ITMI962241A1 - USE OF TETRAIDRO-BETA-CARBOLINIC DERIVATIVES AS ANTIMETASTATIC AGENTS - Google Patents

Description

"USE OF TETRAHYDRO-BETA-CARBOL NI CI DERIVATIVES AS ANTI-METASTATIC AGENTS"

The present invention relates to the use of beta-carboline derivatives for the preparation of pharmaceutical compositions having antimetastatic properties.

The cells of metastasizing tumors are able to migrate from the original tumor to other target organs by means of a mechanism that involves the penetration through the walls of the blood capillaries, the entry of tumor cells into the bloodstream, followed by a subsequent re-crossing of the walls of the vessels until they reach the target organ.

The penetration through the connective tissue of the vessels occurs thanks to the degradation of the extracellular matrix by metalloproteinases released by cells of the resident connective tissue and activated by the tumor cells This mechanism, common also to non-tumor tissues, is however normally in dynamic equilibrium with the regeneration of the connective tissue, while it manifests itself in an uncontrolled manner in invading cells such as tumor or inflammatory cells and is involved in numerous other diseases such as rheumatoid arthritis, osteoarthritis, septic arthritis, corneal, epidermal or gastric ulcerations, coronary thrombosis, proteinuria ( WO 95/13289). _ _ Three types of metallo proteinases are involved in these processes: collagenase, gelatinase and stromelysins. Under normal conditions their release and activity are strictly regulated by endogenous proteinase inhibitors, such as a2-macroglobulin.

Metalloproteinase inhibitors can therefore be useful in the treatment of the pathological conditions described above as well as the pathological consequences of trauma or even as contraceptive agents, since metalloproteinases are involved in the phenomena of ovulation and subsequent implantation of the ovum on the uterine wall. In particular, re-inhibition of tumor metastasis by metalloproteinase inhibitors is described in Matrisian et al, PNAS USA, 83, 9413-7 (1986); Wilhelm et al., PNAS USA, 84, 6725-29 (1987); Werb et al., J. Cell Biol., 109, 872-89 (1989); Liotta et al, Lab. Invest., 49, 636-49 (1983).

Metal proteinase inhibitors are disclosed in US 4,511,504, US 4,568,666, US 4,771,037, WO 95/13289.

Beta-carboline derivatives are described to have various pharmaceutical activities, such as for example antitumor activities [Anticancer Res., 13 (6A1, 2301-8 (1993); J. Antibiot., 46 (111, 1672-7 (1993); EP 357.122], anti-ulcer [WO 92/04348 (19.03.92)], anti-malarial [J. Nat. Prod., 54 (51, 1360-7 (1991)] or are described as agents promoting the absorption of anticancer drugs ( JP 04275221).

However, for none of these molecules an antimetastatic activity is described.

We have surprisingly found that the tetrahydro-beta-carbolines of formula (I) are endowed with a remarkable activity of inhibition of the metastatic process:

U)

R is selected from the group comprising hydrogen, (Ci-Cj) linear or branched alkyl, phenyl (optionally substituted by a (Ci-C5) alkoxy group), - (CH2) n-COOH, where n is an integer from 1 to 3 ;

Ri is hydrogen or a -COOR4 group, where R4 is hydrogen or (C1-Csjalkyl;

R2 is hydrogen or a -COOR4 group as defined above;

R3 is selected from the group comprising hydrogen, halogen (chlorine, bromine, fluorine or iodine), (Cj-C4) alkoxy, benzyloxL

The object of the present invention is the use of the compounds of formula (I), as antimetastatic agents and inhibitors of the tumor invasion process.

Also included in the present invention are the enantiomers, racemates and diastereoisomes of the compounds of formula (I), as well as their salts with pharmaceutically acceptable acids or bases.

Preferred examples of compounds of formula (I) are:

6-methoxy-1,2,3,4-tetrahydronorharmane;

1,2,3, 4-tetrahydronorharman-3-carb or silicon acid;

6-methoxy-1,2, 3, 4-tetrahydronorharman-1-carboxylic acid;

1- (4-methoxyphenyl) - 1,2,3, 4-tetrahydronorharman-3-carboxylic acid;

1-methyl-1,2, 3, 4-tetrahydronorharman-3-carboxylic acid;

1-methyl-1,2,3, 4-tetrahydronorharman-1,3-dicarboxylic acid;

1- (diethylmethyl) -1, 2,3,4-tetrahydronorharman-3-carboxylic acid;

(6-bromo- 1,2, 3, 4-tetrahydronorharman- 1 -yl) -3-propionic acid;

1-isobutyl-1,1,3,4-tetrahydronorharman-3-carboxylic acid;

1-phenyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

1-propyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

1-methyl-1-methoxycarbonyl-6-benzyloxy-1,2,3, 4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-6-methoxy-1,2, 3, 4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-6-hydroxy-1,1,3,4-tetrahydronorhannane;

1-methyl-1-methoxycarbonyl-6-chloro-1,2,3, 4-tetrahydronorharmane;

1-methyl- 1 -methoxycarbonyl-6-bromo- 1, 2,3, 4-tetrahydronorharmane;

1-methyl- 1-methoxycarbonyl- 1,2,3,4-tetrahydronorphiannane.

The compounds included in the present invention are known compounds and are commercially available or can be obtained by extraction from vegetable matrices or synthesized according to methods reported in the literature (see for example WO 92/04348).

The compounds of the present invention were assayed in an "in vitro" pharmacological test for inhibition of MMP8 (human neutrophil collagenase). The test involves the determination by fluorescence of the inhibition of the degradation activity of a fluorescent substrate (DNP-Pro-Leu-Gly-Leu-Tip-Ala-D-Arg-NH2, M1855 Bachem) by the catalytic domain of MMP8 .

Reagents:

1) DNP- sub layer = DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (MI 855 Bachem), P.M.

977.1 g / mol, concentration 25 μΜ in DMSO; 2) measuring buffer - 50 mM TRIS / 100 mM NaCl / 10 mM CaCl2.2H20, adjusted to pH 7.6 with hydrochloric acid. 3) Enzyme = catalytic domain of MMP8 (92 KDa), concentration 0.055 mg / ml in TRIS buffer. Substrate and enzyme are kept at 0 ° C with ice.

Inhibition test:

Total volume = 1 ml of solution kept under stirring in a cuvette.

Control: 0. 98 mi DMSO

0.01 ml of DNP-substrate

0.01 ml of enzyme

Experiment: 0.98 mi DMSO

0.01 mi DNP- sub layer

0.01 ml enzyme

0.01 mi inhibitor (10 pg / ml)

The fluorescence at 346 nm of both the control solution (without inhibitor) and the solution containing the inhibitor is measured. The inhibition of the catalytic activity of MMP8 causes a decrease in the lysis of DNP-substrate, with a consequent decrease in fluorescence.

The percentage of inhibition is expressed by the following formula:

Inhibition% = 100 - (unit rekt./tempOcon bibit / relative unit/tempOcon<>iio x 100) Repeating the experiment at various concentrations of inhibitor it is possible to calculate the respective ICso-Table I shows the enzyme inhibition data for some representative compounds of the invention.

Table I.

The compounds of the present invention were also found to be active in an "in vivo" chemoinvasion test. In the chemoinvasion test the Costar Transwell cell culture chambers (diameter: 6.5 mm; pore diameter: 8 μιη) are coated on the bottom with 100 μΐ of type IV collagen (diluted solution 50 pg / ml, then evaporation overnight). With the same procedure, the chambers are covered with a second layer of type IV collagen (100 μ of solution at a concentration of 50 pg / ml). Before use, the chambers are washed twice with sterile water and incubated for about 1 hour at 37 ° C in a medium (DMEM) deprived of serum. HT1080 human fibrosarcoma cells are harvested by trypsin-EDTA treatment, washed with DMEM 10% FCS and incubated for at least 30 minutes at 37 ° C in the same medium. The cells are then washed with serum-deprived DMEM and resuspended in serum-deprived DMEM added with 0.1% BSA (fraction V), counted and diluted to obtain a final density of 3x105 cells / ml.

The serum-free medium is aspirated from the pre-incubated chambers. The lower compartment of the chambers is filled with 600 μΐ of DMEM 20% FCS 1% BSA (fraction V) compound to be tested. 200 μΐ of cell suspension (6x104 cells) containing the compound to be tested are added to the upper compartment and the chambers are incubated at 37 ° C in a humid atmosphere with C02. After a first 24 hour incubation, the media from the lower and upper compartments are replaced with fresh suspensions and the chambers incubated for another 24 hours.

The incubated filters are then washed with PBS, the cells are fixed for 15 minutes in 4% paraformaldehyde, penneabilized in methanol (10 minutes, -20 ° C) and stained with May-Grunwald-Giemsa. The cells adhering to the upper face of the filters are removed with a cotton swab, the filters are detached from the bottom of the chambers and analyzed under a microscope to determine the number of cells on the lower face of the filters.

In a control experiment, in the absence of a metalloproteinase inhibitor, HT1080 cells, which hyper-metallo-proteinase, are able to degrade type IV collagen and migrate to the underside of the filters. In the experiment with the inhibitor, on the other hand, the activity of the metallo-proteinases is partially or totally inhibited, with a consequent lower number of cells that migrate on the lower face of the filters. bottom of the filter in the control experiment and in the inhibitor experiment.

Table Π shows the data relating to two compounds representative of the invention.

Table II

From the above it is evident that the compounds of the invention can also be used in the treatment of all conditions associated with the action of matrix metallo-proteinases, such as rheumatoid arthritis, osteoarthritis, septic arthritis, corneal, epidermal or gastric ulcerations, coronary thrombosis, proteinuria, pathological consequences of trauma or even as contraceptive agents.

The compounds of the present invention can be administered in amounts ranging from 0.01 mg to 0.4 g per kilogram of body weight per day. A preferred dosage regimen in order to obtain optimal results is that which involves the use of about 1 mg to about 50 mg per kilogram of body weight per day, using unit doses so as to administer from about 70 mg to about 3.5 g of the active compound. to a subject of about 70 kg of body weight in a period of 24 hours. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, divided doses may be administered according to the needs of the therapeutic situation. The active compound can be administered orally, intravenously, intramuscularly or subcutaneously.

The pharmaceutical compositions to which the present invention refers contain at least one compound of the invention in therapeutically effective quantities in admixture with pharmaceutically compatible excipients.

Oral compositions will generally include an inert diluent or an edible carrier. They can be included in gelatin capsules or tablets in tablets. Other forms of oral administration are capsules, pills, elixirs, suspensions or syrups.

The tablets, pills, capsules and similar compositions may contain the following ingredients (in addition to the active ingredient): a binder such as microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, primogel, corn starch and the like; a lubricant such as magnesium stearate; a fluidifier such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin or a flavoring agent such as mint flavor, methyl salicylate or orange flavor. When the selected composition is in capsule form, it may additionally contain a liquid carrier such as a fatty oil. Other compositions may contain various materials that alter their physical form, such as coating agents (for tablets and pills) such as sugar and shellac. The materials used in the preparation of the compositions should be pharmaceutically pure and non-toxic at the dosages employed.

For the preparation of pharmaceutical compositions for parenteral administration, the active ingredient can be embedded in solutions or suspensions, which may additionally include the following components: a sterile diluent such as water for injections, saline, oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting the tonicity of the solution such as sodium chloride or dextrose.

The parenteral preparation can be included in ampoules, disposable syringes, or glass or plastic vials.

Claims (8)

CLAIMS: 1. Use of compounds of fomula (I): (i) R R in which: R is selected from the group comprising hydrogen, (Ci-Cs) linear or branched alkyl, phenyl (optionally replaced by a (Ci-Cj) alkoxy group), - (CH2) B-COOH, where n is an integer from 1 to 3 ; R1 is hydrogen or a -COOR4 group, where R4 is hydrogen or (Ci-Cs) alkyl; R2 is hydrogen or a -COOR4 group as defined above; R3 is selected from the group comprising hydrogen, halogen (chlorine, bromine, fluorine or iodine), (C r C4) alkoxy, benzyloxy, their enantiomers, racemates, diastereoisomers and their salts with pharmaceutically acceptable acids and bases, for the preparation of a pharmaceutical composition having antimetastatic or tumor invasion inhibition activity. Use according to claim 1, wherein said compounds are: 6-methoxy- 1,2, 3, 4-tetrahydronoiharmane; 1,3,4-tetrahydronorharmano-3-carboxylic acid; 6-methoxy-1,2,3, 4-tetrahydronorharman-1-carboxylic acid; 1- (4-methoxyphenyl) -1, 2,3,4-tetrahydronorharman-3-carboxylic acid; 1-methyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid; 1-methyl-1,2,3,4-tetrahydronorharman-1,3-dicarboxylic acid; 1- (diethylmethyl) - 1,2,3,4-tetrahydronorharman-3-carboxylic acid; (6-bromo-1,2,3,4-tetrahydronorharman-1-yl) -3-propionic acid; 1-isobutyl-1,2,3, 4-tetrahydronorharman-3-carboxylic acid; 1-phenyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid; 1-propyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid; 1-methyl-1-methoxycarbonyl-6-benzyloxy-1,2,3, 4-tetrahydronorharmane; 1-methyl-1-methoxycarbonyl-6-methoxy-1,2,3, 4-tetrahydronorhannane; / 1-methyl-1-methoxycarbonyl-6-hydroxy-1,2, 3, 4-tetrahydronorharmane; 1-methyl-1-methoxycarbonyl-6-chloro- 1,2, 3, 4-tetrahydronorharmane; 1-methyl-1-methoxycarbonyl-6-bromo-1,2, 3, 4-tetrahydronorharmane; 1-methyl- 1-methoxycarbonyl- 1, 2, 3, 4-tetrahydronorharmane. Use according to claim 2, wherein the active ingredients are: 6-methoxy-1, 2, 3, 4-tetrahydronorharmane; 1,3,4-tetrahydronorharmano-3-carboxylic acid; 6-methoxy- 1,2,3,4-tetrahydronorharman- 1-carboxylic acid. 4. Use of compounds of phonatyl (I): (i) R is selected from the group comprising hydrogen, (Ci-C5) linear or branched alkyl, phenyl (optionally substituted by a (CrC5) alkoxy group), - (CH2) n-COOH, where n is an integer from 1 to 3; R1 is hydrogen or a -COOR4 group, where R4 is hydrogen or (Ci-Cs) alkyl; R2 is hydrogen or a -COOR4 group as defined above; R3 is selected from the group comprising hydrogen, halogen (chlorine, bromine, fluorine or iodine), (Ci-C4) alkoxy, benzyloxy, their enantiomers, racemates, diastereoisomers and their salts with pharmaceutically acceptable acids and bases, for the preparation of a pharmaceutical composition for the prevention or treatment of associated conditions. to the action of matrix-metallo proteinases. 5. Use according to claim 4, wherein said compounds are: 6-methoxysi- 1, 2, 3, 4-tetrahydronorharmane; 1,3,4-tetrahydronorhannano-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 1- (4-methoxyphenyl) - 1,2,3, 4-tetrahydronorharman-3-carboxylic acid; 1-methyl-1,3,4-tetrahydronorbarman-3-carboxylic acid; 1-methyl- 1, 2,3,4-tetrahydronorharman-1,3-dicarboxylic acid; 1- (diethylmethyl) - 1,2,3, 4-tetrahydronorharman- 3-carbonic acid; (6-bromo- 1,2, 3, 4-tetrahydronorharman-1-yl) -3-propionic acid; 1-isobutyl-1,2,3, 4-tetrahydronorharman-3-carboxylic acid; 1-phenyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid; 1-propyl-1, 2,3,4-tetrahydronorharman-3-carboxylic acid; 1-methyl-1-methoxycarbonyl-6-benzyloxy-1,2,3, 4-tetrahydronoiharmane; 1-methyl-1-methoxycarbonyl-6-methoxy-1,2,3,4-tetrahydronoiharmane; 1-methyl-1-methoxycarbonyl-6-hydroxy-1,2,3, 4-tetrahydronorharmane; 1-methyl-1-methoxycarbonyl-6-chloro-1,2,3, 4-tetrahydronorharmane; 1-methyl-1-methoxycarbonyl-6-bromo-1,2, 3, 4-tetrahydronorharmane; 1 -methyl- 1 -metus ssicarb onyl- 1, 2,3, 4-tetr aidronorhannano. 6. Use according to claim 5, wherein the active ingredients are: 6-methoxy- 1, 2,3,4-tetrahydronorharmane; 1,2,3,4-tetrahydronorharmano-3 - carboxylic acid; 6-methoxy- 1,2,3, 4-tetrahydronorharman- 1-carboxylic acid. Use according to any one of claims 4-6, wherein the condition to be treated is selected from the group consisting of: rheumatoid arthritis, osteoarthritis, septic arthritis, corneal, epidermal or gastric ulcerations, coronary thrombosis, proteinuria, pathological consequences of trauma . 8. Use according to any of claims 4-6, for the prevention of ovulation or implantation of the ovum in the uterus.