WO2000002904A1 - Inhibiteurs de metalloproteases matricielles contenant des derives d'acide aminomalonique et leurs derives a squelette peptidique modifie - Google Patents

Inhibiteurs de metalloproteases matricielles contenant des derives d'acide aminomalonique et leurs derives a squelette peptidique modifie Download PDF

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WO2000002904A1
WO2000002904A1 PCT/EP1999/004826 EP9904826W WO0002904A1 WO 2000002904 A1 WO2000002904 A1 WO 2000002904A1 EP 9904826 W EP9904826 W EP 9904826W WO 0002904 A1 WO0002904 A1 WO 0002904A1
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lower alkyl
aryl
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leu
nhoh
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Harald Tschesche
Dirk Krumme
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Definitions

  • 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.
  • Matrix metalloproteinases 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 Tissue inhibitors of metalloproteinases (TIMPs), naturally oc ⁇ curring proteins specifically inhibiting these proteinases, con ⁇ trol MMPs in vivo.
  • Tissue inhibitors of metalloproteinases Tissue inhibitors of metalloproteinases (TIMPs), naturally oc ⁇ curring proteins specifically inhibiting these proteinases, con ⁇ trol MMPs in vivo.
  • 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 .
  • MMP inhibitors 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.
  • 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.
  • C-terminal hy ⁇ droxamic acid inhibitors are succinyl derivatives with various aliphatic or aromatic substituents on either the - or ⁇ -carbon or both and residues at the ⁇ -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.
  • 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:
  • 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;
  • R ⁇ o (R ⁇ o)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;
  • 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
  • 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;
  • aryl means phenyl, which is optionally substituted by lower alkyl, O-lower alkyl and/or halogen, i.e.
  • 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,
  • 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;
  • 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)
  • R 6 represents a N-protecting group, acetyl, -CO-alkyl (C ⁇ -C ) , .a natural amino acid, lower alkyl or H or Ri, wherein Ri have the significance given earlier; and pharmaceutically acceptable salts thereof;
  • R x , R 2 , R 9 , Z and Ccc have the significance given ear ⁇ lier; and pharmaceutically acceptable salts thereof; H
  • 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;
  • 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
  • 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 .
  • the present invention provides the use of compounds according to general formulae I, II, III, IV, V and VI as inhibitors of metalloproteases .
  • the present invention provides the use of compounds according to general formulae I, II, III, IV, V and VI as thera-Guinically active substances, especially in the control or prevention or in the treatment of:
  • the present invention provides the use of com- pounds according to general formulae I, II, III, IV, V and VI in chromatographical procedures.
  • hydroxamic acid-based peptide inhibitor 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.
  • a a aminomalonic acid
  • 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.
  • 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).
  • 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.
  • 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.
  • the glycine-containing derivates 1 to 3 inhibit the tested en ⁇ zymes one to two orders of magnitude less efficiently.
  • 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 .
  • 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 P ⁇ -P ⁇ ' -peptide bond exhibit a high resistance to cleavage by the proteinases.
  • 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.
  • 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 (+) - ⁇ -methylbenzyl- amine, s (-) - ⁇ -methylbenzylamine, and R (-) - ⁇ -methylcyclohexaneme ⁇ thylamine.
  • Ama aminomalonic acid
  • cdMMP catalytic domain of an MMP
  • MMP matrix metalloproteinase
  • DCC dicyclohexyl carbodiimide
  • TIMP tissue inhibitor of metalloproteinases.
  • 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 (-) - ⁇ -methylbenzylamine, R (+) - ⁇ -methylbenzyl- amine or R (-) - ⁇ -methylcyclohexanemethylamine .
  • Peptide coupling reactions were carried out in organic solvents following stan- dard procedures (9).
  • 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).
  • buffer 0.,1 M Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM CaCl 2 , 0.05% Brij 35.
  • substrate approximately 0.1 mg in 1 ml DMSO
  • Ki values were deter- mined by the established method of Dixon (13).
  • MMP-9 latent progelatinase B
  • Activation of the latent progelatinase B (MMP-9) was carried out in assay buffer by adding bo ⁇ vine trypsin (50 ⁇ l, 0.6 mg/ml) to the proenzyme (0.45 ml, 120 ⁇ g/ml) and incubating at 37°C for 10 minutes. The trypsin was then inactivated with aprotinin (50 ⁇ l, 1.2 mg/ml).
  • 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 Boc-Tyr (Bzl) -Leu-NH 2 .
  • Boc-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 is
  • the preparation is performed by acidolysis of the Boc-protected derivate using a water-free saturated solution of HCl in acetic acid.

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  • Psychiatry (AREA)
  • Pulmonology (AREA)
  • Oncology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Immunology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Selon l'invention, on synthétise et caractérise de nouveaux peptides contenant la séquence Pro Leu Ama(NHOH) au moyen de techniques spectroscopiques. On détermine leurs propriétés d'inhibition de la forme activée de la gélatinase humaine native B (MMP-9) ainsi que du domaine catalytique de la collagénase neutrophile (cdMMP-8). L'inhibiteur synthétisé le plus efficace présente des valeurs Ki s'élevant à 2x10-6 M (cdMMP-8) et à 5x10-9 M, ce qui permet d'obtenir une discrimination intéressante entre les métalloprotéases testées. Une des caractéristiques majeures de ce type d'inhibiteur réside dans sa nature peptidique, qui rend les composés similaires à des substrats naturels. Malgré la nature peptidique des inhibiteurs synthétisés, la liaison peptidique P¿1?-P1' présente une résistance élevée au clivage par les protéases.
PCT/EP1999/004826 1998-07-08 1999-07-08 Inhibiteurs de metalloproteases matricielles contenant des derives d'acide aminomalonique et leurs derives a squelette peptidique modifie Ceased WO2000002904A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2000559133A JP2002520333A (ja) 1998-07-08 1999-07-08 アミノマロン酸誘導体及びそのペプチド骨格を変性した誘導体である化合物又はそれを含む化合物
AU50346/99A AU5034699A (en) 1998-07-08 1999-07-08 Matrix metalloproteinase inhibitors containing aminomalonic acid derivatives andpeptide backbone modified derivatives thereof
EP99934642A EP1095057A1 (fr) 1998-07-08 1999-07-08 Inhibiteurs de metalloproteases matricielles contenant des derives d'acide aminomalonique et leurs derives a squelette peptidique modifie

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP98112652.7 1998-07-08
EP98112652 1998-07-08

Publications (1)

Publication Number Publication Date
WO2000002904A1 true WO2000002904A1 (fr) 2000-01-20

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PCT/EP1999/004826 Ceased WO2000002904A1 (fr) 1998-07-08 1999-07-08 Inhibiteurs de metalloproteases matricielles contenant des derives d'acide aminomalonique et leurs derives a squelette peptidique modifie

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EP (1) EP1095057A1 (fr)
JP (1) JP2002520333A (fr)
AU (1) AU5034699A (fr)
WO (1) WO2000002904A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7041787B2 (en) 2000-12-29 2006-05-09 Kimberly-Clark Worldwide, Inc. Design and use of advanced zinc chelating peptides to regulate matrix metalloproteinases
EP2295402A3 (fr) * 2003-01-08 2011-08-03 The University of Washington Agents anti-bactériens
US9403758B2 (en) 2012-05-10 2016-08-02 Achaogen, Inc. Antibacterial agents
US9617256B2 (en) 2007-06-12 2017-04-11 Achaogen, Inc. Antibacterial agents

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2070899A1 (fr) * 2007-12-14 2009-06-17 F. Hoffmann-La Roche Ag Déprotection des composants N-BOC

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CURTIN M L ET AL: "Broad spectrum matrix metalloproteinase inhibitors: an examination of succinamide hydroxamate inhibitors with P1Calpha gem -disubstitution", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 8, no. 12, 16 June 1998 (1998-06-16), pages 1443-1448, XP004137061, ISSN: 0960-894X *
GRAMS E.A.: "Structure determination and analysis of human neutrophil collagenase complexed with a hydroxamate inhibitor", BIOCHEMISTRY, vol. 34, no. 43, 1995, EASTON, PA US, pages 14012 - 14020, XP002120567 *
KRUMME E.A.: "Hydroxamate derivatives of substrate-analogous peptides containing aminomalonic acid are potent inhibitors of matrix metalloproteinases", FEBS LETTERS, vol. 436, no. 2, 2 October 1998 (1998-10-02), AMSTERDAM NL, pages 209 - 212, XP002120566 *
YAMAMOTO E.A.: "Inhibition of membrane type 1 matrix metalloproteinse by hydroxamate inhibitors: an examination of the subsite pocket", JOURNAL OF MEDICINAL CHEMISTRY, vol. 41, no. 8, 9 April 1998 (1998-04-09), WASHINGTON US, pages 1209 - 1217, XP002120568 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7041787B2 (en) 2000-12-29 2006-05-09 Kimberly-Clark Worldwide, Inc. Design and use of advanced zinc chelating peptides to regulate matrix metalloproteinases
EP2295402A3 (fr) * 2003-01-08 2011-08-03 The University of Washington Agents anti-bactériens
US8084615B2 (en) 2003-01-08 2011-12-27 University Of Washington Antibacterial agents
US8101640B2 (en) 2003-01-08 2012-01-24 Novartis Vaccines And Diagnostics, Inc. Antibacterial agents
US8153843B2 (en) 2003-01-08 2012-04-10 University Of Washington Antibacterial agents
US9617256B2 (en) 2007-06-12 2017-04-11 Achaogen, Inc. Antibacterial agents
US9403758B2 (en) 2012-05-10 2016-08-02 Achaogen, Inc. Antibacterial agents
US9701622B2 (en) 2012-05-10 2017-07-11 Achaogen, Inc. Antibacterial agents

Also Published As

Publication number Publication date
AU5034699A (en) 2000-02-01
JP2002520333A (ja) 2002-07-09
EP1095057A1 (fr) 2001-05-02

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