AU7652998A - New cysteine derivatives, processes for their production, and pharmaceuticals containing them - Google Patents

Description

WO 98/50351 PCT/EP98/02654 5 NEW CYSTEINE DERIVATIVES, PROCESSES FOR THEIR PRODUCTION, AND PHARMACEUTICALS CONTAINING THEM The invention comprises new matrix metalloproteinase inhibitors that are based on the 10 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 15 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 20 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 25 are zinc dependent and calcium requiring enzymes which are inhibited by one of the members of the tissue inhibitor of metalloproteinase (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. 30 WO 98/50351 PCT/EP98/02654 2 an N-terminal propeptide of about 80 residues. The propeptide forms a separate smaller domain that contains three a-helices and an extended peptide that occupies the active site. The catalytic domain in all these structures contains two Zn 2+ ions, i.e. a "structural" zinc ion and a "catalytic" zinc ion. The "catalytic" zink ion is coordinated by the side 5 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(1). 10 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 15 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, H O RI A N 1 R I H SH() 20 wherein 25 A denotes -CO-, -SO 2 -, -NH-CO-, or -O-CO R, denotes hydrogen, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a CI-CIs alkyl group substituted by halogen, mercapto, hydroxy, alkoxy, amino or nitro, or by carbocyclic aromatic or non aromatic ring systems which WO 98/50351 PCT/EP98/02654 3 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 5 carbon atoms or a C 1 -C15 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 R, represent a branched saturated or unsaturated 10 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 C 3

-C

6 cycloalkyls such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, or 15 C 6

-C

1 4 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 20 optionally be substituted once or several times for example by halogen-, nitro-, hydroxy-,

C

1

-C

6 alkyl-, C 1

-C

6 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: 25 Formic Acid, Acetic acid, Propionic acid, Hexanoic acid, Lauric 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, 30 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 WO 98/50351 PCT/EP98/02654 4 acid, p-Phenylbenzoic acid, Phenanthroic acid, Nicotinic acid, 3-Aminopyrazin-2 carbonic acid, Pyridine carboxylic acid, Piperazine carboxylic acid, Piperidine carboxylic acid. 5 If A denotes -SO 2 , the residue RISO 2 - 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 10 acid, 2-Chloroethane sulfonic acid, N,N'-Dimethylsulfamic acid, Piperidine sulfonic acid, 5- (2- Aminoethylamino)- 1- naphthalene- sulfonic acid, lodoxyquinolinesulfonic 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 15 [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. 20 If A denotes -NHCO-, the residue R 1 -NH-CO- is preferably selected from the residues of the following urea derivatives: n- Butyl-, R- (+)- alpha- Phenylethyl-, R-(-)-1-(1-Naphthyl)-ethyl-, Ethyl-, Propyl-, Hexyl-, Octyl-, Benzyl-, Chlorobenzyl-, Methylbenzyl-, 3-Picolyl-, 2-(Aminomethyl) pyridyl urea. 25 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 carbamnate, 9-(2,7-Dibromo)fluorenylmethyl carbamate, 4 30 Methoxyphenacyl carbamnate, 2,2,2-Trichloroethyl carbamate, 2-Phenylethyl carbamate, 1-(1-Adamantyl)- 1-methylethyl carbamate, 1,1-Dimethyl-2-haloethyl carbamate, 1 Methyl- 1-(4-biphenyl)- I -methylethyl carbamate, 2-(2'-Pyridyl)ethyl carbamate, 1- WO 98/50351 PCT/EP98/02654 5 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 5 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 10 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: RI-CO R Formyl- Benzyl Phenyl- Naphthyl Naphthylacetyl- 3-Picolyl 4-Biphenyl- 2-Phenylethyl

R

1

-SO

2 R Pyridine-3-sulfonyl- Phenylpropyl p-Toluenesulfonyl- p-Chlorobenzyl

R

1 -NH-CO R Benzyl- Pyridyl

R

I

-O-CO R 2-Phenylethyl- 4-Phenyoxyphenyl Benzyl- 2-Phenylethyl 2-(p-Toluenesulfonyl)ethyl- Benzyl 15 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 2+ ion, 20 the hydrophobic groups R, or R to interact with the hydrophobic S protein pocket as WO 98/50351 PCT/EP98/02654 6 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 5 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 10 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 15 enzymatic metabolism. Similarly, the C-terminal carboxyl function was derivatized as amide instead of esterification to avoid fast clearance rates due to hydrolysis of the esters by lipases. Moreover, N-alkyl and N-aryl amides were selected which are known to be more stable towards peptidases. 20 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 25 or preferentially with phosphines like tributylphosphine, as shown in scheme 1.

WO 98/50351 PCT/EP98/02654 7 0 0 RA NH OH RIA NH NHR H EDC1*HCI1 S \ HOBt S BpR ANH ,R I + 2 N-R ---- + 1 2 A NH S S Triflourethanol 95% HS Aq OH A. NHR R I NH R NH 0 0 H II I 5 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 10 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 15 rheumatoid arthritis (primary chronic polyarthritis, PCP), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis, Sjogren'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 + 20 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 25 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.

WO 98/50351 PCT/EP98/02654 8 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 5 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 10 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 15 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, 20 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 25 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 30 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 WO 98/50351 PCT/EP98/02654 9 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. 5 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, 10 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 15 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. 20 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. 25 Enzyme assay The catalytic domain of MMP8 (Phe 79 -MMP8) and MMP3 were used for the inhibition experiments. Enzyme assays were performed at 250 C in 10 mM CaC1 2 , 100 mM NaCl, 50 mM Tris/HCI (pH 7.6) using 8 nM enzyme concentrations and the fluorogenic 30 substrates Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH 2 (Bachem M-1855, 1.10

-

5 M) for MMP8 and Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH 2 (Bachem M 2110, 4.10 - 6 M) for MMP3: Measurements were performed essentially as described by WO 98/50351 PCT/EP98/02654 10 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). 5 Table I. Examples for inhibition of MMP8 and MMP3 with non-peptidic L-cysteine derivatives of the general formula I 10 Compound Ri-A R K,; MMP8 K,; MMP3 [IM] [MIl] o 10 ao .

0.71 4.9 0 15 o 0.16 12.3 15 Synthesis General methods: A) Amidation: I mmole N,N'-urethanyl-cystine, 2 mmol HOBT (hydroxybenzotriazole) 20 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- WO 98/50351 PCT/EP98/02654 11 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 % NaHCO 3 , 5 % KHSO 4 and water and dried over MgSO 4 . The solvent is evaporated and the residue precipitated from ethyl 5 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 10 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. 15 N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-hydroxamate (Sa) 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, Rf = 0.5). FAB-MS: m/z = 539.2 [M+H+]; Mr= 538.2 calculated for C 22

H

26

N

4 0 8

S

2 20 Benzyloxycarbonyl-L-cysteine-hydroxamate (5) Prepared according to procedure (B) from 5a. Purified by flash chromatography (eluent:

CH

2 C1 2 /MeOH, 95:5 followed by 45:5). Yield: 18 %. 25 H-NMR (d'-DMSO): 10.7 (bs, 1H, NHOH); 8.89 (bs, 1H, OH); 7.50 (d, 1H, NH ureth.); 7.38 (m, 5H, arom. H's); 5.03 (s, 2H, CH 2 v. Z); 3.99 (m, 1H, H-C(a)); 2.75/2.66 (2xm, 2H, 13-CH 2 ); 2.29 (bs, 1H, SH). 30 Bis-tert-butyloxycarbonyl-L-cystine-bis-benzylamide (6a) WO 98/50351 PCT/EP98/02654 12 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). 5 L-Cystine-bis-benzylamide hydrochloride (6b) 13.87 g (22.4 mmol) 6a in 100 ml 4.6 M HCI in dioxane was stirred overnight at room temperature: The precipitate was collected and washed extensively with ether. Yield: 11 g (quantitative). 10 NN'-Bis-acetyl-L-cystine-bis-benzylamide (6c) 300 mg (0.61 mmol) 6b was distributed between ethyl acetate and 40 ml NaHCO 3 (5 %) and then reacted with acetic anhydride (0.27 g, 2.6 mmol). The organic layer was washed with water, dried over MgSO 4 and evaporated to dryness. Yield: 94 %; homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid: 45:45:10, Rf 15 = 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, Rf= 0.7); m.p. 186 - 189 oC; 20 1H-NMR (d 6 -DMSO): 8.56 (t, 1H, NH, amide); .8.23 (d, 1H, NH ureth.); 7.32-7.09 (m, 5H, arom. H's); 4.56, or 4.38 (m, 1H, H-Ca ); 4.28 (mn, 2H, CH 2 -Bn); 3.12/2.89, oder 2.79/2.69 (2xm, 2H, -CH 2 ); 2.30 (bs, 1H, SH); 1.87 (d, 3H, CH 3 ). FAB-MS: m/z = 253.1 [M+H+]; Mr = 252.1 calculated for C 12

H

1 6

N

2 0 2 S 25 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). 30 N-Formyl-L-cysteine-benzylamide (7) WO 98/50351 PCTIEP98/02654 13 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 oC; IH-NMR (d 6 -DMSO): 8.58 (t, 1H11, NH, amide); 8.35 (d, 1H1, NH ureth.); 8.10 (s, 1H, 5 formyl-H); 7.33-7.21 (min, 5H, arom. H's); 4.49, (min, 1H, H-Ca ); 4.30 (d, 2H, CH 2 -Bn); 2.82/2.72 (2xm, 2H, 3-CH 2 ); 2.26 (bs, 1H, SH). FAB-MS: m/z = 239.0 [M+H+]; M,= 238.3 calculated for CIIHI 4

N

2 0 2 S 10 tert-Butyloxycarbonyl-L-cysteine-benzylamide (8) Reduction of 6a according to procedure (B). Yield: 38 %; homogeneous on TLC (solvent system: heptane/t-butanollacetic acid 5:1:1, Re= 0.7); m.p. 97 - 100 'C; IH-NMR (d 6 -DMSO): 8.38 (min, 1H, NH, amide); 7.32-7.21 (m, 51H, arom. H's); 6.95 (d, 1H, NH-ureth.); 4.29 (d, 2H, CH 2 -Bn); 4.08 (min, 1H, H-Ca ); 2.81/2.68 (2xm, 2H11, 13 15 CH 2 ); 2.29 (bs, 1H, SH); 1.40 (s, 9H, t-Bu). FAB-MS: m/z = 311.1 [M+H ]; M = 310.1 calculated for C 15 isH 22

N

2 0 3 S N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-benzylamide (9a) 20 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: 25 cyclohexane/CHC1l3/acetic acid, 45:45:10, Rf = 0.4); m.p. 148 - 152 'C; IH-NMR (d 6 -DMSO): 8.50 (m, 1H, NIH, amide); 7.49 (d, 1H, NH ureth.); 7.37-7.23 (inm, 10H, arom.. H's); 5.06 (dd, 2H, CH 2 v. Z); 4.30 (d, 2H, CH 2 -Bn); 4.16 (min, 1H, H-Ca ) 2.84/2.70 (2xm, 2H1, 1-CH 2 ); 2.33 (bs, 1H, SH). FAB-MS: m/z = 345.0 [M+H+]; Mr= 344.4 calculated for Cs 18

H

20

N

2 0 3 S 30 N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-4-pyridylmethylamide (10a) WO 98/50351 PCT/EP98/02654 14 From N,N'-bis-benzyloxycarbonyl-L-cystine and 4-(aminomethyl)pyridine according to procedure (A). Yield. 59 %; homogeneous on TLC (solvent system: CHCl 3 /MeOH, 4:1, R = 0.65). 5 N-Benzyloxycarbonyl-L-cysteine-4-pyridylmethylamide (10) From 10a according to procedure (B). Yield: 65 %; homogeneous on TLC (solvent system: CHCl3/MeOH, 4:1, Rf= 0.8); m.p. 122 - 125 oC; 1 H-NMR (d 6 -DMSO): 8.61 (t, 1H, NH, amide); 8.48/7.37/7.25 (m, respectively, 9H, arom. H's); 7.56 (d, 1H, NH-ureth.); 5.08 (dd, 2H, CH 2 , Z); 4.32 (d, 2H, CH 2 -Bn); 4.18 10 (mn, 1H, H-Ca); 2.87/2.72 (2xm, 2H, 13-CH 2 ); 2.40 (bs, 11, SH). FAB-MS: mn/z = 346.2 [M+H ]; Mr= 345.1 calculated for CI 7

H!

9

N

3 0 3 S N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-3-pyridylmethylamide (11a) 15 From N,N'-bis-benzyloxycarbonyl-cystine and 3-(aminomethyl)pyridine according to procedure (A). Yield: 69 %; homogeneous on TLC (solvent system: CHC1 3 /MeOH, 4:1, Re= 0.2). N-Benzyloxycarbonyl-L-cysteine-3-pyridylmethylamide (11) 20 Reduction of 1la according to procedure (B). Yield: 14 %; homogeneous on TLC (solvent system: CHCI 3 /MeOH, 4:1, Rf= 0.8); m.p. 125 - 127 oC; IH-NMR (d 6 -DMSO): 8.58 (t, 1H, NH, amide); 8.50/8.45/7.65/7.36 (mn, respectively, 91, arom. H's); 7.52 (d, 1H, NH-ureth.); 5.07 (dd, 21, CH 2 v. Z); 4.42 (d, 2H, CH 2 Bn); 4.15 (min, 11, H-Ca); 2.82/2.71 (2xm, 2H, 13-CH 2 ); 2.36 (bs, 1H, SH). 25 FAB-MS: m/z = 346.1 [M+H+]; M= 345.1 calculated for C 17 HjgN 3 0 3 S N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-2-pyridylmethylamide (12a) From N,N'-bis-benzyloxycarbonyl-cystine and 2-(aminomethyl)pyridine according to 30 (A). Yield: 96 %; homogeneous on TLC (solvent system: CHC1 3 /MeOH, 4:1, Rf = 0.7). N-Benzyloxycarbonyl-L-c.ysteine-2-pyridylmethylamide (12) WO 98/50351 PCT/EP98/02654 15 Reduction of 12a according to (B). Yield: 33 %; homogeneous on TLC (solvent system: CHCl3/MeOH, 4:1, Rf= 0.8); m.p. 129 - 131 'C; IH-NMR (d 6 -DMSO): 8.59 (t, 1H, NH, amide); 8.48/7.72/7.36-7.22 (m,respectively, 9H, arom. H's); 7.52 (d, 1H, NH-ureth.); 5.07 (dd, 21H, CH 2 v. Z); 4.49 (d, 2H, CH2 5 Bn); 4.20 (m, 1H, H-Ca); 2.85/2.72 (2xm, 2H, O-CH 2 ); 2.42 (bs, 1H, SH). FAB-MS: m/z = 346.1 [M+H+]; M= 345.1 calculated for C 17

HI

9

N

3 0 3 S N,N'-Bis-benzoyl-L-cystine-bis-benzylamide (13a) 10 From 6b and benzoic acid according to (A). Yield: 78 %; homogeneous on TLC (solvent system: cyclohexane/CHCl 3 /acetic acid; 45:45:10, Rf = 0.65). N-Benzoyl-L-cysteine-benzylamide (13) By reduction of 13a according to (B). Yield: 57 %; homogeneous on TLC (solvent 15 system: cyclohexane/CHCl 3 /acetic acid; 45:45:10, Rf= 0.55); m.p. 174 - 176 oC; IH-NMR (d 6 -DMSO): 8.56 (t, 1H, NH, amide); 7.92 (d, 1H, NH ureth.); 7.57-7.22 (m, 10H, arom. H's); 4.59, (m, 1H, H-Ca ); 4.31 (d, 21H, CH 2 -Bn); 2.98/2.89 (2xm, 2H, 03

CH

2 ); 2.41 (t, 1H, SH). FAB-MS: m/z = 315.1 [M+H+]; M= 314.1 calculated for C 17 Hi 8

N

2 0 2 S 20 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 25 evaporated to dryness adding toluene and finally tert-butyl methyl ether. Yield: 81 %; homogeneous on TLC (solvent system: CHCI 3 /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 30 system: CHCI 3 /MeOH, 4:1, Rf= 0.6); m.p. 180 - 182 'C; WO 98/50351 PCT/EP98/02654 16 1 H-NMR (d 6 -DMSO): 8.41 (t, 1H, 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, CH 2 -Bn); 3.86 (m, 1H, H-Ca); 2.59 (mn, 21, 13-CH 2 ); 2.38 (s, 3H, CH 3 ); 2.17 (t, 1H, SH). FAB-MS: m/z = 365.1 [M+H']; M= 364.1 calculated for C 1 7

H

20

N

2 0 3 S 5 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: CHCl 3 /MeOH, 19:1, Rf = 0.8). 10 N-Benzyloxycarbonyl-L-cysteine-2-phenylethylamide (15) Reduction of 15a according to (B). Yield: 61 %; homogeneous on TLC (solvent system: cyclohexane/CHCl3/acetic acid, 45:45:10, Rf= 0.6); m.p. 119 - 121 'C; 1 H-NMR (d 6 -DMSO): 8.02 (t, 1H, NH, amnide); 7.39-7.18 (m, 11H, arom. H's, NH ureth.); 5.03 (dd, 2H, CH 2 v. Z); 4.06 (m, 1H, H-Ca, ); 2.72/2.61 (2xm, 41-, CH 2 -CH2); 15 2.22 (bs, 1H, SH). FAB-MS: m/z = 359.1 [M+H+]; Mr= 358.1 calculated for Cl 9

H

22

N

2 0 3 S N,N'-Bis-benzyloxycarbonyl-L-cystine-bis-2-(4-hydroxyphenyl)ethylamide (16a) 20 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, Rf= 0.5). N-Benzyloxycarbonyl-L-cysteine-2-(4-hydroxyphenyl)ethylamid (16) 25 Reduction of 16a according to (B). Yield: 24 %; homogeneous on TLC (solvent system: cyclohexane/CHCl3/acetic acid, 45:45:10, Rf= 0.6); m.p. 133 - 135 'C; 1H-NMR (d 6 -DMSO): 9.11 (s, 1H, phenol. OH); 7.98 (t, 1I, NH, amide); 7.38 (m, 61, arom. v. Z, NH ureth.); 6.99/6.68 (2xd, 41, arom., phenol. H's); 5.04 (dd, 2H, CH 2 v. Z); 4.05 (m, 1H, H-Ca); 2.73/2.60 (2xm, 4H, CH 2

-CH

2 ); 2.26 (bs, 1H, SH). 30 FAB-MS: m/z = 375.2 [M+H+]; Mr= 374.1 calculated for C 1 9

H

22

N

2 0 4 S N, N'-Bis-benzyloxycarbonyl-L-cystine-bis-4-chlorobenzylamide (17a) WO 98/50351 PCT/EP98/02654 17 From N,N'-bis-benzyloxycarbonyl-cystine and 4-chlorobenzylamine according to (A). Yield: 99 %; homogeneous on TLC (solvent system: CHCl 3 /MeOH, 19:1, Rf = 0.8) N-Benzyloxycarbonyl-L-cysteine-4-chlorobenzylamide (17) 5 Reduction of 17a according to (B). Yield: 71 %; homogeneous on TLC (solvent system: cyclohexane/CHC1 3 /acetic acid, 45:45:10, Rr = 0.85); m.p. 137 - 139 'C; IH-NMR (d 6 -DMSO): 8.53 (t, 1H, NH, amide); 7.51 (d, 1H, NH ureth.); 7.38-7.26 (m, 9H, arom.. H's); 5.06 (dd, 2H, CH 2 v. Z); 4.29 (d, 2H, CH 2 -Bn); 4.13 (m, 1H, H-Ca ); 2.82/2.70 (2xm, 21, 13-CH 2 ); 2.53 (bs, 1H, SH). 10 FAB-MS: m/z = 379.1 [M+H+]; M= 378.1 calculated for Cs 18

H

19

CIN

2 0 3 S 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/CHCl 3 /acetic acid, 15 45:45:10, Rf= 0.7). N-Benzyloxycarbonyl-L-cysteine-3-phenylpropylamide (18) By reduction of 18a according to (B). Yield: 76 %; homogeneous on TLC (solvent system: cyclohexane/CHC13/acetic acid, 45:45:10, Re= 0.7); m.p. 104 - 106 oC; 20 H-NMR (d'-DMSO): 8.02 (t, 11H, NH, amide); 7.43 (d, 1H, NH ureth.); 7.38-7.15 (mn, 10H, arom.. H's); 5.05 (dd, 211, CH 2 v. Z); 4.09 (m, 1H, H-Ca, ); 3.12 (m, 21, N-CH 2 ); 2.70/2.69 (2xm, 2H, 13-CH 2 ); 2.58 (t, 2H, CH2-Ph); 2.32 (bs, 1H, SH); 1.70 (m, 21H,

CH

2

.-CH

2

-CH

2 ). FAB-MS: mn/z = 373.2 [M+H+]; M= 372.2 calculated for C2 0

H

24

N

2 0 3 S 25 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/CHCl 3 /acetic acid, 45:45:10, Rf = 30 0.6). Benzyloxycarbonyl-L-cysteine-tryptamide (19) WO 98/50351 PCT/EP98/02654 18 Reduction of 19a according to (B): Yield: 62 %; homogeneous on TLC (solvent system: cyclohexane/CHCl3/acetic acid, 45:45:10, Rf = 0.7); m.p. 150 - 152 'C; IH-NMR (d 6 -DMSO): 10.80 (s, 1H, NH-tryptamine); 8.09 (t, 1H, NH amide); 7.54 6.96 (mi, 11H, arom. H's, ureth. NH); 5.05 (dd, 2H, CH 2 v. Z); 4.08 (m, 1H, H-C(.)); 5 3.32 (mi, 21H, NHCH 2

CH

2 ); 2.82 (t, 2H, NHCH 2

CH

2 ); 2.77/2.65 (2xm, 2H, 3-CH 2 ); 2.26 (m, 1H, SH). FAB-MS: m/z = 398.2 [M+H+]; Mr= 397.2 calculated for C 21

H

23

N

3 0 3 S N,N'-Bis-hexanoyl-L-cystine-bis-benzylamide (20a) 10 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 (d 6 -DMSO): 8.48 (t, 1H, NH, amide); 8.02 (d, 1H, NH-ureth.); 7.3-7.2 (m, 5H, arom.. H's); 4.41 (m, 1H, H-Ca ); 4.28 (d, 2H, CH 2 -Bn); 2.80/2.70 (2xm, 2H1, 03 15 CH 2 ); 2.25 (t, 1H1, SH); 2.17 (m, 2H, -CHr2-CO-); 1.49-1.21 (m, 10H, alkyl), 0.87 (t, 31H, -CH3). N-Hexanoyl-L-cysteine-benzylamide (20) Reduction of 20a according to procedure (B). Yield: 69 %; m.p. 141 - 143 oC; 20 1H-NMR (d 6-DMSO): 'H-NMR (d 6 -DMSO): 8.48 (t, 1H, NH, amide); 8.02 (d, 1H, NH ureth.); 7.31-7.2 (m, 5H, arom.. H's); 4.40 (m, 11H, H-Ca ); 4.22 (d, 21H, CH 2 -Bn); 2.80/2.70 (2xm, 2H, 3-CH 2 ); 2.3 (bs, 1H1, SH); 2.12 (m, 21H, -CH 2 -CO-); 1.50-1.19 (m, 6H, alkyl), 0.85 (t, 31H, -CH 3 ). FAB-MS: m/z = 309.2 [M+H-]; Mr= 308.2 calculated for C 1 6H 24

N

2 0 2 S 25 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). 30 N-Octanoyl-L-cysteine-benzylamide (21) Reduction of 21a according to procedure (B). Yield: 73 %; m.p. 137 - 139 'C; WO 98/50351 PCT/EP98/02654 19 1H-NMR (d 6 -DMSO): 'H-NMR (d 6-DMSO): 8.48 (t, 1H, NH, amide); 8.02 (d, 1H, NH ureth.); 7.3-7.2 (m, 5H, arom.. H's); 4.41 (m, 1H, H-Ca ); 4.28 (d, 2H, CH 2 -Bn); 2.80/2.70 (2xm, 2H, 3-CH 2 ); 2.25 (t, 1H, SH); 2.17 (m, 2H, -CH 2 -CO-); 1.49-1.21 (m, 10H, alkyl), 0.87 (t, 3H, -CH 3 ). 5 FAB-MS: m/z = 337.2 [M+H+]; Mr= 336.2 calculated for Cs 18

H

28

N

2 0 2 S 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 10 = 0.9). N-Decanoic-L-cysteine-benzylamide (22) Reduction of 22a according to procedure (B). Yield: 33 %; Rf= 0.7); m.p. 138 - 140 oC; 1 H-NMR (d 6 -DMSO): 8.46 (t, 1H, NH, amide); 8.02 (d, 1H, NH-ureth.); 7.3-7.2 (m, 15 5H, arom.. H's); 4.4 (m, 1H, H-Ca ); 4.29 (d, 21H, CH 2 -Bn); 2.80/2.70 (2xm, 2H1, 03

CH

2 ); 2.25 (t, 1H, SH); 2.18 (m, 2H, -CH 2 -CO-); 1.49-1.19 (m, 14H, alkyl), 0.85 (t, 31H,

-CH

3 ). FAB-MS: m/z = 365.2 [M+H t ]; Mr= 364.2 calculated for C 20

H

32

N

2 0 2 S 20 1. Beckett, R. P.; Davidson, A. H.; Drummond, A. H.; Huxley, P.; Whittaker, M. Drug Disc. Today 1996 2. Wtinsch, E. in Houben-Weyl, Methoden der Organischen Chemie, Vol. 15/I, Springer Verlag, Stuttgart, 1974. 3. Stack, M. S.; Gray, R. D. J. Biol. Chem. 1989, 264, 4277. 25 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 (5)

1. A compound represented by the general formula I, H O R . tN fR ARN I H 5 SH() 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 10 wherein A denotes -CO-, -SO 2 -, -NH-CO-, or -O-CO R 1 denotes hydrogen, a linear or branched saturated or unsaturated alkyl group of 1 to 15 carbon atoms or a C 1 -C 1 5 alkyl group substituted by halogen, mercapto, hydroxy, alkoxy, amino or nitro, or by carbocyclic non aromatic or aromatic ring systems which 15 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 C 1 -C 1 5s alkyl group substituted by carbocyclic non aromatic or 20 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 25 pharmacologically acceptable salts, or optically active forms therof and pharmaceutically acceptable carriers. WO 98/50351 PCT/EP98/02654 21

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 5 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.