MATRIX METALLOPROTEINASE INHIBITORS CONTAINING AMINOMALONIC ACID DERIVATIVES AND PEPTIDE BACKBONE MODIFIED DERIVATIVES THEREOF

Abstract: Novel peptides containing the sequence -Pro-Leu-Ama(NHOH)- were synthesized and characterized by spectroscopic techniques. Their inhibitory properties towards the activated form of native human gelatinase B (MMP-9) and the catalytic domain of neutrophil colagenase (cdMMP-8) were determined. The most effective inhibitor synthesized exhibits Ki values of 2x10<-6> M (cdMMP-8) and 5x10<-9> M(MMP-9) thus attaining interesting discrimination between the tested metalloproteinases. A most important feature of this type of inhibitor is its peptide nature making the compounds similar to natural substrates. In spite of the peptide character of the inhibitors synthesized, the P1-P1'-peptide bond shows a high resistance to cleavage by the proteinases.

Description: MATRIX METALLOPROTEINASE INHIBITORS CONTAINING AMINOMALONIC ACID DERIVATIVES AND PEPTIDE BACKBONE MODIFIED DERIVATIVES THEREOF The present invention relates to compounds being or containing aminomalonic acid derivatives and peptide backbone modified derivatives thereof acting as inhibitors of matrix metallopro- teinases and thus being useful for the preparation of medica ¬ ments for treating pathophysiological processes connected to ex ¬ tracellular matrix disintegration. Introduction and prior art. Matrix metalloproteinases (MMPs) are a family of at least nine ¬ teen zinc-containing proteinases playing a fundamental role in the degradation and remodelling of connective tissue by hydrolysis of matrix proteins such as collagens, gelatins and proteo- glycans (1) . In this way MMPs are involved in many pathophysiological processes connected to extracellular matrix disintegration e.g. rheumatoid arthritis or tumor invasion and metastasis. Matrix metalloproteinases have similar domain structures includ ¬ ing as major domains an N-terminal pre-sequence, a prodomain, the catalytic domain and, in most cases, a hemopexin domain pre ¬ sumed to be involved in substrate specificity. The metallopro- teinases are produced as zymogens and activated by removal of the prodomain e.g. by organomercuric agents or proteases. MMPs are divided into several subgroups based on their domain structures, sequence homologies and their substrate preferences. Tissue inhibitors of metalloproteinases (TIMPs), naturally oc ¬ curring proteins specifically inhibiting these proteinases, con ¬ trol MMPs in vivo. An imbalance between MMPs and their natural CONFIRMATION COP. antagonists, the TIMPs, can result in pathophysiological destructive processes such as rheumatoid arthritis, parodontosis or fibrosis. This may be compensated with potent synthetic in ¬ hibitors . Numerous synthetic low-molecular weight MMP inhibitors have been synthesized (2, 3) and research is currently being carried out on their in vivo efficiency in different kinds of cancer or de ¬ structive joint diseases. Most of the synthetic MMP inhibitors developed so far are small molecular compounds binding to the catalytic site of the enzymes. Several X-ray crystal structures of the catalytic domains of collagenases complexed with an in ¬ hibitor have been published (4-7). Effective inhibitors are equipped with a zinc chelator group and a peptide or non-peptide backbone mimicking a natural substrate. The hydroxamic acid function is a very good zinc-chelating group thus providing the most potent inhibitors of MMPs. In general the hydroxamic acid-based inhibitors are of the C-terminal type, binding at S'-subsites of the enzyme (nomenclature S n ...S n ', P n ...P n ' according to Schechter and Berger (8)). N-terminal inhibitors are much less effective. For the most part, C-terminal hy ¬ droxamic acid inhibitors are succinyl derivatives with various aliphatic or aromatic substituents on either the - or β-carbon or both and residues at the γ-carboxyl function, attached by a peptide linkage. An object of the invention is to provide a novel type of hydrox- amate-based peptide inhibitors of MMPs. Accordingly, the present invention provides compounds containing aminomalonic acid derivatives and peptide backbone modified de- rivatives thereof of the general formulas I, II and III, IV, V and VI: (I) wherein Ri represents a N-protecting group like tert .butyloxycarbonyl, benzyloxycarbonyl or FMOC; or acetyl, -CO-lower alkyl, -CH 2 -aryl, a natural amino acid, lower alkyl, aryl or H; or an optionally spacer linked: such as a synthetic or natural peptide, glycopro- tein or the like, a solid or macromolecular product used for chromatographical procedures; R 2 represents NH-D-C (Ph) -CH 3 , NH-L-C (Ph) -CH 3 , N (lower alkyl) 2 , NH- lower alkyl, NH-aryl, a natural amino acid, a lower alkyl ester of an amino acid, O-lower alkyl, NHOH or OH, or an optionally spacer linked: such as a synthetic or natural peptide, glycopro- tein or the like, a solid or macromolecular product used for chromatographical procedures; or R 4 , wherein R 4 has the significance given below; or the residue Ccc, wherein Ccc has the significance given be ¬ low, optionally with a bounded residue R 2 , wherein R 2 has the significance given earlier; or Z, wherein Z has the significance given below; R 3 represents lower alkyl or a side chain of a natural amino acid, or R 4 ; R 7 , R 8 and R 9 which may be the same or different from each other represent H, alkyl, aryl, OH, CO-lower alkyl, O-lower alkyl, 0- CH 2 -aryl, O-aryl, or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which is attached via the N-atom or via a C-atom and (a) optionally contains N, 0 and /or S as an additional ring member and (b) is optionally benz-fused or optionally substituted on one or more other C-atoms by lower alkyl, aryl and/or oxo; or an optionally spacer linked: such as synthetic or natural peptide, glycoprotein or the like, a solid or macromolecular product used for chromatographical procedures; (Rιo)n (with n being selected from 0 to 5) which may be the same or different from each other are defined as R 2 or R 4 or -(CH 2 ) ra R 4 (with m being selected from 0 to 6) ; R represents H, alkyl, aryl, OH, O-lower alkyl, 0-CH 2 -aryl, 0- aryl, NH-CO-aryl, NH-CO-NH-aryl, NH-CO-CH 2 -aryl or NH-CO-R 5 , wherein R 5 has the significance given below; NH 2 , NH-lower alkyl, N(lower alkyl) 2 , N(lower alkyl 1) (lower al ¬ kyl 2), NHaryl, N(aryl) 2 , N(aryl l)(aryl 2), also combinations and pharmaceutically acceptable salts thereof; N (lower alkyl) 3 + , N(aryl) 3 + , also combinations thereof; or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which is attached via the N-atom or via a C-atom and (a) optionally contains N, 0 and /or S as an additional ring member and (b) is optionally benz- fused or optionally substituted on one or more other C-atoms by lower alkyl, aryl and/or oxo; or an optionally spacer linked: such as synthetic or natural peptide, glycoprotein or the like, a solid or macromolecular product used for chromatographical procedures; Z represents OH, O-lower alkyl, NHOH, N(CH 3 )OH, NHO-CH 3 , other NHO-lower alkyl, Q represents -(CH 2 ) ra -, -0-(CH 2 ) m -, -CO-(CH 2 ) m -, -(CH 2 ) m -P-, -0- (CH 2 ) ra -P-, -CO- (CH 2 ) m -P- (with m being selected from 0 to 6), wherein P represents cyclopropyl, cyclopentyl, cyclohexyl, 5- or 6-membered aryl, or a 5- or 6-membered, aromatic or aliphatic N- heterocyclic ring which is attached via the N-atom or via a C- atom and (a) optionally contains N, 0 and/or S as an additional ring member and (b) is optionally benzo-fused or optionally sub- stituted on one or more other ring C-atoms by lower alkyl, aryl and/or oxo; and pharmaceutically acceptable salts thereof; and wherein the term "lower alkyl", alone or in combination, means a straight-chain or branched-chain alkyl group containing a maxi ¬ mum of six carbon atoms, such as methyl, ethyl, n-propyl, iso- propyl, n-butyl, isobutyl, tert. butyl, n-pentyl, n-hexyl and the like; the term "aryl" means phenyl, which is optionally substituted by lower alkyl, O-lower alkyl and/or halogen, i.e. fluorine, chlo ¬ rine, bromine or iodine, the term "spacer" means a straigth or branched alkyl-chain, amino alkyl-chain, carboxy alkyl-chain with a maximum length of 12 carbon atoms, or combinated forms, peptides or saccarides, (ID wherein R 2 , R3, R , Re, R9, Rio, Q, Z and n have the significance given earlier and R 5 represents Ri-Proline, lower alkyl, aryl or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which is attached via the N-atom or via a C- atom and (a) optionally contains N, 0 and /or S as an additional ring member and (b) is optionally benz-fused or optionally sub ¬ stituted on one or more other C-atoms by lower alkyl, aryl and/or oxo; or an optionally spacer linked: synthetic or natural peptide, glycoprotein or the like, a solid or macromolecular product used for chromatographical procedures; and pharmaceutically acceptable salts thereof; wherein R 2 , R 9 and Z have the significance given earlier and Aaa represents a peptide bounded natural amino acid; Bbb represents a peptide bounded natural amino acid; Ccc represents a peptide bounded natural amino acid or Thr(Bzl), Ser(Bzl) or the fragment or NR 8 C (QR 10 ) HCO-, wherein R 8 , Rio, Q and n have the significance given earlier; R 6 represents a N-protecting group, acetyl, -CO-alkyl (Cι-C ) , .a natural amino acid, lower alkyl or H or Ri, wherein Ri have the significance given earlier; and pharmaceutically acceptable salts thereof; wherein R x , R 2 , R 9 , Z and Ccc have the significance given ear ¬ lier; and pharmaceutically acceptable salts thereof; H wherein R 2 , R 3 , R 5 , R 7 , R 9 , R 10 , Q, Z and n have the significance given earlier, X-Y represents especially CO-NH, CH 2 -NH, CO-CH 2 , CH 2 -CH 2 CH 2 -S, CH 2 -0, CO-N (lower alkyl), CH 2 -N (lower alkyl) or PH0 2 -NH, and pharmaceutically acceptable salts thereof; VI wherein R 2 , R 3 , R 5 , R 7 , R 10 , Q, Z and n have the significance given earlier; A-B and X-Y which may be the same or different from each other represent especially CO-NH, CH 2 -NH, CO-CH 2 , CH 2 -CH 2 CH 2 -S, CH 2 -0, CO-N (lower alkyl), CH 2 -N (lower alkyl) or PH0 2 -NH; and pharmaceutically acceptable salts thereof; wherein any available hydrogen atom on any carbon or nitrogen atom in any of the above formulae I to VI and any of the corre- sponding substituents or groups as defined for said formulae may be in part or totally and independently from each other substituted by halogen (bromine, chlorine, fluorine or iodine) , alkyl, especially lower alkyl, aryl, OH, CO-lower alkyl, O-lower alkyl, 0-CH 2 -aryl, O-aryl, or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which (a) optionally contains N, 0 and/or S as an additional ring member and (b) is optionally benz-fused or optionally substituted on one or more other C-atoms by lower alkyl, aryl and/or oxo. Further, the compounds according to the general formulae I, II, III, IV, V and VI may have variations of the included chiral centers. All residues and chiral atoms of the compounds specified above may have the L- or D-conformation. Thus, any of the inventive compounds may be in the form of a L-enantiomer or a D- enantiomer or in the form of a diastereomer, including meso forms, or in the form of any mixture thereof, including racemic mixtures . Further, the present invention provides the use of compounds according to general formulae I, II, III, IV, V and VI as inhibitors of metalloproteases . Moreover, the present invention provides the use of compounds according to general formulae I, II, III, IV, V and VI as thera- peutically active substances, especially in the control or prevention or in the treatment of: degenerative joint diseases rheumatoid arthritis osteoarthritis cancer metastasis tumour invasion multiple sclerosis parodontosis fibrosis Alzheimer's desease inflammatory bowel disease neurodegenerative deseases cerebral haemorrhage wound healing degenerative eye desease aneurism artificial joint replacement organ transplantation emphysema Cholesteatom Praeklampsie Additionally, the present invention provides the use of com- pounds according to general formulae I, II, III, IV, V and VI in chromatographical procedures. Detailed description of the invention with results and discussion. In this invention a novel type of hydroxamic acid-based peptide inhibitor is presented. The residue carrying the hydroxamate function is an aminomalonic acid (A a) in position Pi. This al ¬ lows the addition of various residues suitable to gain maximum attachment to the entire binding site of the target enzyme on both S- and S' -subsides. The most important feature of this type of inhibitor is the peptide backbone built up by -amino acids to comprise the positions P 3 - to Pi' at least, giving these compounds the same basic frame as a peptide substrate. In accordance with the specificity of the metalloproteinases tested the P 3 -position of these inhibitors is occupied by a proline, P 2 is an aliphatic amino acid, whereby leucine or alanine is favored. The best inhibitory properties were obtained with the bulky ty- rosine benzylether moiety in Pi'-position (Fig. 1). The Ki values of the synthesized MMP inhibitors determined in quantitative fluorometric assays are shown in Table 1. Some of these compounds exhibit very good inhibitory properties against the enzymes tested. Analogous derivatives with free carboxyl function, methyl- or ethylesters at the aminomalonic acid instead of a hydroxamate in this position show virtually no in- hibitory effect (9) . Thus it is obvious that a zinc-complexing function is essential for a high affinity to the metalloproteinases. This observation confirms the assumption that these inhibitors bind competitively to the catalytic centre, with the hydroxamic acid in a proper position to chelate the zinc ion in the active site. The complexing group, representing the complete side chain of the amino acid in positon P , is fixed as close as possible to the peptide backbone and to the reactive-site bond. Apart from this favorable conformational arrangement of the che- lating group towards the metal ion another relevant criterion for effective inhibition is an optimal adjustment of the back ¬ bone to a natural substrate. The Pi-residue in a natural substrate is glycine. Its substitution by the aminomalonic acid derivative only adds a small chelating function to the peptide. Except for the Ama (NHOH) -residue in P x position this type of pep ¬ tide inhibitor is highly variable on both N- and C-terminal sides, thus allowing designing of the rest of the structure for an optimal fit into to the enzyme subsites. Corresponding to the substrate specificity of the tested metalloproteinases, the P 3 -position of all inhibitors is occupied by a proline. A substitution of the proline in compound 1 by a Boc protecting group results in a decrease of the inhibition constant by about two orders of magnitude for the MMP-9 and one or ¬ der for the cdMMP-8. Compounds 5, 6 and 7 with leucine at P 2 -position have the strongest inhibitory effect toward the catalytic domain of MMP-8, in agreement with the sequences of various synthetic peptide sub ¬ strates of collagenases (14-16) . The inhibition constants of these compounds for the gelatinase B are about two orders of magnitude lower than those for the collagenase with its high substrate discrimination. A more distinct selectivity between MMP-9 and cdMMP-8 is shown by compound 4 with alanine at the P 2 -site (Ki = 5xl0 "9 M for MMP- 9, 1.9xl0 ~6 for cdMMP-8). Whereas the Ki value of 4 for the MMP-9 equals those of the analogous leucine derivatives, a distinct decrease can be observed for the cdMMP-8. There is a nearly 400- fold inhibitory discrimination of 4 between the two enzymes. The glycine-containing derivates 1 to 3 inhibit the tested en ¬ zymes one to two orders of magnitude less efficiently. Thus an amino acid with a chiral center in position P 2 seems to be favorable. This effect is more pronounced for MMP-9, therefore these inhibitors are less selective. The compounds with the bulky -methylcyclohexanemethylamine at position P 2 ' have weaker inhibitory effects than the aromatic analogues . Despite their peptide character the inhibitors were resistant to hydrolysis by the proteases as confirmed by HPLC investigations after incubation of the inhibitors with the metalloproteinases for several hours. None of the possible fragments from cleavage of the Ama-Tyr-bond could be detected. Thus, these inhibitors and especially the Pχ-Pι ' -peptide bond exhibit a high resistance to cleavage by the proteinases. Figure and Table Legends Fig. 1. Partial sequence of a typical substrate of gelatinase B (MMP-9) with the amino acid residues numbered according to Schechter and Berger (8) compared with the structure of an inhibitor of the type R x -Pro-Leu-Ama (NHOH) -Tyr (Bzl) -R 2 presumably binding at the subsites of the enzyme. Ri is a protecting group, H, acetyl or an amino acid, R 2 is an amine, an alcohol, NHOH or a C-terminal protected amino acid or peptide. Table 1. Tested inhibitors and their Ki values determined with gelatinase B (MMP-9) and the catalytic domain of the neutrophil collagenase (MMP-8) in mole/1. NH-R-CH (Ph) CH 3 , NH-SCH (Ph) CH 3 and NH-R-CH (C 6 Hn)CH 3 are N-bounded residues of R (+) -α-methylbenzyl- amine, s (-) -α-methylbenzylamine, and R (-) -α-methylcyclohexaneme ¬ thylamine. Abbreviations: Ama, aminomalonic acid; cdMMP, catalytic domain of an MMP, MMP, matrix metalloproteinase; DCC, dicyclohexyl carbodiimide; TIMP, tissue inhibitor of metalloproteinases. Table 1 compound R 2 R 3 MMP-9 cdMMP-8 no. 1 NH-R-CH (Ph)CH 3 H 4.0x10 " 6.5x10 -6 2 NH-S-CH(Ph)CH 3 H >10 "7 1.8xl0 _ 3 NH-R-CH (c-C 6 Hn)CH 3 H >10 " 1.4xl0 ~5 4 NH-S-CH(Ph)CH 3 CH 3 5.0x10 " 1.9xl0 "6 5 NH-R-CH (Ph)CH 3 CH 2 -CH- (CH 3 ) 2 5.0x10 " 8.0xl0 ~7 6 NH-S-CH(Ph)CH 3 CH 2 -CH- (CH 3 ) 2 5.0x10 " 8.0x10 -7 7 NH-R-CH (c-C 6 Hn)CH 3 CH 2 -CH- (CH 3 ) 2 1.8x10 " 2.5x10 -6 Further Examples with Ki-values (uncorrected) against some enzy ¬ mes, respectively their catalytic domains (cd) : H-Pro-Le -Λma(NHOH)-P e-L(-)MBΛ K nM] cdMMP9 5000 cdMTl-MMP 6000 cdM P8 10000 Λc-Pro-Leu-Λma(NHOH)-Phe-Tyr(Bzl)-NMe- KitnM] M P9 4,5 cdMTl-MMP 1300 cdMMPS 710 cdMTl-MMP 2200 cdMMPS 1000 Analogous PiPi ' -deoxopeptide : K nM] MMP9 0,23 cdMTl-MMP 60 cdMMPδ 200 Table 2: Preliminary K^-values in nM (not corrected) *) Structure see above (cd = catalytic domain) Inhibitor MMP-9 cdMTl-MMP cdMMP-8 Ac- Pr o~Leu-Ama ( NHOH ) - Amp- ( - ) MBA 1500 12000 2500 Ac-Pro-Leu-Ama (NHOH) -Amp(Bz) -L(-)MBA *) 18 580 250 Ac-Pro-Leu-Ama (NHOH) -Amp (CONHPh) -L (- 35 3200 500 Ac-Pro-Leu-Ama ( NHOH ) -Amp ( Z ) -L ( - ) MBA 250 13000 90 Ac-Pro-Leu-Ama (NHOH) -Leu-2, 4-DiMeP 2000 >10000 30000 Ac-Pro-Leu-Ama (NHOH) -2-Nal-NMe 2 8 1300 890 Ac-Pro-Leu-Ama (NHOH) -D-2-Nal-NHMe 76 5800 580 Ac-Pro-Leu-Ama (NHOH) -D-2-Nal-NMe 2 50 2700 100 H-Pro-Leu-Ama (NHOH) -Phe-D (+) MBA 6000 H- Pro-Leu-A a ( NHOH ) -Phe-L ( - ) MBA *) 5000 6000 10000 Ac-Pro-Leu-Ama (NHOH) -Phe-Tyr (Bzl) - *) 4,5 Ac-Pro-Leu-Ama (NHOH) -Ser (Bzl) -L (- *) 3600 1300 710 Ac-Pro-Leu-Ama (NHOH) -Ser (Bzl) -NMe 2 *) 200 2200 1000 Ac-Pro-Leu-Ama (NHOH) -Thr (Bzl) -L (- 29 720 1100 Ac-Pro-Leu-Ama (NHOH) -Trp-NMe 2 180 3700 1500 Ac-Pro-Leu-Ama (NHOH ) -Tyr-L ( - ) MBA 46 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl) -L (- *) 3,5 4 Ac-Pro-D-Leu-Ama (NHOH) -Tyr (Bzl) -L(- 750 12000 1500 Ac-D-Pro-D-Leu-Ama (NHOH) -Tyr (Bzl) - (- 1000 10000 1200 Z-Pro-Leu-Ama (NHOH) -D-Tyr (Bzl) - 4500 2000 Z-Pro-Leu-Ama (NHOH) -D-Tyr (Bzl) -L (- 5000 6400 2000 Ac-Pro-Leu-Ama (N (Me) OH)-Tyr(Bzl)-L(- 1000 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl) -Leu- 0,7 28 21 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl)-NHMe 0,25 49 12 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl) -NHOH *) 2 12 15 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl)-NMe 2 0,3 9,8 6 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl)-Phe- 0,8 35 40 Ac-Pro-Leu-Ama (NHOH) -Tyr(Bzl)-Val- 3 200 Ac-Pro-Leu-Ama (NHOH) -Tyr (Et)-NHMe 0,7 85 52 Ac-Pro-Leu-Ama (NHOH) -Tyr (Et)-NMe 2 1 32 17 Ac-Pro-Leu-Ama (NHOH) -Tyr(Me)-L(-)MBA 2 140 70 Ac-Pro-Leu-Ama (NHOH) -Tyr (Me)-NHMe 4 140 Ac-Pro-Leu-Ama (NHOH) -Tyr (Me) -NMe 2 *) 0,23 60 200 Boc-D/L-2-Pip-Leu-Ama (NHOH) -Tyr (Bzl) - 1500 Bz-Ala-Ama (NHOH) -Tyr (Bzl) -NMe 2 1300 Cp-CO-Leu-Ama (NHOH) -Tyr (Bzl ) -L (-) MBA 140 >10000 9000 Abreviations : 2,4- ■ DiMeP 2, 4-dimethylpentylamine Ac acetyl Ama aminomalonic acid Amp p-aminophenylalanine Bzl benzyl Bz benzoyl Cp cyclopentane Et ethyl MBA methylbenzylamine Me methyl Nal β-naphthylalanine Pip pipecoline carboxylic acid Z benzyloxycarbonyl natural amino acids are in three letter code Table 3: Preliminary K x -values in mole/1 (not corrected) Abreviations as above Inhibitor cdMMP-9 cdMTl-MMP cdMMP-8 Boc-Ama (NHOH) -Tyr (Bzl) - (-) MBA 4,1x10 ' " 3x10 ' HC1 H-Ama (NHOH) -Tyr (Bzl) - (-)MBA 9,lxl0 '6 Boc-Ama (NHOH) -Tyr (Bzl) -NHOH 9xl0- η 2xl0 '5 3,5x10 ' Boc-Ama (NHOH) -Tyr (Bzl ) -Val-Gly-NMe 2 5xl0 '7 2,5x10 ' 2,1x10 ' Table 4: Preliminary Ki-values in mole/1 (not corrected) Abreviations as above Inhibitor cdMMP-9 cdMTl-MMP cdMMP- Ac-Pro-Leu-A a (NHOH) OH 1,4x10 ' * 1x10 ' " l,2xl0 ~5 Z-Pro-Leu-Ama (NHOH) 2 4,2xl0 '5 2,7x10 ' 3xl0 'e Z-Pro-Ala-Ama (NHOH) 2 3,lxl0 '5 2,3x10 ' l,8xl0 '5 Bz-Ala-Ama (NHOH) 2 7x10 ' * 3x10 ' * Table 5: Preliminary Ki-values in nM (not corrected) * structure see above Inhibitor cdMMP-2 cdMMP-13 Ac-Pro-Leu-Ama (NHOH) -Amp- (-) MBA Ac-Pro-Leu-Ama (NHOH) -Amp (Bz)-L(-)MBA *) Ac-Pro-Leu-Ama (NHOH) -Amp (CONHPh) -L (- Ac-Pro-Leu-Ama (NHOH) -Amp(Z) - (-)MBA Ac-Pro-Leu-Ama (NHOH) -Leu-2, 4-DiMeP Ac-Pro-Leu-Ama (NHOH) -2-Nal-NMe 2 190 35 Ac-Pro-Leu-Ama (NHOH) -D-2-Nal-NHMe Ac-Pro-Leu-Ama (NHOH) -D-2-Nal-NMe 2 H-Pro-Leu-Ama(NHOH) -Phe-D(+)MBA H-Pro-Leu-Ama (NHOH) -Phe- (-) MBA *) Ac-Pro-Leu-Ama (NHOH) -Phe-Tyr (Bzl) - *) Ac-Pro-Leu-Ama (NHOH) -Ser (Bzl) -L (- *) Ac-Pro-Leu-Ama (NHOH) -Ser (Bzl) -NMe 2 *) Ac-Pro-Leu-Ama (NHOH) -Thr (Bzl) -L(- Ac-Pro-Leu-Ama(NHOH)-Trp-NMe 2 290 Ac-Pro-Leu-Ama (NHOH) -Tyr-L (- ) MBA Ac-Pro-Leu-Am (NHOH) -Tyr (Bzl) -L (- *) Ac-Pro-D-Leu-Ama (NHOH) -Tyr (Bzl) - (- Ac-D-Pro-D-Leu-Ama (NHOH) -Tyr (Bzl) - (- Z-Pro-Leu-Ama (NHOH) -D-Tyr (Bzl) - Z-Pro-Leu-Ama (NHOH) -D-Tyr (Bzl) -L (- Ac-Pro-Leu-Ama (N (Me) OH)-Tyr (Bzl)- (- Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl)-Leu- Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl)-NHMe 13 Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl) -NHOH *) 3 Ac-Pro-Leu-Ama (NHOH -Tyr(Bzl)-NMe 2 5,4 Ac-Pro-Leu-Ama (NHOH -Tyr (Bzl) -Phe- Ac-Pro-Leu-Ama (NHOH Tyr (Bzl)-Val- 3,8 Ac-Pro-Leu-Ama (NHOH Tyr(Et)-NHMe Ac-Pro-Leu-Ama (NHOH Tyr(Et)-NMe 2 8,5 16 Ac-Pro-Leu-Ama (NHOH Tyr (Me)-L(-)MBA Ac-Pro-Leu-Ama (NHOH Tyr(Me)-NHMe 20 Ac-Pro-Leu-Ama (NHOH Tyr(Me)-NMe 2 *) 9 13 Boc-D/L-2-Pip-Leu-Ama (NHOH) -Tyr (Bzl) - Bz-A] a-Ama (NHOH) -Tyr (Bzl ) -NMe 2 Cp-CO-Leu-Ama (NHOH) -Tyr (Bzl )-L(-) MBA Materials and methods The peptides containing an Ama(OMe) residue were synthesized by segment condensation of N-protected tripeptides of the type R x - Pro-Aaa-Ama (OMe)OH and H-Tyr (Bzl) -R 2 , where R x is a Boc protecting group or acetyl, Aaa is glycine, alanine or leucine and R 2 is an amide-bonded S (-) -α-methylbenzylamine, R (+) -α-methylbenzyl- amine or R (-) -α-methylcyclohexanemethylamine . Peptide coupling reactions were carried out in organic solvents following stan- dard procedures (9). Alternatively an N-protected dipeptide Boc- Aaa-Ama (OMe) OH was coupled with the C-terminal fragment and, after removal of the Boc group, the Ri-Pro was added using an active ester. The conversion of the aminomalonic acid methylester function into the corresponding hydroxamic acid was effected via hydrolysis of the ester to the free malonic acid and treatment with hydroxylamine hydrochloride, a tertiary amine and DCC or directly by aminolysis of the methylester with hydroxylamine. Characterization of the synthesized inhibitors and precursors was carried out using NMR, liquid SIMS and ion spray mass spec- trometry (9) . The inhibition constants Ki of the inhibitors were determined for the proteolytically activated form of native human gelatinase B (MMP-9) (10) , the catalytic domain of neutrophil collagenase (cdMMP-8) (11), and for the additional enzymes of tables 2 to 5 given above using a spectrofluorometer Jasco FP-550 or a luminescence spectrometer Per in-Elmer LS 50B. The substrate used, (7-methoxycumarin-4-yl) acetyl-Pro-Leu-Gly-Leu- (3- (2, -dinitro- phenyl) -L-2, 3-diamino-propionyl) -Ala-Arg-NH 2 (12), is an inter- nally quenched fluorescent peptide, which is cleaved at the Gly- Leu bond by the tested metalloproteinases. The resulting in- crease of fluorescence was measured at an excitation wavelength of 328 nm and an emission wavelength of 393 nm. The assay mixtures contained constant concentrations of the en- zyme and 3% DMSO (as solvent for inhibitors and substrate) in 2 ml of buffer (0,1 M Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM CaCl 2 , 0.05% Brij 35). For at least three series with constant concentrations of substrate (approx. 0.2 to 1 μM) and different con ¬ centrations of the inhibitor every initial fluorescence in- crease, which is proportional to the substrate concentration and corresponds to the remaining proteolytic activity of the enzyme, was measured at 25°C. The substrate (predissolved 0.1 mg in 1 ml DMSO) was added after incubation of the enzyme with the inhibi ¬ tor in the buffer for at least ten minutes. Ki values were deter- mined by the established method of Dixon (13). Activation of the latent progelatinase B (MMP-9) , isolated from human neutrophils, was carried out in assay buffer by adding bo ¬ vine trypsin (50 μl, 0.6 mg/ml) to the proenzyme (0.45 ml, 120 μg/ml) and incubating at 37°C for 10 minutes. The trypsin was then inactivated with aprotinin (50 μl, 1.2 mg/ml). Examples Preparation of Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl) - eu-NH 2 . Ac-Pro-Leu-OH . 30 mmole of each of Ac-Pro-OH, H-Leu-OMe hydrochloride, HOBT and N-ethylmorpholine are suspended in a mixture of 100 ml THF and 80 ml dichloromethane, and the resulting mixture is cooled to 0°C. Thereafter, 32 mmole of DCC are added in portions under stirring for 2 h at 0°C, and stirring is continued at 5°C until completion of the reaction. After filtration the solution is evaporated and the residue taken into ethylacetate. The solution is than extracted sequentally with aqueous 10% NaCl/0,5 M HC1, 5% Na 2 C0 3 and 5% NaHC0 3 solutions and is dried over sodium sul- fate. After this, the solvent is removed in vaccuo. Finally, the residue is taken into a small volume dichloromethane, and the solution stored over night at 5°C and than filtered and evaporated. Ac-Pro-Leu-OH . 20 mmole of Ac-Pro-Leu-OMe are dissolved in 40 ml methanol and added with 20 ml of aqueous 2 N NaOH at 0°C. After completion of the reaction it is acidified with a concentrated solution of KHS0 4 , and the product is extracted with ethylacetate. Further purification can be conducted by additional extraction steps. Ac-Pro-Leu-Ama (OMe) 2 . 15 mmole of each of Ac-Pro-Leu-OH, aminomalonic acid dimethyles- ter hydrochloride, HOBT and N-methylmorpholine are suspended in 100 ml of a mixture of THF, ethylacetate and dichloromethane (3:1:1). Thereafter, 15 mmole of DCC are added at 0°C, and it is stirred at 0-5°C until the reaction is nearly complete. The mixture is filtered, the solvent removed in vaccuo, and the residue taken into ethylacetate. The solution is extracted with 10% NaHS0 4 , 5% Na 2 C0 3 and 5% NaHC0 3 solutions and than dried over so ¬ dium sulfate. After this, the solvent is removed in vaccuo, and the residue taken into a small volume of dichloromethane. Fi ¬ nally, the solution is stored over night at 5°C and than fil ¬ tered and evaporated. Ac-Pro-Leu-Ama (OMe) OH . 10 mmole of Ac-Pro-Leu-Ama (OMe) 2 are completely dissolved in about 25 ml of absolute methanol, and under trapping any mois- ture and vigorous stirring at 0°C the solution is added dropwise with a solution of 9,5 mmole of KOH in about 10 ml of absolute methanol. The mixture is left stand over night at 0-5°C, option ¬ ally reduced in volume in vaccuo, and than under cooling with ice diluted with a 5% solution of NaHC0 3 . After this, the aqueous solution is extracted several times with ethylacetate or a mix ¬ ture of ethylacetate and diethylether and than slighly acidified under cooling with ice using concentrated NaHS0 4 . The free malo ¬ nic acid is extracted several times with etylacetate, and the combined organic phases are dried over sodium sulfate. Finally, the solvent is removed in vaccuo at max. 30 °C. Boc-Tyr (Bzl) -Leu-NH 2 . 2 mmole of each of Boc-Tyr (Bzl) -OH, leucineamide hydrochloride, HOBT and N-ethylmorpholine are suspended in 10 ml THF. Than 1,05 mmole of EDC are added at 0°C, and it is stirred at 5°C until completion of the reaction. The reaction mixture is evaporated, the residue taken into ethylacetate and the solution purified by extraction. H-Tyr (Bzl) -Leu-NH 2 1,5 mmole of the Boc-protected derivatives are dissolved in about 6 ml of trifluoroethanol and added with 1 ml of concen- trated HCl. After completion of the reaction 5% Na 2 C0 3 solution is added and the product extracted with ethylacetate. Ac-Pro-Leu-Ama (OMe) -Tyr (Bzl) -Leu-NH 2 . 0,3 mmole of each of H-Tyr (Bzl) -Leu-NH 2 and HOBT and 0,33 mmole of Ac-Pro-Leu-Ama (OMe) OH are suspended in a mixture of 5 ml di ¬ chloromethane, 5 ml dimethoxyethane, 3 ml ethylacetate and 1 ml of methanol and added with a solution of 0,3 mmole of DCC in 2 ml dichloromethane at 0°C. The suspension is stirred at 5°C, added with 5 ml acetone and filtered, and the precipitate is re- cristallized several times from methanol. Ac-Pro-Leu-Ama (NHOH) -Tyr (Bzl) -Leu-NH 2 . 0,1 mmole of the methylester are suspended in 3 ml of absolute methanol, and the mixture is added with hydroxylamine hydrochlo- ride and, under trapping any moisture, at 0°C with sodium metha- nolate until the suspension reacts clearly alkaline. After com ¬ pletion of the reaction it is acidified with 10% aqueous NaHS0 4 solution and extracted with ethylacetate. The organic phases are dried over sodium sulfate and evaporated. Further purification can be performed by chromatographic methods. Preparation of Ac-Pro-Leu-Ama (NHOH) -Tyr (Me) -NMe 2 . Boc-Tyr (Me) -NMe 2 . The preparation is performed by condensation of Boc-Tyr (Me) -OH with dimethylamine similar to the synthesis of Boc-Tyr (Bzl) -Leu- NH 2 . H-Tyr (Me) -NMe 2 hydrochloride . The preparation is performed by acidolysis of the Boc-protected derivate using a water-free saturated solution of HCl in acetic acid. Ac-Pro-Leu-Ama (OMe) -Tyr (Me) -NMe 2 . 0,25 mmole of H-Tyr (Me) -NMe 2 hydrochloride, 0,27 mmole of Ac-Pro- Leu-Ama (OMe) OH and 0,27 mmole of bromo-tris- (dimethylamino) - phosphonium-hexafluorophosphate are taken into 2,5 ml of water- free dichloromethane. While trapping any moisture and using a blanketing inert atmosphere, the mixture is added with 0,9 mmole diethylisopropylamine at 0°C and than stirred at 0-5°C until completion of the reaction. After this, the mixture is taken into ethylacetate and than extracted sequentally with aqueous 10% NaCl/0,5 M HCl, 5% Na 2 C0 3 and 5% NaHC0 3 solutions and dried over sodium sulfate. Finally, the solvent is removed in vaccuo. The product is further purified chromatographically using Sephadex LH20 in methanol. Ac-Pro-Leu-Ama (NHOH) -Tyr (Me) -NMe 2 . The preparation is analogous to the synthesis of Ac-Pro-Leu- Ama (NHOH) -Tyr (Bzl) -Leu-NH 2 . References (1) Birkedal-Hansen, H., Moore, W.G.I., Bodden, M.K., Windsor, L.J., Birkedal-Hansen, B., DeCarlo, A., Engler, J.A. (1993) Crit. Rev. Oral. Biol. Med. 4, 197-250. (2) Johnson, W.H., Roberts, N.A., Borkakoti, N. (1987) J. Enzyme Inhibition 2, 1-22. (3) Schwartz, M.A., Van Wart, H.E. (1992) Progress in Medicinal Chemistry 29, 271-334. (4) Bode, W. , Reinemer, P., Huber, R., Kleine, T., Schnierer, S., Tschesche, H. (1992) EMBO J. 13, 1263-1269. (5) Grams, F., Crimmin, M. , Hinnes, L., Huxley, P., Pieper, M. , Tschesche, H., Bode, W. (1995) Biochemistry 34, 14012-14020. (6) Borkakoti, N., Winkler, F.K., Williams, D.H., D ' Arcy, A., Broadhurst, M.J., Brown, P. A., Johnson, W.H., Murray, E.J. (1994) Struct. Biol. 1, 106-110. (7) Spurlino, J.C., Smallwood, A.M., Carlton, D.D., Banks, T.M., Vavra, K.J., Johnson, J.S., Cook, E.R., Falvo, J. , Wahl, R.C., Pulvino, K.J., Wendoloski, J.J., Simth, D.L. (1994) Proteins Struct. Funct. Genet. 19, 98-109. (8) Schechter, I., Berger, A. (1967) Biochem. Biophys. Res. Com ¬ mun. 27, 157-162. (9) Krumme, D. (1995) Diplomarbeit, Universitat Bielefeld. (10) Tschesche, H., Knauper, V., Kramer, S., Michaelis, J. , Oberhoff, R. , Reinke, H. (1992) Matrix Supplement 1, 245-255. (11) Schnierer, S., Kleine, T., Gote, T., Hillemann, A., Knau ¬ per, V., Tschesche, H. (1993) Biochem. Biophys. Res. Commun. 191, 319-326. (12) Knight, C.G., Willenbrock, F. , Murphy, G. (1992) FEBS Lett. 296, 263-266. (13) Dixon, M. (1953) Biochem. J. 55, 170-171. (14) Weingarten, H., Martin, R. , Feder, J. (1985) Biochemistry 24, 6730-6734. (15) Fields, G.B., Van Wart, H.E., Birkedal-Hansen, H. (1973) J. Biol. Chem. 262, 6221-6226. (16) Netzel-Arnet, S., Fields, G., Birkedal-Hansen, H., Van Wart, H.E. (1991) J. Biol. Chem. 266, 6747-6755.

Claims: Claims 1. Compounds containing aminomalonic acid derivatives and pep ¬ tide backbone modified derivatives thereof of the general formu- las I, II and III, IV, V and VI: (I) wherein Ri represents a N-protecting group like tert .butyloxycarbonyl, benzyloxycarbonyl or FMOC; or acetyl, -CO-lower alkyl, -CH 2 -aryl, a natural amino acid, lower alkyl, aryl or H; or an optionally spacer linked: such as a synthetic or natural peptide, glycopro- tein or the like, a solid or macromolecular product used for chromatographical procedures; R 2 represents NH-D-C (Ph) -CH 3 , NH-L-C (Ph) -CH 3 , N (lower alkyl) 2 , NH- lower alkyl, NH-aryl, a natural amino acid, a lower alkyl ester of an amino acid, O-lower alkyl, NHOH or OH, or an optionally spacer linked: such as a synthetic or natural peptide, glycopro- tein or the like, a solid or macromolecular product used for chromatographical procedures; or R, wherein R 4 has the significance given below; or the residue Ccc, wherein Ccc has the significance given be ¬ low, optionally with a bounded residue R 2 , wherein R 2 has the significance given earlier; or Z, wherein Z has the significance given below; R 3 represents lower alkyl or a side chain of a natural amino acid, or R ; R 7 , R 8 and R 9 which may be the same or different from each other represent H, alkyl, aryl, OH, CO-lower alkyl, O-lower alkyl, 0- CH 2 -aryl, O-aryl, or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which is attached via the N-atom or via a C-atom and (a) optionally contains N, 0 and /or S as an additional ring member and (b) is optionally benz-fused or optionally substituted on one or more other C-atoms by lower alkyl, aryl and/or oxo; or an optionally spacer linked: such as synthetic or natural peptide, glycoprotein or the like, a solid or macromolecular product used for chromatographical procedures; (Rιo)n (with n being selected from 0 to 5) which may be the same or different from each other are defined as R 2 or R or -(CH 2 ) m R 4 (with m being selected from 0 to 6) ; R represents H, alkyl, aryl, OH, O-lower alkyl, 0-CH 2 -aryl, 0- aryl, NH-CO-aryl, NH-CO-NH-aryl, NH-CO-CH 2 -aryl or NH-CO-R 5 , wherein R 5 has the significance given below; NH 2 , NH-lower alkyl, N(lower alkyl) 2 , N(lower alkyl 1) (lower al ¬ kyl 2), NHaryl, N(aryl) 2 , N(aryl l) (aryl 2), also combinations and pharmaceutically acceptable salts thereof; N (lower alkyl) 3 + , N(aryl) 3 + , also combinations thereof; or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which is attached via the N-atom or via a C-atom and (a) optionally contains N, 0 and /or S as an additional ring member and (b) is optionally benz- fused or optionally substituted on one or more other C-atoms by lower alkyl, aryl and/or oxo; or an optionally spacer linked: such as synthetic or natural peptide, glycoprotein or the like, a solid or macromolecular product used for chromatographical procedures; Z represents OH, O-lower alkyl, NHOH, N(CH 3 )OH, NH0-CH 3 , other NHO-lower alkyl, Q represents -(CH 2 ) m -, -0-(CH 2 ) m -, -CO-(CH 2 ) m -, -(CH 2 ) ra -P-, -0- (CH 2 ) m -P-, -CO- (CH 2 ) m -P- (with m being selected from 0 to 6), wherein P represents cyclopropyl, cyclopentyl, cyclohexyl, 5- or 6-membered aryl, or a 5- or 6-membered, aromatic or aliphatic N- heterocyclic ring which is attached via the N-atom or via a C- atom and (a) optionally contains N, 0 and/or S as an additional ring member and (b) is optionally benzo-fused or optionally sub ¬ stituted on one or more other ring C-atoms by lower alkyl, aryl and/or oxo; and pharmaceutically acceptable salts thereof; and wherein the term "lower alkyl", alone or in combination, means a straight-chain or branched-chain alkyl group containing a maximum of six carbon atoms, such as methyl, ethyl, n-propyl, iso- propyl, n-butyl, isobutyl, tert. butyl, n-pentyl, n-hexyl and the like; the term "aryl" means phenyl, which is optionally substituted by lower alkyl, O-lower alkyl and/or halogen, i.e. fluorine, chlo ¬ rine, bromine or iodine, the term "spacer" means a straigth or branched alkyl-chain, amino alkyl-chain, carboxy alkyl-chain with a maximum length of 12 carbon atoms, or combinated forms, peptides or saccarides, (ID wherein R 2 , R 3 , R 7 , R 8 , R 9 , R 10 , Q, Z and n have the significance given earlier and R 5 represents Ri-Proline, lower alkyl, aryl or cyclopropyl, cy ¬ clopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which is attached via the N-atom or via a C- atom and (a) optionally contains N, 0 and /or S as an additional ring member and (b) is optionally benz-fused or optionally sub ¬ stituted on one or more other C-atoms by lower alkyl, aryl and/or oxo; or an optionally spacer linked: synthetic or natural peptide, glycoprotein or the like, a solid or macromolecular product used for chromatographical procedures; and pharmaceutically acceptable salts thereof; wherein R 2 , R 9 and Z have the significance given earlier and Aaa represents a peptide bounded natural amino acid; Bbb represents a peptide bounded natural amino acid; Ccc represents a peptide bounded natural amino acid or Thr(Bzl), Ser (Bzl) or the fragment or NR 8 C (QRio) HCO-, wherein Rβ r Rio, Q and n have the significance given earlier; ε represents a N-protecting group, acetyl, -CO-alkyl (C1-C4) , a natural amino acid, lower alkyl or H or Ri, wherein Ri have the significance given earlier; and pharmaceutically acceptable salts thereof; wherein R X R 2 , R 9 , Z and Ccc have the significance given ear ¬ lier; and pharmaceutically acceptable salts thereof; V wherein R 2 , R 3/ R 5 , R 7 , R 9 , Rio, Q, Z and n have the significance given earlier, X-Y represents especially CO-NH, CH 2 -NH, CO-CH 2 , CH 2 -CH 2 CH 2 -S, CH 2 -0, CO-N (lower alkyl), CH 2 -N (lower alkyl) or PH0 2 -NH, and pharmaceutically acceptable salts thereof; VI wherein R 2 , R 3 , R 5 , R 7 , Rio, Q, Z and n have the significance given earlier; A-B and X-Y which may be the same or different from each other represent especially CO-NH, CH 2 -NH, CO-CH 2 , CH 2 -CH 2 CH 2 -S, CH 2 -0, CO-N (lower alkyl), CH 2 -N (lower alkyl) or PH0 2 -NH; and pharmaceutically acceptable salts thereof; wherein any available hydrogen atom on any carbon or nitrogen atom in any of the above formulae I to VI and any of the corresponding substituents or groups as defined for said formulae may be in part or totally and independently from each other substituted by halogen (bromine, chlorine, fluorine or iodine) , alkyl, especially lower alkyl, aryl, OH, CO-lower alkyl, O-lower alkyl, 0-CH 2 -aryl, O-aryl, or cyclopropyl, cyclopentyl, cyclohexyl, a 5- or 6-membered, aromatic or aliphatic N-heterocyclic ring which (a) optionally contains N, 0 and/or S as an additional ring mem ¬ ber and (b) is optionally benz-fused or optionally substituted on one or more other C-atoms by lower alkyl, aryl and/or oxo. 2. Compounds according to claim 1, in the form of a L- enantiomer or a D-enantiomer or in the form of a diastereomer, including meso forms, or in the form of any mixture thereof, including racemic mixtures. 3. Use of compounds as defined in any preceeding claim as in- hibitors of metalloproteases . 4. Use of compounds as defined in any preceeding claim as therapeutically active substances, especially in the control or prevention or in the treatment of: degenerative joint diseases rheumatoid arthritis osteoarthritis cancer metastasis tumour invasion multiple sclerosis parodontosis fibrosis Alzheimer's desease inflammatory bowel disease neurodegenerative deseases cerebral haemorrhage wound healing degenerative eye desease aneurism artificial joint replacement organ transplantation emphysema Cholesteatom Praeklampsie 5. Use of compounds as defined in any preceeding claim in chro ¬ matographical procedures.

Inventors: KRUMME DIRK, TSCHESCHE HARALD, KRUMME, DIRK, TSCHESCHE, HARALD
Applicants: KRUMME DIRK, TSCHESCHE HARALD, KRUMME, DIRK, TSCHESCHE, HARALD
Application year: 1999
Main classification: C07K5
IPC codes: A61K38, A61P1, A61P9, A61P11, A61P17, A61P19, A61P25, A61P27, A61P29, A61P35, A61P43, C07K5, C07K7