MXPA98000014A - Analogue compounds of thalidomide from the class of piperidin-2,6-dio - Google Patents

Abstract

The present invention relates to: Analogous compounds of thalidomide of the class of piperidine-2,6-diones, processes for their preparation as well as their use in medicament are described.

Description

ANALOGUE COMPOUNDS OF THALIDOMIDE FROM THE CLASS OF PIPERIDIN-2. ß-DIONAS Description of the invention The invention relates to thalidomide analogs of the piperidin-2,6-diones class, with a process for their preparation as well as their use in medicaments. Excessive formation of zytokine TNF-a (tumor necrosis factor A) plays a major role in the pathogenesis of Graft versus Host syndrome, in multiple sclerosis, in transplant rejection, in aphthous stomatitis, in erythema nodosum leprous, in the Morbus Boeck, in rheumatoid arthritis and in a series of other diseases that are accompanied by inflammatory symptoms. A starting point for the therapy of these diseases consists in the directed suppression of TNF-a release by the administration of immunosuppressive or suppressive active substances, such as dexamethazone, pentoxifylline or thalidomide. However, it is necessary to differentiate between the indications that require a general immunosuppression, and those in which it is necessary to evaluate the advantages and disadvantages (the pros and cons) of an immunosuppression. Thalidomide was found to be superior in the treatment of aphthous stomatitis compared to the classic immunosuppressants. Other examples of diseases in which thalidomide showed good activity without causing general immunosuppression are cutaneous lupus erythematodes (H + G __, 816 to 822 (1994)), gangrenous polymorphism and orogenital ulcer in Morbus Behcet (The Lancet, 20.05 .89, 1093 to 1095). The endogenous mediators with activity on the endothelium and circulating leukocytes are considered as pathogenetic factors of these lesions that are limited to the skin and mucous membranes. Under the influence of TNF-a and other zytokines, the adhesiveness of the endothelium increases focally with respect to leukocytes, which contributes greatly to the formation of vasculitides. In the case of systemic disease pictures, the activity of thalidomide itself is limited to the skin and mucous membranes, which requires immunosuppression (additional). Examples of this are systemic lupus erythematosus, which in addition to skin manifestations also presents changes with danger of death of internal vessels, especially the kidney; Reaction to type II leprosy involving the eyes and / or joints, as well as Morbús Behcet involving the eyes and / or joints.

Substances such as thalidomide suppress this modification of the endothelium, but simultaneously block all or part of the reactions of specific cellular immune resistance may represent a significant progress for the therapy of the aforementioned systemic diseases. A key messenger substance of the cellular immune response is interleukin 2, on which the proliferation of specific antigen-specific lymphocytes depends. In the development of new drugs it is therefore a goal to highlight the anti-inflammatory properties of thalidomide together with immunosuppressive active components that thalidomide alone does not have in clinical application. The task on which the invention was based consisted in the development of thalidomide analogs of the piperidin-2,6-diones class, which inhibit the release by inflammation of TNF-α as well as the synthesis induced by antigens of the interleukin 2. It was found that the compounds according to the invention meet the formulated requirements.

Accordingly, piperidine-2, 6-diones substituted in position 3 and 5 of the general formula (I) R ° are the subject of the invention. where Z represents one of the groups RA R * RA / / -C- -CH2- -C = CH- wherein the carbon atom is linked to the carboxyl group with the R substituent and in which R stands for the phthalimide radical (when Z is = a -C (RR) -CH2-) or represents a phthalimide radical substituted in a single or double form with hydroxy, methoxy or amino groups (when Z represents -C (R) = CH-), R2 is hydrogen or C-alkyl. s (straight or branched chain), R represents hydrogen, a Cx_g alkyl group (straight or branched chain), or an aromatic or heteroaromatic ring system, and R4 means a C_6 alkyl group (straight or branched chain), or an aromatic or heteroaromatic ring system.

Of the piperidin-2, 6-diones of the formula (I) wherein Z = -C (RV) -CH2-), R1 means phthalimide and R2 and R3 signify hydrogen, the compound in which R4 is particularly suitable is phenyl. Of the piperidine-2, 6-diones of the formula (I) in which Z = -CÍR1) = CH- and R3 signifies ethyl and R 4 signifies phenyl, the compound in which R is 3,4-dimethoxyphthalimide is particularly suitable. . Another additional object of the invention is a process for the preparation of the thalidomide analogs of the class of piperidin-2,6-diones of the general formula (I). The compounds of the general formula (I) in which Z = -C ^ R2) -CH2-) can be prepared by the condensation of phthalic acid anhydride with a substituted glutamic acid, such as for example 4-phenylglutamic acid or 4-phenylglutamic acid. -methylglutamic acid in an organic solvent, preferably pyridine, cyclization of the product in acetic anhydride and subsequent transformation into the imide. The transformation of the anhydride into the imide is carried out for this by melting with urea. These target compounds of the formula (I) can also be obtained by reacting phthalic anhydride with a 5-substituted 3-amino-glutarimide, preferably by heating in acetic acid.

The compounds of the general formula (I) in which Z = -C (R) = CH- can be prepared by the condensation of substituted phthalic anhydride, such as, for example, 3, 4-dimethyloxyphthalic anhydride with 3-amino 3,4-substituted 4-dehydropiperidine-2,6-diones, such as for example 3-amino-5-ethyl-5-phenyl-glutaconimide in an organic solvent, for example acetic acid.

Example 1 2- (5-methyl-2,6-dioxo-piperidin-3-yl) -1,3-dihydro-2H-isoindole-1,3-dione (1) 2.00 g (11 mmol) of 4-acid Methylglutamic acid and 1.95 g (13 mmol) of phthalic anhydride were heated for 6 h to reflux in 15 ml of dry pyridine. After removal of the solvent by distillation, the residue was heated to boiling for 1 h in 10 ml of acetic anhydride. The solid that precipitated during the cooling was sucked and the filtrate was concentrated. After mixing the filtrate with ether, the precipitate that formed was suctioned and the combined precipitates were recrystallized with absolute toluene. 2.00 g (7 mmol) of the crystallized product and 0.23 g (3.8 mmol) of urea were mixed thoroughly and melted in an oil bath for 30 min. at approximately 200 ° C. The solidified melt was briefly heated to boiling successively with 4 ml of acetic anhydride and 6 ml of ethanol. The precipitated solid was separated by suction and recrystallized with DMF / water. Obtained 1.35 g (67% of theory) of 2- (5-methyl-2,6-dioxo-piperidin-3-yl) -1,3-dihydro-2H-isoindol-1,3-dione (1) with a melting point of 270 to 272 ° C.

Example 2 2- (5-pheny1-2,6-dioxo-piperidin-3-yl) -1,3-dihydro-2H-isoindole-1,3-dione (2) 3.00 g (12 mmol) of 4-acid Phenylglutamic acid and 2.12 g (14 mmol) of phthalic anhydride were heated for 6 h to reflux in 40 ml of dry pyridine. After the solvent was distilled off, the residue was taken up in 50 ml of 5% HCl and extracted with ethyl acetate. The organic phase was washed with water, de-colored with activated charcoal and dried over sodium sulfate. After removal by distillation of the solvent, the residue was heated for 1 h at reflux in 40 ml of acetic anhydride. The solution was then concentrated and mixed with ether. The precipitate that formed was suctioned and recrystallized with dry toluene. 2.00 g (6 mmoles) of the crystallized product and 0.19 g (3 mmoles) of urea were melted in an oil bath for 30 min. at approximately 200 ° C. The solidified melt was heated to boiling successively with 4 ml of acetic anhydride and 8 ml of ethanol. The precipitated solid was recrystallized with DMF / water. 0.80 g (40% of theory) of 2- (5-phenyl-2,6-dioxo-piperidin-3-yl) -1,3-dihydro-2H-isoindol-1,3-dione (2) were obtained. with a melting point of 228 to 231 ° C.

Example 3 2- (5-ethyl-5-phenyl-2,6-dioxo-piperidin-3-yl) -1,3-dihydro-2H-isoindol-1,3-dione (3) 1.0.0 g (4 mmoles) of 3-amino-5-ethyl-5-phenyl-glutaconimide were dissolved in 40 ml of anhydrous ethanol, the solution was mixed with 0.1 g of palladium on carbon (10% Pd / C) and stirred under hydrogen atmosphere for 8.5 h. The catalyst was then removed by filtration and the filtrate was concentrated to dryness. The residue was refluxed for 4 h with 0.70 g (5 mmoles) of phthalic acid anhydride in 40 ml of glacial acetic acid. After removal by distillation of the solvent, the residue was recrystallized from ethanol. 0.99 g (63% of theory) of 2- (5-ethyl-5-phenyl-2,6-dioxo-piperidin-3-yl) -1,3-dihydro-2H-isoindol-3, 3- were obtained dione (3) with a melting point of 174-177 ° C.

Example 4 2- (5-ethyl-5-phenyl-2,6-dioxo-1, 2,5,6-tetrahydropyridin-3-yl) -4,5-dimethoxy-1,3-dihydro-2H-isoindol- 1, 3- dione (4) 0.45 g (2 mmoles) of 3-amino-5-ethyl-5-phenyl-glutaconimide and 0.45 g (2 mmoles) of 4,5-dimethoxyphthalic acid anhydride were heated for 5 h reflux in 15 ml of glacial acetic acid. It was then concentrated to dryness and the residue was recrystallized from ethanol. 0.55 g (67% of theory) of 2- (5-ethyl-5-phenyl-2,6-dioxo-1, 2,5,6-tetrahydropyridin-3-yl) -4,5-dimethoxy- were obtained. 1,3-dihydro-2H-isoindol-l, 3-dione (4) with a melting point of 203-205 ° C.

The compounds according to the invention are toxicologically harmless and therefore are suitable as pharmacological active substances. Accordingly, the invention also relates to the use of thalidomide analogs of the class of piperidine-2,6-diones of the formula (I) as active substances in medicaments, preferably as suppressants of the release of TNF-α triggered by inflammation as well as the synthesis induced by antigens of interleukin 2.

The medicaments according to the invention contain in addition to at least one compound of the formula (I) carrier materials (carriers), fillers, solvents, diluents, colorants and / or binders. The selection of auxiliary materials as well as the amounts to be used are established depending on the form of administration of the drug, whether oral, intravenous, intraperitoneal, intradermal, intramuscular, intranasal, buccal or local. For the oral application, preparations in the form of tablets, chewable tablets, dragees, capsules, granules, drops, juices or syrups are suitable for parenteral applications, topical and inhalative solutions, suspensions anhydrous preparations of easy reconstitution, as well as preparations for spraying . The compounds according to the invention in dissolved form in a reservoir, in a carrier sheet or in an adhesive bandage, optionally with the addition of means promoting penetration into the skin, are suitable examples of percutaneous application forms. From forms of preparation to be used orally or percutaneously, the compounds according to the invention can be released in deferred form. The amount of active substance to be administered to patients varies according to the weight of the patient, the method of application, the indications and the degree of severity of the disease. In general, from 1 to 150 mg / kg of at least one thalidomide-like compound of the formula (I) are applied.

Pharmacological investigations The release of TNF-α can be checked in vitro in human peripheral blood mononuclear cells (T cells, B cells, monocytes) after lipopolysaccharide (LPS) estimation. LPS is a component of the bacterial cell wall and stimulates monocytes and macrophages. In addition to stimulation with LPS, it is also possible to provoke the release of TNF-a by stimulating human peripheral blood mononuclear cells with monoclonal antibodies against T cell-specific Activation Antigens (AntiCD2-AntiCD28)., or with the bacterial superantigen Toxin Shock Syndrome Toxin 1 TSST-1. Apart from the release of TNF-a, these stimulants lead inter alia to the formation of interleukin 2 (11-2). Stimulation of mononuclear cells with LPS: Effect on TNF-a The release of TNF-a can be investigated __ vitro in human peripheral blood mononuclear cells, specifically T cells, B cells and monocytes, after the lipopolysaccharide (LPS) estimation . LPS is a component of the bacterial cell wall and stimulates monocytes and macrophages. Mononuclear cells were obtained from the heparinized blood of at least three volunteer donors. For this purpose 20 ml of blood were separated in each case on a Ficoll-Paque gradient according to known methods, the cells were harvested and washed three times with a cell culture medium. This cell culture medium consisted of RPMI 1640 medium supplemented with 2 mM glutamine (Life Technologies, Eggenstein), 10% fetal calf serum (Life Technologies), 50 μg / ml streptomycin (Sigma, Deisenhofen), 50 IU / ml of penicillin (Sigma) and 100 μM of β-mercaptoethanol (Merck, Darmstadt). Finally, the mononuclear cells were incorporated in 15 ml of cell culture medium and distributed in 1 ml tanks in sterile 24-well incubation plates (Sigma). 1 μl of dimethylsulfoxide (DMSO, Merck) or 1 μl of a solution of the test substance (in DMSO) were added to the 1 ml tanks in each case.; final concentration in the assay: 0.5; 5; 12.5 and 50 μg / ml), and the deposits were incubated for one hour in a C02 incubator (5% C02, 90% air humidity). Except for the control samples, then 2.5 μg of LPS (from E. coli 0127: B8, Sigma) was added in each case. The incubation of the cultures was continued for 20 h. The concentration of TNF-a in the supernatants of the cell cultures was determined following the incubation by commercial ELISA assays (Boehringer, Mannheim). The inhibitory potency of TNF-α was calculated from the measured values of the control tanks not treated with active substance and the deposits incubated with the test compounds. With the aid of a regression line, the concentrations that led to a 50% inhibition of TNF-a libreation (IC50 values) were calculated. The inhibitory influence of the compounds according to the invention on the release of TNF-α induced by LPS is shown in Table 1: Table 1 T cell stimulation: Inhibition of 11-2 The release of Interleukin 2 can be verified by in vitro stimulation of human peripheral blood mononuclear cells, which apart from T cells also contains B cells and monocytes. By means of polyclonal stimulation on constant epitopes of the T-cell receptor or so-called surface molecules transmitting accessory signals, a more marked measurement path is obtained than by the antigenic stimulation of small populations of T cells. The combination of 2 such signals was used accessory, specifically those through the surface molecules CD2 and CD28. Mononuclear cells were obtained from the heparinized blood of at least three volunteer donors. For this purpose, in each case 20 ml of blood were separated on a Ficoll-Paque gradient according to known methods, the cells were harvested and washed three times with a cell culture medium. This cell culture medium consisted of RPMI 1640 medium supplemented with 2 mM glutamine (Life Technologies, Eggenstein), 10% fetal calf serum (Life Technologies), 50 μg / ml streptomycin (Sigma, Deisenhofen), 50 IU / ml of penicillin (Sigma) and 100 μM of β-mercaptoethanol (Merck, Darmstadt). Finally, the mononuclear cells were incorporated in 15 ml of cell culture medium and distributed in 1 ml tanks in sterile 24-well incubation plates (Sigma). To the 1 ml tanks were added in each case 1 μl of dimethylsulfoxide (DMSO, Merck) or 1 μl of a solution of the test substance (in DMSO, final concentration in the assay: 0.5, 5, 12.5 and 50). μg / ml), and the deposits were incubated for one hour in a CO2 incubator (5% C02, 90% air humidity). With the exception of the control samples, 0.1 μg / ml of the monoclonal antibodies were added in each case (Clone No. AICD2.M1 of Prof. Dr. Meuer, anti-CD28 of CLB, Amsterdam). The incubation of the cultures was continued for 20 h. The concentration of TNF-a in the supernatants of the cell cultures was determined following the incubation by commercial ELISA assays (Boehringer, Mannheim). From the measured values of the control tanks not treated with active substance and the deposits incubated with the test compounds, the inhibition potency of 11-2 was calculated. Under these conditions, at a concentration of 50 μg / ml, the substance of example 4 inhibited by 86 ± 6% the synthesis of 11-2 stimulated by CD2 / CD28. In the case of the use of the Estrafilococo Superantigen (from E. coli 0127: B8; Sigma, Deisenhofen) TSST-1 (0.1 μg / ml) as a T-cell stimulant, the synthesis of 11-2 was inhibited in 77 ± 20%. The investigations carried out above show that the thalidomide analogs of the piperidin-2,6-diones class of the formula (I) inhibit both the release of TNF-a caused by inflammation, as well as the synthesis of Interleukin. 2 induced by antigen.

Claims (1)

1. CLAIMS Piperidin-2, 6-substituted diones of the formula (I), which are characterized by the fact that Z means -C (R ^ 2) -CH2- or -C (RX) = CH-, R1 means phthalimide (when Z = -C (R ^ 2) -CH2-) or represents a phthalimide radical substituted in a simple or double manner with hydroxy, methoxy or amino groups (when Z = -CÍR1) = CH-), R2 means hydrogen or an alkyl group ^ g R is hydrogen, a Cx_6 alkyl group or an annular system (hetero) aromatic or heteroaromatic, and R4 represents C1_6 alkyl or an annular (hetero) aromatic system. Piperidine-2, 6-substituted diones of the formula (I) according to claim 1, characterized in that Z is -C (RXR2) -CH2-, R means phthalimide, R represents hydrogen, R means hydrogen or ethyl, and R represents methyl or phenyl. Piperidin-2, 6-substituted diones of the formula (I) according to claim 1, characterized by the fact that Z is -CÍR1) = CH-, R means 3,4-dimethoxyphthalimide, R represents ethyl, and R represents phenyl. Process for the preparation of substituted piperidin-2,6-diones of the formula (I) according to claim 1 and 2, characterized in that an anhydride of phthalic acid is condensed with a substituted glutamic acid, the product is cyclized to obtain anhydride, and this is transformed into the imide. Process for the preparation of substituted piperidin-2,6-diones of the formula (I) according to claim 1 to 3, characterized in that an anhydride of phthalic acid is condensed with substituted 3-aminoglutamimides or with 5-substituted aminoglutarimides. Use of a substituted piperidine-2,6-dione of the formula (I) according to claim 1 to 3 as active substance in a medicament. Use according to claim 6, characterized in that the medicament is a modulator of immunity.