WO2003089416A1

WO2003089416A1 - Substituted pyrimidine-2,4(1h,3h)-diones for use as matrix metalloproteinase (mmp) inhibitors

Info

Publication number: WO2003089416A1
Application number: PCT/EP2003/004085
Authority: WO
Inventors: Jochen Heinicke; Uwe Klausmeier
Original assignee: Ibfb Gmbh
Priority date: 2002-04-22
Filing date: 2003-04-17
Publication date: 2003-10-30
Also published as: AU2003227648A1

Abstract

The invention relates to substituted condensed pyrimidine-2,4(1H,3H)-diones of general formula (I), to their tautomers, stereoisomers, blends, prodrugs and salts, whereby in formula (I) the variables are defined as cited in the description. Said compounds constitute matrix metalloproteinase inhibitors and are therefore suitable for producing medicaments for treating cancer, rheumatism, inflammation and allergic reactions.

Description

Substituted pyrimidine-2,4 (1H, 3H) diones as matrix metalloproteinase (MMP)

inhibitors

The invention relates to substituted condensed pyrimidine-2,4 (1 H, 3H) -diones of the general formula I,

their tautomers, their stereoisomers, mixtures, prodrugs and salts, where the indices in the formula I are:

n = the numbers 0, 1 or 2,

A = a fused benzene ring or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with at least one oxygen or sulfur atom or a heteroaromatic bonded via two carbon atoms with one, two or three nitrogen, oxygen and / or sulfur atoms as heteroatoms,

R1, R2, R3 = independently of one another hydrogen, halogen, C1-C6 alkyl, C1-C3 alkylthio, C6-C12 aryl, nitro, carbamoyl, C1-C4 alkoxy, -CN, CF ₃ , NH ₂ -, Car-boxy-, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl or C1-C5 alkenyl R4 = hydrogen, C1-C8 alkyl, C2-C5 alkenyl, C2-C8 alkynyl, C3-C10 cycloalkyl -, C3-C10 cycloalkenyl-, C3-C10 cycloalkynyl-, [- (CH ₂ ) ι- ₅ -Z- (C1-C10 alkyl) with Z = O, S, C = 0, S = 0, S0 ₂ ] , C6-C12 aryl, C7-C12 aralkyl. R5 = hydrogen, methyl The alkyl, alkylthio, alkoxy, alkoxycarbonyl, N-alkylcarbamoyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl groups and the Z group defined for R1 to R4 and the Z group can preferably be mono- or disubstituted by leftovers from the group:

Carbamoyl, hydroxy, C1-C4 alkoxy, -CN, S0 ₃ H, CF ₃ , NH ₂ , carboxy, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl, C1-C4 N-dialkyl-carbamoyl, phenyl or piperazinylcarboxy or a heterocycle where the above-mentioned alkyl groups can be substituted by one or two hydroxyl groups, SO ₃ H, NH ₂ , C1-C4 alkylamino, C1-C4 dialkylamino, or a heterocycle.

Intensive research is being carried out worldwide in the field of the development of efficient, low-molecular and non-proteinogenic MMP (matrix metalloproteinase) inhibitors. It is known that physiologically, the enzymatic activity of the MMPs is under a strict, coordinated regulation between activation and inhibition. For this purpose, there are special proteins in the organism, the so-called tissue inhibitors of matrix metalloproteinases (TIMPs), which are able to inhibit the activity of MMPs quickly and efficiently (Nagase, H. et al .: Engineering of selective TIMPs. 1 -11; In: Inhibition of Matrix Metalloproteinases - Therapeutic Application (Eds. Greenwal.d RA., Zucker, S., Golub, LM.) Ann NYAcad Sei 878 (1999). An unchecked enzymatic activity of these enzymes is particularly helpful rheumatic diseases to break down the cartilage and - pathologically significant - to chronic painful changes in the joints (Goldbach-Mansky, R. et al .: Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis. Arthritis Res 2 (2000) 145-153).

Another example of the pathological effect of MMPs is given in the invasion and metastasis of tumors. Released and activated MMPs cut a path through the dense collagenous connective tissue and in particular also through the basement membrane of the vessels and thereby enable the cancer cells to migrate out of the tumor association, to immigrate into the vascular system and to form daughter tumors elsewhere. MMPs play another crucial role in the supply of blood to the growing tumor by cutting the way for the newly formed blood vessels through the collagenous connective tissue and thus responsible for this vascularization of the growing tumor. are literal (Shapiro, SD .: Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Current Opinion in Cell Biology 10 (1998) 602-608).

Another relevant medical result of inadequately inhibited effects of MMPs is UV-induced erythema, which among other things occurs as a result of intense sun exposure. The high-energy UV rays of sunlight and tanning devices, among others, activates the inactive procollagenases in the irradiated skin, which subsequently split collagen in the connective tissue and the blood capillaries and are therefore responsible for the symptoms of sunburn.

With these exemplary pathological effects of the unchecked enzymatic action of MMPs, their sequelae can be prevented or substantially alleviated by stable MMP inhibitors. It is fascinating to realize the idea of specifically inhibiting these enzymes with specific inhibitors, for example to interrupt the progressive cartilage destruction in rheumatic diseases or to prevent the growth or metastasis of tumors.

Numerous processes for obtaining compounds with an MMP-inhibiting effect are already known. The active substances of the first generation generally have a proteinogenic structure and are structurally related to natural inhibitors, which are special proteins. These proteinogenic or pseudoproteinogenic substrate analogues have a zinc-binding group as structural element, which chelates the zinc ion in the active center of the MMPs.

With regard to therapeutic use, all such proteinogenic or pseudoproteinogenic active substances have a number of disadvantages, such as inadequate absorbability, generally short half-lives, only low stability, and frequently undesirable side effects (inhibition of matrix metalloproteinases - therapeutic application (Eds. Greenwald, RA., Zucker, S., Golub, LM.) Ann NYAcad Sci8∑8 (1999)). Further developments in this field have resulted, for example, from phosphonamide inhibitors, piperazine inhibitors, sulfonamide inhibitors, carbamate inhibitors, diazepine inhibitors, tetracycline inhibitors and last but not least hydroxamate inhibitors (Skotnick JS et al .: design and synthetic considerations of matrix metalloproteinase inhibitors. 61-72 In: Inhibition of Matrix Metalloproteinases - Therapeutic Application (Eds. Greenwald, RA., Zucker, S., Golub, LM.) Ann NY Acad Sei 878 (1999)). Most of these inhibitors developed have impressive inhibitory effects and specificities in vitro, but have a number of serious disadvantages in in vivo animal and human applications. The focus here was on cytotoxic reactions to a large number of cells, poor bioavailability and undesirable side effects, especially negative effects on the musculoskeletal system.

Different classes of substances have also been investigated for the effect of their representatives as MMP inhibitors. It has already been suggested that thiazolo [2,3-b] quinazolinones, oxazolo [2,3-b] quinazolinones, imidazo [2,1-b] quinazolinones, benzothiadiazine-5,5-dioxide (DE 10046728), 1 -Dimercaptoalkylquinazoline-2,4 (1H, 3H) - diones (DE 101 01 324), multi-cyclic pyrimidine-2,4 (1H, 3H) -diones with functionalized alkyl radicals in the 1- and / or 3-position (WO 01 / 14344) as MMP inhibitors.

Nevertheless, there is still a need for drugs with a non-proteinogenic structure which do not have the disadvantages of the active ingredients which have hitherto been on the market. In particular, there is a need for new active substances with an MMP-inhibiting effect which are sufficiently stable and readily absorbable, have better pharmacokinetic properties and, above all, do not have any undesirable side effects and cytotoxic reactions.

There is therefore the task of finding new chemical substances of non-proteinogenic structure which are suitable as matrix metalloproteinase inhibitors. They are said to differ advantageously from the previously known inhibitors by virtue of their favorable inhibitory action with few side effects. It is also the aim of the invention to develop pharmaceuticals based on MMP inhibitors for the treatment of numerous diseases, such as cancer, rheumatism, non-specific inflammatory reactions including sunburn and allergic reactions.

The object is achieved in that pyrimidinediones of the general formula I are prepared and it has been found that the new compounds have the desired activity as MMP inhibitors.

The object is achieved in particular by substituted fused pyrimidine-2,4 (1 H, 3H) -diones of the general formula I,

whose tautomers, their stereoisomers, mixtures, prodrugs and salts are dissolved, the indices in formula I denoting:

n = the numbers 0, 1 or 2,

A = a fused benzo ring or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with an oxygen or sulfur atom or a heteroaromatic bonded via two carbon atoms with one, two or three nitrogen, oxygen and / or Sulfur atoms as heteroatoms,

R1, R2, R3 = independently of one another hydrogen, halogen, C1-C6 alkyl, 01 -03-alkylthio, C6-C12-aryl, nitro, carbamoyl, 01-C4 alkoxy, -CN, CF ₃ , NH ₂ -, Car-boxy-, C1-C4 alkoxycarbonyl, 01-04 N-alkyl-carbamoyl or 01 -C5 alkenyl R4 = hydrogen, C1-C8 alkyl, C2-C5 alkenyl, C2-C8 alkynyl, C3-C10 cycloalkyl-, C3-C10 cycloalkenyl-, C3-C10 cycloalkynyl-, [- (CH ₂ ) ι- ₅ -Z- ( C1-C10 alkyl) with Z = O, S, C = 0, S = 0, S0 ₂ ], C6-C12 aryl, C7-C12 aralkyl, R5 = hydrogen, methyl

The alkyl, alkoxy, alkylthio, alkoxycarbonyl, N-alkylcarbamoyl, alkenyl, alkylcarboxy or alkylsulfonyl groups mentioned as radicals R1, R2, R3 or R4 can be shown or undisplayed or open-chain or cyclic. Furthermore, they can preferably be substituents, e.g. Halogen atoms, cyano, carboxy, carbonyl, nitrile, carbamoyl, C1-C4 alkoxy, phenyl, C1-C4 alkoxycarbonyl, C1- C4 alkyl carboxy, 01 -C4 N-alkyl carbamoyl, 01 -C4 N-Dialkylcarbamoyl-, Hydroxy-, Nitro.-, SO3H-, Ether-, Sulfamoyl-, 01 -C4 N-Alkylsulfamoyl, 01 -C4 Dialkylsulfamoyl, -CO-R (with: R = OH, O-Alkyl, NH- Alkyl), trifluoromethyl groups or further open-chain or cyclic groups with heteroatoms (for example with N, S and / or O), in which the heteroatoms can optionally be part of a heterocycle. The possible presence of the aforementioned substituents applies to all groups mentioned as radicals R1, R2, R3 or R4 (insofar as they can be substituted), in particular for the alkyl, cycloalkyl, alkenyl, cycloalkyl and C6-C12 aryl groups mentioned there and the Grouping containing "Z" group.

“Halogen” means fluorine, chlorine, bromine or iodine.

A fused-on benzene or thiophene ring is preferred as radical "A". Furthermore, pyrrole, thiazole, furan, imidazole, pyrazole, pyridine, pyrimidine or pyrazine rings / ring systems are preferred.

The terms "fused" and "fused" are used synonymously.

Among C1-C4 alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl., Sec. Butyl. and to understand tert-butyl.

C1-C8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. Butyl., Tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3- Methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3,3-dimethylpentyl, 3-ethylpentyl, 2,2,3-trimethylbutyl, n-octyl, 2-methylhepyl, 3 - Methylhepyl, 4-methylhepyl, 2,2-dimethylhexyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 3,3-dimethylhexyl, 3,4-dimethylhexyl, 4,4-dimethylhexyl, 2 - ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2,2,3-trimethylpentyl, 2,3,3-

Trimethylpentyl, etc. to understand and their cyclic equivalents.

C2-C5 alkenyl includes Ethene, propene, 1-butene, (cis / trans) -2-butene, 2-methyl propene, 1-pentene, 2-pentene, 2-methyl-1-butene, 3-methyl-1-butene and 2-methyl- To understand 2-butene and their cyclic equivalents.

Among C2-C8 alkynyl include Acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, 1-hexyne, 2-hexyne, 3-hexyne, 3,3-dimethyl-1-butyne To understand 1-heptin, 2-heptin, 3-heptin, 1-octin, 2-0ctin, 3-octin and 4-0ctin and their cyclic equivalents.

C1-C4 alkoxy means methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy.

Among C6-C12 aryl e.g. To understand phenyl, tolyl, xylyl, naphthyl, biphenyl ring systems.

The following preferred compounds exist within the above formula (I):

(a) dimercaptoalkyl-substituted fused pyrimidine-2,4 (1 H, 3H) -diones of the general formula (Ia),

(la)

where in the formula (la) the indices mean:

n = the numbers 0 or 1

R4 = hydrogen, C1 -C8 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, [- (CH ₂ ) ι- ₃ -Z- (C1 -05 alkyl) with Z = O, S, 0 = 0, S = 0, S0 ₂ ], C7-C12 aralkyl, where the alkyl, alkenyl, alkynyl groups and the Z group can be mono or disubstituted by radicals from the group: carbamoyl, hydroxy, C1-C2 alkoxy, -CN, S0 ₃ H, CF ₃ ,, carboxy, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl, C1-C4 N-dialkyl-carbamoyl or piperazinylcarboxy and the above-mentioned alkyl groups can be substituted by one or two hydroxyl groups, SO3H, NH ₂ , 01 -C4 alkylamino, 01 -C4 dialkylamino, or a heterocycle.

R5 = a hydrogen atom or a methyl group

A = a fused benzo ring unsubstituted or monosubstituted, disubstituted or trisubstituted with radicals which may be the same or different from the group: halogen, 01 to C3-alkyl, 01 to C3 alkoxy, trifluoromethyl, nitro-, carbamoyl-, -NH ₂ , Carboxy-, C1-C4 alkoxycarbonyl, C1-C4 N-alkylcarbamoyl, arylalkylcarbamoyl, arylalkyloxycarbonyl, het-alkylcarbamoyl and het-alkyloxycarbonyl with aryl = phenyl, tolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolol and het , Piperazinyl, Piperidinyl, Pyrrolidinyl, Imidazolidinyl or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with an oxygen or sulfur atom or a heteroaromatic bonded via two carbon atoms, this six-membered with one, two or three nitrogen atoms as heteroatoms or five-membered with a nitrogen atom or five-membered with an oxygen or sulfur atom or five-membered with a nitrogen atom and one oxygen or one sulfur atom or five-membered with two nitrogen atoms as heteroatoms or five-membered with two nitrogen atoms and one sulfur atom or five-membered with three nitrogen atoms as heteroatoms, where the heteroaromatics defined above for A are mono-, di- or tri-substituted by radicals R6 and the substituents can be identical or different

R6 = halogen, C1 to C6-alkyl, C1 to C3-alkylthio, cyano, trifluoromethyl, benzyl, phenyl, where the latter two aromatic radicals can be substituted as follows:

C1 to C3-alkyl, halogen, COR7, S02R7, 0C0R7, 0R8, C00R8, CONR8R9, S02NR8R9, NR8R9 with R7 = 01 to C4-alkyl, C3 to C6-cycloalkyl, phenyl, R8 / R9 = independently of one another H, phenyl, Benzyl, the latter unsubstituted or mono-, di- or trisubstituted by methoxy, 01 to C4-alkyl or

R8 / R9 = as a straight-chain alkyl group together forming a ring with a nitrogen atom, a five-membered ring which may optionally contain a further heteroatom N, O, S, or together forming a six-membered heterocycle with a nitrogen atom and, if appropriate, a further nitrogen atom or an oxygen atom or a sulfur atom, it being possible for five and / or six rings in the carbon skeleton to be substituted by C1 to 04 alkyl or by benzyl; R6 = in the event that A is thieno, also a fused 04--07 cycloalkyl ring, which may contain an oxygen, a sulfur or a nitrogen atom, the nitrogen atom being unsubstituted or substituted by 01 to 04 alkyl, phenyl or Benzyl and the latter two aromatics can be substituted with 01 to 03 alkyl, C1 to 03 alkoxy or halogen, furthermore for the case A = thieno R6 may also be: carboxy, a heterocycle-substituted alkyl group C1-C5, a heterocycle and a C1 -C5 N-alkylcarbamoyl group which may be substituted by amino, sulfo, hydroxy, dihydroxy or a heterocycle.

The above explanations regarding the chemical groupings and possible substitutions apply correspondingly to the compounds of the formula I (a).

The invention also includes compounds of the general formulas I and I (a) in the form of their stereoisomers, racemates, enantiomers, diastereomers, or their physiologically tolerable salts with inorganic or organic acids or bases, and mixtures of these compounds. The present invention also relates to prodrugs of the compounds of the general formulas I or l (a) in which the mercapto function is carried out by processes known per se to a person skilled in the art (cf.; Higuchi T., Stella,]., Prodrugs as Novel Delivery Systems, Vol 14 oftheA.CS Symposium Series; Roche.EB, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987) is substituted with a residue that can be split off in vivo. Examples of residues which can be split off in vivo are acyl groups, acyloxycarbonyl groups, alkoxycarbonyl groups, carbamoyl groups, phosphate residues. Also suitable are the disulfides, isothiuronium salts, thiosulfates (BUNTE salts) of compounds of the general formulas I or I (a) as prodrugs which can be converted into the free mercaptans in vivo. The mercaptans of the general formula I, in particular of the formula I (a), can contain one or more centers of asymmetry, which is responsible for their occurrence as stereoisomers or racemates. The compounds of general formula I or I (a) obtained can, if appropriate, be converted into their enantiomers and according to methods known per se (cf. for example Allinger, NL and Elliel EL in "Topics in Stereochemistry" Vol. 6, Wiley Interscience, 1971) / or diastereomers are separated.

Preferred compounds of the formula I (a) are:

(R, S) -3- (2,3-dimercaptopropyl) pyrido [3,2-d] pyrimidine-2,4 (1H, 3H) -dione (R, S) -3- (2,3-dimercapto- 2-methyl-propyl) pyrido [3,2-d] pyrimidine-2,4 (1H, 3H) -dione (R, S) -3- (3,4-dimercaptobutyl) pyrido [3,2-d] pyrimidine -2.4 (1H, 3H) -dione (R, S) -3- (3,4-dimercapto-3-methylbutyl) pyrido [3,2-d] pyrimidine-2,4 (1H, 3H) -dione (R, S) -3- (2,3-dimercaptopropyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercapto-2-methyl-propyl) pyrido [2,3-d] pyrimidine-2,4 (1H ₎ 3H) -dione

(R, S) -3- (3,4-dimercaptobutyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercaptopropyl) -6,7-dimethylpyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercapto-2-methyl-propyl) -6,7-dimethylpyrido [2,3-d] pyrimidine-

2.4 (1H, 3H) dione

(R, S) -3- (3,4-dimercaptobutyl) -6,7-dimethylpyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl) 6,7-dimethylpyrido [2,3-d] pyrimidine-

2.4 (1H, 3H) dione

(R, S) -3- (2,3-dimercaptopropyl) -6-pyridin-4-ylpyrido [2,3-d] pyrimidin-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercapto-2-methyl-propyl)) -6-pyridin-4-yl pyridof 2,3-d] pyrimidine-

2.4 (1H, 3H) dione

(R, S) -3- (3,4-dimercaptobutyl)) -6-pyridin-4-yl pyrido [2,3-d] pyrimidin-2,4 (1H, 3H) -dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl)) -6-pyridin-4-yl pyrido [2,3-d] pyrimidin-

2.4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercaptopropyl) pteridine-2,4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercapto-2-methyl-propyl) pteridine-2,4 (1H, 3H) dione

(R, S) - 3- (3,4-dimercaptobutyl) pteridine-2,4 (1H, 3H) dione

(R, S) - 3- (3,4-dimercapto-3-methylbutyl) pteridine-2,4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercaptopropyl) -6,7-dimethylpteridine-2,4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercapto-2-methyl-propyl) -6,7-dimethylpteridine-2,4 (1H, 3H) -dione

(R, S) - 3- (3,4-dimercaptobutyl) -6,7-dimethylpteridine-2,4 (1H, 3H) dione

(R, S) - 3- (3,4-dimercapto-3-methylbutyl) -6,7-dimethylpteridine-2,4 (1H, 3H) dione

(R, S) -5- (2,3-dimercaptopropyl) -2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercapto-2-methyl-propyl) -2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) - dione

(R, S) -5- (3,4-dimercaptobutyl) -2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (3,4-dimercapto-3-methyl-butyl) -2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercaptopropyl) -2-methyl-2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercapto-2-methylpropyl) -2-methyl -2H-pyrazolo [3,4-d] pyrimidine-

4,6 (5H, 7H) -dione

(R, S) -5- (3,4-dimercaptobutyl) -2-methyl -2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) dione (R, S) -5- (3,4-dimercapto-3-methylbutyl) -2-methyl -2H-pyrazolo [3,4-d] pyrimidine-

4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercaptopropyl) -1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercapto-2-methyl-propyl) -1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) - dione

(R, S) -5- (3,4-dimercaptobutyl) -1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (3,4-dimercapto-3-methyl-butyl) -1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercaptopropyl) -1-methyl-1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (2,3-dimercapto-2-methyl-propyl) -1-methyl-1 H-pyrazolo [3,4-d] pyrimidine-

4,6 (5H, 7H) -dione

(R, S) -5- (3,4-dimercaptobutyl) -1-methyl-1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

(R, S) -5- (3,4-dimercapto-3-methyl-butyl) -1-methyl-1H-pyrazolo [3,4-d] pyrimidine

4,6 (5H, 7H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -5-methyl-1H-pyrrolo [3,2-d] pyrimidine-2,4 (3H, 5H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) -5-methyl-1H-pyrrolo [3,2-d] pyrimidine

2,4 (3H, 5H) -dione

(R, S) -3- (3,4-dimercaptobutyl) -5-methyl-1H-pyrrolo [3,2-d] pyrimidine-2,4 (3H, 5H) -dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl) -5-methyl-1H-pyrrolo [3,2-d] pyrimidine

2,4 (3H, 5H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -5,7-dimethyl-1H-pyrrolo [3,2-d] pyrimidine-2,4 (3H, 5H) - dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) -5,7-dimethyl-1H-pyrrolo [3,2-d] pyrimidine

2,4 (3H, 5H) -dione

(R, S) -3- (3,4-dimercaptobutyl) -5,7-dimethyl-1H-pyrrolo [3,2-d] pyrimidine-2,4 (3H, 5H) - dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl) -5,7-dimethyl-1H-pyrrolo [3,2-d] pyrimidine

2,4 (3H, 5H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -5,6,7-trimethyl-1H-pyrrolo [3,2-d] pyrimidine-2,4 (3H, 5H) - dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) -5,6,7-trimethyl-1H-pyrrolo [3,2-d] pyrimidine

2,4 (3H, 5H) -dione

(R, S) -3- (3,4-dimercaptobutyl) -5,6,7-trimethyl-1H-pyrrolo [3,2-d] pyrimidine-2,4 (3H, 5H) - dione (R, S) -3- (3,4-dimercapto-3-methylbutyl) -5,6,7-trimethyl-1H-pyrrolo [3,2-d] pyrimidine

2,4 (3H, 5H) -dione

(R, S) -3- (2,3-dimercaptopropyl) furo [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) furo [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (3,4-dimercaptobutyl) furo [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (3,4-dimercapto-3-methyl-butyl) furo [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -1-methyl-thieno [3,2-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) -1-methylthieno [3,2-d] pyrimidine-2,4 (1H, 3H) - dione

(R, S) -3- (3,4-dimercaptobutyl) -1-methyl-thieno [3,2-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl) -1-methylthieno [3,2-d] pyrimidine-2,4 (1H, 3H) - dione

(R, S) -3- (2,3-dimercaptopropyl) -1-methyl-thieno [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) -1-methylthieno [2,3-d] pyrimidine-2,4 (1H, 3H) - dione

-dione (R, S) -3- (3,4-dimercaptobutyl) -1-methyl-thieno [2,3-d] pyrimidine-2,4 (1H, 3H)

(R, S) -3- (3,4-dimercapto-3-methylbutyl) -1-methylthieno [2,3-d] pyrimidine-2,4 (1H, 3H) - dione

(R, S) -3- (2,3-dimercaptopropyl) -1-methylquinazolin-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) -1-methylquinazolin-2,4 (1H, 3H) -dione

(R, S) -3- (3,4-dimercaptobutyl) -1-methylquinazolin-2,4 (1H, 3H) -dione

(R, S) -3- (3,4-dimercapto-3-methylbutyl) -1-methylquinazolin-2,4 (1H, 3H) -dione

Particularly preferred compounds of the formula I (a) are:

(S R) -3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) quinazoline-2,4- (1H, 3H) dione

(S R) -7-chloro-3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(S R) -6-chloro-3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(S R) -3- (2,3-dimercaptopropyl) -5-methyl-quinazoline-2,4 (1H, 3H) -dione

(S R) -3- (2,3-dimercaptopropyl) -8-methyl-quinazoline-2,4 (1H, 3H) -dione

(S R) -3- (2,3-dimercaptopropyl) -6,7-dimethoxy-quinazoline-2,4 (1H, 3H) -dione

(R, S) -N-benzyl-3- (2,3-dimercaptopropyl) -2,4-dioxo-1, 2,3,4-tetrahydroquinazoline-7-carboxamide (R, S) -3- (2,3-dimercaptopropyl) pyrido [3,2-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercapto-2-methyl-propyl) pyrido [3,2-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercaptopropyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercapto-2-methyl-propyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) - 3- (2,3-dimercaptopropyl) pteridine-2,4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercapto-2-methyl-propyl) pteridine-2,4 (1H, 3H) dione

2,4 (3H, 5H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -1-methylquinazolin-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercaptopropyl) thieno [3,2-d] pyrimidine-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -5,6,7,8-tetrahydro [1] benzothieno [2,3-d] pyrimidine

2.4 (1H, 3H) dione

(R, S) -3- (2,3-dimercaptopropyl) -6-phenyl-thieno [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

The dimercaptans according to the invention are accessible by acidic or alkaline hydrolysis of monomer captaptans of the general formula II.

(II)

n = the numbers 0, 1 or 2, A = a fused benzene ring or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with an oxygen or sulfur atom or a five- to seven-membered heteroaromatic bonded with two carbon atoms with one, two or three nitrogen or oxygen atoms - and / or sulfur atoms as heteroatoms, where A is unsubstituted, mono-, di- or trisubstituted with halogen, C1-C6 alkyl, C1-C3-alkylthio-, C6- C12-aryl-, nitro-, carbamoyl-, C1- C4 alkoxy, -CN, CF ₃ , NH ₂ -, carboxy-, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl or C1-C5 alkenyl R5 = hydrogen, methyl

Monomercaptans of the general formula II are already known for the quinazoline system (A = benzo) (DD 10046728).

Monomercaptans of the general formula II with a heterocyclically condensed pyrimidine base (A = heterocycle) have already been proposed.

The structure of precursors II with A = heterocycle is generally carried out according to the patterns well known from the chemistry of the corresponding quinazolines. Some of the starting compounds or intermediates in the form of the corresponding heterocyclic anthranilic acid analogs are commercially available, some are known from the literature or are accessible to the person skilled in the art by methods known from the literature. For summary literature see eg; Methods of Organic Chemistry (Houben Weyl) Thieme Verlag, Stuttgart, Vol. E8b (1994) pp. 1 ff. And Vol. E8a (1993) pp. 668 ff. (Thiazole); Vol. E 6a (1994) pp. 556 ff. (Pyrroles); Vol. E 8b (1994) pp. 399 ff. (Pyrazole); Vol. E 6 (1994) pp. 186 ff. (Thiophene); Vol. 9b / 1 (1998) pp. 8 ff. (Pyrimidine); Vol. 9b / 1 (1998) pp. 250 ff. (Pyrazine); O. Meth-Cohn (Ed.), Comprehensive heterocyclic Chemistry, Pergamon Press, Oxford, 1996; The Chemistry of heterocyclic Compounds, Wiley Interscience, New York, Vol. I (1950) u. ff. Examples of special approaches to synthetic building blocks of the heterocyclic ß-enaminocarboxylic acid derivative type are: RW Sabnis, The Gewald-Synthesis, Sulfur Reports 6 (1994) p. 1 ff. (Thiophene); RW Sabnis, DW Ragnekar, ND Sonawane, 2-Aminothiophenes by the Gewald Reaction, J. Heterocyclic Chem. 36 (1999) 333 ff. (Thiophene); Briel, D., Maschke, T., Wagner, G., Synthesis of Thieno [3,3-d] - and [3,4-d] pyrimidines by Alternative Ring Closure Reactions, Pharmazie 47 (1992) 577 ff. (Thiophene); Punto, IL, Jarvest, RL, Serafinowska, HT, The Syntesis of 5-Alkoxy and 5-Amino Substituted Thiophenes, Tetrahedron Lett. 41 (2000) 1597-1699, Dave, OG, Synthesis and Reactions of Fluoroaryl Substituted 2-Amino-3-cyanopyrroles and Pyrrolo [2,3-d] pyrimidines, J. Heterocyclic Chem. 36 (1999) 729 ff. (pyrroles); Lim.M., Klein, RS, Fox, JJ, A New Synthesis of Pyrrolo [3,2-d] pyrimidines via 3-Amino-2-carboalkoxypyrroles, J. Org. Chem. 44 (1979) 3826 ff. (Pyrrole ); Hanning, E., Beyer, G., Laban, G., Pharmazie 41 (1986) 381 ff. (Pyrazole); Bridson, PK, Wang, X., 1-Substituted Xanthines, Synthesis 1995 pp. 855 ff. (Imidazole) as well as WO 99/11631 (Thiophene) and US 4,835,157 (Thiophene, Furan).

In addition, various syntheses for heterocyclically fused pyrimidines are known in which the basic multi-cyclic system is built up from appropriately substituted pyrimidines, e.g .: El-Ahl, A.A.S. et al., A One-Pot Synthesis of Pyrido [2,3-d] - and Quinolino [2,3-d] pyrimidines, Heterocycles 55 (2001) 1315 ff .; Falch, E., Synthesis and Structure Determination of Thiazolo- and Thiazinopteridinones, Acta Chemica Scandinavica B 3J. (1977) 167 ff .; Cottam, H.B. et al., Substituted Xanthines, Pteridinediones and Related Compounds as Potential Anti-inflammatory Agents. Synthesis and Biological Evaluation of Inhibitors of Tumor Necrosis Factor α, J. Med. Chem. 39 (1996) 2 ff .; Bhuyan, Studies on Uracils: A Facile One-Pot Synthesis of Pyrazolo [3,4-d] pyrimidines, Tetrahedron Lett. 43 (2002), WO 00/00491 (thiadiazoles, imidazoles, pyrazines).

It is already known to arrive at the corresponding mercaptans by acid- or base-catalyzed ring opening of thiazolo- or thiazinopyrimidines (eg; Falch, E., Synthesis and Structure Determination of Thiazolo- and Thiazinopteridones, Acta Chem. Scand. B 3 ± (1977) 167 ff; Gütschow.M .; Drössler.K .; Leistner, S., Polycyclic Azines with Heteroatoms in the 1- and 3-Position. 40. Synthesis of Heterocyclic Immunomodulators. 2. 3-Mercaptolakylthieno (2, 3-d) pyrimidine-2,4 (1H, - 3H) - diones: Synthesis and Testing of Immunostimulant Activity, Arch Pharm 328: 3 (1995) 231 ff). So far no attempt has been made to apply the catalytic ring opening to pyrimidinediones of the general formula II. Surprisingly, however, the ring opening succeeds under conditions which are adapted to the nature of the substituent. The general working procedure described below gives the method dik again, according to which the ring can be opened, while the examples show a particularly favorable synthetic route to the pyrimidinediones according to the invention.

The substitution of N1 (radical R4 in formula (I) and l (a)) is preferably carried out by subsequent alkylation of the correspondingly protected tricyclic mercaptan II. Suitable processes for this subsequent alkylation (for example using methyl iodide or bromoacetic acid esters in the system K ₂ COs / DMF) are well known to the person skilled in the art.

The protective group chemistry which may be used in the synthesis of the compounds of the general formulas (I) or (Ia) is sufficiently known to the person skilled in the art and is mastered by him.

With regard to the compounds described here, the underlying structure-activity relationships are still unclear and the enzyme-inhibitory properties of the compounds found are therefore to be regarded as new and surprising.

General working instructions for the preparation of the dimercaptans according to the invention:

Acid hydrolysis (variant A)

5 mmol mercaptomethyl precursor (general formula II) are concentrated in a 0.6 ml. Sulfuric acid, 1.5 ml glacial acetic acid and 66 ml water prepared acid mixture entered and heated under reflux under chromatographic control (TLC or HPLC) until complete conversion. The average response time is 8-10 hours. The precipitate after cooling (possibly diluting with water) is separated off, washed with water, dried and, if necessary, recrystallized.

Alkaline hydrolysis (variant B)

5 mmol mercaptomethyl precursor (general formula II) are introduced under nitrogen in 50 ml of a mixture of 3N NaOH (nitrogen-saturated) / ethanol (10: 1) and 100 mg zinc dust. The batch is stirred while continuously introducing nitrogen at 80 to 85 ° C. until conversion is complete (TLC or HPLC control). The average response time is 5-8 hours. After the reaction is over the cooled reaction mixture is filtered with direct ice cooling with vigorous stirring and nitrogen blowing into 10% hydrochloric acid. After storage overnight at 4 ° C., the precipitate is filtered off with suction, washed with water, dried and, if necessary, recrystallized.

In the context of the invention it has been possible to open up classes of substances whose representatives have the desired effect and which are also easily accessible synthetically, which are stable in substance and in solution, easy to handle and can be formulated into possible medicaments. By testing the new compounds of the general formula I, in particular the formula I (a), on different human MMPs (MMP-2, recombinant catalytic domain of MMP-3, recombinant catalytic domain of MMP-8, MMP-9, recombinant catalytic domain of MMP-14) the effectiveness and specificities of the compounds of the invention are demonstrated. Specific inhibitors, especially when used as medicines, would only influence the desired target enzymes and would not interfere with the balanced interplay of the other MMPs and thus help to avoid possible undesirable side effects.

The invention is described in more detail below with the aid of the examples.

example 1

(S R) -3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(general formula (la), A = benzo, n = 0, R4, R5 = H)

The target compound was derived from (R, S) -2-mercaptomethyl-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo; n = 0; R5 = H; DD 100 46 728) obtained according to variant B.

Yield 58%

Colorless solid

F .: 181-185 ° C

CnH ₁₂ N ₂ 0 ₂ S ₂ (268)

MS m / e (% B): 268 (2, M ⁺ ), 234 (41), 201 (70), 163 (100), 146 (85) Example 2

(R, S) -3- (2,3-dimercapto-2-methylpropyl) quinazoline-2,4 (1H, 3H) -dione

(general formula (la), A = benzo, n = 0, R4 = H, R5 = methyl)

The target compound was derived from (R, S) -2-mercaptomethyl-2-methyl-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo, n = 0, R5 = methyl; DD 100 46 728) obtained according to variant B.

Yield 45%

Colorless solid

F .: 128-131 ° C

MS m / e (% B): M ⁺ 282 (2, M ⁺ ), 217 (8), 201 (18), 137 (22), 119 (76), 43 (100)

Example 3

(S R) -7-chloro-3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(general formula (la), A = benzo with 7-chlorine, n = 0, R4, R5 = H)

The target compound was obtained from (R, S) -8-chloro-2-mercaptomethyl-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo with 8 -Chlor, n = 0, R5 = H; DD 10046728) obtained according to variant B.

Yield 70%

Colorless solid

F .: 183-186 ° C

CιιHιιCIN ₂ 0 ₂ S2 (302.9)

MS m / e (% B): 302 (1, M ⁺ ), 268 (29), 235 (75), 197 (100), 180 (95) Example 4

(S R) -6-chloro-3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(general formula (la), A = benzo with 6-chlorine, n = 0, R4, R5 = H)

The target compound was obtained from (R, S) -7-chloro-2-mercaptomethyl-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo with 7 -Chlor, n = 0, R5 = H; F.

162-166 ° C), which is accessible analogously to the precursor of Example 3, obtained according to variant B.

Yield 76%

Colorless solid

F .: 212-218 ° C

C11H11CIN2O2S2 (302.9)

MS m / e (% B): 268 (5), 236 (80), 221 (100), 196 (35), 180 (90)

Example 5

(S R) -3- (2,3-dimercaptopropyl) -5-methyl-quinazoline-2,4 (1H, 3H) -dione

(general formula (la), A = benzo with 5-methyl, n = 0, R4, R5 = H)

The target compound was derived from (R, S) -2-mercaptomethyl-6-methyl-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo with R6 = 6-methyl, n = 0, R5 = H;; F. 203 ° C), which is accessible analogously to the precursor of Example 1, obtained according to variant B.

Yield 65%

Colorless solid

F .: 176-178 ° C

MS m / e (% B): 282 (1, M ⁺ ), 264 (70), 217 (100), 177 (28) Example 6

(S R) -3- (2,3-dimercaptopropyl) -8-methyl-quinazoline-2,4 (1H, 3H) -dione

(general formula (la), A = benzo with 8-methyl, n = 0, R4, R5 = H)

The target compound was obtained from (R, S) -2-mercaptomethyl-9-methyl-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo with 9 -Methyl, n = 0, R5 = H; F. 180- 184 ° C), which is accessible analogously to the precursor of Example 1, obtained according to variant B.

Yield 52%

Colorless solid

F .: 169-171 ° C

MS m / e (% B) 282 (10, M ⁺ ); 249 (40); 192 (10); 177 (100); 161 (20)

Example 7

(S R) -3- (2,3-dimercaptopropyl) -6,7-dimethoxy-quinazoline-2,4 (1H, 3H) -dione

(general formula I (a), A = benzo with 6,7-dimethoxy, n = 0, R4, R5 = H)

The target compound was derived from (R, S) -2-mercaptomethyl-7,8-dimethoxy-2,3-dihydro-5H-thiazolo [2,3-b] quinazolin-5-one (general formula II, A = benzo with 7,8-dimethoxy, n = 0, R5 = H; 180-184 ° C), which is accessible analogously to the precursor of Example 1, obtained according to variant A.

Yield 79%

Colorless solid

F .: 197-204 ° C

Cι ₃ H ₁₆ N ₂ 0 ₄ S ₂ (328.5)

MS m / e (% B): 328 (30, M ⁺ ), 295 (55), 261 (70), 223 (100) Example 8

(R, S) -N-benzyl-3- (2,3-dimercaptopropyl) -2,4-dioxo-1,2,3,4-tetrahydroquinazoline-7-carboxamide

(general formula (la), A = benzo with 7-N-benzylcarboxamide, n = 0, R4, R5 = H)

The target compound was obtained from (R, S) -N-benzyl-2-mercaptomethyl-5-oxo-2,3-dihydro-5H-thiazolo [2,3-b] quinazoline-8-carboxamide (general formula II, A = 8-N-benzylcarboxamide, n = 0, R5 = H; mp 174-176 ° C.), which is accessible analogously to the precursor of Example 1, obtained according to variant A.

Yield 19.5%

Colorless solid

F .: 214-218 ° C (methanol)

MS m / e (% B): 368 (8), 335 (35), 295 (20), 216 (55)

Example 9

(R, S) -3- (2,3-dimercaptopropyl) thieno [3,2-d] pyrimidine-2,4 (1H, 3H) -dione

(general formula (la), A = Thieno, n = 0, R4, R5 = H)

The target compound was derived from the (R, S) -6-mercaptomethyl-6,7-dihydro-thiazolo [3,2-a] thieno [3,2-d] pyrimidin-9-one (general formula II, A = Thieno, n = 0, R5 = H) obtained according to variant B.

Yield 68%

Pale yellow solid

F .: 170-174 ° C

C ₉ H ₁₀ N ₂ O ₂ S ₃ (274)

MS m / e (% B): 274 (3, M ⁺ ), 240 (45), 207 (70), 169 (97), 152 (98), 126 (100) Example 10

(R, S) -3- (2,3-dimercaptopropyl) -5,6,7,8-tetrahydro [1] benzothieno [2,3-d] pyrimidine-2,4 (1H, 3H) -dione

(general formula (la), A = thieno with R6 = fused cyclohexyl ring, n = 0, R4, R5 = H)

The target compound was derived from the (R, S) -2-mercaptomethyl-2,3,6,7,8,9-hexahydro-5H-benzo [4,5] thieno [2,3-d] thiazolo already proposed by us [3,2- a] pyrimidin-5-one (general formula II, A = thieno with R6 = fused cyclohexyl ring, n = 0, R5 = H) obtained according to variant B.

Yield 56%

Cream colored solid

F .: 97-100 ° C

Cι ₃ Hι ₆ N ₂ 0 ₂ S ₃ (328)

MS m / e (% B): 328 (2, M ⁺ ), 310 (100), 282 (40), 263 (27), 235 (20)

Example 11

(general formula (la), A = thieno with R6 = 6-phenyl, n = 0, R4, R5 = H)

The target connection was derived from the (R, S) -2-

Mercaptomethyl-7-phenyl-2,3-dihydro-5H-thiazolo [3,2-a] thieno [2,3-d] pyrimidin-5-one

(general formula II, A = thieno with 7-phenyl, n = 0, R5 = H) obtained according to variant B.

Yield 47%

Light beige powder

F .: 197-205 ° C (chloroform / ethanol / heptane)

MS m / e (% B): 350 (10, M ⁺ ), 324 (40), 317 (10), 283 (15), 245 (20), 202 (100) Example 12

Preparation of Matrix Metalloproteinase-2 (MMP-2)

MMP-2 (gelatinase) is released from cultured dermal fibroblasts in considerable quantities into the culture medium and is therefore easily accessible. The secreted and inactive proform of the enzyme is converted into the enzymatically active form by treatment with organic mercury compounds.

For this purpose, human dermal fibroblasts are obtained and cultivated according to established standard methods. Human MMP-2 is obtained from the conditioned cell medium by the following method:

500 ml of cell medium are 30 min. Centrifuged at 10000 g and the supernatant was mixed with a volume of 2-fold binding buffer (40 mM Tris-HCl pH 7.5, 10 mM CaCl ₂ , 1 M NaCl, 0.2% (v / v) Triton X-100) , 6 ml of gelatin agarose, which was equilibrated with binding buffer, are added and the mixture is shaken on ice for 1 hour. The loaded gelatin agarose is transferred to a column and rinsed protein-free with at least 10 vol. Binding buffer. The bound MMP-2 is eluted with 2 gel volumes of binding buffer plus 5% (v / v) DMSO. The eluate contains the human MMP-2 as an inactive zymogen in a highly enriched form. Foreign proteins are TIMP-2 and fibronectin; the latter can be separated by gel filtration on Sephacryl S-200. Running buffer is 100mM Tris-HCl pH 7.5, 10mM CaCl ₂ , 100mM NaCl, 0.05% (v / v) Brij 35.

The inactive human MMP-2 is activated by incubation for two hours at 37 ° C. in the presence of 1 mM p-aminophenyl mercuric acetate (APMA). To remove APMA after activation, the sample is filtered on Sephadex G-25 in running buffer (see above).

Preparation of the human collagenase MMP-9

Native MMP-9 was obtained reproducibly and in good yield and purity from human buffy coat. For this, Buffy Coat with 10% (v / v) Triton X-100 is brought to a final concentration of 0.4%, 30 min. Shaken on ice and then 1 vol. 2-fold binding buffer (40 mM Tris-HCl pH 7.5, 10 mM CaCl ₂ , 1 M NaCl, 0.2% (v / v) Triton X-100) was added and another 30 minute shaken on ice. The solution is 15 min. centrifuged at 16,000 rpm on an SS-34 rotor at 4 ° C and filtered over glass wool. The filtrate is batched with gelatin agarose, which has been equilibrated with binding buffer, and shaken on ice for 1 hour. The loaded gelatin agarose is transferred to a column and rinsed protein-free with at least 10 vol. Binding buffer. The bound MMP-9 is eluted with 2 gel volumes of binding buffer plus 5% (v / v) DMSO.

The eluate can be separated by gel filtration on Sephadex G-75 for buffer exchange and simultaneous removal of small impurities on MMP-2. Buffer I (20 mM Tris-HCl pH 7.5, 5 mM CaCl ₂₎ 100 mM NaCl, 0.1% (v / v) Triton X-100) is used for this.

The eluate obtained in this way contains MMP-9 in the three known states: monomer, homodimer, heterodimer. The purity of the enzyme is about 90%, with the remaining foreign proteins being fibronectin and extremely small amounts of TIMP's.

The latent enzyme is 30-60 min. Incubation at 37 ° C with 1/100 vol. Trypsin (10 mg / ml) activated. Trypsin is inhibited by adding 1 mM PMSF or with a specific inhibitor (TKI).

Cloning and expression of the MMP-8 catalytic domain

The use of the catalytic domain of the MMP-8 as a further test enzyme was chosen because it has a high stability and is also present as an active enzyme. It therefore does not have to be activated, which is one of the most common causes of errors, since the mercury compounds used for activation often interfere with the test system or the enzyme and will thereby falsify the measurement results. We designed the cloning strategy so that we only cloned the enzymatically active catalytic domain in E. coli instead of the entire enzyme. With the constructed catalytic domain of the MMP-8 we achieved a stable, enzymatically active and high-purity enzyme, which was very well suited for routine tests of the inhibitory activity of the synthesized inhibitors and made the measurement results of the individual measurement series comparable.

The cloning and expression of the catalytic domain of the MMP-8 was carried out in accordance with the information provided by SCHNIERER et al. (Schnierer S, Kleine T, Gote T, Hilemann A, Knäuper V, Tschesche H: The recombinant catalytic domain of human neutrophil collagenase lacks type I collagen Substrate specificity. Biochem Biophys Res Comm (1993) vol. 191 No. 2, 319-326).

Cloning and expression of the MMP-3 catalytic domain

The catalytic domain of human MMP-3 was criticized according to the information in the following publication:

Ye Q-Z, Johnson LL, Hupe DJ, Baragi V: Purification and Characterization of the Human Stromelysin Catalytic Domain Expressed in Escherichia coli. Biochemistry (1992), 31, 11231-11235).

After refolding the enzyme and gel filtration on Sephadex G-50 (running buffer 100 mM Tris-HCl pH 7.5, 10 mM CaCI, 100 mM NaCI, 0.05% Brij 35) we received a 95% pure preparation.

Cloning and expression of the MT1-MMP catalytic domain

The catalytic domain of the human MT1-MMP was cloned according to the information in the following publication:

Lichte A, Kolkenbrock H, Tschesche H: The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2. FEBS letters (1996), 397, 277-282.

After refolding the enzyme, autoprocessing to the active form and gel filtration on Sephadex G-50 (running buffer 20 mM Tris-HCl pH 7.5, 5 mM CaCl ₂ , 100 mM NaCl, 0.1 mM ZnCl ₂ , 0.02% (w / v) sodium azide) we received a 95% pure preparation.

Quantitative fluorescence assay for matrix metalloproteinases

By enzymatic cleavage of the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH ₂ by the respective collagenase, the fluorescent group Mca ((7-methoxycoumarin-4-yl) acetyl) is removed from the internal quencher Dpa ((N-3- (2,4-dinitrophenyl) -L-2,3-diaminopropienyl) separated. There is a strong increase in the fluorescence in the measurement batch, which can be quantified on the fluorimeter (λ _θx 328 nm, λ _β 393 nm ) and runs linearly within the first minutes. On the one hand, a certain specificity of the test for matrix metalloproteinases results from the amino nucleic acid sequence -Pro-Leu-Gly-Leu- in the substrate and on the other hand by the chosen incubation conditions. Matrix metalloproteinases cleave the substrate at the Gly-Leu bond. The proteolytic residual activity of preincubated batches of enzyme and inhibitor is measured, the substrate and enzyme concentration being kept constant and the inhibitor concentration varied. Three series of measurements with different substrate concentrations were recorded for each inhibitor tested. The enzyme activity is calculated in fluorescence units per minute from the time-dependent increase in fluorescence. The K _r values were determined graphically according to the DIXON (1953) method by plotting the reciprocal reaction rate 1 / v (y-axis) against inhibitor concentration (x-axis). Assay

1984 μ \ measuring buffer (100mM Tris-HCl pH 7.5, 100mM NaCl, 10mM CaCl ₂ , 0.05% Brij 35 or 50mM MES-NaOH, pH 6.0, 10mM CaCl2, 100mM NaCl, 0.05% Brij 35 in the case of KaDo MMP-3)

2 μ \ inhibitor, dissolved in DMSO, or DMSO alone (non-inhibitory approach) 4 μ \ enzyme

• 5 min. Preincubate the enzyme inhibitor mixture at room temperature with stirring

• Start the reaction with 10 μ \ substrate dissolved in DMSO

• Recording the time-dependent increase in fluorescence over 2 - 10 min.

Inhibition of human MMPs by the inhibitors of the general formula I according to the invention

Claims

1. Substituted condensed pyrimidine-2,4 (1H, 3H) -diones of the general formula (I)

where in formula I the indices are:

n = the numbers 0, 1 or 2,

R1. R2, R3 = independently of one another hydrogen, halogen, C1-C6 alkyl, C1-C3-alkylthio, C6-C12 aryl, nitro, carbamoyl, C1-C4 alkoxy, -CN, CF ₃ , NH ₂ -, carboxy -, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl or C1-C5 alkenyl R4 = hydrogen, C1-C8 alkyl, C2-C5 alkenyl, C2-C8 alkynyl, C3-C10 cycloalkyl-, C3-C10 cycloalkenyl- , C3-C10 cycloalkynyl, [- (CH ₂ ) ι- ₅ -Z- (C1-C10 alkyl) with Z = O, S, C = 0, S = 0, S0 ₂ ], C6-C12 aryl, C7 -C12 aralkyl, R5 = hydrogen, methyl

2. Substituted condensed pyrimidine-2,4 (1H, 3H) -diones according to claim 1, wherein the alkyl, alkylthio, alkoxy, alkoxycarbonyl, N-alkylcarbamoyl, alkenyl, alkynyl defined for R1 to R4 -, Cycloalkyl-, Cycloalkenyl- and Cycloalkinylgruppen as well as the Z group are mono or disubstituted by residues from the group: Carbamoyl, hydroxy, C1-C4 alkoxy, -CN, SO3H, CF ₃ , NH ₂ , carboxy, C1-C4 alkyl, C1-C4 N-alkyl-carbamoyl, C1-C4 N-dialkyl-carbamoyl, phenyl or Pi - Perazinylcarboxy or a heterocycle, where the alkyl groups mentioned above can be substituted by one or two hydroxyl groups, S0 ₃ H, NH ₂ , C1-C4 alkylamino, C1-C4 dialkylamino, or a heterocycle.

3. pyrimidine-2,4 (1 H, 3H) -dione according to claim 1 or 2 in the form of a 3-dimercaptoalkylpyrimidine-2,4 (1 H, 3H) -dione of the general formula I (a)

(la)

n = the numbers 0 or 1

R4 = hydrogen, C1 -C8 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, [- (CH ₂ ) ι-3 -Z- (C1 -C5 alkyl) with Z = O, S, 0 = O, S = 0, S0 ₂ ], C7-C12 aralkyl, where the alkyl, alkenyl, alkynyl groups and the Z group can preferably be mono or disubstituted by radicals from the group: carbamoyl, hydroxy, C1-C2 alkoxy, CN, S0 ₃ H, CF ₃ ,, carboxy, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl, C1-C4 N-dialkyl-carbamoyl or piperazinylcarboxy and the abovementioned alkyl groups can preferably be substituted by one or two hydroxyl groups , S0 ₃ H, NH ₂₎ C1 -C4 alkylamino, 01-04 dialkylamino, or a heterocycle. R5 = a hydrogen atom or a methyl group

A = a fused benzo ring unsubstituted or monosubstituted, disubstituted or trisubstituted with radicals which may be the same or different from the group: halogen, 01 to C3-alkyl, 01 to C3 alkoxy, trifluoromethyl, nitro-, carbamoyl-, -NH ₂ , Carboxy-, 01 -C4 alkoxycarbonyl, 01 -C4 N-alkyl- carbamoyl, arylalkylcarbamoyl, arylalkyloxycarbonyl, het-alkylcarbamoyl and

Het-alkyloxycarbonyl with aryl = phenyl, tolyl, pyridyl, pyrazinyl, pyrimidinyl,

Pyrrolyl, pyrazolyl, imidazolyl and Het = morpholinyl, piperazinyl, piperidinyl,

Pyrrolidinyl, imidazolidinyl or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with a

Oxygen or sulfur atom or a heteroaromatic bonded via two carbon atoms, this six-membered with one, two or three nitrogen atoms as heteroatoms or five-membered with a nitrogen atom or five-membered with an oxygen - or sulfur atom or five-membered with a nitrogen atom and an oxygen or a sulfur atom or five-membered with two nitrogen atoms as heteroatoms or five-membered with two nitrogen atoms and one sulfur atom or five-membered with three nitrogen atoms as heteroatoms, where the heteroaromatics defined above for A are mono-, di- or tri-substituted by radicals R6 and the substituents can be identical or different

01 to C3-alkyl, halogen, COR7, S02R7, OCOR7, OR8, C00R8, C0NR8R9, S02NR8R9, NR8R9 with R7 = C1 to C4-alkyl, C3 to C6-cycloalkyl, phenyl, R8 / R9 = independently of one another H, phenyl, Benzyl, the latter unsubstituted or mono-, di- or tri-substituted by methoxy, 01 to 04-alkyl or

R8 / R9 = as a straight-chain alkyl group together forming a ring with a nitrogen atom, a five-membered ring which may optionally contain a further heteroatom N, O, S, or together forming a six-membered heterocycle with a nitrogen atom and, if appropriate, a further nitrogen atom or an oxygen atom or a Sulfur atom, where five and / or six rings in the carbon skeleton can be substituted by C1 to 04 alkyl or by benzyl R6 = if A is thieno, also a fused 04- to 07-cycloalkyl ring which is an oxygen, a sulfur or can contain a nitrogen atom, where the nitrogen atom is unsubstituted or substituted by C1 to C4 alkyl, phenyl or benzyl and the latter two aromatics can be substituted by C1 to 03 alkyl, C1 to C3 alkoxy or halogen, furthermore for the case A = Thieno R6 also be: carboxy, an alkyl group 01 -C5 substituted with a heterocycle, a heterocycle and a 01 -C5 N-alkylcarbamoyl group which can be substituted with amino, sulfo, hydroxy, dihydroxy or a heterocycle.

4. 3-dimercaptoalkylpyrimidine-2,4 (1H, 3H) -dione according to claim 1, 2 or 3, characterized in that it is the following compounds

(S R) -3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercapto-2-methylpropyl) quinazoline-2,4- (1H, 3H) dione

(S R) -7-chloro-3- (2,3-dimercaptopropyl) quinazoline-2,4 (1H, 3H) -dione

(R, S) -6-chloro-3- (2,3-dimercaptopropyl) quinazoline-2,4- (1H, 3H) dione

(S R) -3- (2,3-dimercaptopropyl) -5-methyl-quinazoline-2,4 (1H, 3H) -dione

(S R) -3- (2,3-dimercaptopropyl) -8-methyl-quinazoline-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -6,7-dimethoxy-quinazoline-2,4- (1 H, 3H) dione

(R, S) -3- (2,3-dimercaptopropyl) pyrido [3,2-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) -3- (2,3-dimercaptopropyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercaptopropyl) pteridine-2,4 (1H, 3H) dione

(R, S) - 3- (2,3-dimercapto-2-methyl-propyl) pteridine-2,4 (1H, 3H) dione

(R, S) -5- (2,3-dimercapto-2-methyl-propyl) -2H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) - dione (R, S) -5- (2,3-dimercaptopropyl) -1H-pyrazolo [3,4-d] pyrimidine-4,6 (5H, 7H) -dione

2,4 (3H, 5H) -dione

(R, S) -3- (2,3-dimercaptopropyl) -1-methylquinazolin-2,4 (1H, 3H) -dione

(R, S) -3- (2,3-dimercaptopropyl) thieno [3,2-d] pyrimidine-2,4 (1H, 3H) dione

2.4 (1H, 3H) dione

5. A process for the preparation of 3-dimercaptoalkylpyrimidine-2,4 (1H, 3H) -dions according to one of claims 1 to 4, characterized in that

Monomercaptans of the general formula II

(II)

n = the numbers 0, 1 or 2,

A = a fused benzene ring or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with an oxygen or sulfur atom or a five- to seven-membered heteroaromatic bonded with two carbon atoms with one, two or three nitrogen or oxygen atoms - and or sulfur atoms as heteroatoms, where A is unsubstituted, mono-, di- or tri-substituted with halogen, C1-C6 alkyl, C1-C3-alkylthio-, C6- C12-aryl-, nitro-, carbamoyl-, C1-C4 alkoxy, -CN, CF ₃ , NH ₂ -, carboxy-, C1-C4 alkoxycarbonyl, C1 -C4 N-alkyl-carbamoyl or 01 -C5 alkenyl R5 = hydrogen , Methyl

be subjected to acidic hydrolysis in such a way that a monomer capaptan compound of the general formula II is introduced into an acid mixture of sulfuric acid, glacial acetic acid and water and heated for 8 to 10 hours, water is added after cooling and the precipitate is separated.

6. A process for the preparation of 3-dimercaptoalkylpyrimidine-2,4 (1H, 3H) -diones according to one of claims 1 to 3, characterized in that monomer captaptans of the general formula II

(left)

n = the numbers 0, 1 or 2,

A = a fused benzene ring or a fused five- to seven-membered cycloalkyl ring or a fused five- to seven-membered cycloheteroalkyl ring with an oxygen or sulfur atom or a five- to seven-membered heteroaromatic bonded with two carbon atoms with one, two or three nitrogen or oxygen atoms and / or sulfur atoms as heteroatoms, where A is unsubstituted, mono-, di- or tri-substituted with halogen, 01 -C6 alkyl, 01 -C3 alkylthio, 06- C12 aryl, nitro, carbamoyl, C1- C4 alkoxy, -CN, CF ₃ , NH ₂ -, carboxy-, C1-C4 alkoxycarbonyl, C1-C4 N-alkyl-carbamoyl or C1-C5 alkenyl R5 = hydrogen, methyl

be subjected to an alkaline hydrolysis such that a monomer capaptan compound of the general formula II is introduced in an inert atmosphere into a mixture of 3N NaOH / ethanol (10: 1) and zinc dust, the mixture is stirred at 80 to 85 ° C. until the reaction is complete, with further introduction of nitrogen, and the cooled reaction mixture is cooled under cooling and in an inert atmosphere Filtered 10% hydrochloric acid and the precipitate is separated.

7. The method according to claim 5 or 6, wherein the respective process products subsequently at the N1 position with a radical R4 = C1-C8 alkyl, C2-C5 alkenyl, C2-C8 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl , C3-C10 cycloalkynyl, [- (CH ₂ ) ι- ₅ - Z- (C1-C10 alkyl) with Z = O, S, 0 = O, S = 0, S0], C6-C12 aryl, C7- C12 aralkyl can be substituted.

8. Use of substituted condensed pyrimidine-2,4 (1H, 3H) -dions according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of cancer, rheumatism, inflammation and allergic reactions.

9. Use according to claim 8, wherein it is the 3-dimercaptoalkylpyrimidine-2,4 (1 H, 3H) -diones according to claim 3 or 4.