Hydroxy-ethylene derivatives for the treatment of arthrosis
10202418 ยท 2019-02-12
Assignee
Inventors
Cpc classification
C07K5/0207
CHEMISTRY; METALLURGY
C07K7/02
CHEMISTRY; METALLURGY
A61P25/14
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K5/02
CHEMISTRY; METALLURGY
C07K7/02
CHEMISTRY; METALLURGY
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to compounds of the formula I and in particular medicaments comprising at least one compound of the formula I for use in the treatment and/or prophylaxis of physiological and/or pathophysiological conditions in the triggering of which cathepsin D is involved, in particular for use in the treatment and/or prophylaxis of arthrosis, traumatic cartilage injuries, arthritis, pain, allodynia or hyperalgesia.
Claims
1. Compounds of the formula I, ##STR00045## in which I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5 each, independently of one another, denote CR, R.sup.1 denotes ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, cyclobutyl, tert-butyl, phenyl, benzyl, 2-oxetanyl, 3-oxetanyl, tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, cyclopentyl, pentyl, methylsulfanylmethyl, ethylsulfanylmethyl, 2-methylsulfanylethyl or 1-methylsulfanylethyl, A.sup.1 denotes 0 to 3 identical or different amino acid radicals (NHCHRCO) connected to one another in a peptide-like manner or CO, OCO, NRCO, SO.sub.2 or NRSO.sub.2, L.sup.1 denotes a single bond or a linear or branched alkyl linker having 1-10 C atoms, in which 1-5 CH.sub.2 groups may be replaced, independently of one another, by 0, S, CXX, SO, SO.sub.2, NR, CO, OCO, NRCONR, NRCO, NRSO.sub.2R, COO, CONR, CC groups and/or by CHCH groups and/or, in addition, 1-20 H atoms may be replaced by F and/or Cl, L.sup.2 denotes a single bond, NR, NRCRR or NRCRRCRR, X, X, Y, each, independently of one another, denote T, R, R, independently of one another, denote H, T, OT or a linear or branched alkyl having 1-10 C atoms, in which one, two or three CH.sub.2 groups may be replaced, independently of one another, by O, S, SO, SO.sub.2, NH, NCH.sub.3, OCO, NHCONH, NHCO, NRSO.sub.2R, COO, CONH, NCH.sub.3CO, CONCH.sub.3, CC groups and/or by CHCH groups and/or, in addition, 1-20 H atoms may be replaced by F and/or Cl, and which is unsubstituted, mono-, di- or trisubstituted by S, NR, O, Hal, OH, NH.sub.2, SO.sub.2CH.sub.3, SO.sub.2NH.sub.2, CN, CONH.sub.2, NHCOCH.sub.3, and/or NHCONH.sub.2, or a cyclic alkyl having 3-7 C atoms, in which one, two or three CH.sub.2 groups may be replaced, independently of one another, by O, S, SO, SO.sub.2, NH, NCH.sub.3, OCO, NHCONH, NHCO, NRSO.sub.2R, COO, CONH, NCH.sub.3CO, CONCH.sub.3 and/or by CHCH groups and/or, in addition, 1-11 H atoms may be replaced by F and/or Cl, and which is unsubstituted or mono-, di- or trisubstituted by S, NR, O, OH, NH.sub.2, SO.sub.2CH.sub.3, SO.sub.2NH.sub.2, CN, CONH.sub.2, NHCOCH.sub.3, and/or NHCONH.sub.2, T denotes a phenyl or naphthyl, which is unsubstituted or mono-, di- tri- or tetrasubstituted by R, or a mono- or bicyclic saturated, unsaturated or aromatic heterocycle having 1 to 4 N, O and/or S atoms, which may be mono-, di- or trisubstituted by R, S, NR and/or O, m denotes 0-4, n denotes 0-2 and Hal denotes F, Cl, Br or I, and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
2. Compounds according to claim 1 in which R.sup.1 denotes isopropyl, 2-butyl or isobutyl and I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, A.sup.1, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal have the meanings indicated in claim 1, and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
3. Compounds according to claim 1 in which R.sup.1 denotes isopropyl and I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, A.sup.1, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal have the meanings indicated in claim 1, and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
4. Compounds according to claim 1 in which R.sup.1 denotes isopropyl, having an S configuration of the chiral centre to which the isopropyl group is bonded, and I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, A.sup.1, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal have the meanings indicated in claim 1, and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
5. Compounds according to claim 1 in which A.sup.1 denotes 0 to 1 amino acid radicals, selected from alanine, glycine, cyclopropylglycine, cyclobutylglycine, cyclopentylglycine, cyclohexylglycine, 3-oxetanylglycine, 3-oxetanylglycine, tetrahydrofuran-3-ylglycine, tetrahydrofuran-2-ylglycine, ethylsulfanylmethylglycine, 2-methylsulfanylethylglycine, 1-methylsulfanylethylglycine, valine, norvaline, aminobutyric acid, leucine, isoleucine, proline, tert-leucine, norleucine, methionine, phenylalanine, naphthylalanine, O-methylserine and O-ethylserine, which are connected to one another in a peptide-like manner, or OCO, NRCO, SO.sub.2, or NRSO.sub.2, and I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, R, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal have the meanings given in claim 1, and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
6. Compounds according to claim 1 in which A.sup.1 denotes 0 to 1 amino acid radicals, selected from valine, norvaline, leucine, isoleucine, norleucine, phenylalanine and naphthylalanine, which are connected to one another in a peptide-like manner, and I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, R, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal have the meanings indicated in claim 1, and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
7. Compound according to claim 1 selected from the group consisting of: c) (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-[2-(3-trifluoromethoxyphenyl)-acetylamino]butyrylamino}-6-phenylhexanoic acid phenethylamide; d) (4S,5S)-4-Hydroxy-5-{(S)-2-[2-(2-methoxy-5-trifluoromethoxyphenyl)-acetylamino]-3-methylbutyrylamino}-6-phenylhexanoic acid phenethylamide; e) (4S,5S)-5-{(S)-2-[2-(3,4-Dimethoxyphenyl)acetylamino]-3-methyl-butyrylamino}-4-hydroxy-6-phenylhexanoic acid phenethylamide; f) (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-[2-(3-phenoxyphenyl)acetylamino]-butyrylamino}-6-phenylhexanoic acid phenethylamide; g) (4S,5S)-4-Hydroxy-5-{(S)-2-[2-(4-methoxyphenyl)acetylamino]-3-methyl-butyrylamino}-6-phenylhexanoic acid phenethylamide; h) (4S,5S)-4-Hydroxy-5-{(S)-2-[2-(3-methoxyphenyl)acetylamino]-3-methyl-butyrylamino}-6-phenylhexanoic acid phenethylamide; i) (4S,5S)-4-Hydroxy-5-{(S)-2-[2-(2-methoxyphenyl)acetylamino]-3-methyl-butyrylamino}-6-phenylhexanoic acid phenethylamide; j) (4S,5S)-5-{(S)-2-[2-(3,4-Dimethylphenoxy)acetylamino]-3-methylbutyrylamino}-4-hydroxy-6-phenylhexanoic acid phenethylamide; k) (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-(2-naphthalen-2-ylacetylamino)-butyrylamino}-6-phenylhexanoic acid phenethylamide; l) (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-(2-naphthalen-1-ylacetylamino)-butyrylamino}-6-phenylhexanoic acid phenethylamide; m) (4S,5S)-5-((S)-2-Diphenylacetylamino-3-methylbutyrylamino)-4-hydroxy-6-phenylhexanoic acid phenethylamide; n) (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-[2-(3-phenoxyphenyl)acetylamino]-butyrylamino}-6-phenylhexanoic acid (2,6-dimethylphenyl)amide; o) (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-[2-(3-phenoxyphenyl)acetylamino]-butyrylamino}-6-phenylhexanoic acid benzylamide; p) (S)N-[(1S,2S)-1-Benzyl-5-(2,3-dihydroindol-1-yl)-2-hydroxy-5-oxopentyl]-3-methyl-2-[2-(3-phenoxyphenyl)acetylamino]butyramide; s) (4S,5S)-5-{(S)-2-[2-(3-Ethoxyphenyl)acetylamino]-3-methylbutyrylamino}-4-hydroxy-6-phenylhexanoic acid phenethylamide; and t) (4S,5S)-4-Hydroxy-5-[(S)-3-methyl-2-(4-phenylbutyrylamino)butyrylamino]-6-phenylhexanoic acid phenethylamide; and physiologically acceptable salts, derivatives, prodrugs and stereoisomers thereof, and mixtures thereof in all ratios.
8. Process for the preparation of a compound of the formula I of claim 1, comprising preparing a compound of the formula III from a compound of the formula II by hydrolysis, reacting the compound of the formula Ill with a compound of the formula IV to give a compound of the formula V, converting the compound of the formula V into a compound of the formula VI, reacting the compound of the formula VI with a compound of the formula VII to give a compound of the formula VIII and converting the compound of the formula VIII into a compound of the formula I by removal of protecting groups, by the following scheme, wherein I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, R.sup.1, A.sup.1, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal in the compounds of the formula I, II; III; IV, V, VI; VII and VIII have the meanings indicated in claim 1 and A.sup.2 is a single bond: ##STR00046##
9. Process for the preparation of a compound of the formula I claim 1, comprising reacting a compound of the formula IX with a compound of the formula X to give a compound of the formula XI, converting the compound of the formula XI into a compound of the formula XII by removal of protecting groups and reacting the compound of the formula XII with a compound of the formula XIII to give a compound of the formula I, by the following scheme, wherein I.sup.1, I.sup.2, I.sup.3, I.sup.4, I.sup.5, R.sup.1, A.sup.1, L.sup.1, L.sup.2, X, X, Y, T, R, R, m, n and Hal in the compounds of the formula I, IX, X, XI, XII and XIII have the meanings indicated in claim 1 and A.sup.2 is a single bond: ##STR00047##
10. Process for the preparation of a compound of the formula I claim 1, comprising: a) converting the base of a compound of the formula I into one of its salts by treatment with an acid, or b) converting an acid of a compound of the formula I into one of its salts by treatment with a base.
11. Pharmaceutical composition comprising at least one compound according to claim 1 and/or a physiologically acceptable salt, derivative, prodrug or stereoisomer thereof, and mixtures thereof in all ratios.
12. Pharmaceutical composition according to claim 11 comprising one or more excipients and/or adjuvants.
13. Pharmaceutical composition comprising at least one compound according to claim 1 and/or a physiologically acceptable salt, derivative, prodrug or stereoisomer thereof, and mixtures thereof in all ratios, and at least one further medicament active compound.
14. Process for the preparation of a pharmaceutical composition, comprising bringing a compound according to claim 1 and/or a physiologically acceptable salt, derivative, prodrug or stereoisomer thereof, and mixtures thereof in all ratios, into a suitable dosage form together with a solid, liquid or semi-liquid excipient or adjuvant.
15. A kit consisting of separate packs of a) an effective amount of a compound according to claim 1 and/or a physiologically acceptable salt, derivative, prodrug or stereoisomer thereof, and mixtures thereof in all ratios, and b) an effective amount of a further medicament active compound.
16. A diagnostic method which comprises contacting a compound according to claim 1 or a physiologically acceptable salt, derivative, prodrug or stereoisomer thereof, and mixtures thereof in all ratios, with cathepsin D to inhibit cathepsin D.
Description
EXAMPLE 1
Illustrative Compounds of the Formula I
(1) Table 2
(2) The following compounds are in accordance with the invention
(3) TABLE-US-00002 TABLE 2a Com- pound Structure A1
(4) TABLE-US-00003 TABLE 2b Ret. Cath D Renin time M + Compound MW total IC.sub.50 [nM] IC.sub.50 [nM] [min] Method H A1 768.9946 4.30E07 3.20E07 2.17 polar 769.5 A2 761.9953 4.10E08 1.10E06 2.72 polar 762.5 A3 627.7 >30 M 2.47 polar 628.3 A4 657.7258 4.00E06 >30 M 2.50 polar 658.3 A5 603.7555 >30 M 2.15 polar 604.3 A6 635.8005 1.20E05 >30 M 2.53 polar 636.3 A7 573.7297 >30 M 2.25 polar 574.3 A8 573.7297 >30 M 2.26 polar 574.3 A9 573.7297 >30 M 2.30 polar 574.3 A10 587.7565 >30 M 2.50 polar 588.3 A11 593.7637 2.43 polar 594.3 A12 593.7637 >30 M 2.43 polar 594.3 A13 619.8015 4.30E06 >30 M 2.53 polar 620.3 A14 635.8005 >30 M 2.54 polar 636.3 A15 621.7737 >30 M 2.49 polar 622.3 A16 633.7847 >30 M 2.63 polar 634.3 A17 780.0135 2.55E07 >30 M 2.45 polar 780.5 A18 647.8115 >30 M 2.58 polar 648.3 A19 587.7565 >30 M 2.36 polar 588.3 A20 571.7575 >30 M 2.41 polar 572.3 A21 731.9695 2.40E08 1.20E05 2.28 polar 732.4 A22 736.8239 3.50E07 >30 M 2.35 polar 737.3
and physiologically acceptable salts, derivatives, solvates, prodrugs and stereoisomers thereof, including mixtures thereof in all ratios.
(5) Stable: Recovery 75% after 4 h.
(6) For the avoidance of any doubt, in all cases where the chemical name of a compound according to the invention and the depiction of the chemical structure of a compound according to the invention mistakenly do not agree, the compound according to the invention is defined unambiguously by the depiction of the chemical structure.
(7) The retention times were determined:
(8) Polar method: Chromolith Speed Rod RP 18e 50-4.6 mm LCMS; polar.m, 2.4 ml/min, 220 nm, buffer A 0.05% of HCOOH/H2O, buffer B 0.04% of HCOOH/ACN, 0.0-3.0 min 5%-100% of buffer B; 3.0-3.5 min 100% of buffer B
(9) TABLE-US-00004 TABLE 3 Com- pound NMR data peak lists A1 1H NMR (500 MHz, DMSO-d6) ppm = 8.39 (t, J = 6.1, 1H), 7.84 (s, 1H), 7.78 (d, J = 8.7, 1H), 7.68 (d, J = 8.9, 1H), 7.44 (d, J = 9.0, 1H), 7.24-7.17 (m, 4H), 7.15-7.11 (m, 1H), 7.02 (d, J = 8.4, 1H), 6.65 (s, 2H), 4.85 (d, J = 5.5, 1H), 4.20- 4.14 (m, 1H), 4.12-4.07 (m, 1H), 4.06-3.98 (m, 2H), 3.97-3.88 (m, 2H), 3.43-3.35 (m, 1H), 2.83 (dd, J = 13.8, 6.2, 1H), 2.61 (dd, J = 13.6, 8.1, 1H), 2.27 (s, 3H), 2.22- 2.10 (m, 2H), 1.95-1.86 (m, 1H), 1.75-1.64 (m, 1H), 1.62-1.47 (m, 3H), 1.46-1.27 (m, 14H), 1.11-0.99 (m, 1H), 0.89-0.81 (m, 6H), 0.80-0.71 (m, 10H). A2 1H NMR (500 MHz, DMSO-d6) ppm = 8.06 (d, J = 7.8, 1H), 7.75-7.63 (m, 2H), 7.44 (d, J = 9.0, 1H), 7.26-7.16 (m, 4H), 7.16-7.11 (m, 1H), 7.02 (d, J = 8.5, 1H), 4.85 (d, J = 5.4, 1H), 4.29-4.22 (m, 1H), 4.20-4.14 (m, 1H), 4.14- 4.08 (m, 1H), 3.98-3.84 (m, 2H), 3.60 (s, 3H), 3.43-3.33 (m, 1H), 2.83 (dd, J = 13.7, 6.2, 1H), 2.61 (dd, J = 13.7, 8.1, 1H), 2.21-2.09 (m, 2H), 2.07-1.96 (m, 1H), 1.95-1.85 (m, 1H), 1.73-1.62 (m, 1H), 1.60-1.46 (m, 3H), 1.45- 1.28 (m, 12H), 1.10-0.98 (m, 1H), 0.90-0.82 (m, 12H), 0.81-0.74 (m, 12H). A3 1H NMR (400 MHz, DMSO-d6) ppm = 8.07 (d, J = 9.1, 1H), 7.82 (t, J = 5.6, 1H), 7.67 (d, J = 9.0, 1H), 7.45-7.39 (m, 1H), 7.31-7.25 (m, 4H), 7.23-7.16 (m, 8H), 7.15-7.11 (m, 1H), 4.89 (d, J = 5.6, 1H), 4.16 (dd, J = 9.0, 6.7, 1H), 3.98- 3.88 (m, 1H), 3.66-3.46 (m, 2H), 3.42-3.36 (m, 1H), 3.24-3.17 (m, 2H), 2.82 (dd, J = 13.6, 5.7, 1H), 2.69-2.60 (m, 3H), 2.19-2.07 (m, 1H), 2.07-1.97 (m, 1H), 1.97- 1.87 (m, 1H), 1.57-1.48 (m, 2H), 0.79-0.72 (m, 6H). A4 1H NMR (400 MHz, DMSO-d6) ppm = 7.81-7.75 (m, 2H), 7.60 (d, J = 8.9, 1H), 7.30-7.25 (m, 2H), 7.23-7.17 (m, 8H), 7.17-7.16 (m, 1H), 7.16-7.12 (m, 1H), 7.05-7.00 (m, 1H), 4.87 (d, J = 5.6, 1H), 4.17 (dd, J = 9.0, 6.6, 1H), 3.97-3.90 (m, 1H), 3.74 (s, 3H), 3.57-3.42 (m, 2H), 3.42- 3.36 (m, 1H), 3.24-3.17 (m, 2H), 2.83 (dd, J = 13.6, 5.9, 1H), 2.68-2.62 (m, 3H), 2.17-2.07 (m, 1H), 2.07-1.98 (m, 1H), 1.93 (h, J = 6.8, 1H), 1.56-1.49 (m, 2H), 0.80- 0.75 (m, 6H). A5 1H NMR (400 MHz, DMSO-d6) ppm = 7.82 (d, J = 9.0, 1H), 7.78 (t, J = 5.6, 1H), 7.58 (d, J = 9.0, 1H), 7.30-7.25 (m, 2H), 7.22-7.17 (m, 6H), 7.17-7.16 (m, 1H), 7.16-7.11 (m, 1H), 6.90 (d, J = 2.0, 1H), 6.85 (d, J = 8.2, 1H), 6.76 (dd, J = 8.2, 2.0, 1H), 4.86 (d, J = 5.6, 1H), 4.14 (dd, J = 9.0, 6.7, 1H), 3.97-3.88 (m, 1H), 3.70 (d, J = 2.6, 6H), 3.48-3.32 (m, 3H), 3.24-3.17 (m, 2H), 2.81 (dd, J = 13.7, 5.9, 1H), 2.68-2.60 (m, 3H), 2.17-2.07 (m, 1H), 2.06-1.98 (m, 1H), 1.93 (h, J = 6.7, 1H), 1.56-1.48 (m, 2H), 0.78-0.73 (m, 6H). A6 1H NMR (400 MHz, DMSO-d6) ppm = 7.92 (d, J = 9.0, 1H), 7.78 (t, J = 5.6, 1H), 7.59 (d, J = 9.0, 1H), 7.39-7.33 (m, 2H), 7.31-7.25 (m, 3H), 7.21-7.17 (m, 6H), 7.17-7.15 (m, 1H), 7.15-7.09 (m, 2H), 7.05-7.01 (m, 1H), 7.00-6.94 (m, 3H), 6.87-6.83 (m, 1H), 4.85 (d, J = 5.6, 1H), 4.14 (dd, J = 9.0, 6.7, 1H), 3.96-3.88 (m, 1H), 3.57-3.35 (m, 3H), 3.24-3.17 (m, 2H), 2.85-2.78 (m, 1H), 2.68-2.59 (m, 3H), 2.17-1.97 (m, 2H), 1.90 (h, J = 6.7, 1H), 1.57-1.46 (m, 2H), 0.76-0.71 (m, 6H). A7 1H NMR (400 MHz, DMSO-d6) ppm = 7.83 (d, J = 9.0, 1H), 7.78 (t, J = 5.6, 1H), 7.56 (d, J = 9.0, 1H), 7.31-7.24 (m, 2H), 7.23-7.12 (m, 10H), 6.86-6.81 (m, 2H), 4.87 (d, J = 5.5, 1H), 4.13 (dd, J = 9.0, 6.8, 1H), 3.97-3.88 (m, 1H), 3.71 (s, 3H), 3.48-3.33 (m, 3H), 3.24-3.17 (m, 2H), 2.85-2.78 (m, 1H), 2.68-2.60 (m, 3H), 2.17-1.97 (m, 2H), 1.91 (h, J = 6.8, 1H), 1.57-1.46 (m, 2H), 0.75 (d, J = 6.7, 6H). A8 1H NMR (400 MHz, DMSO-d6) ppm = 7.90 (d, J = 9.0, 1H), 7.78 (t, J = 5.6, 1H), 7.58 (d, J = 9.0, 1H), 7.31-7.25 (m, 2H), 7.22-7.17 (m, 7H), 7.17-7.16 (m, 1H), 7.16-7.10 (m, 1H), 6.87-6.80 (m, 2H), 6.79-6.75 (m, 1H), 4.86 (d, J = 5.6, 1H), 4.14 (dd, J = 9.0, 6.8, 1H), 3.97-3.88 (m, 1H), 3.71 (s, 3H), 3.53-3.38 (m, 3H), 3.24-3.17 (m, 2H), 2.85- 2.78 (m, 1H), 2.69-2.58 (m, 3H), 2.17-1.98 (m, 2H), 1.93 (h, J = 6.7, 1H), 1.58-1.46 (m, 2H), 0.76 (d, J = 6.7, 6H). A9 1H NMR (400 MHz, DMSO-d6) ppm = 7.78 (t, J = 5.6, 1H), 7.55 (dd, J = 20.5, 8.9, 2H), 7.31-7.12 (m, 12H), 6.97- 6.92 (m, 1H), 6.87 (td, J = 7.4, 1.1, 1H), 4.89 (d, J = 5.5, 1H), 4.16 (dd, J = 8.9, 6.4, 1H), 3.98-3.89 (m, 1H), 3.72 (s, 3H), 3.53-3.36 (m, 3H), 3.24-3.17 (m, 2H), 2.83 (dd, J = 13.6, 5.9, 1H), 2.68-2.60 (m, 3H), 2.20-1.97 (m, 2H), 1.92 (h, J = 6.7, 1H), 1.57-1.47 (m, 2H), 0.76 (d, J = 6.7, 3H), 0.74 (d, J = 6.7, 3H). A10 1H NMR (400 MHz, DMSO-d6) ppm = 7.85-7.72 (m, 2H), 7.61 (d, J = 9.0, 1H), 7.33-7.24 (m, 2H), 7.24-7.15 (m, 7H), 7.14-7.08 (m, 1H), 7.02 (d, J = 8.4, 1H), 6.75 (d, J = 2.7, 1H), 6.65 (dd, J = 8.2, 2.8, 1H), 4.90 (d, J = 5.4, 1H), 4.54-4.39 (m, 2H), 4.24 (dd, J = 9.0, 6.4, 1H), 4.03-3.86 (m, 1H), 3.47-3.36 (m, 1H), 3.25-3.15 (m, 2H), 2.86- 2.78 (m, 1H), 2.70-2.61 (m, 3H), 2.20-2.09 (m, 7H), 2.08-1.91 (m, 2H), 1.60-1.46 (m, 2H), 0.77 (d, J = 6.7, 3H), 0.73 (d, J = 6.8, 3H). A11 1H NMR (400 MHz, DMSO-d6) ppm = 8.01 (d, J = 9.0, 1H), 7.88-7.85 (m, 1H), 7.85-7.81 (m, 2H), 7.81-7.75 (m, 2H), 7.60 (d, J = 9.0, 1H), 7.51-7.42 (m, 3H), 7.30-7.24 (m, 2H), 7.22-7.15 (m, 7H), 7.13-7.08 (m, 1H), 4.88 (d, J = 5.5, 1H), 4.17 (dd, J = 9.0, 6.8, 1H), 3.98-3.90 (m, 1H), 3.76-3.57 (m, 2H), 3.43-3.36 (m, 1H), 3.24-3.17 (m, 2H), 2.82 (dd, J = 13.6, 5.7, 1H), 2.65 (t, J = 7.3, 3H), 2.17- 1.99 (m, 2H), 1.94 (h, J = 6.8, 1H), 1.57-1.49 (m, 2H), 0.80- 0.74 (m, 6H). A12 1H NMR (400 MHz, DMSO-d6) ppm = 8.12-8.07 (m, 1H), 8.03 (d, J = 9.0, 1H), 7.92-7.88 (m, 1H), 7.83-7.75 (m, 2H), 7.60 (d, J = 8.9, 1H), 7.49 (dd, J = 6.4, 3.3, 2H), 7.45- 7.41 (m, 2H), 7.30-7.25 (m, 2H), 7.23-7.19 (m, 5H), 7.19-7.15 (m, 3H), 4.88 (d, J = 5.6, 1H), 4.17 (dd, J = 9.0, 6.6, 1H), 4.06 (d, J = 15.0, 1H), 3.99-3.92 (m, 1H), 3.90 (d, J = 15.0, 1H), 3.43-3.36 (m, 1H), 3.24-3.17 (m, 2H), 2.82 (dd, J = 13.7, 6.0, 1H), 2.68-2.61 (m, 3H), 2.19-2.00 (m, 2H), 1.99-1.90 (m, 1H), 1.57-1.49 (m, 2H), 0.76 (d, J = 6.7, 6H). A13 1H NMR (400 MHz, DMSO-d6) ppm = 8.10 (d, J = 9.0, 1H), 7.78 (t, J = 5.6, 1H), 7.65 (d, J = 8.8, 1H), 7.34-7.04 (m, 20H), 5.17 (s, 1H), 4.89 (d, J = 5.5, 1H), 4.22 (dd, J = 9.0, 6.8, 1H), 3.95-3.86 (m, 1H), 3.40-3.36 (m, 1H), 3.25- 3.16 (m, 2H), 2.81 (dd, J = 13.7, 5.8, 1H), 2.70-2.55 (m, 3H), 2.17-2.07 (m, 1H), 2.06-1.97 (m, 1H), 1.96-1.84 (m, 1H), 1.58-1.45 (m, 2H), 0.77-0.67 (m, 6H). A14 1H NMR (500 MHz, DMSO-d6) ppm = 9.13 (s, 1H), 7.98 (d, J = 9.1, 1H), 7.70 (d, J = 9.0, 1H), 7.40-7.33 (m, 2H), 7.29 (t, J = 7.9, 1H), 7.23-7.17 (m, 4H), 7.17-7.09 (m, 2H), 7.06-6.92 (m, 7H), 6.89-6.83 (m, 1H), 4.91 (d, J = 5.8, 1H), 4.20-4.13 (m, 1H), 3.99-3.92 (m, 1H), 3.54 (d, J = 13.7, 1H), 3.50-3.44 (m, 1H), 3.40 (d, J = 13.7, 1H), 2.84 (dd, J = 13.6, 6.1, 1H), 2.63 (dd, J = 13.5, 8.3, 1H), 2.44- 2.34 (m, 1H), 2.32-2.23 (m, 1H), 2.05 (s, 6H), 1.96- 1.86 (m, 1H), 1.68-1.60 (m, 2H), 0.79-0.70 (m, 6H). A15 1H NMR (500 MHz, DMSO-d6) ppm = 8.26 (t, J = 6.0, 1H), 7.96 (d, J = 9.1, 1H), 7.66 (d, J = 9.0, 1H), 7.40-7.33 (m, 2H), 7.29 (t, J = 7.6, 3H), 7.25-7.16 (m, 7H), 7.16-7.09 (m, 2H), 7.03 (d, J = 7.7, 1H), 7.00-6.92 (m, 3H), 6.88- 6.82 (m, 1H), 4.89 (d, J = 5.6, 1H), 4.21 (d, J = 6.0, 2H), 4.16- 4.11 (m, 1H), 3.98-3.88 (m, 1H), 3.54 (d, J = 13.7, 1H), 3.44-3.36 (m, 2H), 2.86-2.75 (m, 1H), 2.67-2.56 (m, 1H), 2.27-2.17 (m, 1H), 2.16-2.06 (m, 1H), 1.95-1.83 (m, J = 7.1, 1H), 1.64-1.49 (m, 2H), 0.76-0.69 (m, 6H). A16 1H NMR (500 MHz, DMSO-d6) ppm = 8.04 (d, J = 8.0, 1H), 7.96 (d, J = 9.1, 1H), 7.70 (d, J = 9.0, 1H), 7.40-7.32 (m, 2H), 7.28 (t, J = 7.9, 1H), 7.24-7.16 (m, 5H), 7.15-7.08 (m, 3H), 7.06-7.00 (m, 1H), 7.00-6.92 (m, 4H), 6.88- 6.80 (m, 1H), 4.92 (d, J = 5.6, 1H), 4.20-4.11 (m, 1H), 4.03 (t, J = 8.5, 2H), 4.00-3.91 (m, 1H), 3.57-3.47 (m, 2H), 3.39 (d, J = 13.7, 1H), 3.11 (t, J = 8.5, 2H), 2.84 (dd, J = 13.7, 5.4, 1H), 2.64 (dd, J = 13.7, 8.9, 1H), 2.50-2.38 (m, 1H), 1.96-1.84 (m, J = 6.8, 1H), 1.70-1.56 (m, 2H), 1.17 (s, 1H), 0.76-0.69 (m, 6H). A17 1H NMR (400 MHz, DMSO-d6) ppm = 8.13 (d, J = 8.2, 1H), 7.97-7.88 (m, 2H), 7.57-7.48 (m, 2H), 7.29-7.08 (m, 10H), 4.82 (d, J = 5.5, 1H), 4.63-4.54 (m, 1H), 4.35-4.26 (m, 1H), 4.20-4.05 (m, 2H), 3.94-3.84 (m, 1H), 3.62 (s, 3H), 3.35-3.33 (m, 1H), 2.99-2.91 (m, 1H), 2.84-2.69 (m, 2H), 2.64-2.56 (m, 1H), 2.19-2.09 (m, 1H), 2.08- 1.92 (m, 5H), 1.91-1.82 (m, 1H), 1.61-1.50 (m, 1H), 1.48-1.35 (m, 4H), 0.91-0.79 (m, 18H), 0.76-0.69 (m, 6H). A18 1H NMR (400 MHz, DMSO-d6) ppm = 8.12-8.01 (m, 1H), 7.93 (d, J = 9.0, 1H), 7.65-7.57 (m, 1H), 7.41-7.33 (m, 2H), 7.32-7.26 (m, 1H), 7.25-7.07 (m, 10H), 7.04 (d, J = 7.5, 1H), 7.00-6.93 (m, 3H), 6.88-6.82 (m, 1H), 5.23 (q, J = 8.0, 1H), 4.90-4.82 (m, 1H), 4.20-4.10 (m, 1H), 3.99-3.88 (m, 1H), 3.54 (d, J = 13.8, 1H), 3.48-3.37 (m, 2H), 2.95-2.70 (m, 3H), 2.69-2.58 (m, 1H), 2.38-2.27 (m, 1H), 2.25-2.04 (m, 2H), 1.99-1.85 (m, 1H), 1.78- 1.65 (m, 1H), 1.63-1.53 (m, 2H), 0.79-0.70 (m, 6H). A19 1H NMR (500 MHz, DMSO-d6) ppm = 7.94 (d, J = 9.0, 1H), 7.83 (t, J = 5.6, 1H), 7.64 (d, J = 8.9, 1H), 7.28 (t, J = 7.5, 2H), 7.23-7.12 (m, 9H), 6.87-6.70 (m, 3H), 4.89 (d, J = 5.6, 1H), 4.19-4.11 (m, 1H), 4.02-3.86 (m, 3H), 3.50 (d, J = 13.7, 1H), 3.42-3.36 (m, 2H), 3.24-3.15 (m, 2H), 2.87- 2.76 (m, 1H), 2.68-2.56 (m, 3H), 2.17-2.08 (m, 1H), 2.06-1.98 (m, 1H), 1.96-1.86 (m, 1H), 1.61-1.43 (m, 2H), 1.29 (t, J = 6.9, 3H), 0.76 (d, J = 6.8, 6H). A20 1H NMR (500 MHz, DMSO-d6) ppm = 7.82 (t, J = 5.6, 1H), 7.74 (d, J = 9.0, 1H), 7.53 (d, J = 9.0, 1H), 7.31-7.24 (m, 4H), 7.22-7.13 (m, 10H), 7.12-7.05 (m, 1H), 4.92 (d, J = 5.5, 1H), 4.16-4.08 (m, 1H), 3.97-3.87 (m, 1H), 3.42- 3.37 (m, 1H), 3.25-3.14 (m, 2H), 2.84-2.77 (m, 1H), 2.69-2.58 (m, 3H), 2.57-2.52 (m, 2H), 2.21-2.08 (m, 3H), 2.06-1.97 (m, 1H), 1.93-1.86 (m, 1H), 1.82-1.72 (m, 2H), 1.57-1.46 (m, 2H), 0.82-0.72 (m, 6H). A21 1H NMR (500 MHz, DMSO-d6) ppm = 12.53 (s, 1H), 8.00 (d, J = 8.3, 1H), 7.92-7.54 (m, 4H), 7.29-7.07 (m, 5H), 5.14-4.76 (m, 1H), 4.39-4.17 (m, 2H), 4.16-3.99 (m, 2H), 3.97-3.84 (m, 1H), 3.43-3.39 (m, 1H), 2.87-2.77 (m, 1H), 2.68-2.58 (m, 1H), 2.26-2.08 (m, 2H), 2.08- 1.81 (m, 3H), 1.77-1.64 (m, 1H), 1.61-1.32 (m, 8H), 1.09-0.98 (m, 1H), 0.91-0.67 (m, 30H). A22 1H NMR (400 MHz, DMSO-d6) ppm = 12.47 (s, 1H), 8.04 (d, J = 9.0, 1H), 7.85 (d, J = 8.1, 1H), 7.75-7.62 (m, 2H), 7.42 (t, J = 8.1, 1H), 7.32-7.25 (m, 2H), 7.24-7.09 (m, 6H), 4.85 (s, 1H), 4.31-4.21 (m, 1H), 4.20-4.05 (m, 2H), 3.98-3.87 (m, 1H), 3.62 (d, J = 13.9, 1H), 3.49 (d, J = 13.9, 1H), 3.40 (s, 1H), 2.89-2.75 (m, 1H), 2.67-2.57 (m, 1H), 2.26-2.11 (m, 2H), 2.08-1.97 (m, 1H), 1.96-1.86 (m, 1H), 1.76-1.63 (m, 1H), 1.58-1.31 (m, 3H), 1.12-0.98 (m, 1H), 0.92-0.84 (m, 6H), 0.83-0.71 (m, 12H).
EXAMPLE 2
Preparation of the Compounds of the Formula I According to the Invention
(10) The compounds according to the invention can be prepared, for example, by the following synthesis sequences by methods known to the person skilled in the art. The examples indicated describe the synthesis, but do not restrict this to the examples.
(11) Synthesis Sequence:
(12) ##STR00028##
(13) Starting from the diprotected building block A-2, which is obtainable from A1 by hydrolysis, the new peptide bond to corresponding amines, amino acid derivatives, dipeptides or tripeptides (typically protected on the C terminal) is built up with the aid of an amide coupling method known to the person skilled in the art, such as, for example, a DAPECI coupling.
(14) In the second step, the BOC protecting group and the acetal are cleaved off under suitable conditions (for example by HCl/dioxane or TFA/DCM), and the building block obtained is coupled to a corresponding acid, amino acid derivatives, dipeptides or tripeptides (typically in each case protected on the N terminal). Particular preference is given in this step to coupling reagents which are suitable for suppressing racemisation, such as, for example, HATU or similar reagents. In further steps, the removal of further protecting groups may follow, for example the hydrogenolysis of Z protecting groups or of benzyl esters to give the free acid in the presence of Pd/C.
(15) The acids or amino acid derivatives required are either commercially available, accessible by methods of peptide synthesis known to the person skilled in the art or accessible, for example, by the following route:
(16) ##STR00029##
(17) where in this case L.sup.1=L.sup.1-CO.
(18) Alternatively, the compounds of the formula I claimed can also be prepared by the following sequence:
(19) ##STR00030##
EXAMPLE 3
Preparation of (4S,5S)-4-benzyl-5-(2-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester
(20) ##STR00031##
(21) (S)-4-Benzyl-5-(2-cyanoethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (30.0 g; 87 mmol; 100.00 mol %) was dissolved in 300 ml of methanol, and potassium hydroxide (300 ml, 40% solution in water) was added. The batch was refluxed until the nitrile had hydrolysed completely. The mixture was subsequently rendered acidic with cooling using citric acid solution (1.5 equiv. based on KOH). The water phase was extracted three times with dichloromethane, the organic phases were combined, washed with NaCl solution until neutral, dried, and the solvent was removed, giving 25.3 g of (4S,5S)-4-benzyl-5-(2-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester as oil (yield 73.5%, content 92%). Chromatographic purification on silica gel (eluent: DCM/methanol) starting from 5.0 g of crude product gave 4.6 g of an oil. MS-FAB (M+H.sup.+-BOC)=264.1 R.sub.f (polar method): 2.50 min.
EXAMPLE 4
Preparation of (4S,5S)-4-hydroxy-5-{(S)-2-[2-(2-methoxy-5-trifluoromethoxyphenyl)acetylamino]-3-methylbutyrylamino}-6-phenylhexanoic acid N-phenethylamide (A4)
Step 1: (4S,5S)-4-Benzyl-2,2-dimethyl-5-(2-phenethylcarbamoylethyl)-oxazolidine-3-carboxylic acid tert-butyl ester
(22) ##STR00032##
(23) 2.50 g of (4S,5S)-4-benzyl-5-(2-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester, 0.975 ml of phenethylamine, 465 mg of 1-hydroxybenzotriazole hydrate, 1.50 ml of 4-methylmorpholine and 1.45 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (DAPECI) were dissolved in about 40 ml of DMF in a flask with ice-cooling and stirred overnight. The reaction mixture was poured into saturated sodium hydrogencarbonate solution and stirred for 15 min. The precipitate formed was filtered off with suction, dissolved in DCM and washed a number of times firstly with hydrogencarbonate solution, then with dilute formic acid solution and water. The solvent was removed, and the oily residue was lyophilised, giving 3.25 g of (4S,5S)-4-benzyl-2,2-dimethyl-5-(2-phenethylcarbamoylethyl)oxazolidine-3-carboxylic acid tert-butyl ester as oil (yield 97.1%, content 94%). MS-FAB (M+H.sup.+-BOC)=367.2 R.sub.f (polar method): 2.76 min (MS track).
Step 2 (4S,5S)-5-Amino-4-hydroxy-6-phenylhexanoic acid N-(phenethyl)-amide (hydrochloride)
(24) ##STR00033##
(25) 3.15 g of (4S,5S)-4-benzyl-2,2-dimethyl-5-(2-phenethylcarbamoylethyl)-oxazolidine-3-carboxylic acid tert-butyl ester were dissolved in 45 ml of methanol and 110 ml of HCl (4M) in dioxane in a flask and stirred at RT for a few hours. Removal of the solvent and lyophilisation gave 2.32 g of (4S,5S)-5-amino-4-hydroxy-6-phenylhexanoic acid N-(phenethyl)amide as hydrochloride. (Yield 96%, content >95%). MS-FAB (M+H.sup.+)=327.2 R.sub.f (polar method): 1.46 min (MS track).
(26) The following unknown compounds, for example, can be prepared by this method:
(27) ##STR00034##
Step 3 [(S)-1-((1S,2S)-1-Benzyl-2-hydroxy-4-phenethylcarbamoylbutylcarbamoyl)-2-methylpropyl]carbamic acid tert-butyl ester
(28) ##STR00035##
(29) 1.80 g of (4S,5S)-5-amino-4-hydroxy-6-phenylhexanoic acid N-(phenethyl)-amide hydrochloride, 1.13 g of (S)-2-tert-butoxycarbonylamino-3-methylbutyric acid, 325 mg of 1-hydroxybenzotriazole hydrate, 1.05 ml of 4-methylmorpholine and 1.01 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (DAPECI) were dissolved in about 20 ml of DMF in a flask with ice-cooling and stirred overnight. The reaction mixture was poured into saturated sodium hydrogencarbonate solution and stirred for 15 min. The precipitate formed was filtered off with suction and rinsed with copious dilute formic acid and water. Lyophilisation gave 2.12 g of [(S)-1-((1S,2S)-1-benzyl-2-hydroxy-4-phenethylcarbamoylbutylcarbamoyl)-2-methylpropyl]-carbamic acid tert-butyl ester as white solid (yield 85.4%, content 100%). MS-FAB (M+H.sup.+)=526.3 R.sub.f (polar method): 2.36 min (MS track).
Step 4 (4S,5S)-5-((S)-2-Amino-3-methylbutyrylamino)-4-hydroxy-6-phenylhexanoic acid N-phenethylamide hydrochloride
(30) ##STR00036##
(31) 2.12 g of [(S)-1-((1S,2S)-1-benzyl-2-hydroxy-4-phenethylcarbamoylbutylcarbamoyl)-2-methylpropyl]carbamic acid tert-butyl ester were dissolved in 75 ml of HCl solution in dioxane (4 M) and 30 ml of methanol in a flask and stirred at RT for 1 h. Excess HCl was removed in the house vacuum, the solvent was removed in vacuo, and the residue was lyophilised overnight, giving 2.23 g of (4S,5S)-5-((S)-2-amino-3-methylbutyrylamino)-4-hydroxy-6-phenylhexanoic acid N-phenethylamide hydrochloride as white solid. (Yield 100%, content 86.6%). MS-FAB (M+H.sup.+)=426.2 R.sub.f (polar method): 1.52 min (MS track).
Step 5 (4S,5S)-4-Hydroxy-5-{(S)-2-[2-(2-methoxy-5-trifluoromethoxy-phenyl)acetylamino]-3-methylbutyrylamino}-6-phenylhexanoic acid N-phenethylamide
(32) ##STR00037##
(33) 150 mg of (4S,5S)-5-((S)-2-amino-3-methylbutyrylamino)-4-hydroxy-6-phenylhexanoic acid N-phenethylamide hydrochloride (content 86.6%), 79 mg of (2-methoxy-5-trifluoromethoxyphenyl)acetic acid, 97.6 l of ethyldiisopropylamine 118 mg of HATU were dissolved in about 5 ml of DMF in a flask with ice-cooling and stirred overnight. The reaction mixture was poured into saturated sodium hydrogencarbonate solution and stirred for 15 min. The precipitate formed was filtered off with suction and rinsed with copious dilute formic acid and water. Lyophilisation gave 135 mg of (4S,5S)-4-hydroxy-5-{(S)-2-[2-(2-methoxy-5-trifluoromethoxyphenyl)acetylamino]-3-methylbutyrylamino}-6-phenylhexanoic acid N-phenethylamide as white solid (yield 73%, content 100%). MS-FAB (M+H.sup.+)=658.3 R.sub.f (polar method): 2.50 min (MS track).
(34) Compounds A13, A14, and A15, for example, can be prepared analogously to this sequence (without restricting the method to these compounds).
EXAMPLE 5
Preparation of (4S,5S)-4-hydroxy-5-{(S)-3-methyl-2-[2-(3-trifluoromethoxyphenyl)acetylamino]butyrylamino}-6-phenylhexanoic acid N-phenethylamide (A3)
Step 1: (S)-3-Methyl-2-[2-(3-trifluoromethoxyphenyl)acetylamino]butyric acid benzyl ester
(35) ##STR00038##
(36) 1.50 g of (S)-2-amino-3-methylbutyric acid benzyl ester hydrochloride, 1.36 g of (3-trifluoromethoxyphenyl)acetic acid, 416 mg of 1-hydroxybenzotriazole hydrate, 1.35 ml of 4-methylmorpholine and 1.18 g of N-(3-dimethylamino-propyl)-N-ethylcarbodiimide hydrochloride (DAPECI) were dissolved in about 10 ml of DMF in a flask with ice-cooling and stirred overnight. The reaction mixture was poured into saturated sodium hydrogencarbonate solution and stirred for 15 min. The suspension formed was extracted a number of times with DCM, the organic phases were combined and rinsed with dilute formic acid and water. Drying using sodium sulfate, removal of the solvent and lyophilisation of the residue gave 2.39 g of (S)-3-methyl-2-[2-(3-trifluoromethoxyphenyl)acetylamino]butyric acid benzyl ester. (Yield 92.5%, content 97.5%). MS-FAB (M+H.sup.+)=410.1 R.sub.f (polar method): 2.69 min (MS track).
Step 2: (S)-3-Methyl-2-[2-(3-trifluoromethoxyphenyl)acetylamino]butyric acid
(37) ##STR00039##
(38) 2.39 g of (S)-3-methyl-2-[2-(3-trifluoromethoxyphenyl)acetylamino]butyric acid benzyl ester were hydrogenated in a suitable vessel in the presence of 1.00 g of Pd/C (5% of Pd, moist) in 30 ml of THF at RT at atmospheric pressure until the starting material had reacted completely (overnight). The reaction mixture was diluted with THF and filtered off with suction in order to remove the catalyst. The filtrate was evaporated in vacuo, and the residue was lyophilised, giving 1.91 g of (S)-3-methyl-2-[2-(3-trifluoromethoxy-phenyl)acetylamino]butyric acid as white solid (yield 97.2%, content 92%). MS-FAB (M+H.sup.+)=320.1 R.sub.f (polar method): 2.11 min (MS track).
(39) The synthesis of the following products can be carried out analogously to this preparation:
(40) ##STR00040## ##STR00041##
Step 3 (4S,5S)-4-Hydroxy-5-{(S)-3-methyl-2-[2-(3-trifluoromethoxyphenyl)-acetylamino]butyrylamino}-6-phenylhexanoic acid N-phenethylamide
(41) ##STR00042##
(42) 100 mg of (4S,5S)-5-((S)-2-amino-3-methylbutyrylamino)-4-hydroxy-6-phenylhexanoic acid N-phenethylamide hydrochloride (content 95.5%), 100 mg of (S)-3-methyl-2-[2-(3-trifluoromethoxyphenyl)acetylamino]butyric acid, 90.9 l of ethyldiisopropylamine, 109.5 mg of HATU were dissolved in about 5 ml of DMF in a flask with ice-cooling and stirred overnight. The reaction mixture was poured into saturated sodium hydrogencarbonate solution and stirred for 15 min. The precipitate formed was filtered off with suction and rinsed with copious dilute formic acid and water. Lyophilisation gave 101.6 mg of (4S,5S)-4-hydroxy-5-{(S)-3-methyl-2-[2-(3-trifluoro-methoxyphenyl)acetylamino]butyrylamino}-6-phenylhexanoic acid N-phenethylamide as white solid (yield 60%, content 97%). MS-FAB (M+H.sup.+)=628.3 R.sub.f (polar method): 2.47 min (MS track).
(43) The products A1, A2, A5-A12, A16-A20, but also the following compounds, can be prepared by this process by suitable combination of the building blocks from Example 2, step 2 or similar compounds and the building blocks from Example 3, step 2 or similar compounds:
(44) ##STR00043##
EXAMPLE 6
Preparation of (S)-2-[(2S,3S)-2-((4S,5S)-4-hydroxy-5-{(S)-3-methyl-2-[(S)-4-methyl-2-(3-methylbutyrylamino)pentanoylamino]-butyrylamino}-6-phenylhexanoylamino)-3-methylpentanoylamino]-3-methylbutyric acid (A21)
(45) ##STR00044##
(46) 106 mg of (S)-2-[(2S,3S)-2-((4S,5S)-4-hydroxy-5-{(S)-3-methyl-2-[(S)-4-methyl-2-(3-methylbutyrylamino)pentanoylamino]butyrylamino}-6-phenylhexanoylamino)-3-methylpentanoylamino]-3-methylbutyric acid benzyl ester were hydrogenated in a suitable vessel in the presence of 100 mg of Pd/C (5% of Pd, moist) in 40 ml of THF at RT at atmospheric pressure until the starting material had reacted completely (overnight). The reaction mixture was diluted with water and THF and filtered off with suction in order to remove the catalyst. The filtrate was evaporated in vacuo, the residue was dissolved in DMF and precipitated using water. Drying gave 2.45 g of (S)-2-[(2S,3S)-2-((4S,5S)-4-hydroxy-5-{(S)-3-methyl-2-[(S)-4-methyl-2-(3-methyl-butyrylamino)pentanoylamino]butyrylamino}-6-phenylhexanoylamino)-3-methylpentanoylamino]-3-methylbutyric acid as white powder (yield 70%, content 98%). MS-FAB (M+H.sup.+)=732.4 R.sub.f (polar method): 2.28 min.
(47) Compound A22 can be prepared analogously.
(48) Abbreviations: DAPECI=N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride DCM=dichloromethane DMA=dimethylacetamide DMF=dimethylformamide EA=ethyl acetate h=hours HATU=(2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) MTBE=methyl tert-butyl ether PE=petroleum ether RT=room temperature SPhos=2-dicyclohexylphosphino-2,6-dimethoxybiphenyl TFA=trifluoroacetic acid
EXAMPLE 7
In-Vitro Fluorescence Assay for Identification of Cathepsin D Inhibitors
(49) In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCA-GKPILFFRLK(Dnp)d-RNH.sub.2 (Enzo Life Sciences, Lor-rach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 l of each substance solution with serially diluted substance concentration were added to in each case 4 l of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 l of substrate solution (final concentration 5 M). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
(50) The IC.sub.50 values of the compounds according to the invention can be obtained from Table 2 from Example 1.
EXAMPLE 8
Cartilage Explant Assay
(51) In order to investigate the effect of potential cathepsin D inhibitors on cartilage degradation, a pH-induced model based on bovine explants is used. The pH of the medium in which the explants are cultivated is matched here to the pathophysiological pH of an arthrotic knee. This pH is pH 5.5. In this ex vivo model, potential cathepsin D inhibitors are subsequently investigated for their action with respect to stopping of the cartilage degradation process. If the cartilage is destroyed, glycosaminoglycans (GAGs) are released into the cell culture supernatant. The amount of GAGs liberated can be determined quantitatively with the aid of DMMB (dimethylmethylene blue hydrochloride). If sulfated GAGs are detected using dimethylmethylene blue hydrochloride, the decrease in the absorption at 633 nm is utilised. Since work can also be carried out at very low GAG concentrations, a dye/GAG complex does not precipitate out even after extended incubation of DMMB with GAG, which sometimes happens after only a short time in other measurement methods. In order to determine the concentration, a calibration line is also recorded using chondroitin sulfate. The GAG values can be used to calculate an IC.sub.50 value, i.e. a concentration at which a substance exhibits 50% of its action.
(52) Solutions:
(53) Incubation Medium, pH 7.4:
(54) DMEM without FBS, addition of 1% of Pen/Strep and 30 g/ml of ascorbic acid, the medium is not stored.
(55) Incubation Medium, pH 5.5:
(56) DMEM without FBS, the pH is adjusted by addition of MES and monitored using a pH meter, addition of 1% of Pen/Strep and 30 g/ml of ascorbic acid.
(57) Solutions for the GAG Measurement:
(58) DMMB Colouring Solution (V=500 ml):
(59) Dissolve 8 mg of DMMB (dimethylmethylene blue) in 2.5 ml of ethanol+1 g of sodium formate+1 ml of formic acid, make up to 500 ml with bidistilled water.
(60) Incubation medium: FBS (medium without FBS)
(61) Chondroitin Sulfate Solutions (Standard Curve)
(62) Preparation of standard solutions with the following concentrations: 50 g/ml; 25 g/ml; 12.5 g/ml; 6.25 g/ml; 3.125 g/ml; 1.56 g/ml; 0.78 g/ml and a blank control of the medium. The preparation of the standard solution is carried out in the medium with which the experiment was also carried out.
(63) 1.) Procedure: pH-Induced Cartilage Degradation of Bovine Explants
(64) The bovine explants are firstly prepared. The induction of the cartilage degradation is carried out in 96-multiwell plates. One explant is cultivated per well. In each case, 200 l of DMEM (incubation medium pH 5.5) without FBS+30 g/ml of ascorbic acid are added. Thus negative control, explants (n=4) are incubated at pH 7.4 (without FBS). This control is not included in the calculation of the data, but instead ensures that the pH change has the desired effect on the liberation of GAG. At this point, the substances to be tested are added. No pre-incubation of the explants is carried out. The explants are cultivated with the corresponding substances for 3 days in the incubator at 37 C. and 7.5% CO.sub.2.
(65) 2.) Incubation Procedure
(66) In order to investigate the effect of cathepsin D inhibitors on the liberation of GAG (glycosaminoglycan), the substances are employed in the desired concentration and cultivated for 3 days. The compounds to be tested are tested in a first experiment in a concentration of 1 M and 1% of DMSO. Substances which have an effect of >50% on the liberation of GAG (this corresponds to <50% of the control in the Assay Explorer) are tested in the next experiment at 100 nM and 1% of DMSO. Substances which have an effect of >50% on the liberation of GAG under these conditions (this corresponds to <50% of the control in the Assay Explorer) are tested in a concentration/effect relationship. The compounds here are investigated in the following concentrations: 30 M, 10 M, 3 M, 1 M, 0.3 M, 0.1 M, 0.03 M, 0.01 M.
(67) The positive control used is pepstatin A with a concentration of 0.01 M. The assay window is defined by the control (pH 5.5), defined as 0% effect, and the control pH 5.5+0.01 M pepstatin A, defined as 100% effect. After incubation for 3 days, the cell culture supernatants are collected and stored at 20 C. or measured directly. The amount of liberated GAG is measured photometrically.
(68) The effect (1 value) of the respective substance in % based on the positive control (pH 5.5+0.01 M pepstatin A) and the negative control (pH 5.5) is reported for concentrations of 1 M and 100 nM. The value represents the average of 4 replicants. In the determination of a concentration/effect relationship, an IC.sub.50 value is reported to the database (Assay Explorer).
(69) 4.) Measurement
(70) The cell culture supernatants (200 l) are either measured directly or stored at 20 C. In order to ensure an accurate determination of the concentration (g/ml of GAG in the supernatant) of GAG, the measurement values must be located in the linear region of the standard curve. In order to ensure this, various dilutions are routinely introduced (1/5, 1/10, 1/20, 1/40). The dilutions are prepared with medium and introduced automatically (Hamilton) into a 384-well plate (15 l). 60 l of DMMB solution are likewise added automatically (or using a multichannel pipette). A rapid colour reaction occurs, which is subsequently measured at 633 nm using a plate reader (for example Envision).
(71) Depending on the amount of sample present, at least one double determination is carried out.
(72) The data are provided by the MTP reader as csv or xls files and stored as raw data based on this format (xls) or used for the calculation of the percentage effect of the particular compound.
(73) 5.) Quality Controls
(74) As control for the induction of the pH-induced cartilage degradation, 4 explants are incubated at pH 7.4. This corresponds to the physiological pH of the cartilage, and no effect on the liberation of GAG is thus expected here. These GAG values (g/ml of supernatant) are thus always significantly lower than the GAG values for incucation at pH 5.5.
(75) A further control, which both serves for checking of the experiment, but is also important for the definition of the assay window, is the pepstatin control (pH 5.5+0.01 M pepstatin A). This substance non-specifically blocks the activity of most proteases and thus determines the maximum possible effect of a compound.
(76) 6.) Results
(77) All compounds measured exhibited an IC.sub.50 value of 10.sup.8 to 10.sup.10 M in the GAG assay. (1) Klompmakers, A. & Hendriks, T. (1986) Anal. Biochem. 153, 80-84, Spectrophotometric Determination of Sulfated Glycosaminoglycans. (2) Groves, P. J. et al. (1997) Anal. Biochem. 245, 247-248 Polyvinyl alcohol-stabilised binding of sulfated GAGs to dimethylmethylene blue.
EXAMPLE 9
Investigation of the Anti-Hyperalgesic Effect in Animals
(78) In order to induce an inflammation reaction, a carrageenan solution (CAR, 1%, 50 l) was injected intra-articularly on one side into a rat knee joint. The uninjected side was used for control purposes. Six animals per group were used. The threshold was determined by means of a micrometer screw (medial-lateral on the knee joint), and the thermal hyperalgesia was determined by means of a directed infrared light source by the Hargreaves method (Hargreaves et al., 1988) on the sole of the foot. Since the site of inflammation (knee joint) is different from the site of measurement (paw sole), use is made here of the term secondary thermal hyperalgesia, the mechanism of which is of importance for the discovery of effective analgesics.
(79) Experimental description of thermal hyperalgesia (Hargreaves test): the experimental animal is placed in a plastic chamber on a quartz sheet. Before testing, the experimental animal is firstly given about 5-15 minutes time to familiarise itself with the environment. As soon as the experimental animal no longer moves so frequently after the familiarisation phase (end of the exploration phase), the infrared light source, whose focus is in the plane of the glass bottom, is positioned directly beneath the rear paw to be stimulated. An experiment run is then started by pressing the button: infrared light results in an increase in the skin temperature of the rear paw. The experiment is terminated either by the experimental animal raising the rear paw (as an expression of the pain threshold being reached) or by automatic switching-off of the infrared light source when a pre-specified maximum temperature has been reached. Light reflected by the paw is recorded as long as the experimental animal sits still. Withdrawal of the paw interrupts this reflection, after which the infrared light source is switched off and the time from switching on to switching off is recorded. The instrument is calibrated in such a way that the infrared light source increases the skin temperature to about 45 degrees Celsius in 10 s (Hargreaves et al. 1988). An instrument produced by Ugo Basile for this purpose is used for the testing.
(80) CAR was purchased from Sigma-Aldrich. Administration of the specific cathepsin D inhibitors according to the invention was carried out intra-articularly 30 minutes before the CAR. Triamcinolone (TAC) in an amount of 10 g/joint was used as positive control, and the solvent (vehicle) was used as negative control. The hyperalgesia is quoted as the difference in the withdrawal times between the inflamed and non-inflamed paw.
(81) Result: TAC was capable of reducing the CAR-induced swelling, but the specific cathepsin D inhibitors according to the invention were not. In contrast, the specific cathepsin D inhibitors according to the invention were able to reduce the extent of thermal hyperalgesia as a function of the dose. Assessment: it has been shown that the compounds of the present invention exert an anti-hyperalgesic action. This can be postulated since the compounds exhibited no influence on inflammatory swelling and thus on the hyperalgesia trigger. It can thus be assumed that the compounds develop a pain-reducing action in humans.
EXAMPLE 10
Stability of the Compounds According to the Invention in Bovine Synovial Fluid
(82) 1.) Extraction of Bovine Synovial Fluid
(83) In the preparation of bovine explants (for the diffusion chamber or other assays), either cow hoof (metacarpal joints) or cow knee is used. The synovial fluid can be obtained from both joints. To this end, the synovial fluid is carefully removed from the open joint using a 10 ml syringe and a cannula and transferred into prepared 2 ml Eppendorf vessels. The Eppendorf vessels are labelled depending on the animal (cow passport is available). It must be ensured here that blood does not enter the joint gap during preparation of the joints. If this is the case, the synovial fluid will become a reddish colour and must consequently be discarded. The synovial fluid is basically highly viscous and clear to yellowish in colour. The removal together with a macroscopic analysis of the synovial fluid is documented.
(84) 2.) Batch for Stability Testing of Substances in SF
(85) In order to check the stability of individual compounds, a pool of four different bovine synovial fluids is mixed. To this end, about 1 ml per SF is used. The mixture is prepared directly in a 5 ml glass vessel. The SFs are mixed thoroughly, but carefully. No air bubbles or foam should form. To this end, a vortex unit is used at the lowest speed. The compounds to be tested are tested in an initial concentration (unless required otherwise) of 1 M. After addition of the substance, the batch is again mixed thoroughly and carefully. For visual monitoring, all SF batches are photographed, and the pictures are filed in the eLabBio file for the corresponding experiment. FIG. 1 shows photodocumentation of this type by way of example. The batches are incubated in the incubator for 48 h at 37 C. and 7.5% CO.sub.2.
(86) 3.) Sampling
(87) The sampling is carried out after the pre-agreed times (unless required otherwise, see below). 200 l of the SF are removed from the mixture per time and transferred directly into a 0.5 ml low-binding Eppendorf vessel. Low-binding Eppendorf vessels are used in order to minimise interaction of the substances with the plastic of the vessels. 200 l of acetonitrile have already been introduced into the Eppendorf vessel, so that a 1+1 mixture of the SF forms thereafter. This simplifies the subsequent analysis, but precipitation of protein may occur immediately after addition of the SF. This should be noted on the protocol. The 0 h sample is taken immediately after addition of the substance. This corresponds to the 100% value in the stability calculation. Ideally, the concentration employed should be retrieved here. The samples can be frozen at 20 C. 0 h 6 h 24 h 48 h
(88) The negative control used is SF without substance. The positive control used is SF with 1 M of substance. This corresponds to the 0 h value and thus 100% stability.
(89) The samples are stored in low-binding Eppendorf vessels at 20 C. The samples are subsequently measured quantitatively.
(90) 4.) Data Processing
(91) The concentrations measured (ng/ml) are plotted against the time in a graph
(92) (Graph Pad Prism). The percentage stability of the substance is determined here. The 100% value used is the initial value in the SF at time 0 h. The data are stored in eLabBio under the respective experiment number and reported in the MSR database (as percent stability after the corresponding incubation times).
(93) 5.) Results
(94) All compounds measured remained stable.
EXAMPLE 11
In-Vitro Fluorescence Assay for Identification of Renin-Inhibitory Activity
(95) In order to identify modulators of renin activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group Edans (=(5-(aminoethyl)aminonaphthalenesulfonate) which is quenched by energy transfer from a Dabcyl (4-dimethylaminoazobenzene-4-carboxylate) group on the same molecule, in Greiner 384-well microtitre plates. Cleavage of the peptidic substrate by renin causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was renin inhibitor 2 (Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His N-Boc-Lys methyl ester Z) (Sigma-Aldrich). The substrate used was renin FRET substrate I (DABCYL-g-Abu-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr EDANS) (Anaspec, Fremont Calif.). The enzyme employed was recombinant human renin (Proteos, Kalamazoo, Mich.) in a final concentration of 10 nM. The test was carried out in 50 mM Mops buffer, 1.5% (v/v) of DMSO, 0.1% (w/v) of Igepal, pH 7.2, 0.5% (w/v) of BSA. 2 l of each substance solution with serially diluted substance concentration were added to in each case 4 l of renin solution and incubated at room temperature for 15 min. The reaction was started by addition of 4 l of substrate solution (final concentration 5 M). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 495 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at 37 C. for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 495 nm (excitation wavelength 340 nm).
(96) Result: Apart from compounds A1 and A2, all compounds measured have an IC.sub.50 of the renin selectivity of >30 M.
EXAMPLE 12
Injection Vials
(97) A solution of 100 g of a compound of the formula I and 5 g of disodium hydrogenphosphate in 3 l of bidistilled water is adjusted to pH 6.5 using 2 N hydrochloric acid, filtered under sterile conditions, transferred into injection vials, lyophilised under sterile conditions and sealed under sterile conditions. Each injection vial contains 5 mg of a compound of the formula I.
EXAMPLE 13
Solution
(98) A solution is prepared from 1 g of a compound of the formula I, 9.38 g of NaH.sub.2PO.sub.42H.sub.2O, 28.48 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.1 g of benzalkonium chloride in 940 ml of bidistilled water. The pH is adjusted to 6.8, and the solution is made up to 1 l and sterilised by irradiation. This solution can be used in the form of eye drops.
EXAMPLE 14
Ointment
(99) 500 mg of a compound of the formula I are mixed with 99.5 g of Vaseline under aseptic conditions.
EXAMPLE 15
Ampoules
(100) A solution of 1 kg of a compound of the formula I in 60 l of bidistilled water is filtered under sterile conditions, transferred into ampoules, lyophilised under sterile conditions and sealed under sterile conditions. Each ampoule contains 10 mg of a compound of the formula I.