml each time of ethyl acetate. The combined organic phases were dried over sodium sulphate, filtered and concentrated. The residue was taken up in dichloromethane and purified by means of column chromatography (25 g silica gel, eluent: cyclohexane/ethyl acetate 7:3). 138 mg (46% of theory, purity 100%) of the title compound were obtained.

    [0388] .sup.1H NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.52 (s, 1H), 8.45 (s, 2H), 7.93 (d, 2H), 7.04 (d, 2H), 4.60-4.50 (m, 2H), 4.15-4.07 (m, 3H), 3.82 (dd, 2H), 3.64-3.47 (m, 2H), 3.31-3.18 (m, 3H), 2.97-2.83 (m, 1H), 2.19-2.06 (m, 1H), 2.06-1.94 (m, 1H), 1.81-1.49 (m, 6H), 1.29-1.14 (m, 3H), 0.36-0.22 (m, 2H), 0.18 (s, 9H).

    [0389] LC/MS (Methode 1, ESIpos): R.sub.t=1.53 min, m/z=674 [M+H].sup.+

    Example 11A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3 (4H)-yl]methyl}-5-(4-{[(trifluoromethyl)sulphonyl]oxy}benzoylicyclopentanecarboxylate (racemate)

    [0390] ##STR00044##

    [0391] To a solution of 1.00 g (1.78 mmol) of the compound from Example 8A in 5.0 ml of dichloromethane under argon were added, at 0 C., first 0.25 ml (3.12 mmol) of pyridine and then, gradually, 0.45 ml (2.67 mmol) of trifluoromethanesulphonic anhydride. The mixture was stirred at 0 C. for 1 h, then dichloromethane was added and the mixture was washed once each with water and saturated sodium hydrogencarbonate solution. The organic phase was dried over magnesium sulphate, filtered and concentrated. 1.21 g (98% of theory, 100% purity) of the title compound were obtained.

    [0392] LC/MS (Method 2, ESIpos): R.sub.t=3.41 min, m/z=694 [M+H].sup.+.

    Example 12A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-(4-sulphanylbenzoyl)cyclopentanecarboxylate (racemate)

    [0393] ##STR00045##

    [0394] To a solution of 800 mg (1.15 mmol) of the compound from Example 11A in 10 ml of dioxane were successively added 264 mg (1.38 mmol) of triisopropylsilanethiol, 298 mg (2.31 mmol) of N,N-diisopropylethylamine, 26 mg (0.03 mmol) of tris(dibenzylideneacetone)dipalladium and 33 mg (0.06 mmol) of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos). Subsequently, the mixture was degassed, purged with argon and stirred under reflux for 2.5 h. After cooling to RT, the mixture was admixed with ethyl acetate and washed once with water. After the aqueous phase had been extracted once with ethyl acetate, the combined organic phases were washed once with saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was purified by means of preparative HPLC (Method 4). The product-containing fractions were combined, neutralized with saturated aqueous sodium hydrogencarbonate solution and concentrated down to a small residual volume of water. After this aqueous phase had been extracted twice with dichloromethane, the combined organic phases were dried over magnesium sulphate, filtered and concentrated, and the residue was dried under reduced pressure. 350 mg (35% of theory, 67% purity) of the title compound were obtained. According to LC/MS, the corresponding disulphide (dimerized product, (+/)-bis[2-(trimethylsilyflethyl]2,2-[disulphanediylbis(benzene-4,1-diylcarbonyl)]bis(5-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}cyclopentanecarboxylate) was present to an extent of 25%.

    [0395] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.52 (s, 1H), 8.44 (s, 2H), 7.83 (d, 2H), 7.42 (d, 2H), 6.03 (br. s, 1H), 4.60-4.48 (m, 2H), 4.08 (q, 1H), 3.64-3.48 (m, 2H), 3.23 (t, 1H), 2.97-2.81 (m, 1H), 2.19-2.06 (m, 1H), 2.06-1.94 (m, 1H), 1.79-1.67 (m, 1H), 1.62-1.48 (m, 1H), 0.37-0.22 (m, 2H), 0.18 (s, 9H).

    [0396] LC/MS (Method 1, ESIpos): R.sub.t=1.44 min, m/z=578 [M+H].sup.+.

    Example 13A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-{[4-oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]-methyl}-5-{4-[(tetrahydro-2H-pyran-4-ylmethyl)sulphanyl]benzoyl}cyclopentanecarboxylate (racemate)

    [0397] ##STR00046##

    [0398] To a solution of 200 mg of the compound from Example 12A (0.35 mmol, not corrected for purity, about 25% corresponding disulphide present) in 14 ml of DMF were added 96 mg (0.69 mmol) of potassium carbonate, and the mixture was stirred at RT for 2 min Subsequently, 136 mg (0.76 mmol) of 4-(bromomethyl)tetrahydropyran and 123 mg (1.04 mmol) of sodium hydroxymethan-esulphinate were added and the mixture was stirred at RT for a further 30 min The mixture was then concentrated, and the residue was admixed with water and extracted twice with ethyl acetate. The combined organic phases were washed once with saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. 248 mg (100% of theory, purity 95%) of the title compound were obtained.

    [0399] LC/MS (Method 1, ESIpos): R.sub.t=1.50 min, m/z=676 [M+H].sup.+.

    Example 14A

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-[4-(benzyloxy)benzoyl]-5-[(6-methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylate (racemate)

    [0400] ##STR00047##

    [0401] To a suspension of 9.60 g (20.06 mmol, 95% purity) of the compound from Example 3A/Step 5 in 110 ml of toluene under argon were added 3.88 g (24.07 mmol) of the compound from Example 2A. Subsequently, 25.1 ml (100.30 mmol) of tributylphosphine and 27.4 ml (60.18 mmol) of a 40% solution of diethyl azodicarboxylate in toluene were added dropwise at 0 C. After stirring at RT for 2 h, the mixture was diluted with ethyl acetate and washed once with water. The aqueous phase was reextracted once with ethyl acetate. The combined organic phases were washed once with saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was purified by means of flash chromatography (silica gel, eluent: cyclohexane/ethyl acetate 85:15.fwdarw.80:20). 6.28 g (51% of theory, 98% purity) of the title compound were obtained.

    [0402] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.12-8.05 (m, 2H), 7.97-7.88 (m, 3H), 7.48-7.27 (m, 5H), 7.12 (d, 2H), 5.20 (s, 2H), 4.56-4.42 (m, 2H), 4.08 (q, 1H), 3.61-3.46 (m, 2H), 3.22 (t, 1H), 2.96-2.83 (m, 1H), 2.55 (s, 3H), 2.17-2.05 (m, 1H), 2.03-1.92 (m, 1H), 1.78-1.67 (m, 1H), 1.59-1.47 (m, 1H), 0.38-0.23 (m, 2H), 0.17 (s, 9H).

    [0403] LC/MS (Method 1, ESIpos): R.sub.t=1.49 min, m/z=598 [M+H].sup.+.

    Example 15A

    2-(Trimethylsilyl)ethyl (1RS,2RS,5SR)-2-(4-hydroxybenzoyl)-5-[(6-methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]cyclopentanecarboxylate (racemate)

    [0404] ##STR00048##

    [0405] To a solution of 6.25 g (10.25 mmol, 98% purity) of the compound from Example 14A in a mixture of 50 ml of ethyl acetate and 50 ml of ethanol under argon were added 273 mg (0.26 mmol) of palladium on activated carbon (10% Pd) and 969 mg (15.37 mmol) of ammonium formate. The mixture was then stirred at 70 C. for 2 h. After cooling to RT, the mixture was filtered through kieselguhr, the filter residue was washed with ethyl acetate and ethanol, the filtrate was concentrated and the residue was dried under reduced pressure. 5.20 g (97% of theory, 97% purity) of the title compound were obtained.

    [0406] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=10.40 (br. s, 1H), 8.12-8.05 (m, 2H), 7.92 (dd, 1H), 7.84 (d, 2H), 6.84 (d, 2H), 4.57-4.42 (m, 2H), 4.03 (q, 1H), 3.62-3.46 (m, 2H), 3.21 (t, 1H), 2.96-2.83 (m, 1H), 2.55 (s, 3H), 2.17-2.04 (m, 1H), 2.03-1.91 (m, 1H), 1.78-1.66 (m, 1H), 1.60-1.48 (m, 1H), 0.39-0.25 (m, 2H), -0.17 (s, 9H).

    [0407] LC/MS (Method 1, ESIpos): R.sub.t=1.28 min, m/z=508 [M+H].sup.+.

    Example 16A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-[(6-methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentancarboxylat (racemate)

    [0408] ##STR00049##

    [0409] To a solution of 500 mg (0.96 mmol, 97% purity) of the compound from Example 15A in 5.3 ml of DMF under argon were added 129 mg (1.15 mmol) of potassium tert-butoxide. After stirring at RT for 5 min, 205 mg (1.15 mmol) of 4-(bromomethyl)tetrahydro-2H-pyran were added, and the mixture was stirred at bath temperature 100 C. for 1 h. After the mixture had been cooled and left to stand overnight, 60 ml of water and 60 ml of ethyl acetate were added. After the phases had been separated, the aqueous phase was extracted once with 30 ml of ethyl acetate. The combined organic phases were washed once with saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was purified by means of column chromatography (90 g of silica gel, eluent: cyclohexane/ethyl acetate 7:3). 290 mg (41% of theory, purity 82%) of the title compound were obtained.

    [0410] LC/MS (Method 1, ESIpos): R.sub.t=1.43 min, m/z=606 [M+H].sup.+.

    Example 17A

    2-(Trimethylsilyl)ethyl (1RS,2SR,5RS)-2-[(6-methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylate (racemate)

    [0411] ##STR00050##

    [0412] To a solution of 200 mg (0.38 mmol, 97% purity) of the compound from Example 15A in 2.1 ml of DMF under argon were added 51 mg (0.46 mmol) of potassium tert-butoxide. After stirring at RT for 5 min, 89 mg (0.46 mmol) of 4-(2-bromoethyl)tetrahydro-2H-pyran were added, and the mixture was stirred at bath temperature 100 C. for 2 h. After cooling to RT, 60 ml of water and 60 ml of ethyl acetate were added to the mixture. After the phases had been separated, the aqueous phase was extracted once with 30 ml of ethyl acetate. The combined organic phases were washed once with saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was purified by means of column chromatography (40 g of silica gel, eluent: cyclohexane/ethyl acetate 7:3). 142 mg (60% of theory, purity 100%) of the title compound were obtained.

    [0413] LC/MS (Method 1, ESIpos): R.sub.t=1.46 min, m/z=620 [M+H].sup.+.

    WORKING EXAMPLES

    Example 1

    (+/)-(1RS,2SR,5RS)-2-[(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (racemate)

    [0414] ##STR00051##

    [0415] To a solution of 83 mg (0.14 mmol) of the compound from Example 5A in 0.5 ml of dichloromethane was added, at 0 C., 0.25 ml (3.24 mmol) of trifluoroacetic acid. The mixture was stirred at 0 C. for 2.5 h and then concentrated. The residue was taken up in acetonitrile and purified by means of preparative HPLC (Method 4). 60 mg (85% of theory, 98% purity) of the title compound were obtained.

    [0416] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.12 (s, 1H), 8.26 (dd, 1H), 8.20 (d, 1H), 8.08 (td, 1H), 7.99-7.90 (m, 3H), 7.04 (d, 2H), 4.59-4.46 (m, 2H), 4.14-4.05 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.38-3.32 (partly concealed, 2H), 3.23 (t, 1H), 2.94-2.81 (m, 1H), 2.17-1.95 (m, 2H), 1.95-1.83 (m, 1H), 1.72-1.61 (m, 3H), 1.57-1.45 (m, 1H), 1.33 (qd, 2H).

    [0417] LC/MS (Method 1, ESIpos): R.sub.t=1.06 min, m/z=492 [M+H].sup.+.

    [0418] Separation of the Enantiomers:

    [0419] 30 mg of the racemic compound from Example 1 were dissolved in 12 ml of hot methanol/acetonitrile and separated into the enantiomers by means of preparative SFC on a chiral phase (see Examples 2 and 3) [column Daicel Chiralpak AZ-H, 5 m, 250 mm20 mm; flow rate: 80 ml/min; detection: 210 nm; injection volume: 1.0 ml; temperature: 40 C.; eluent: 60% carbon dioxide/40% ethanol].

    Example 2

    (+)-(1RS,2SR,5RS)-2-[(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (enantiomer 1)

    [0420] Yield: 14 mg; chem. purity=100%; ee=99%

    [0421] [].sub.D.sup.20=+66.9, 589 nm, c=0.27 g/100 ml, chloroform

    [0422] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.10 (br. s, 1H), 8.26 (d, 1H), 8.20 (d, 1H), 8.11-8.05 (m, 1H), 7.99-7.89 (m, 3H), 7.04 (d, 2H), 4.59-4.46 (m, 2H), 4.14-4.05 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.32-3.19 (m, partly concealed, 3H), 2.94-2.80 (m, 1H), 2.18-1.96 (m, 2H), 1.95-1.84 (m, 1H), 1.72-1.61 (m, 3H), 1.58-1.44 (m, 1H), 1.33 (qd, 2H).

    [0423] LC/MS (Method 1, ESIpos): R.sub.t=1.04 min, m/z=492 [M+H].sup.+.

    Example 3

    ()-(1RS,2SR,5RS)-2-[(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-yl-methoxy)benzoyl]cyclopentanecarboxylic acid (enantiomer 2)

    [0424] Yield: 17 mg; chem. purity=100%; ee=99%

    [0425] [].sub.D.sup.20=56.4, 589 nm, c=0.28 g/100 ml, chloroform

    [0426] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.07 (br. s, 1H), 8.26 (dd, 1H), 8.20 (d, 1H), 8.08 (td, 1H), 7.99-7.89 (m, 3H), 7.04 (d, 2H), 4.53 (dd, 2H), 4.14-4.05 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.34-3.28 (m, partly concealed, 2H), 3.24 (t, 1H), 2.98-2.81 (m, 1H), 2.17-1.96 (m, 2H), 1.95-1.83 (m, 1H), 1.73-1.60 (m, 3H), 1.57-1.45 (m, 1H), 1.33 (qd, 2H).

    [0427] LC/MS (Method 1, ESIpos): R.sub.t=1.04 min, m/z=492 [M+H].sup.+.

    Example 4

    (+/)-(1RS,2SR,5RS)-2-[(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (racemate)

    [0428] ##STR00052##

    [0429] To a solution of 2.91 g (4.75 mmol, purity 99%) of the compound from Example 6A in 16 ml of dichloromethane were added, at 0 C., 8.0 ml (104 mmol) of trifluoroacetic acid, and the mixture was stirred at 0 C. for 2 h. Subsequently, the mixture was concentrated and the residue was dried under reduced pressure. After adding a little ethyl acetate, a solid was obtained, which was filtered off, washed once with a little ethyl acetate and pentane, and dried under reduced pressure. In this way, 2.11 g (88% of theory, 100% purity) of a first batch of the title compound were obtained. The remaining mother liquor was concentrated and the residue was purified by means of preparative HPLC [column: Kinetix C18, 5 m, 100 mm21.2 mm; flow rate: 25 ml/min; detection: 210 nm; injection volume: 0.5 ml; temperature: 40 C.; eluent: 44% water/45% acetonitrile/11% formic acid in water, isocratic over 8 min] In this way, 52 mg (2% of theory, 100% purity) of a second batch of the title compound were obtained.

    [0430] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.14 (br. s, 1H), 8.26 (d, 1H), 8.20 (d, 1H), 8.11-8.05 (m, 1H), 7.99-7.90 (m, 3H), 7.04 (d, 2H), 4.58-4.47 (m, 2H), 4.15-4.05 (m, 3H), 3.83 (dd, 2H), 3.34-3.19 (m, 3H), 2.94-2.81 (m, 1H), 2.16-2.04 (m, 1H), 1.95-1.83 (m, 1H), 1.75-1.58 (m, 6H), 1.57-1.45 (m, 1H), 1.29-1.14 (m, 2H).

    [0431] LC/MS (Method 1, ESIpos): R.sub.t=1.05 min, m/z=506 [M+H].sup.+.

    [0432] Separation of the Enantiomers:

    [0433] 2.00 g of the racemic compound from Example 4 were partly dissolved in 20 ml of dioxane, 180 ml of a methanol/acetonitrile mixture were added, and the mixture was converted to a solution by heating and then separated into the enantiomers by means of preparative SFC on a chiral phase (see Examples 5 and 6) [column Daicel Chiralpak AY-H, 5 m, 250 mm20 mm; flow rate: 80 ml/min; detection: 210 nm; injection volume: 1.2 ml; temperature: 40 C.; eluent: 70% carbon dioxide/30% ethanol, run time 16 min].

    Example 5

    (+)-(1RS,2SR,5RS)-2-[(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (enantiomer 1)

    [0434] 910 mg (chem. purity=97%, ee=100%) of the title compound were obtained, which were taken up in 20 ml of acetonitrile and purified once again by chromatography [column: Kinetix C 18, 5 m, 100 mm30 mm; flow rate: 60 ml/min; detection: 210 nm; injection volume: 1.0 ml; temperature: 30 C.; eluent: 45% water/50% acetonitrile/5% formic acid in water, isocratic over 4 min] In this way, 850 mg of the title compound were obtained in a chem. purity of 100%.

    [0435] [].sub.D.sup.2=+71.0, 589 nm, c=0.37 g/100 ml, chloroform

    [0436] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): [ppm]=12.13 (br. s, 1H), 8.26 (dd, 1H), 8.20 (d, 1H), 8.08 (td, 1H), 7.98-7.90 (m, 3H), 7.04 (d, 2H), 4.58-4.47 (m, 2H), 4.14-4.05 (m, 3H), 3.83 (dd, 2H), 3.32-3.20 (m, partly concealed, 3H), 2.88 (sext, 1H), 2.15-2.05 (m, 1H), 1.93-1.84 (m, 1H), 1.76-1.58 (m, 6H), 1.56-1.47 (m, 1H), 1.27-1.16 (m, 2H).

    [0437] LC/MS (Method 1, ESIpos): R.sub.t=1.07 min, m/z=506 [M+H].sup.+.

    Example 6

    ()-(1RS,2SR,5RS)-2-[(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (enantiomer 2)

    [0438] Yield: 903 mg; chem. purity=100%; ee=100%

    [0439] [].sub.D.sup.20=70.1, 589 nm, c=0.35 g/100 ml, chloroform

    [0440] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=8.26 (d, 1H), 8.20 (d, 1H), 8.11-8.05 (m, 1H), 7.99-7.90 (m, 3H), 7.04 (d, 2H), 4.59-4.47 (m, 2H), 4.14-4.06 (m, 3H), 3.83 (dd, 2H), 3.32-3.20 (m, 3H), 2.87 (sext, 1H), 2.17-2.04 (m, 1H), 1.95-1.83 (m, 1H), 1.75-1.58 (m, 6H), 1.57-1.45 (m, 1H), 1.29-1.15 (m, 2H).

    [0441] LC/MS (Method 1, ESIpos): R.sub.t=1.05 min, m/z=506 [M+H].sup.+.

    Example 7

    (+/)-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (racemate)

    [0442] ##STR00053##

    [0443] To a solution of 585 mg (0.89 mmol) of the compound from Example 9A in 3 ml of dichloromethane were added, at 0 C., 1.5 ml (19.47 mmol) of trifluoroacetic acid. The mixture was stirred at 0 C. for 5.5 h and then concentrated. The residue was taken up in 5 ml of acetonitrile. A solid precipitated out, which was filtered off and dried under reduced pressure. 468 mg (95% of theory, purity 100%) of the title compound were obtained.

    [0444] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.13 (s, 1H), 8.51 (s, 1H), 8.46-8.38 (m, 2H), 7.96 (d, 2H), 7.04 (d, 2H), 4.57 (d, 2H), 4.15-4.05 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.38-3.28 (concealed, 2H), 3.24 (t, 1H), 2.94-2.79 (m, 1H), 2.18-1.87 (m, 3H), 1.73-1.61 (m, 3H), 1.59-1.46 (m, 1H), 1.33 (qd, 2H).

    [0445] LC/MS (Method 1, ESIpos): R.sub.t=1.17 min, m/z=660 [M+H].sup.+.

    [0446] Separation of the Enantiomers:

    [0447] 465 mg of the racemic compound from Example 7 were dissolved in 15 ml of DMSO and 30 ml of ethanol and separated into the enantiomers by means of preparative SFC on a chiral phase (see Examples 8 and 9) [column Daicel Chiralpak AY, 20 m, 250 mm30 mm; flow rate: 175 ml/min; detection: 210 nm; injection volume: 1.3 ml; temperature: 38 C.; eluent: 75% carbon dioxide/25% ethanol, run time 16.5 min].

    Example 8

    (+)-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-[4-(tetra-hydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (enantiomer 1)

    [0448] Yield: 239 mg; chem. purity=100%; ee=100%

    [0449] [].sub.D.sup.20=+80.2, 589 nm, c=0.31 g/100 ml, chloroform

    [0450] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.13 (br. s, 1H), 8.51 (s, 1H), 8.46-8.38 (m, 2H), 7.96 (d, 2H), 7.04 (d, 2H), 4.57 (d, 2H), 4.15-4.05 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.37-3.28 (concealed, 2H), 3.24 (t, 1H), 2.94-2.80 (m, 1H), 2.17-1.88 (m, 3H), 1.72-1.61 (m, 3H), 1.58-1.46 (m, 1H), 1.33 (qd, 2H).

    [0451] LC/MS (Method 1, ESIpos): R.sub.t=1.17 min, m/z=660 [M+H].sup.+.

    Example 9

    ()-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-[4-(tetra-hydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (enantiomer 2)

    [0452] Yield: 228 mg; ee=100%

    [0453] [].sub.D.sup.20=88.9, 589 nm, c=0.31 g/100 ml, chloroform

    [0454] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.13 (br. s, 1H), 8.51 (s, 1H), 8.46-8.37 (m, 2H), 7.96 (d, 2H), 7.04 (d, 2H), 4.57 (d, 2H), 4.14-4.05 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.37-3.28 (concealed, 2H), 3.23 (t, 1H), 2.94-2.80 (m, 1H), 2.17-1.87 (m, 3H), 1.73-1.61 (m, 3H), 1.58-1.46 (m, 1H), 1.33 (qd, 2H).

    [0455] LC/MS (Method 1, ESIpos): R.sub.t=1.17 min, m/z=660 [M+H].sup.+.

    Example 10

    (+/)-(1RS ,2S R,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (racemate)

    [0456] ##STR00054##

    [0457] To a solution of 135 mg (0.20 mmol) of the compound from Example 10A in 0.7 ml of dichloromethane was added, at 0 C., 0.35 ml (4.54 mmol) of trifluoroacetic acid. The mixture was stirred at 0 C. for 2.5 h and then concentrated. The residue was taken up in 2 ml of acetonitrile and purified by means of preparative HPLC (Method 5). 91 mg (80% of theory, purity 100%) of the title compound were obtained.

    [0458] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.13 (s, 1H), 8.51 (s, 1H), 8.46-8.38 (m, 2H), 7.96 (d, 2H), 7.04 (d, 2H), 4.57 (d, 2H), 4.14-4.05 (m, 3H), 3.83 (dd, 2H), 3.32-3.20 (m, 3H), 2.93-2.81 (m, 1H), 2.17-2.03 (m, 1H), 2.00-1.88 (m, 1H), 1.76-1.45 (m, 7H), 1.29-1.14 (m, 2H).

    [0459] LC/MS (Method 1, ESIpos): R.sub.t=1.21 min, m/z=574 [M+H].sup.+.

    [0460] Separation of the Enantiomers:

    [0461] 83 mg of the racemic compound from Example 10 were dissolved in 2 ml of ethanol and separated into the enantiomers by means of preparative HPLC on a chiral phase (see Examples 11 and 12) [column: Daicel Chiralpak AY-H, 5 m, 250 mm20 mm; flow rate: 15 ml/min; detection: 220 nm; injection volume: 1 ml; temperature: 45 C.; eluent: t=0-15 min 25% isohexane/75% ethanol+0.2% acetic acid].

    Example 11

    (+)-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (enantiomer 1)

    [0462] Yield: 38 mg; ee=100%

    [0463] [].sub.D.sup.20=+70.7, 589 nm, c=0.10 g/100 ml, chloroform

    [0464] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.13 (s, 1H), 8.51 (s, 1H), 8.46-8.38 (m, 2H), 7.96 (d, 2H), 7.04 (d, 2H), 4.57 (d, 2H), 4.14-4.06 (m, 3H), 3.83 (dd, 2H), 3.32-3.20 (m, 3H), 2.91-2.83 (m, 1H), 2.16-2.04 (m, 1H), 1.99-1.88 (m, 1H), 1.75-1.58 (m, 6H), 1.57-1.47 (m, 1H), 1.29-1.15 (m, 2H).

    [0465] LC/MS (Method 1, ESIpos): R.sub.t=1.22 min, m/z=574 [M+H].sup.+.

    Example 12

    ()-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (enantiomer 2)

    [0466] Yield: 45 mg; ee=100%

    [0467] [].sub.D.sup.20=77.1, 589 nm, c=0.37 g/100 ml, chloroform

    [0468] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.13 (s, 1H), 8.51 (s, 1H), 8.46-8.38 (m, 2H), 7.96 (d, 2H), 7.04 (d, 2H), 4.57 (d, 2H), 4.15-4.05 (m, 3H), 3.83 (dd, 2H), 3.32-3.20 (m, 3H), 2.93-2.81 (m, 1H), 2.17-2.04 (m, 1H), 1.99-1.87 (m, 1H), 1.74-1.58 (m, 6H), 1.58-1.47 (m, 1H), 1.29-1.15 (m, 2H).

    [0469] LC/MS (Method 1, ESIpos): R.sub.t=1.22 min, m/z=574 [M+H].sup.+.

    Example 13

    (+/)-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3 (4H)-yl]methyl}-5-{4-[(tetrahydro-2H-pyran-4-ylmethyl)sulphanyl]benzoyl}cyclopentanecarboxylic acid (racemate)

    [0470] ##STR00055##

    [0471] To a solution of 249 mg (0.35 mmol, purity 95%) of the compound from Example 13A in 3.5 ml of dichloromethane were added, at 0 C., 1.75 ml of trifluoroacetic acid. The mixture was first stirred at 0 C. for 15 min and then at RT for 1 h, and then concentrated. The residue was purified by means of preparative HPLC (Method 4). 163 mg (81% of theory, purity 100%) of the title compound were obtained.

    [0472] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.16 (br. s, 1H), 8.51 (s, 1H), 8.46-8.37 (m, 2H), 7.90 (d, 2H), 7.41 (d, 2H), 4.62-4.52 (m, 2H), 4.15-4.06 (m, 1H), 3.83 (dd, 2H), 3.30-3.19 (m, 3H), 3.02 (d, 2H), 2.94-2.81 (m, 1H), 2.20-2.04 (m, 1H), 2.01-1.87 (m, 1H), 1.84-1.61 (m, 4H), 1.60-1.45 (m, 1H), 1.35-1.17 (m, 2H).

    [0473] LC/MS (Method 1, ESIpos): R.sub.t=1.20 min, m/z=576 [M+H].sup.+.

    [0474] Separation of the Enantiomers:

    [0475] 150 mg of the racemic compound from Example 13 were dissolved in 3 ml of acetonitrile/ethanol and separated into the enantiomers by means of preparative HPLC on a chiral phase (see Examples 14 and 15) [column Daicel Chiralpak AS-H, 5 m, 250 mm4.6 mm; flow rate: 20 ml/min; detection: 230 nm; injection volume: 0.06 ml; temperature: 25 C.; eluent: t=0-16 min 20% ethanol/76% acetonitrile/4% of 5% strength acetic acid in acetonitrile].

    Example 14

    ()-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-{4-[(tetrahydro-2H-pyran-4-ylmethyl)sulphanyl]benzoyl}cyclopentanecarboxylic acid (enantiomer 1)

    [0476] Yield: 59 mg; chem. purity =100%; ee =100%

    [0477] [].sub.D.sup.20=85.6, 589 nm, c=0.39 g/100 ml, chloroform

    [0478] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.15 (br. s, 1H), 8.51 (s, 1H), 8.46-8.37 (m, 2H), 7.90 (d, 2H), 7.41 (d, 2H), 4.63-4.51 (m, 2H), 4.16-4.03 (m, 1H), 3.83 (dd, 2H), 3.29-3.19 (m, 3H), 3.02 (d, 2H), 2.94-2.81 (m, 1H), 2.18-2.05 (m, 1H), 2.02-1.86 (m, 1H), 1.83-1.61 (m, 3H), 1.59-1.44 (m, 1H), 1.36-1.19 (m, 2H).

    [0479] LC/MS (Method 1, ESIpos): R.sub.t=1.21 min, m/z=576 [M+H].sup.+.

    Example 15

    (+)-(1RS,2SR,5RS)-2-{[4-Oxo-6-(trifluoromethyl)-1,2,3-benzotriazin-3(4H)-yl]methyl}-5-{4-[(tetrahydro-2H-pyran-4-ylmethyl)sulphanyl]benzoyl}cyclopentanecarboxylic acid (enantiomer 2)

    [0480] Yield: 61 mg; chem. purity=100%; ee=99%

    [0481] [].sub.D.sup.20=+53.1, 589 nm, c=0.16 g/100 ml, chloroform

    [0482] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.14 (br. s, 1H), 8.51 (s, 1H), 8.46-8.37 (m, 2H), 7.90 (d, 2H), 7.41 (d, 2H), 4.63-4.50 (m, 2H), 4.16-4.05 (m, 1H), 3.83 (dd, 2H), 3.29-3.17 (m, 3H), 3.02 (d, 2H), 2.94-2.77 (m, 1H), 2.18-2.04 (m, 1H), 2.02-1.86 (m, 1H), 1.83-1.62 (m, 3H), 1.60-1.45 (m, 1H), 1.35-1.20 (m, 2H).

    [0483] LC/MS (Method 1, ESIpos): R.sub.t=1.21 min, m/z=576 [M+H].sup.+.

    Example 16

    (+/)-(1RS,2SR,5RS)-2-[(6-Methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (racemate)

    [0484] ##STR00056##

    [0485] To a solution of 290 mg (0.39 mmol, purity 82%) of the compound from Example 16A in 1.3 ml of dichloromethane was added, at 0 C., 0.7 ml (8.65 mmol) of trifluoroacetic acid. The mixture was stirred at RT for 1 h and then concentrated. The residue was purified by means of preparative HPLC (Method 4). 153 mg (77% of theory, purity 100%) of the title compound were obtained.

    [0486] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.12 (br. s, 1H), 8.12-8.04 (m, 2H), 7.96 (d, 2H), 7.90 (dd, 1H), 7.04 (d, 2H), 4.51 (d, 2H), 4.13-4.04 (m, 1H), 3.93 (d, 2H), 3.88 (dd, 2H), 3.37-3.28 (m, 2H), 3.23 (t, 1H), 2.93-2.80 (m, 1H), 2.55 (s, 3H), 2.16-1.95 (m, 2H), 1.93-1.82 (m, 1H), 1.71-1.61 (m, 3H), 1.50 (dd, 1H), 1.33 (qd, 2H).

    [0487] LC/MS (Method 1, ESIpos): R.sub.t=1.06 min, m/z=506 [M+H].sup.+.

    [0488] Separation of the Enantiomers:

    [0489] 144 mg of the racemic compound from Example 16 were dissolved in 11 ml ethanol and separated into the enantiomers by means of preparative SFC on a chiral phase (see Examples 17 and 18) [column: Phenomenex Amylose II, 5 m, 250 mm20 mm; flow rate: 100 ml/min; detection: 210 nm; injection volume: 0.40 ml; temperature: 40 C.; eluent: 65% carbon dioxide/35% ethanol, run time 15 min].

    Example 17

    (+)-(1RS,2SR,5RS)-2-[(6-Methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (enantiomer 1)

    [0490] Yield: 63 mg; chem. purity=94%; ee=100%

    [0491] [].sub.D.sup.20=+68.4, 589 nm, c=0.39 g/100 ml, chloroform

    [0492] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.12 (br. s, 1H), 8.11-8.04 (m, 2H), 7.96 (d, 2H), 7.90 (dd, 1H), 7.04 (d, 2H), 4.51 (d, 2H), 4.14-4.04 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.37-3.28 (partly concealed, 2H), 3.23 (t, 1H), 2.92-2.80 (m, 1H), 2.55 (s, 3H), 2.16-1.94 (m, 2H), 1.93-1.82 (m, 1H), 1.67 (d, 3H), 1.56-1.44 (m, 1H), 1.33 (qd, 2H).

    [0493] LC/MS (Method 1, ESIpos): R.sub.t=1.07 min, m/z=506 [M+H].sup.+.

    Example 18

    ()-(1RS,2SR,5RS)-2-[(6-Methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-[4-(tetrahydro-2H-pyran-4-ylmethoxy)benzoyl]cyclopentanecarboxylic acid (enantiomer 2)

    [0494] Yield: 63 mg; chem. purity=100%; ee=100%

    [0495] [].sub.D.sup.20=63.7, 589 nm, c=0.37 g/100 ml, chloroform

    [0496] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.12 (br. s, 1H), 8.12-8.04 (m, 2H), 7.96 (d, 2H), 7.90 (dd, 1H), 7.04 (d, 2H), 4.51 (d, 2H), 4.14-4.04 (m, 1H), 3.93 (d, 2H), 3.87 (dd, 2H), 3.37-3.28 (partly concealed, 2H), 3.22 (t, 1H), 2.93-2.79 (m, 1H), 2.55 (s, 3H), 2.16-1.96 (m, 2H), 1.94-1.81 (m, 1H), 1.72-1.60 (m, 3H), 1.56-1.43 (m, 1H), 1.33 (qd, 2H).

    [0497] LC/MS (Method 1, ESIpos): R.sub.t=1.07 min, m/z=506 [M+H].sup.+.

    Example 19

    (+/)-(1RS,2SR,5RS)-2-[(6-Methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(letrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (racemate)

    [0498] ##STR00057##

    [0499] To a solution of 142 mg (0.23 mmol) of the compound from Example 17A in 0.8 ml of dichloromethane was added, at 0 C., 0.4 ml (5.03 mmol) of trifluoroacetic acid. The mixture was stirred at RT for 1 h and then concentrated. The residue was purified by means of preparative HPLC (Method 6). 84 mg (71% of theory, purity 100%) of the title compound were obtained.

    [0500] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.12 (br. s, 1H), 8.11-8.04 (m, 2H), 7.96 (d, 2H), 7.90 (dd, 1H), 7.04 (d, 2H), 4.51 (d, 2H), 4.15-4.05 (m, 3H), 3.83 (dd, 2H), 3.32-3.20 (m, 3H), 2.93-2.80 (m, 1H), 2.55 (s, 3H), 2.16-2.04 (m, 1H), 1.93-1.82 (m, 1H), 1.76-1.57 (m, 6H), 1.57-1.44 (m, 1H), 1.30-1.12 (m, 2H).

    [0501] LC/MS (Method 1, ESIpos): R.sub.t=1.14 min, m/z=520 [M+H].sup.+.

    [0502] Separation of the Enantiomers:

    [0503] 69 mg of the racemic compound from Example 19 were dissolved in 10 ml ethanol/acetonitrile and separated into the enantiomers by means of preparative SFC on a chiral phase (see Examples 20 and 21) [column: Phenomenex Amylose II, 5 m, 250 mm20 mm; flow rate: 100 ml/min; detection: 210 nm; injection volume: 0.40 ml; temperature: 40 C.; eluent: 70% carbon dioxide/30% ethanol, run time 18 min]

    Example 20

    (+)-(1RS,2SR,5RS)-2-[(6-Methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (enantiomer 1)

    [0504] Yield: 22 mg; chem. purity=100%; ee=100%

    [0505] [].sub.D.sup.20=+50.6, 589 nm, c=0.32 g/100 ml, chloroform

    [0506] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.10 (br. s, 1H), 8.12-8.04 (m, 2H), 7.96 (d, 2H), 7.90 (d, 1H), 7.04 (d, 2H), 4.51 (d, 2H), 4.16-4.04 (m, 3H), 3.83 (dd, 2H), 3.32-3.19 (m, partly concealed, 3H), 2.93-2.79 (m, 1H), 2.55 (s, 3H), 2.17-2.04 (m, 1H), 1.94-1.82 (m, 1H), 1.76-1.58 (m, 6H), 1.56-1.44 (m, 1H), 1.29-1.11 (m, 2H).

    [0507] LC/MS (Method 1, ESIpos): R.sub.t=1.10 min, m/z=520 [M+H].sup.+.

    Example 21

    ()-(1RS,2SR,5RS)-2-[(6-Methyl-4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-5-{4-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]benzoyl}cyclopentanecarboxylic acid (enantiomer 2)

    [0508] Yield: 20 mg; chem. purity=95%; ee=100%

    [0509] [].sub.D.sup.20=50.6, 589 nm, c=0.31 g/100 ml, chloroform

    [0510] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): [ppm]=12.10 (br. s, 1H), 8.11-8.04 (m, 2H), 7.96 (d, 2H), 7.90 (dd, 1H), 7.04 (d, 2H), 4.51 (d, 2H), 4.15-4.04 (m, 3H), 3.83 (dd, 2H), 3.32-3.19 (m, partly concealed, 3H), 2.93-2.79 (m, 1H), 2.55 (s, 3H), 2.16-2.04 (m, 1H), 1.94-1.81 (m, 1H), 1.74-1.57 (m, 6H), 1.56-1.44 (m, 1H), 1.29-1.14 (m, 2H).

    [0511] B. Assessment of Pharmacological Efficacy

    [0512] 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.

    [0513] Abbreviations and Acronyms:

    [0514] APMA 4-aminophenylmercuric acetate

    [0515] Brij-35 polyoxyethylene lauryl ether

    [0516] BSA bovine serum albumin

    [0517] CYP cytochrome P450

    [0518] Dap (or Dpa) L-2,3-diaminopropionic acid (-amino-L-alanine)

    [0519] DMSO dimethyl sulphoxide

    [0520] Dnp 2,4-dinitrophenyl

    [0521] EDTA ethylenediaminetetraacetic acid

    [0522] HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulphonic acid

    [0523] HME human macrophage elastase

    [0524] IC inhibition concentration

    [0525] Mca (7-methoxycoumarin-4-yl)acetyl

    [0526] MMP matrix metallopeptidase

    [0527] MTP microtitre plate

    [0528] NADP nicotinamide adenine dinucleotide phosphate (oxidized form)

    [0529] NADPH nicotinamide adenine dinucleotide phosphate (reduced form)

    [0530] Nval norvaline

    [0531] PBS phosphate-buffered salt solution

    [0532] PEG polyethylene glycol

    [0533] Tris tris(hydroxymethyl)aminomethane

    [0534] v/v volume to volume ratio (of a solution)

    [0535] w/w weight to weight ratio (of a solution)

    [0536] B-1. In Vitro HME Inhibition Test:

    [0537] The potency of the compounds according to the invention with respect to HME (MMP-12) is determined in an in vitro inhibition test. The HME-mediated amidolytic cleavage of a suitable peptide substrate leads to an increase in fluorescent light therein. The signal intensity of the fluorescent light is directly proportional to the enzyme activity. The active concentration of a test compound at which half the enzyme is inhibited (50% signal intensity of the fluorescent light) is reported as the IC.sub.50 value.

    [0538] Standard In Vitro HME Inhibition Test:

    [0539] In a 384 hole microtiter plate, in a total test volume of 41 l, 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; from 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 a solution in DMSO) at 37 C. for two hours. The fluorescence intensity of the test mixtures is measured (excitation 323 mm, emission 393 nm). The IC.sub.50 values are ascertained by plotting the fluorescent light intensity against the active ingredient concentration.

    [0540] High-Sensitivity In Vitro HME Inhibition Test:

    [0541] If sub-nanomolar IC values are found for highly potent test substances in the standard HME inhibition test described above, a modified test is used to determine them more accurately. In this case, an enzyme concentration ten times lower is used (final concentration, for example, 0.05 nM) in order to achieve an elevated test sensitivity. The incubation period chosen for the test is correspondingly longer (for example 16 hours).

    [0542] In Vitro HME Inhibition Test in the Presence of Serum Albumin in the Reaction Buffer:

    [0543] This test corresponds to the standard HME inhibition test described above, except using a modified reaction buffer. This reaction buffer additionally contains bovine serum albumin (BSA, fatty acid-free, A6003, from Sigma-Aldrich) of final concentration 2% (w/w), which corresponds to about half 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).

    [0544] Table 1A below shows, for individual working examples of the invention, the IC.sub.50 values from the standard or high-sensitivity HME inhibition test (in some cases as mean values from two or more independent individual determinations and rounded to two significant figures):

    TABLE-US-00001 TABLE 1A Inhibition of human macrophage elastase (HME/hMMP-12) Example No. HME/hMMP-12 IC.sub.50 [nM] 1 0.040 2 0.071 4 0.37 5 0.085 6 80 7 0.031 8 0.016 9 42 10 0.021 11 0.19 12 90 13 0.48 14 10 15 0.026 16 0.17 17 0.030 18 3.3 19 0.12 20 0.053 21 78

    [0545] In Table 1B below, for representative working examples of the invention, the IC.sub.50 values from the HME inhibition test in the absence (cf. data in Table 1A) and in the presence of serum albumin are compared (in some cases as mean values from a plurality of independent individual measurements, rounded to two significant figures):

    TABLE-US-00002 TABLE 1B Inhibition of human macrophage elastase (HME/hMMP-12) in the absence () or the presence (+) of serum albumin (BSA) HME IC.sub.50 [nM] HME IC.sub.50 [nM] Example No. (BSA) (+BSA) 1 0.040 6.45 2 0.071 6.71 4 0.37 37.3 5 0.085 4.06 8 0.016 2.45 11 0.19 39.2 15 0.026 16.0 19 0.12 32.0 20 0.053 12.2

    [0546] On comparison of the data shown in Table 1B, it is found that the compounds according to the invention, even in the presence of serum albumin, still have high inhibitory potency (frequently in the nanomolar range) with respect to HME. This indicates a less significant unspecific interaction of the compounds according to the invention with blood plasma constituents and means that an elevated free fraction of these compounds in the blood can be expected, which should have a favourable effect on in vivo efficacy.

    [0547] B-2. In Vitro MMP Inhibition Tests

    [0548] The potency of the compounds according to the invention with respect to other MMPs (and hence their selectivity) is likewise determined in in vitro inhibition tests. The MMP-mediated amidolytic cleavage of a suitable peptide substrate leads to an increase in fluorescent light here too. The signal intensity of the fluorescent light is directly proportional to the enzyme activity. The active concentration of a test compound at which half the enzyme is inhibited (50% signal intensity of the fluorescent light) is reported as the 1050 value.

    [0549] a) Human MMPs:

    [0550] In Vitro MMP-1 Inhibition Test:

    [0551] Recombinant MMP-1 (from R&D Systems, 901-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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-1 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0552] In Vitro MMP-2 Inhibition Test:

    [0553] Recombinant MMP-2 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-2 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0554] In Vitro MMP-3 Inhibition Test:

    [0555] Recombinant MMP-3 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-002), so as to result in a total test volume of 50 l. The course of the MMP-3 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0556] In Vitro MMP-7 Inhibition Test:

    [0557] Recombinant MMP-7 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-7 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0558] In Vitro MMP-8 Inhibition Test:

    [0559] Recombinant MMP-8 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-8 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0560] In Vitro MMP-9 Inhibition Test:

    [0561] Recombinant MMP-9 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-9 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0562] In Vitro MMP-10 Inhibition Test:

    [0563] Recombinant MMP-10 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-002), so as to result in a total test volume of 50 l. The course of the MMP-10 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0564] In Vitro MMP-13 Inhibition Test:

    [0565] Recombinant MMP-13 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-13 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0566] In Vitro MMP-14 Inhibition Test:

    [0567] Recombinant MMP-14 (from R&D Systems, 918-MP) is enzymatically activated in accordance with the manufacturer's instructions using recombinant furin (from 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 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 Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-010), so as to result in a total test volume of 50 l. The course of the MMP-14 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0568] In Vitro MMP-16 Inhibition Test:

    [0569] Recombinant MMP-16 (from R&D Systems, 1785-MP) is enzymatically activated in accordance with the manufacturer's instructions using recombinant furin (from 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 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 Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-010), so as to result in a total test volume of 50 l. The course of the MMP-16 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0570] Tables 2A and 2B below show, for representative working examples of the invention, the 1050 values from these tests relating to inhibition of human MMPs (in some cases as mean values from two or more independent individual determinations and rounded to two significant figures):

    TABLE-US-00003 TABLEs 2A Inhibition of human MMPs Example MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 No. IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] 1 26000 140 1100 1200 11 2 10000 60 710 640 4.7 4 >40000 360 3200 98 5 >40000 44 360 600 15 7 6500 380 190 130 1.0 8 1800 140 100 45 0.6 10 8800 220 120 96 3.9 11 27000 510 260 190 5.6 15 11000 330 95 80 0.9 19 >40000 930 1400 1100 28 20 >40000 300 440 290 10

    TABLE-US-00004 TABLE 2B Inhibition of human MMPs Example MMP-9 MMP-10 MMP-13 MMP-14 MMP-16 No. IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] 1 450 120 110 160 1500 2 360 80 49 99 550 4 5000 170 460 2700 7100 5 460 13 67 250 940 7 120 11000 140 170 830 8 55 12 51 100 280 10 310 5100 230 600 1800 11 660 18 470 1200 3800 15 150 9 220 150 760 19 960 58 590 2700 2500 20 370 31 170 1000 3100

    [0571] On comparison of the inhibition data shown in Tables 1A and 2A/2B, it is found that the compounds according to the invention in general and the more active stereoisomers thereof in particular have very high inhibitory potency (frequently in the sub-nanomolar range) with respect to HME, and simultaneously high to very high selectivity (generally one to three orders of magnitude) with respect to related human MMPs.

    [0572] b) Rodent MMPs:

    [0573] In Vitro Mouse MMP-2 Inhibition Test:

    [0574] Recombinant mouse MMP-2 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-2 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0575] In Vitro Mouse MMP-3 Inhibition Test:

    [0576] Recombinant mouse MMP-3 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-002), so as to result in a total test volume of 50 l. The course of the MMP-3 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0577] In Vitro Mouse MMP-7 Inhibition Test:

    [0578] Recombinant mouse MMP-7 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-010), so as to result in a total test volume of 50 l. The course of the MMP-7 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0579] In Vitro Mouse MMP-8 Inhibition Test:

    [0580] Recombinant mouse MMP-8 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-010), so as to result in a total test volume of 50 l. The course of the MMP-8 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0581] In Vitro Mouse MMP-9 Inhibition Test:

    [0582] Recombinant mouse MMP-9 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-9 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0583] In Vitro Mouse MMP-12 Inhibition Test:

    [0584] Recombinant mouse MMP-12 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-010), so as to result in a total test volume of 50 l. The course of the MMP-12 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0585] High-Sensitivity In Vitro Mouse MMP-12 Inhibition Test:

    [0586] If sub-nanomolar IC values are found for highly potent test substances in the mouse MMP-12 inhibition test described above, a modified test is used to determine them more accurately. In this case, an enzyme concentration ten times lower is used (final concentration, for example, 0.1 nM) in order to achieve an elevated test sensitivity. The incubation period chosen for the test is correspondingly longer (for example 16 hours).

    [0587] In Vitro Rat MMP-2 Inhibition Test:

    [0588] Recombinant rat MMP-2 (from 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 10 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-2 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0589] In Vitro Rat MMP-8 Inhibition Test:

    [0590] Recombinant rat MMP-8 (from R&D Systems, 3245-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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Lys-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-010), so as to result in a total test volume of 50 l. The course of the MMP-8 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0591] In Vitro Rat MMP-9 Inhibition Test:

    [0592] Recombinant mouse MMP-9 (from R&D Systems, 5427-MM) 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, for example, 1 nM to 30 M) is pipetted into 24 l of activated enzyme (final concentration, for example, 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 M; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-9 reaction is measured by measuring the fluorescence intensity (excitation 320 nm, emission 410 nm) over a suitable period of time (for example over 120 mM at a temperature of 32 C.).

    [0593] In Vitro Rat MMP-12 Inhibition Test:

    [0594] Rat MMP-12 (Uniprot NP_446415.1; construct L96-V277) is expressed with an additional N-terminal His target and a consecutive TEV cleavage sequence by means of a pDEco7 vector in E. coli (BL21). The protein thus expressed in recombinant form forms an intracellular insoluble protein compartment (called an inclusion body). This is solubilized after separation and intensive washing under denaturing conditions. For this purpose, the inclusion body pellet fragment 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 dialysing 60 ml batches of the sample repeatedly 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 refolded protein is obtained in the supernatant with a yield of 3.7 mg per 250 ml of E. coli culture. The protein thus obtained is enzymatically active without further purifying operations or protease-mediated cleavage processes.

    [0595] 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 Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH.sub.2 substrate (final concentration, for example, 5 mol; R&D Systems, ES-001), so as to result in a total test volume of 50 l. The course of the MMP-12 reaction is measured by measuring the fluorescence intensity (excitation 320 mm, emission 410 nm) over a suitable period of time (for example over 120 min at a temperature of 32 C.).

    [0596] Table 3 below shows, for representative working examples of the invention, the 1050 values from the tests relating to inhibition of mouse MMPs (in some cases as mean values from two or more independent individual determinations and rounded to two significant figures):

    TABLE-US-00005 TABLE 3 Inhibition of mouse MMPs 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] 1 170 570 85 40 560 3.3 2 58 710 52 21 280 0.85 4 490 4000 1400 370 1500 7.7 5 45 270 130 54 210 0.67 10 300 120 130 14 930 2.8 11 640 620 210 34 1900 9.4 19 840 3100 630 150 2200 7.6 20 340 670 200 46 800 1.7

    [0597] On comparison of the inhibition data shown in Table 3, it is found that the compounds according to the invention in general and the more active stereoisomers thereof in particular have very high inhibitory potency (frequently in the nanomolar or even sub-nanomolar range) with respect to mouse MMP-12, and simultaneously high selectivity (generally one to two orders of magnitude with respect to related murine MMPs.

    [0598] B-3. Animal Model of Pulmonary Emphysema

    [0599] Elastase-induced pulmonary emphysema in mice, rats and hamsters is a widely used 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 on the day of the instillation of the porcine pancreas elastase and extends over a period of 3 weeks. At the end of the study, lung compliance is determined and alveolar morphometry is conducted.

    [0600] B-4. Animal Model of Silica-Induced Pulmonary Inflammation

    [0601] Orotracheal administration of silica in mice, rats or hamsters leads to 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 with the test substance of the day of instillation of the silica. After 24 hours, a bronchio-alveolar lavage is carried out to determine the cell content and the biomarker.

    [0602] B-5. Animal Model of Silica-Induced Pulmonary Fibrosis

    [0603] Silica-induced pulmonary fibrosis in mice, rats or hamsters is a widely used 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 biomarkers and a histological assessment of pulmonary fibrosis are carried out.

    [0604] B-6. Animal Model of ATP-Induced Pulmonary Inflammation

    [0605] Intratracheal administration of ATP (adenosine triphosphate) in mice 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. 9:79 (2008)]. On the day of the instillation of ATP, the animals are treated with the test substance for a duration of 24 h (by gavage, by addition to the feed or drinking water, using an osmotic minipump, by subcutaneous or intraperitoneal injection or by inhalation). At the end of the experiment, a bronchio-alveolar lavage is conducted to determine the cell content and the pro-inflammatory markers.

    [0606] B-7. CYP Inhibition Test

    [0607] The ability of substances to inhibit the CYP enzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in humans is examined 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 studied at six different concentrations of the test compounds [2.8, 5.6, 8.3, 16.7, 20 (or 25) and 50 M) and compared with the extent of the CYP-specific metabolite formation of the standard substrates in the absence of the test compounds, and the corresponding IC50 values are calculated. A standard inhibitor that specifically inhibits an individual CYP isoform is always included in the incubation, in order to make results comparable between different series.

    [0608] The incubation of phenacetin, diclofenac, tolbutamide, dextromethorphan or midazolam with human liver microsomes in the presence of six different concentrations of each test compound (as potential inhibitor) is carried out on a workstation (Tecan, Genesis, Crailsheim, Germany). Standard incubation mixtures contain 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 with a suitable internal standard present therein. Precipitated proteins are removed by centrifugation, and the supernatants are combined and analysed by LC-MS/MS.

    [0609] B-8. Hepatocyte Assay for Determination of Metabolic Stability

    [0610] 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 maximum linearity of kinetic conditions in the experiment. Seven samples from the incubation solution are taken for the LC-MS analysis within a fixed time pattern, 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).

    [0611] 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 minimize the influence of the organic solvent on the enzymes in the incubation batches, the concentration thereof is generally limited to 1% (acetonitrile) or 0.1% (DMSO).

    [0612] For all species and breeds, a hepatocyte cell count in the liver of 1.1*10.sup.8 cells/g of liver is expected. CL parameters calculated on the basis of half-lives which extend considerably beyond the incubation time (typically 90 minutes) can only be regarded as rough guide values.

    [0613] The parameters calculated and the meanings thereof are: [0614] F.sub.max well-stirred [%] maximum possible bioavailability after oral administration [0615] Calculation: (1-CL.sub.blood well-stirred/QH)*100 [0616] CL.sub.blood well-stirred [L/(h*kg)] calculated blood clearance (well-stirred model) [0617] Calculation: (QH*CL.sub.intrinsic)/(QH+CL.sub.intrinsic) [0618] 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) [0619] Calculation: CL.sub.intrinsic, apparent* species-specific hepatocyte count [1.1* 10.sup.8/g liver]*species-specific liver weight [g/kg] [0620] CL.sub.intrinsic, apparent[ml/(min*mg)] normalizes the elimination constant by dividing it by the hepatocyte cell count x used (x*10.sup.6/ml) [0621] Calculation: k.sub.cl[1/min]/(cell count [x*10.sup.6]/incubation volume [ml]) [0622] (QH=species-specific liver blood flow).

    [0623] Table 4 below shows, for representative working examples of the invention, the CL and F.sub.max values from this assay after incubation of the compounds with rat hepatocytes (some as mean values from two or more independent individual determinations):

    TABLE-US-00006 TABLE 4 Calculated blood clearance and bioavailability after incubation with rat hepatocytes Example CL.sub.blood F.sub.max No. [L/(h*kg)] [%] 1 1.02 75.7 2 0.44 89.5 5 0.87 79.2 8 0.29 93.1 11 0.3 92.9 15 0.16 96.1 20 0.77 81.8

    [0624] B-9. Metabolic Study

    [0625] To determine the metabolic profile of the compounds according to the invention, 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.

    [0626] The compounds according to the invention 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 were prepared, and then pipetted with a 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 was centrifuged off at about 15 000g. The samples thus stopped are either analysed directly or stored at 20 C. until analysis.

    [0627] 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 the mass spectrometry data serve for identification, structural elucidation and quantitative estimation of the metabolites, and for quantitative determination of the metabolic decrease in the compounds according to the invention in the incubation mixtures.

    [0628] B-10. Pharmacokinetic Studies In Vivo

    [0629] The substance to be examined is administered to rats or mice 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 tylose), in each case via a gavage. After administration of the substance, blood is obtained from the animals at fixed time points. It is heparinized, then plasma is obtained from it by centrifugation. The test substance is quantified analytically in the plasma by LC-MS/MS. The plasma concentration/time plots determined in this way are used to calculate, 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).

    [0630] B-11. Determination of Solubility

    [0631] Test Procedure:

    [0632] The test substance is dissolved in DMSO. An aliquot is taken from this solution and introduced into PBS buffer pH 6.5 (DMSO content: 1%). This solution/suspension is agitated at room temperature for 24 h. After ultracentrifugation at 114000 g for 30 min, the supernatant is removed, diluted with acetonitrile/water 8:2 and analysed by LC-MSMS. Quantification is effected by means of a five-point calibration curve of the test compound in DMSO.

    [0633] Instruments for LC-MSMS Quantification:

    [0634] AB Sciex TRIPLE QUAD 4500; Agilent 1260 with primary pump (G1312B Infinity), degasser (G4225A Infinity), column thermostat (G1316C Infinity); CTC Analytics PAL injection system THC-xt.

    [0635] HPLC Method:

    [0636] Eluent A: 0.5 ml formic acid/litre of water, eluent B: 0.5 ml formic acid/litre of acetonitrile; gradient: 0 min 90% A.fwdarw.0.5 min 5% A.fwdarw.0.84 min 5% A.fwdarw.0.85 min 90% A.fwdarw.1.22 min 90%

    [0637] A; flow rate: 2.5 ml/min; injection volume: 15 l; column: Waters OASIS HLB, 2.120 mm, 25; column temperature: 30 C.; splitter (before MS): 1:20.

    [0638] MS Methods:

    [0639] Flow injection analysis (FIA) for optimization, multiple reaction monitoring (MRM) for quantification; eluent A: 0.5 ml formic acid/litre of water, eluent B: 0.5 ml formic acid/litre of acetonitrile; flow rate: 0.25 ml/min; injection volume: 15 l; column: stainless steel capillary; capillary temperature: 25 C.

    [0640] Table 5 below shows the solubility values thus determined for representative working examples in PBS buffer pH 6.5:

    TABLE-US-00007 TABLE 5 Solubility in PBS buffer pH 6.5 Example Solubility No. [mg/litre] 1 52.2 4 51.4 5 338.1 6 354.9 8 4.5 10 33.4 11 210 12 240 14 80 15 100 17 250 18 330 19 17 20 425.2

    [0641] C. Working Examples of Pharmaceutical Compositions

    [0642] The compounds according to the invention can be converted to pharmaceutical preparations as follows:

    [0643] Tablet:

    [0644] Composition:

    [0645] 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) (BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.

    [0646] Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm

    [0647] Production:

    [0648] The mixture of compound according to the invention, 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 using a conventional tabletting press (see above for format of the tablet). The guide value used for the pressing is a pressing force of 15 kN.

    [0649] Suspension for Oral Administration:

    [0650] Composition:

    [0651] 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.

    [0652] 10 ml of oral suspension correspond to a single dose of 100 mg of the compound according to the invention.

    [0653] Production:

    [0654] 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 until the swelling of the Rhodigel is complete.

    [0655] Solution for Oral Administration:

    [0656] Composition:

    [0657] 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.

    [0658] Production:

    [0659] 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.

    [0660] i.v. Solution:

    [0661] 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.