WO1998050351A1

WO1998050351A1 - New cysteine derivatives, processes for their production, and pharmaceuticals containing them

Info

Publication number: WO1998050351A1
Application number: PCT/EP1998/002654
Authority: WO
Inventors: Juliane Constanze Desirée MÜLLER; Erich GRAF VON RÖDERN; Luis Moroder
Original assignee: Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin
Priority date: 1997-05-07
Filing date: 1998-05-06
Publication date: 1998-11-12
Also published as: AU7652998A; JP2001525800A; CN1261878A; BR9808758A; ZA983792B; CA2289094A1; TR199903120T2; KR20010012321A; EP1034163A1

Abstract

A compound represented by general formula (I), which binds and inhibits matrix metalloproteinases (MMPs), wherein the cysteine moiety contains an unprotected thiol group, the cysteine moiety is in the L- or D-configuration wherein A denotes -CO-, SO2-, -NH-CO-, or -O-CO-, R1 denotes hydrogen, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a C1-C15 alkyl group substituted by halogen, mercapto, hydroxy, alkoxy, amino or nitro, or by carbocyclic non aromatic or aromatic ring systems which are optionally substituted once or several times or aromatic or non aromatic heterocycles, optionally substituted, their pharmacologically acceptable salts, or optically active forms thereof. R denotes hydroxy, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a C1-C15 alkyl group substituted by carbocyclic non aromatic or aromatic ring systems which are optionally substituted once or several times or aromatic or non aromatic heterocycles, optionally substituted, their pharmacologically acceptable salts, or optically active forms thereof, processes for the preparation, pharmaceutical compositions and their use in medicine.

Description

NEW CYSTEINE DERIVATIVES, PROCESSES FOR THEIR PRODUCTION, AND PHARMACEUTICALS CONTAINING THEM

The invention comprises new matrix metailoproteinase inhibitors that are based on the use of the amino acid L- or D-cysteine which is derivatized at the amino and carboxyl function with non-peptidic type groups (formula I). The invention comprises methods for the production of the inhibitors and their use in the field of therapeutics.

The family of matrix metalloproteases (MMPs) has become a major target for drug design, since these enzymes are involved in tissue remodeling and connective tissue turnover, and thus in several diseases where (i) rapid extracellular matrix degradation is taking place, e.g. during congestive heart failure and extravasion of highly metastatic tumor cells, or (ii) slow extracellular matrix degradation is occurring, e.g. artherosclerotic lesion formation and rupture, cartilage matrix loss in osteoarthritis, bone matrix degradation in osteoporosis, gingival degradation in periodontal disease, matrix remodeling and deposition in Alzheimer plaque formation and rheumatoid arthritis.

The MMP family currently includes fourteen members, ten of which are secreted from the cells in a soluble form and four members are membrane-bound enzymes. The MMPs are zinc dependent and calcium requiring enzymes which are inhibited by one of the members of the tissue inhibitor of metailoproteinase (TIMP) family. Synthetic inhibitors of this class of enzymes have been developed as hydroxamates, N-carboxyalkyl derivatives, phosphonamidates and phosphinates as well as by using thiol groups as ligands for the active-site zinc atom. an N-terminal propeptide of about 80 residues. The propeptide forms a separate smaller domain that contains three α-helices and an extended peptide that occupies the active site. The catalytic domain in all these structures contains two Zn ⁺ ions, i.e. a "structural" zinc ion and a "catalytic" zinc ion. The "catalytic" zink ion is coordinated by the side chains of three histidyl residues of the consensus sequence HEXXHXXGXXH. The fourth ligand of the "catalytic" zink in the inhibited enzymes is a coordinating group of the inhibitors like the hydroxamate or carboxylate; in the pro-MMP propeptide it is the thiol group of the cysteine residue(l).

Correspondingly, the thiol-based collagenase inhibitors, proposed so far, are generally of peptidic structure containing cysteine or cysteine-like amino acids and their design was based on the binding mode of the substrate and more recently, of the the cysteine- containing propeptide. The present invention relates to a new class of MMP inhibitors which were derived from cysteine in a non-peptidic manner as shown in the general formula I, processes for the preparations, and pharmaceutical compositions containing these compounds and their therapeutical use in medicine,

wherein A denotes -CO-, -SO₂-, -NH-CO-, or -O-CO-

Ri denotes hydrogen, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a Ci-Cu alkyl group substituted by halogen, mercapto, hydroxy, alkoxy, amino or nitro, or by carbocyclic aromatic or non aromatic ring systems which are optionally substituted once or several times or aromatic or non aromatic heterocycles, optionally substituted, their pharmacologically acceptable salts, or optically active forms thereof.

R denotes hydroxy, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a Ci-Cu alkyl group substituted by carbocyclic aromatic or non aromatic ring systems which are optionally substituted once or several times or aromatic or non aromatic heterocycles, optionally substituted, their pharmacologically acceptable salts, or optically active forms thereof.

With respect to formula I R or/and Ri represent a branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms selected from methyl, ethyl, propyl, n-butyl, tert- butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc., vinyl, etc. as well as the corresponding alkinyl groups e. g. acetylene.

The carbocyclic aromatic or non aromatic substituents for said alkyl groups are selected from C₃-Cβ cycloalkyls such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, or Cβ-Cι carbocyclic aromatic substituents such as phenyl, naphtyl, or anthranyl, or heterocyclic non aromatic substituents such as pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, or heterocyclic aromatic substituents such as pyrrolyl, pyridinyl, furyl, thienyl, thiazolyl, imidazolyl, pyrimidinyl, purinyl, indolyl, quinolyl, carbazolyl.

The carbocyclic aromatic or non aromatic ring systems respectively heterocycles can optionally be substituted once or several times for example by halogen-, nitro-, hydroxy-,

Cι-C₆ alkyl-, Ci-Cβ alkoxy-, amino-, mercapto-, carboxyl-, cyano-, or sulfonyl groups.

If A denotes -CO-, the residue RiCO- is preferably selected from the residues of the following carboxyclic acids: Formic Acid, Acetic acid, Propionic acid, Hexanoic acid, Laurie acid, Myrisic acid,

Palmitic acid, Stearic acid, Arachidonic acid, Behenic acid, Octadecenoic acid, Linoleic acid, Linolenic acid, 3-Mercaptopropionic acid, Glyoxylic acid, Malonic acid, Succinic acid, 4-Aminobutanoic acid, 6-Aminocaproic acid, 3-Nitropropionic acid, Naphthylacetic acid, 4-Aminophenylacetic acid, Acrylic acid, Cinnamic acid, 4-Amino-cinnamic acid, Aminocrotonic acid, Fumaric acid, Maleinic acid, Phthalic acid, Benzoic acid,

Nitrobenzoic acid, 3-Aminobenzoic acid, 4-Aminobenzoic acid, Anthranilic acid, Salicilic acid, 3-Amino-salicilic acid, 4-Amino-salicilic acid, 5-Amino-salicilic acid, Naphthoic acid, p-Phenylbenzoic acid, Phenanthroic acid, Nicotinic acid, 3-Aminopyrazin-2- carbonic acid, Pyridine carboxylic acid, Piperazine carboxylic acid, Piperidine carboxylic acid.

If A denotes -SO₂, the residue RιSO₂- is preferably selected from the residues of the following sulfonic acids:

Methanesulfonic acid, Ethanesulfonic acid, Propane sulfonic acid, 3-Hydroxypropane sulfonic acid, Orthanilic acid (aniline- 2- sulfonic acid), Naphthalenesulfonic acid, Naphthylamine sulfonic acid, Aminomethane sulfonic acid, 2-Mercaptoethane sulfonic acid, 2-Chloroethane sulfonic acid, N,N'-Dimethylsulfamic acid, Piperidine sulfonic acid, 5- (2- Aminoethylamino)- 1- naphthalene- sulfonic acid , Iodoxyquinolinesulfonic acid, Pyridine- 3- sulfonic acid, p- Toluenesulfonic acid, 2- (p- Toluidino)naphthalene- 6- sulfonic acid, Decyl methanesulfonic acid, 2- [(2- Amino- 2- oxoethyl)amino] ethanesulfonic acid, 2- (N- Cyclohexylamino)ethanesulfonic acid, 2- [bis(2- Hydroxyethyl)amino]ethanesulfonic acid, N- 2- Hydroxyethylpiperazine- N^ 2- ethanesulfonic acid, N- tris(Hydroxymethyl)methyl- 2- amino- ethanesulfonic acid, 2- (N- Morpholino)ethanesulfonic acid, Piperazine- N,N- bis(2- ethanesulfonic acid), 3- (2- Pyridyl)- 5,6- bis(4- phenylsulfonic acid)- 1,2,4- triazine.

If A denotes -NHCO-, the residue Ri-NH-CO- is preferably selected from the residues of the following urea derivatives: n- Butyl-, R- (+)- alpha- Phenylethyl-, R-(-)-l-(l-Naphthyl)-ethyl-, Ethyl-, Propyl-,

Hexyl-, Octyl-, Benzyl-, Chlorobenzyl-, Methylbenzyl-, 3-Picolyl-, 2-(Aminomethyl)- pyridyl urea.

If A denotes -O-CO-, the residue Ri-O-CO- is preferably selected from the residues of the following Carbamates:

Methyl carbamate, Ethyl carbamate, 9-Fluorenylmethyl carbamate, 9-(2-

Sulfo)fluorenylmethyl carbamate, 9-(2,7-Dibromo)fluorenylmethyl carbamate, 4- Methoxyphenacyl carbamate, 2,2,2-Trichloroethyl carbamate, 2-Phenylethyl carbamate, l-(l-Adamantyl)-l-methylethyl carbamate, l,l-Dimethyl-2-haloethyl carbamate, 1-

Methyl-l-(4-biphenyl)-l-methylethyl carbamate, 2-(2^'-Pyridyl)ethyl carbamate, 1- Adamantyl carbamate, Vinyl carbamate, Allyl carbamate, 1-Isopropylallyl carbamate, 4- Nitrocinnamyl carbamate, 8-Quinolyl carbamate, Cyclohexyl carbamate, Benzyl carbamate, p-Methoxybenzyl carbamate, p-Nitrobenzyl carbamate, p-Bromobenzyl carbamate, 9-Anthrylmethyl carbamate, diphenylmethyl carbamate, 2-Methylsulfonylethyl carbamate, 2-(p-Toluenesulfonyl)ethyl carbamate, 4-Methylthiophenyl carbamate.

R is preferably selected from the following residues:

Ethyl-, Propyl-, Hexyl-, Octyl-, Benzyl-, 4-Chlorobenzyl-, 4-Methylbenzyl-, 3-Picolyl-, 2-(methyl)-pyridyl-, 4-(methyl)pyridyl-, 3-Phenylpropyl-, 4-Phenylbutyl-, 2-(p- Tolyl)ethyl- , 3-Nitrobenzyl-, Benzylethyl-, 2-Phenylethyl-, Adamantyl-, Pyridyl-, Phenyl, Cholestenyl-, Naphthyl-, 4-Phenoxy-phenyl or indolylethyl.

Preferred compounds according to formula I are compounds of example 5-22, and of the following table:

The compounds according to the formula I consist of three parts which have different

2+ ^■ structures and different properties. The chelating group SH for the active-site Zn ion, the hydrophobic groups R} or R to interact with the hydrophobic S ι protein pocket as well as to contribute to additional hydrophobic interactions along the P' substrate binding cleft.

Inhibitors of the general formula I allow for developing selective inhibitors of the different MMPs as required for their differentiated pathological implications e.g. osteoporosis rheumatoid arthritis, periodontal disease, artherosclerosis, congestive heart failure, tumor invasion and metastasis and angiogenesis.

It is known that there are similar compounds described in the literature. However, these are peptidic compounds and they display a worse half life time in human plasma.

The chemical structure of the cysteine derivatives was choosen in view of increased metabolic stability. Correspondingly, the amino group was acylated with carboxylic acids to produce amides, but preferentially urethanyl derivatives known to be more stable to enzymatic metabolism. Similarly, the C-terminal carboxyl fiinction was derivatized as amide instead of esterification to avoid fast clearance rates due to hydrolysis of the esters by Upases. Moreover, N-alkyl and N-aryl amides were selected which are known to be more stable towards peptidases.

For the synthesis of the inhibitors classical procedures of organic synthesis were applied (2). Related L- and D-cystine derivatives of formula II were prepared according to standard procedures of peptide chemistry and then amidated with EDCI/HOBt according to Scheme 1. Subsequent reduction of the cystine compounds of formula III to the cysteine derivatives of Formula I was performed with reducing agents like mercaptanes or preferentially with phosphines like tributylphosphine, as shown in scheme 1.

π m

Scheme 1. General route for the synthesis of the MMP inhibitors of the present invention

The compounds of the present invention, which specifically inhibit MMPs, are pharmacologically useful in the treatment of rheumatoid arthritis and related diseases in which collagenolytic activity is a contributing factor, such as, for example, corneal ulceration, osteoporosis, periodontitis, Paget's disease, gingivitis, tumor invasion, dystrophic epidermolysis, bullosa, systemic ulceration, epidermal ulceration, gastric ulceration, and the like. These compounds are particularly useful in the treatment of rheumatoid arthritis (primary chronic polyarthritis, PCP), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis, Sjόgren's syndrome (RA + sicca syndrome), polyarteritis nodosa and related vasculities, e. g. Wegener's granulomatosis, giant-cell arteritis, Goodpasture's syndrome, hypersensitiveness angiitis, polymyositis and dermatomyositis, progressive system sclerosis, M. Behcet, Reiter syndrome (arthtritis + urethritis + conjunctivitis), mixed connective tissue disease (Sharp's syndrome), spondylitis ankylopoetica (M. Bechterew).

The compounds of the present invention may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route and in dose effective for the treatment intended. Therapeutically effective doses of the compounds of the present invention required to prevent or arrest the progress of the medical condition are readily ascertained by one of ordinary skill in the art. Accordingly, the invention provides a class of novel pharmaceutical compositions comprising one or more compounds of the present invention, in association with one or more non-toxic pharmaceutically acceptable carriers and/or adjuvants (collectively referred to herein as „carrier materials") and, if desired, other active ingredients, the compounds and compositions may, for example, be administered intravascularly, intraperitoneally, subcutaneously, intramuscularly or topically.

For all administrations, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit contained in a particular amount of the active ingredient. Examples of such dosage units are tablets or capsules. A suitable daily dose for a mammal may vary widely depending on the condition of the patient and other factors. However, a dose from about 0.1 to 300 mg/kg body weight, particularly from about 1 to 30 mg kg body weight may be appropriate. The active ingredient may also be administered by injection.

The dose regimen for treating a disease condition with the compounds and/or compositions of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex and medical conditions of the patient. Severity of the infection and the role of administration and the particular compound employed and thus may vary widely.

For therapeutic purposes, the compounds of the invention are ordinarily combined with one ore more adjuvants appropriate to the indicated route of administation. If per os, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl ester, talc, stearic acid, magnesium stearat, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, gelatine, acacia, sodium alginate, polyvinyl-pyrrolidone and/or polyvinyl alcohol, and thus tabletted or encapsulated for convenient administration. Alternatively, the compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cotton seed oil, peanut oil, sesam oil, benzyl alcohol, sodium chloride and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. Appropriate dosages in any given instance, of course, depend upon the nature and severity of the condition treated, the route of administration and the species of mammal involved, including its size and any individual idiosyncracies.

Representative carriers, dilutions and adjuvants include, for example, water, lactose, gelatine starch, magnesium stearate, talc, vegetable oils, gums, polyalkylene glycols, petroleum gelly, etc. The pharmaceutical compositions may be made up in a solid form, such as granules, powders or suppositories, or in liquid form, such as solutions, suspensions or emulsions. The pharmaceutical compositions may be subjected to conventional pharmaceutical adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

For use in the treatment of rheumatoid arthritis, the compounds of this invention can be administered by any convenient route, preferably in the form of a pharmaceutical composition adpted to such route and in a dose effective for the intended treatment. In the treatment of arthritis, administration may be conveniently be by the oral route or by injection intra-articularly into the affected joint.

As indicated, the dose administered and the treatment regimen will be dependent, for example, on the disease, the severity thereof, on the patient being treated and his response to treatment and, therefore, may be widely varied.

Enzyme assay

The catalytic domain of MMP8 (Phe 79 -MMP8) and MMP3 were used for the inhibition experiments. Enzyme assays were performed at 25° C in 10 mM CaCl , 100 mM NaCl, 50 mM Tris/HCl (pH 7.6) using 8 nM enzyme concentrations and the fluorogenic substrates Dnρ-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH₂ (Bachem M-1855, 1.10"⁵ M) for MMP8 and Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnρ)-NH₂ (Bachem M- 2110, 4.10^-6 M) for MMP3: Measurements were performed essentially as described by Stack et al. (3) for MMP8 and by Nagase et al. (4) for MMP3. The increase in fluorescence at 350 nm (MMP8) or 390 nm (MMP3) was monitored over a period of 100 sec to determine initial rates of hydrolysis. Evaluation of the kinetic data was performed as reported by Copeland et al. (5).

Table I. Examples for inhibition of MMP8 and MMP3 with non-peptidic L-cysteine derivatives of the general formula I

Compound R,-A R KJ; MMP8 K--i; MMP3 [μM] [μM]

10 i 0.71 4.9

15 JO 0.16 12.3

Synthesis

General methods:

A) Amidation. 1 mmole N,N^'-urethanyl-cystine, 2 mmol HOBT (hydroxybenzotriazole) and 2.08 mmole EDCI (N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride) are dissolved or suspended in 10 ml THF. The amine is added in excess

(2.5-5 mmol) and in the case of the hydrochloride equivalent amounts of N- methylmorpholine are added. The reaction mixture is stirred overnight at room temperature, concentrated to a small volume and distributed between ethyl acetate and water. The organic layer is washed twice with 5 % NaHCO₃, 5 % KHSO₄ and water and dried over MgSO₄. The solvent is evaporated and the residue precipitated from ethyl acetate with suitable solvents like petroleum ether, diisopropylether, tert-butyl methyl ether or pentane.

B) Reduction: the cystine compound (1 mmol) is reacted in 10 ml 95 % trifluorethanol with 1.5 mmol tributylphosphine. The reaction mixture is stirred overnight at room temperature, evaporated to small volume and upon dilution with ethyl acetate the product is precipitated with suitable solvents like petroleum ether, diisopropylether, tert- butyl methyl ether or pentane.

N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-hydroxamate (5a)

Prepared from N,N'-bis-benzyloxycarbonyl-L-cystine (2) and hydroxylamine according to procedure (A). Yield: 22 %; homogeneous on TLC (solvent system: chloroform/methanol; 4 : 1, R_f = 0.5).

FAB-MS: m/z = 539.2 [M+H⁺]; M,= 538.2 calculated for C₂₂H₂₆N₄O₈S₂

Benzyloxycarbonyl-L-cysteine-hydroxamate (5)

Prepared according to procedure (B) from 5a. Purified by flash chromatography (eluent: CH₂Cl₂/MeOH, 95:5 followed by 45:5). Yield: 18 %. 1H-NMR (d⁶-DMSO): 10.7 (bs, IH, NHOH); 8.89 (bs, IH, OH); 7.50 (d, IH, NH ureth.); 7.38 (m, 5H, arom. H's); 5.03 (s, 2H, CH-. v. Z); 3.99 (m, IH, H-C(α)); 2.75/2.66 (2xm, 2H, β-CH₂); 2.29 (bs, IH SH).

Bis-tert-butyloxycarbonyl-L-cystine-bis-benzylamide (6a) Prepared from N,N'-bis-tert-butyloxycarbonyl-L-cystine (12) and benzylamine according to (A). Yield: 77 %. Homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid, 45:45:10, Rf = 0.7).

L-Cystine-bis-benzylamide hydrochloride (6b)

13.87 g (22.4 mmol) 6a in 100 ml 4.6 M HC1 in dioxane was stirred overnight at room temperature: The precipitate was collected and washed extensively with ether. Yield: 11 g (quantitative).

N,N '-Bis-acetyl-L-cystine-bis-benzylamide (6c)

300 mg (0.61 mmol) 6b was distributed between ethyl acetate and 40 ml NaHCO₃ (5 %) and then reacted with acetic anhydride (0.27 g, 2.6 mmol). The organic layer was washed with water, dried over MgSO and evaporated to dryness. Yield: 94 %; homogeneous on TLC (solvent system: cyclohexane/chloroform acetic acid: 45:45:10, Rf = 0.4).

N-Acetyl-L-cysteine-benzylamide (6)

6c was reduced according to procedure (B). Yield: 65 %; homogeneous on TLC (solvent system: CHCl₃/MeOH; 4 : 1, R_f = 0.7); m.p. 186 - 189 °C; 1H-NMR (d⁶-DMSO): 8.56 (t, IH, NH, amide); 8.23 (d, IH, NH ureth.); 7.32-7.09 (m, 5H, arom. H's); 4.56, or 4.38 (m, IH, H-C_α ); 4.28 (m, 2H, CH₂-Bn); 3.12/2.89, oder 2.79/2.69 (2xm, 2H, β-CH₂); 2.30 (bs, IH, SH); 1.87 (d, 3H, CH₃). FAB-MS: m/z = 253.1 [M+H⁺]; M_r= 252.1 calculated for C₁₂H₁₆N₂O₂S

N, N '-Bis-formyl-L-cystine-bis-benzylamide (7a)

Prepared from 6b and formic acid according to procedure (A). Yield: 59 %; homogeneous on TLC (solvent system: cyclohexane/CHCl₃/acetic acid, 45:45: 10; Rf =

0.1).

N-Formyl-L-cysteine-benzylamide (7) Reduction of 7a according to procedure (B). Yield: 62 %; homogeneous on TLC (solvent system: cyclohexane/CHCl₃/acetic acid; 45:45:10; Rf = 0.45); m.p. 180 - 183

°C;

1H-NMR (d⁶-DMSO): 8.58 (t, IH, NH, amide); 8.35 (d, IH, NH ureth.); 8.10 (s, IH, formyl-H); 7.33-7.21 (m, 5H, arom. H's); 4.49, (m, IH, H-C_α ); 4.30 (d, 2H, CH₂-Bn); 2.82/2.72 (2xm, 2H, β-CH₂); 2.26 (bs, IH, SH). FAB-MS: m/z = 239.0 [M+H⁺]; M_r= 238.3 calculated for C_nHι₄N₂O₂S

tert-Butyloxycarbonyl-L-cysteine-benzylamide (8)

Reduction of 6a according to procedure (B). Yield: 38 %; homogeneous on TLC (solvent system: heptane/t-butanol acetic acid 5:1:1, R_f = 0.7); m.p. 97 - 100 °C; 1H-NMR (d⁶-DMSO): 8.38 (m, IH, NH, amide); 7.32-7.21 (m, 5H, arom. H^'s); 6.95 (d, IH, NH-ureth.); 4.29 (d, 2H, CH₂-Bn); 4.08 (m, IH, H-C_α ); 2.81/2.68 (2xm, 2H, β- CH₂); 2.29 (bs, IH, SH); 1.40 (s, 9H, t-Bu).

FAB-MS: m/z = 311.1 [M+H^ M^ 310.1 calculated for C₁₅H₂₂N₂O₃S

N, N '-Bis-benzyloxycarbonyl-L-cystine-bis-benzylamide (9a) From N,N'-bis-benzyloxycarbonyl-cystine and benzylamine according to (A). Yield: 90 %

N-Benzyloxycarbonyl-L-cysteine-benzylamide (9)

Reduction of 9a according to (B). Yield: 41 %; homogeneous on TLC (solvent system: cyclohexane/CHCU/acetic acid, 45:45:10, R_f = 0.4); m.p. 148 - 152 °C;

1H-NMR (d⁶-DMSO): 8.50 (m, IH, NH, amide); 7.49 (d, IH, NH ureth.); 7.37-7.23 (m, 10H, arom.. H's); 5.06 (dd, 2H, CH₂ v. Z); 4.30 (d, 2H, CH₂-Bn); 4.16 (m, IH, H-C_α ); 2.84/2.70 (2xm, 2H, β-CH₂); 2.33 (bs, IH, SH). FAB-MS: m z = 345.0 [M+H⁺]; M,= 344.4 calculated for C₁₈H₂oN₂O₃S

N, N '-Bis-benzyloxycarbonyl-L-cystine-bis-4-pyridylmethylamide (10a) From N,N'-bis-benzyloxycarbonyl-L-cystine and 4-(aminomethyl)pyridine according to procedure (A). Yield. 59 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 4:1, Rf = 0.65).

N-Benzyloxycarbonyl-L-cysteine-4-pyridylmethylamide (10)

From 10a according to procedure (B). Yield: 65 %; homogeneous on TLC (solvent system: CHCb MeOH, 4:1, R_f = 0.8); m.p. 122 - 125 °C;

1H-NMR (d⁶-DMSO): 8.61 (t, IH, NH, amide); 8.48/7.37/7.25 (m, respectively, 9H, arom. H's); 7.56 (d, IH, NH-ureth.); 5.08 (dd, 2H, CH₂, Z); 4.32 (d, 2H, CH₂-Bn); 4.18 (m, IH, H-C_α ); 2.87/2.72 (2xm, 2H, β-CH₂); 2.40 (bs, IH, SH).

FAB-MS: m/z = 346.2 [M+H⁺]; M-= 345.1 calculated for C₁₇H₁₉N₃O₃S

N,N'-Bis-ben∑yloxycarbonyl-L-cystine-bis-3-pyridylmethylamide (11a) From N,N'-bis-benzyloxycarbonyl-cystine and 3-(aminomethyl)pyridine according to procedure (A). Yield: 69 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 4: 1, Rf= 0.2).

N-Benzyloxycarborryl-L-cysteine-3-pyridylmethylamide (11) Reduction of 11a according to procedure (B). Yield: 14 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 4: 1, R_f = 0.8); m.p. 125 - 127 °C; 1H-NMR (d⁶-DMSO): 8.58 (t, IH, NH, amide); 8.50/8.45/7.65/7.36 (m, respectively, 9H arom. H's); 7.52 (d, IH, NH-ureth.); 5.07 (dd, 2H, CH₂ v. Z); 4.42 (d, 2H, CH₂- Bn); 4.15 (m, IH, H-C_a ); 2.82/2.71 (2xm, 2H, β-CH₂); 2.36 (bs, IH, SH). FAB-MS: m/z = 346.1 [M+H⁺]; M,= 345.1 calculated for C₁₇H₁₉N₃O₃S

N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-2-pyridylmethylamide (12a) From N,N^'-bis-benzyloxycarbonyl-cystine and 2-(aminomethyl)pyridine according to (A). Yield: 96 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 4:1, Rf = 0.7).

N-Ben∑yloxycarbonyl-L-cysteine-2-pyridylmetlτylamide (12) Reduction of 12a according to (B). Yield: 33 %; homogeneous on TLC (solvent system: CHCb/MeOH, 4:1, Rf = 0.8); m.p. 129 - 131 °C;

1H-NMR (d⁶-DMSO): 8.59 (t, IH, NH, amide); 8.48/7.72/7.36-7.22 (irrespectively, 9H, arom. H's); 7.52 (d, IH, NH-ureth.); 5.07 (dd, 2H, CH₂ v. Z); 4.49 (d, 2H, CH₂- Bn); 4.20 (m, IH, H-C_« ); 2.85/2.72 (2xm, 2H, β-CH₂); 2.42 (bs, IH, SH). FAB-MS: m/z = 346.1 [M+H⁺]; M,= 345.1 calculated for C,₇H₁₉N₃O₃S

N,N'-Bis-benzoyl-L-cystine-bis-benzylamide (13a) From 6b and benzoic acid according to (A). Yield: 78 %; homogeneous on TLC (solvent system: cyclohexane/CHCWacetic acid; 45:45:10, R_f = 0.65).

N-Benzoyl-L-cysteine-benzylamide (13)

By reduction of 13a according to (B). Yield: 57 %; homogeneous on TLC (solvent system: cyclohexane/CHCVacetic acid; 45:45:10, R = 0.55); m.p. 174 - 176 °C;

1H-NMR (d⁶-DMSO): 8.56 (t, IH, NH, amide); 7.92 (d, IH, NH ureth.); 7.57-7.22 (m,

10H, arom. H's); 4.59, (m, IH, H-C_α ); 4.31 (d, 2H, CH₂-Bn); 2.98/2.89 (2xm, 2H, β-

CH₂); 2.41 (t, 1H SH).

FAB-MS: m/z = 315.1 [M+H⁺]; M,= 314.1 calculated for C₁₇H₁₈N₂O₂S

N,N'-Bis-tosyl-L-cystine-bis-benzylamide (14a)

390 mg (0.794 mmol) 6b were reacted with 180 mg (0.952 mmol) tosyl chloride in 6 ml pyridine. After 12 h stirring at room temperature the solid was filtered off and the filtrate evaporated to dryness adding toluene and finally tert-butyl methyl ether. Yield: 81 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 4: 1, Rf = 0.7).

N-Tosyl-L-cysteine-benzylamide (14)

By reduction of 14a according to (B). Yield: 54 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 4: 1, R_f = 0.6); m.p. 180 - 182 °C; 1H-NMR (d⁶-DMSO): 8.41 (t, IH, NH, amide); 7.98/7.68/7.35-7.14 (m, respectively, and d's, 10H, arom. H's, NH ureth.); 4.13 (d, 2H, CH₂-Bn); 3.86 (m, IH, H-C_α); 2.59 (m, 2H, β-CH₂); 2.38 (s, 3H, CH₃); 2.17 (t, IH, SH). FAB-MS: m/z = 365.1 [M+H⁺]; H= 364.1 calculated for Cι₇H₂₀N₂O₃S

N, N '-Bis-benzyloxycarbonyl-L-cystine-bis-2-phenylethylamide (15a)

From N,N'-bis-benzyloxycarbonyl-L-cystine and 2-phenylethylamine according to (A).

Yield: 34 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 19: 1, Rf = 0.8).

N-Ben∑yloxycarbonyl-L-cysteιne-2-phenylethylamide (15)

Reduction of 15a according to (B). Yield: 61 %; homogeneous on TLC (solvent system: cyclohexane/CHCl₃/acetic acid, 45:45:10, R_f= 0.6); m.p. 119 - 121 °C; 1H-NMR (d⁶-DMSO): 8.02 (t, IH, NH, amide); 7.39-7.18 (m, 11H, arom. H^'s, NH ureth.); 5.03 (dd, 2H, CH₂ v. Z); 4.06 (m, IH, H-C_α ); 2.72/2.61 (2xm, 4H, CH₂-CH₂); 2.22 (bs, IH, SH).

FAB-MS: m/z = 359.1 [M+H⁺]; M,= 358.1 calculated for C₁₉H₂₂N₂O₃S

N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-2-(4-hydroxyphenyl)ethylamιde (16a) From N,N^'-bis-benzyloxycarbonyl-cystine and 2-(4-hydroxyphenyl)ethylamine according to (A). Yield: 71 %; homogeneous on TLC (solvent system: cyclohexane/CHCl₃/acetic acid, 45:45: 10, R_f = 0.5).

N-Ben∑yloxycarbonyl-L-cysteme-2-(4-hydroxyphenyl)ethylamιd (16) Reduction of 16a according to (B). Yield: 24 %; homogeneous on TLC (solvent system: cyclohexane CHC acetic acid, 45:45:10, Rf = 0.6); m.p. 133 - 135 °C;

1H-NMR (d⁶-DMSO): 9.11 (s, IH, phenol. OH); 7.98 (t, IH, NH, amide); 7.38 (m, 6H, arom. v. Z, NH ureth.); 6.99/6.68 (2xd, 4H arom., phenol. H's); 5.04 (dd, 2H, CH₂ v.

Z); 4 05 (m, IH, H-C_α ); 2.73/2.60 (2xm, 4H, CH₂-CH₂); 2.26 (bs, IH, SH). FAB-MS: m/z = 375.2 [M+H⁺]; M_r= 374.1 calculated for C₁₉H₂₂N₂O₄S

N,N^'-Bis-benzyloxycarbonyl-L-cysttne-bιs-4-chlorobenzylamιde (17a) From N,N'-bis-benzyloxycarbonyl-cystine and 4-chlorobenzylamine according to (A). Yield: 99 %; homogeneous on TLC (solvent system: CHCl₃/MeOH, 19: 1, Rf = 0.8)

N-Benzyloxycarbonyl-L-cysteine-4-chlorobenzylamide (17) Reduction of 17a according to (B). Yield: 71 %; homogeneous on TLC (solvent system: cyclohexane/CHCl₃/acetic acid, 45:45:10, R_f = 0.85); m.p. 137 - 139 °C;

1H-NMR (d⁶-DMSO): 8.53 (t, IH NH, amide); 7.51 (d, IH, NH ureth.); 7.38-7.26 (m,

9H, arom.. H's); 5.06 (dd, 2H, CH₂ v. Z); 4.29 (d, 2H, CH₂-Bn); 4.13 (m, IH, H-C_α );

2.82/2.70 (2xm, 2H, β-CH₂); 2.53 (bs, IH, SH). FAB-MS: m/z = 379.1 [M+H⁺]; M,= 378.1 calculated for Cι₈H₁₉ClN₂O₃S

N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-3-phenylpropylamide (18a) From N,N'-bis-benzyloxycarbonyl-cystine and 3-phenylpropylamine according to (A). Yield: 94 %; homogeneous on TLC (solvent system: cyclohexane/CHCVacetic acid,

N-Benzyloxycarbonyl-L-cysteine-3-phenylpropylamide (18)

By reduction of 18a according to (B). Yield: 76 %; homogeneous on TLC (solvent system: cyclohexane/CHCVacetic acid, 45:45: 10, Rf = 0.7); m.p. 104 - 106 °C; 1H-NMR (d⁶-DMSO): 8.02 (t, IH NH, amide); 7.43 (d, IH, NH ureth.); 7.38-7.15 (m,

10H, arom.. H^'s); 5.05 (dd, 2H, CH₂ v. Z); 4.09 (m, IH, H-C_α ); 3.12 (m, 2H N-CH₂);

2.70/2.69 (2xm, 2H, β-CH₂); 2.58 (t, 2H, CH₂-Ph); 2.32 (bs, IH, SH); 1.70 (m, 2H,

CH₂.-CH₂-CH₂).

FAB-MS: m/z = 373.2 [M+H⁺]; M,= 372.2 calculated for C₂₀H₂₄N₂O₃S

N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-tryptamide (19a)

From N,N'-bis-benzyloxycarbonyl-cystine and tryptamine according to (A). Yield: 75 %; homogeneous on TLC (solvent system: cyclohexane/CHC /acetic acid, 45:45:10, Rf = 0.6).

Benzyloxycarbonyl-L-cysteine-tryptamide (19) Reduction of 19a according to (B): Yield: 62 %; homogeneous on TLC (solvent system: cyclohexane/CHCb/acetic acid, 45:45:10, R = 0.7); m.p. 150 - 152 °C; 1H-NMR (d⁶-DMSO): 10.80 (s, IH, NH-tryptamine); 8.09 (t, IH, NH amide); 7.54 - 6.96 (m, 11H, arom. H's, ureth. NH); 5.05 (dd, 2H, CH₂ v. Z); 4.08 (m, IH, H-C(α)); 3.32 (m, 2H, NHCH₂CH₂); 2.82 (t, 2H NHCH₂CH₂); 2.77/2.65 (2xm, 2Η, β-CH₂); 2.26 (m, IH, SH). FAB-MS: m/z = 398.2 [M+H⁺]; M,= 397.2 calculated for C₂₁H₂₃N₃O₃S

N,N^'-Bis-hexanoyl-L-cystine-bis-benzylamide (20a) Prepared from 6b and hexanoic acid according to procedure (A). Yield: 86 %; homogeneous on TLC (solvent system: cyclohexane/chloroform acetic acid, 45:45:10; Rf

= 0.6).

1H-NMR (d⁶-DMSO): 8.48 (t, IH NH, amide); 8.02 (d, IH, NH-ureth.); 7.3-7.2 (m,

5H, arom.. H's); 4.41 (m, IH, H-C_α ); 4.28 (d, 2H, CH₂-Bn); 2.80/2.70 (2xm, 2H, β- CH₂); 2.25 (t, IH, SH); 2.17 (m, 2H, -CH CO-); 1.49-1.21 (m, 10Η, alkyl), 0.87 (t, 3Η,

-CH₃).

N-Hexanoyl-L-cysteine-benzylamide (20)

Reduction of 20a according to procedure (B). Yield: 69 %; m.p. 141 - 143 °C; 1H-NMR (d⁶-DMSO): 1H-NMR (d⁶-DMSO): 8.48 (t, IH, NH, amide); 8.02 (d, IH NH- ureth.); 7.31-7.2 (m, 5H, arom.. H's); 4.40 (m, IH H-C_α ); 4.22 (d, 2H CH₂-Bn); 2.80/2.70 (2xm, 2H, β-CH₂); 2.3 (bs, IH SH); 2.12 (m, 2H -CH,-CO-); 1.50-1.19 (m, 6H, alkyl), 0.85 (t, 3H, -CH₃). FAB-MS: m/z = 309.2 [M+H⁺]; M,= 308.2 calculated for C₁₆H₂₄N₂O₂S

N,N^'-Bis-octanoyl-L-cystine-bis-benzylamide (21a)

Prepared from 6b and octanoic acid according to procedure (A). Yield: 86 %; homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid, 45:45: 10; Rf = 0.6).

N-Octanoyl-L-cysteine-benzylamide (21)

Reduction of 21a according to procedure (B). Yield: 73 %; m.p. 137 - 139 °C; 1H-NMR (d⁶-DMSO): 1H-NMR (d⁶-DMSO): 8.48 (t, IH NH amide); 8.02 (d, IH NH- ureth.); 7.3-7.2 (m, 5H, arom.. H's); 4.41 (m, IH H-C_α ); 4.28 (d, 2H CH₂-Bn); 2.80/2.70 (2xm, 2H, β-CH₂); 2.25 (t, IH, SH); 2.17 (m, 2H -CH CO-); 1.49-1.21 (m, 10Η, alkyl), 0.87 (t, 3Η -CH₃). FAB-MS: m z = 337.2 [M+H⁺]; M-= 336.2 calculated for C₁₈H₂₈N₂O₂S

N,N'-Bis-decanoyl-L-cystine-bis-benzylamide (22a)

Prepared from 6b and decanoic acid according to procedure (A). Yield: quantitative; homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid, 45:45:10; Rf = 0.9).

N-Decanoic-L-cysteine-benzylamide (22)

Reduction of 22a according to procedure (B). Yield: 33 %; Rf = 0.7); m.p. 138 - 140 °C; 1H-NMR (d⁶-DMSO): 8.46 (t, IH, NH, amide); 8.02 (d, IH, NH-ureth.); 7.3-7.2 (m, 5H, arom.. H's); 4.4 (m, IH, H-C_α ); 4.29 (d, 2H, CH₂-Bn); 2.80/2.70 (2xm, 2H, β- CH₂); 2.25 (t, IH, SH); 2.18 (m, 2H, -CH₂-CO-); 1.49-1.19 (m, 14Η, alkyl), 0-85 (t, 3H, -CH₃). FAB-MS: m/z = 365.2 [M+H⁺]; M_r= 364.2 calculated for C₂₀H₃₂N₂O₂S

1. Beckett, R P.; Davidson, A. H; Drummond, A. H.; Huxley, P.; Whittaker, M. Drug Disc. Today 1996

2. Wϋnsch, E. in Houben-Weyl, Methoden der Organischen Chemie, Vol. 15/1, Springer Verlag, Stuttgart, 1974.

3. Stack, M. S.; Gray, R. D. J. Biol. Chem. 1989, 264, 4277. 4. Nagase, H; Fields, C. G.; Fields, G. B. J. Biol. Chem. 1994, 269, 20952.

5. Copeland, R A; Lombardo, D.; Giannaras, J.; Decicco, C. P. Bioorg. Med Chem. Lett. 1995, 5, 1947.

Claims

Patent Claims

1. A compound represented by the general formula I,

which binds and inhibits matrix metalloproteinases (MMPs), wherein the cysteine moiety contains an unprotected thiol group, the cysteine moiety is in the L- or D-configuration wherein

A denotes -CO-, -SO₂-, -NH-CO-, or -O-CO-

Ri denotes hydrogen, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a C1-C₁₅ alkyl group substituted by halogen, mercapto, hydroxy, alkoxy, amino or nitro, or by carbocyclic non aromatic or aromatic ring systems which are optionally substituted once or several times or aromatic or non aromatic heterocycles, optionally substituted, their pharmacologically acceptable salts, or optically active forms thereof.

R denotes hydroxy, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a Ci-Cu alkyl group substituted by carbocyclic non aromatic or aromatic ring systems which are optionally substituted once or several times or aromatic or non aromatic heterocycles, optionally substituted, their pharmacologically acceptable salts, or optically active forms thereof.

2. A pharmaceutical composition containing a compound as claimed in claim 1, or the pharmacologically acceptable salts, or optically active forms therof and pharmaceutically acceptable carriers.

3. A therapeutical composition according to claim 2, for the treatment of rheumatoid arthritis and related diseases in which collagenolytic activity is a contributing factor.

4. Use according to claim 3, wherein the dose of the therapeutic agent is 0.1 to 300 mg/kg body weight.

5. Use according to claim 3 or 4, wherein the therapeutic agent is administered orally, intravascularly, intraperitoneally, subcutaneously, intramuscularly or topically.