DD294711A5 - NEW IRREVERSIBLE INHIBITORS FOR INHIBITING THE CATALYTIC ACTIVITY OF ENZYMES AND METHOD FOR THE PRODUCTION THEREOF - Google Patents

Abstract

The invention is for development; Preparation and application of potent, irreversible inhibitors to inhibit the undesirable activity of enzymes that, as they catalyze substrates, cleave and link ester bonds, and can cleave, isomerize, and synthesize peptide bonds. The invention relates to diacyl hydroxylamine derivatives of the general formula R ind 1-CO-NHO-CO-R 2. The object of the invention is an easily applicable method for controlling the activity of enzymes in their purified form and in normal and pathologically altered body fluids. Organs, tissues, cells and cell cultures of human, animal, microbial viral origin both in vitro and in vivo and for the synthetic recovery of the inhibitors. The radical R ind 1-CO- may represent amino acid- or N-protected amino acid residues and N-protected peptidyl residues or N-unprotected peptidyl residues, where the N-protecting groups are alkoxycarbonyl, aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl , Arylsulphonyl, Monoaralkylcarbamoyl, cinnamoyl, alpha-Aralkoxycarbonylaminoalkanoyl groups or together with the nitrogen atom formed cyclic amides and where the amino acids or the components of Peptidylreste proteinogical L and D-amino acids or other non-proteinogenic or substituted amino acids may be or aliphatic branched, aromatic and aromatic substituted and heterocyclic acyl radicals such as aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkonoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl; monoaralkylcarbamoyl; Cinnamoyl, alpha-aralkoxycarbonylaminoalkanoyl moieties. R ind 2-CO- represents aliphatic, branched, aromatic, aromatic, substituted acyl radicals, such as e.g. Benzoylreste with substituents in ortho, meta or para position or substituent combinations in these positions, preferably the atoms or groups F, Cl, Br, NO ind 2, NH ind 2, CH ind 3, CH ind 3 O and substituted and unsubstituted heterocyclic acyl radicals such as aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, monoaralkylcarbamoyl, cinnamoyl, alpha- ..). The invention is of importance for the medical, immunobiochemical and pharmaceutical industries for use as diagnostics, therapeutics or pharmaceuticals. {Inhibitors; enzymes; Hydrolases; proteases; Amino acid derivatives; Peptide derivatives; irreversible inhibition; diagnostics; therapeutics; pharmaceuticals}

Description

Field of application of the invention

The invention relates to the development of mechanism-oriented, irreversibly acting inhibitors of enzymes which, in the course of their catalytic action on substrates, can cleave, isomerize and bond ester bonds and peptide bonds.

The compounds of the invention are effective i.a. to the activity of peptide hydrolases (proteases, proteinases, peptidases), lipases, esterases, synthetases, transacylases, which form both covalent and noncovalent intermediates with their substrates during catalysis and to express their activity and the like. a. contain catalytically active serine, cysteine and Aspartatreste or are catalytically active with the aid of metal ions.

Inhibitors of such enzymes serve to effectively suppress their undesirable activity in various pathological conditions, in fermentation processes in the food industry, under laboratory conditions.

The invention is suitable for use as pharmaceuticals in the therapy of various diseases in human medicine, veterinary medicine, pharmacology, as fine chemicals in biotechnology, biochemistry, immunobiochemistry, molecular biology, genetics, pharmacy, food industry and for the pharmaceutical industry.

Characteristic of the known technical solutions

The selective inhibition of enzymes (in particular of peptide hydrolases) for therapeutic, scientific and technical purposes has hitherto been achieved, in particular, by the use of reversible inhibitors which have to be isolated by lengthy processes from microbial, animal and plant material (Proteinase Inhibitors, Katunuma, N., Umezawa , H., Holzer, K., ed., Springer-Verlag, Berlin, 1883) and by synthetic substances such as statin peptides, cephalosporins, haloenolactones, peptide boronic acids and peptide aldehydes (Design of Enzyme Inhibitors as Drugs, Sandler, M., Smith, HJ , Ed., Oxford University Press, Oxford (1989).

In order to achieve a selective inhibition, often very complicated synthetic methods must be used. Thus, the synthesis of an immunosuppressive effective inhibitor of cyclophilin by loss-rich condensation of four structural segments to a 23-membered ring system Stinson, S., Chem.Eng. News. 67,6,1989,29-30). On the other hand, have various inhibitors such as peptidyl halomethyl ketones, peptide aldehydes, peptidyl-N-nitrosamides activated carbonyl or methylene groups, which severely limit the stability of the compounds in biological material and beyond unspecific u.a. very mutagenic (Methods in Enzymology, Vol.XLVI, Academic Press, New York 1977, 3-240 and Fittkau, S., Jahreis, G., Peters, K., and L. Balaspiri, J. Prakt. Chem.328 , 1986.5? 9-538). Subsequently, we developed an easily applicable, robust method which leads to the specific inhibition of the catalytic activity of peptide hydrolases which do not require C-terminally free carboxyl groups in the substrate during their function and contain catalytically active serine or cysiein residues (Fischer, G. , Demuth, H.-U., Barth, A., DD158109, 1982).

With the (corresponding to this method) compounds of the structure N-peptidyl-O-Benzovl hydroxylamine, the selective inhibition of serine proteases (Demuth, H.-U., Baumgrau, R., Schaper, C, Fischer, G., and Barth , AJ Enzyme Inhibition 2,1988,129-142) and cysteine proteases (Smith, RA, Coles, PJ, Spencer, RW, Copp, LJ, Jones, CS, and Krantz, A., Biochem. Biophys. 155,1988,1201-1207).

Object of the invention

The aim of the invention is to provide highly effective, irreversible inhibitors for the inhibition of the activity of enzymes (preferably peptide hydrolases, lipases, esterases, synthetases, transacylases) based on the diacyl hydroxylamines, which are easy to produce, highly effective and selective acting substances by varying their R _n earmarked, for gradual gradation of their stability, applicability, bioavailability and inhibitory potency can be used especially in medical therapy and diagnosis.

Explanation of the essence of the invention

The invention has for its object to develop new irreversible inhibitors of enzymes (especially peptide hydrolases) of the type 'Jer Diacyl Hydroxylamine.

The method we have described above defined the N-peptidyl residue of the compounds as α-amino acid, N-protected α-amino acid or peptidyl residue and a benzoyl residue with substituents or substituent combinations in ortho, meta or para. Position, preferably F, Cl, Br, NO 2, CHj, CH ₃ O.

Investigations on the mechanism of inactivation of Sorinpopi'dpse dipeptidyl pdptidase IV by means of inhibitors of the above structure, but can be expected that the mode of action goes far beyond our structures and target enzymes described in the dor patent DD158109 (Demuth, H.-U., Neumann , U., and Barth, A., J. Enzyme Inhibition, 2.1989, 239-248) and thus the usability of compounds a) further structural variations in ancestors for the inhibition of enzymes with b) aucr. other properties than those of the covalently catalyzing peptide hydrolases with catalytically active cysteine and serine residues. This also includes enzymes whose substrates carry C-terminal free carboxyl groups which have been expressly excluded by the original patent. For the preparation of these structures, novel synthesis processes have been developed which, in comparison to the previous ones, continue to have the following advantages, through the reduction of the synthesis steps and easier accessibility of the structural variations and their products:

1. Simple molecular structure

2. Easy synthetic and therefore cheap accessibility

3. Ability to achieve high selectivity through the simplest structural variations

4. Ability by appropriate choice of substituents

a) the life of the compounds

b) the bioavailability of the compounds

c) influence the transportability of the compounds

5. More universal applicability to different classes of enzymes, not just proteases that require cysteine and serine residues for catalysis.

These properties are inventively compounds of general formula I.

R ₁ -C-NH-OCR ₂ OO

wherein the radical R ₁ -CO- stands for:

Amino acid- or N-protected amino acid residues and N-protected poptidyl residues or N-unprotected peptidyl residues; these N-protecting groups may be alkoxycarbonyl, aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, monoaralkylcarbamoyl, cinnamoyl, a-aralkoxycarbonylaminoalkanoyl, or cyclic amides formed together with the nitrogen atom; and wherein the amino acids or the constituents of the peptidyl residues may be proteinogenic L and D amino acids or other nonproteinogenic or substituted amino acids.

- Aliphatic, branched, aromatic and aromatic substituted and heterocyclic acyl radicals such as aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, Hotorocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, monoaralkylcarbamoyl, cinnamoyl, a-Aralkoxycarbonylaminoalkanoyl groupings.

R ₂ -CO-stands for:

- aliphatic, branched, aromatic and aromatic substituted acyl radicals, such as. B. Benzoylreste with substituents in the ortho, meta or para position or Substitue.ntenkombinationon in these positions, preferably the atoms or groups F, Cl, Br, NO}, NH ₂ , CH ₃ , CH ₃ O and substituted and unsubstituted heterocyclic acyl radicals such as aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, monoaralkylcarbamoyl, cinnamoyl, a-aralkoxycarbonylaminoalkanoyl moieties.

- R _{2 can} also stand for:

primary or secondary amines (as for aliphatic, branched aliphatic or heterocyclic or aromatic, substituted and unsubstituted amines, including C-terminal protected and unprotected amino acids and peptides, D and other eg non-proteinogenic or substituted amino acids, the may also be components of the peptides such as sarcosine, N-mothylalanine, dehydroalanine, aziridinecarboxylic acid, azetidinecarboxylic acid, dehydroproline, aminobenzoic acid).

In the action of the aqueous solution of a substance of the general formula with such radicals R _t and R ₂ , which are recognized by the relevant target enzyme as substrate-analogous structures on the relevant, in the isolated state or in biological material enzyme is a time-dependent inhibition of enzyme activity achieved under conditions that are characteristic of the usual expression of the enzyme activity, depending on the inhibitor concentration and the structure of the residues Ri and R ₂ .

The compounds of the invention develop their effects such that after binding of the inhibitors to the target enzyme a radical R ₂ -COO "is cleaved, and a remaining reactive intermediate with the possible structures R) -CON, R ₁ -CONH ⁺ or R ₂ -NCO the further catalytic activity of the particular enzyme is blocked by reaction with groups in or in the vicinity of the active site This type of inhibition is referred to as mechanism-oriented inhibition (Demuth, H.-U., Neumann, U., Pharmacy 44, The process is based on the fact that the chemical reaction leading to the modification of the enzyme is predominantly due to the catalytic action of the target enzyme on the inhibitor, so that unspecific reactions with other biomolecules can be avoided as far as possible.

Inhibitors of the general formula are distinguished by high selectivity between the target enzymes. Variation of this specificity, rate of inhibition, lifetime in biological solutions and their penetration properties can be made by the appropriate choice and combination of R ₁ and R ₂ and their substituents. The necessary synthetic operations for providing the grids R) -COOH and R ₂ -COOH or R ₁ and R ₂ are carried out according to the usual peptide chemical methods (Wünsch, E "Synthesis of peptides, Houben-Weyl, Volume 15 / I, Methods of organic chemistry, Müller, E "ed., Goorg-Thiemo-Vorlag Stuttgart 1974). For the connection of these starting materials to inhibitors analogous to formula I, new synthesis methods are used.

The erfir. Diacyl hydroxylamino corresponding to the general formula I or their pharmaceutically applicable formulation complexes obtained according to the invention can be used as irreversible inhibitors for inhibiting the catalytic activity of enzymes such as proteinases, peptidases, lipases, esterases, synthotases, transacylases in their purified form as well as in

inhibit normal and pathologically altered human or animal body fluids, organs and tissues as well as cells and cell cultures, viral, microbial, animal or plant origin both in vitro and in vivo and thus as highly effective diagnostics, cytostatics, immunosuppressants, anti-tumor drugs, antiviral drugs, Anti-HIV-1 or generally used as therapeutics in metabolic or regulatory processes in which unwanted activity of specified enzymes above induce pathological changes in the respective organism.

embodiments example 1 The acylhydroxamic acids as starting materials are obtained by treating the acid R ₁ -COOH with an equimolar amount Carbonyldiimidazole and subsequent addition of hydroxylamine hydrochloride (Staab, H.A., Lüking, M., Dürr, F. H.,

Chem.Ber.951962,1275-1283).

Without further purification operations is then by the addition of another Equivalents carbonyldiimidazole and a Equivalents of the radical R ₂ -COOH using catalyzing base (pyridine, N-dimethylpyridine) the desired Obtained end product. The column chromatographic purification of the reaction solution on silica gel or Sephadox LH-20 and

subsequent crystallization of the product from ethanol / ethyl acetate, or ethyl acetate / petroleum ether leads to the desired

End product of general formula I. Example 2 The synthesis method serves the selective introduction of carbamic acid residues (R2 corresponds to amines, C-terminal protected

or unprotected α-amino acids or peptides) into the structure of general formula I. Here, acylhydroxamic acid

R ₁ -CO-NHOH used purified. By reaction between 4-nitrophenyl chloroformate with the amino component R ₂ , an activated Carbaminsäureesterderivat the general formula: Np-CO-NH-R 'or Np- ^ CO-NR ¹ R ", wherein NH-R' and NR'R" the radical R ₂

correspond to the general formula and may represent aliphatic, branched, heterocyclic or aromatic amines in addition to amino acids and peptide derivatives. Np- is the 4-nitrophenyl radical. Stir one equivalent

Acylhydroxamic acid (R ₁ -CO-NHOH) with one equivalent of Np-CO-R ₂ at O ⁰ C in THF leads to the desired Diacylhydroxamic acid, which can be purified analogously to the methods described in Example 1. Example 3 N-t-butyloxycarbonyl-alanyl-phenylalanyl-O-acetyl Hydroxylamine (concentration range 1-100μΜ) at 25 ⁰ C (in 5OmM tricine buffer, pH 7.6) with the enzyme subtilisin

incubated and the inactivation of the enzyme using the test substrate N-succinyl-alanyl-alanyl-alanyl-nitroanilide (10-500μΜ) followed.

The determined inactivation parameters are: Ki = 47μΜ and kinact = 0.003sec "'. Example 4 N-carbobenzoxy-phenylalanyl-phenylalanyl-O-methacryloyl Hydroxylamine (concentration range 10-100OnM) at 25 ⁰ C (in 5OmM acetate buffer, pH 5.5) with the enzyme Cathepsin L

incubated and the inactivation of the enzyme by means of the test substrate N-carbobenzoxy-phenylalanyl-arginyl-

Methylcoumarinamide (3-8 μΜ) was followed. The determined inactivation parameters are: Ki = 9CnM and kinact = 0.11 see " ¹ .

Claims (4)

1. Use of novel inhibitors for inhibiting the catalytic activity of enzymes and as diagnostics for these enzymes, characterized by compounds of general formula I. R _{1 is} -CO-NH-O-CO-R ₂ , the radical R ₁ -CO-for: Amino acid- or N-protected amino acid residues and N-protected peptidyl residues or N-unprotected peptidyl residues, wherein these N-protecting groups alkoxycarbonyl, aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, Monoaralkylcarbamoyl, cinnamoyl, a-Aralkoxycarbonylaminoalkanoyl groups or together with the nitrogen atom formed cyclic amides can represent, and wherein the amino acids or the constituents the peptidyl residues may be proteinogenic L and D amino acids or other non-proto-ingenic or substituted amino acids; - aliphatic, branched, aromatic and aromatic substituted and heterocyclic acyl radicals such as aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, monoaralkylcarbamoyl, cinnamoyl, a-aralkoxycarbonylaminoalkanoyl groupings; R ₂ -CO-for: - aliphatic, branched, aromatic and aromatic substituted acyl radicals such. B. Benzoylreste with substituents in ortho, meta or para position or substituent combinations in these positions, preferably the atoms or groups F, Cl, Br, NO ₂ , NH ₂ , CH ₃ , CH ₃ O and substituted and unsubstituted heterocyclic acyl radicals such Aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, monoaralkylcarbamoyl, cinnamoyl, a-aralkoxycarbonylaminoalkanoyl groupings, or for: primary or secondary amines, such as aliphatic, branched aliphatic or heterocyclic or aromatic, substituted and unsubstituted amines, including C-terminal protected and unprotected amino acids and peptides, D and others, e.g. B. nonproteinogenic or substituted amino acids, which may also be components of the peptides such. Sarcosine, N-methylalanine, dehydroalanine, aziridinecarboxylic acid, azetidinecarboxylic acid, dehydroproline, aminobenzoic acid. 2. A method for influencing the activity of enzymes by novel, mechanically-oriented inhibitors of the general Formal I, characterized in that the inhibitory active form of the inhibitor is generated during the interaction between the enzyme and the inhibitor and either a carbonyl-active isocyanate, a Carbonylnitrenium Represents carbonyl nitrene or an acyl radical. 3. A process for the preparation of novel, mechanism-oriented inhibitors of enzymes, characterized in that their representation starting from Ri-CO-NHOH preferably by introducing the O-acyl function (R ₂ -CO-) by means of carbonyldiimidazole as coupling reagent and the corresponding carboxylic acid R ₂ -COOH and in the case of introduction of carbamic acid derivatives (as O-acyl groups, R ₂ -CO-) via the preparation of an activated Carbaminsäurenitrophenylesters and its subsequent reaction with the purified acyl hydroxamic R ₁ -CO-NHOH to the desired diacyl hydroxylamine of general formula I. he follows. 4. Use of novel, mechanism-oriented inhibitors of enzymes according to claim 1, characterized in that disse or pharmaceutically formulated complexes for the irreversible inhibition of the catalytic activity of Peptidhydrolasen, lipases, esterases, synthetases, transacylases, both covalent and noncovalent intermediates with their substrates during catalysis form and to the expression of their activity and others contain catalytically active serine, cysteine or Aspartatreste or are catalytically active with the aid of metal ions, in their purified form or in normal and pathologically altered body fluids, organs, tissues, cells and cell cultures of human, animal, microbial, viral origin as diagnostics, therapeutics or pharmaceuticals.