MXPA97005965A - Derivatives of istitiourea biciclicos, useful in tera - Google Patents

Abstract

The present invention relates to novel compounds of the formula (I) wherein D represents alkyl with C1 to 6, T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) m-NXY; (CH2) 2-NH- substituted by - (CH2) m-NXY; or -U- (CH2) aN (X) - (CH2) b-; a, b, m, X and Y are as defined in specification together with processes for their preparation compositions containing the same and their use in therapy. The compounds of the formula (I) are expected to be useful inter alia in the treatment of neurodegenerative disorders.

Description

DERIVATIVES OF ISOTIOUREA BICÍCLICOS, USEFUL IN THERAPY Field of the Invention The present invention relates to novel compounds, to processes for their preparation, to compositions that contain them and to their use as neuroprotective agents.

Background of the Invention The thiourea and isothiourea derivatives have been described above for a variety of therapeutic uses. WO 94/12165 (Wellcome) describes isothiourea derivatives for use in the inter alia treatment of systemic hypotension, septic shock and inflammatory conditions; WO 95/09619 (Wellcome) (published after the priority date of this application) discloses substituted urea and isothiourea derivatives for use in the treatment of cerebral ischemia; U.S. Patent No. 1178242 (Wellcome) discloses bisisothioureas having an anti-inflammatory activity; European Patent Application No. 411615 (Warner Lambert) discloses thiourea derivatives having use in the treatment of Ref.025278 the symptoms of cognitive decline; European Patent Application No. 392802 (Beecham) describes thiourea derivatives for use in the treatment of bronchial, cerebrovascular or neuronal disorders. Isothiourea derivatives are also known as chemical intermediates in the preparation of guanidine derivatives (see U.S. Patent No. 4,211,867 (McNeil Laboratories) and Synthesis (1988) 6, 460-466 (Rasmussen) which describes the 4-dimethylaminophenylcarbamimidothioic acid methyl ester compound and U.S. Patent No. 5,223,498 (Boots) The N-alkoxyphenyl-N'-quinolinyl thiourea derivatives useful as tuberculostatic agents are described in DE- B-1157626 (Hoechst) International Patent Application No. WO 95/05363 (Fisons) (published after the priority date of this application) describes N-phenyl amidine derivatives which are indicated for the inter alia treatment of The neurodegenerative diseases We have now discovered a novel and useful group of isothiourea derivatives In accordance with the invention, the compounds of the formula I are provided H wherein D represents alkyl with Cl at 6 T represents a saturated or unsaturated alkylene chain with C3_5 substituted by - (CH2) m-NXY; -0- (CH2) 2-NH- substituted by - (CH2) m-NXY; or -U- (CH2) a-N (X) - (CH2) b-; U represents NH, 0 or CH2; a and b, which may be the same or different, represent an integer from 0 to 3, provided that a + b is in the range of 1 to 3; X and Y, which may be the same or different, represent hydrogen, alkyl with Cl to 6, or a group - (CH2) nQ, or NXY together represent piperidinyl, pyrrolidinyl, morpholinyl, or tetrahydroisoquinolinyl; Q represents phenyl or phenyl substituted by one or more substituents selected from the group consisting of alkyl with Cl to 6, alkoxy with Cl to 6, trifluoromethyl, halogen, nitro and cyano; m and n independently represent a whole number of 0 to 5; and pharmaceutically acceptable salts thereof. It is preferred that T represents -U- (CH2) a-N (X) - (CH2) b-. It is particularly preferred that T represents -U- (CH2) a-N (X) - (CH2) b- and U represents CH2. It is especially preferred that T represents -U- (CH2) a-N (X) - (CH2) b-. U represents CH2 and a and b each represent 1. It is preferred that D represents alkyl with Cl to 3, particularly methyl or ethyl, especially ethyl. When T represents -U- (CH2) a -N (X) - (CH2) b-, it is preferred that X represents hydrogen, methyl or the group -CH2Q. When T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) prNXY or -0- (CH2) 2-NH- substituted by - (CH2) m-NXY, it is preferred that X and Y independently represent hydrogen, methyl or the group -CH2Q, although it is not preferred that X and Y both represent the group -CH2Q. It is particularly preferred that one of X and Y represents hydrogen or methyl and the other represents the group -CH2Q. It is preferred that n represents 1. It is preferred that Q represents phenyl or phenyl substituted by a substituent selected from the group consisting of alkyl with Cl to 6., alkoxy with Cl a 6, triflmethyl, halogen, nitro or cyano. It is particularly preferred that Q represents phenyl or phenyl substituted by alkyl with Cl to 6 or halogen. According to the invention, there is also provided a process for the preparation of the compounds of the formula I and the pharmaceutically acceptable salts thereof, which comprises: (a) preparing a compound of the formula I in which X or at least one of X and Y represents alkyl with Cl to 6 or the group - (CH2) nQ, by reacting a corresponding compound of the formula I in which X or one or both of X and Y represent hydrogen, with a compound of the formula II Rx-L II where R1 represents alkyl with Cl to 6 or the group - (CH2) nQ and L is a separation group, or (b) preparing a compound of the formula I in which T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) m-NXY or -0- (CH2) 2-NH- substituted by - (CH2) m-NXY by the reaction of a corresponding compound in which T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) mL or -0- (CH2) 2-NH- substituted by - (CH2) mL and L represents a separation group, with a compound of the formula III XYNH III wherein X and Y are as defined above, or (c) reacting a compound of formula IV where T is as defined above, with a compound of the formula V D - L V wherein D is as defined above and L is a separation group, and where it is necessary or desirable, to convert the resulting compound into a salt thereof or vice versa, as described below. In processes (a) and (b), the reactions will be carried out under standard conditions, for example by reacting the two materials in an inert solvent under basic conditions at room temperature for a period of up to 12 hours. It has been found that it is often desirable to treat the amine with NaH before reacting it with the other compound. Suitable L-cleaving groups include thioalkyl, sulfonic acid, trifluorocarbon sulfonic acid, alkyl and aryl alcohols and tosyl groups; others are described in "Advanced Organic Chemistry", 'J. March (1985) 3 / a. Edition, McGraw-Hill on page 315 and are well known in the art. It is preferred that L represents halide, particularly bromide. In the process (c) the reaction will proceed by combining the two reagents in an inert solvent, for example acetone. Suitable spacing groups that L may represent include thioalkyl, sulfonic acid, trifluorocarbon sulfonic acid, halides, alkyl and aryl alcohols and tosyl groups; others are described in "Advanced Organic Chemistry", J. March (1985) 3 / a. Edition, McGraw-Hill on page 315 and are well known in the art. It is preferred to use the iodide, toluenesulfonate or methanesulfonate derivatives. The compounds of formula IV can be prepared following the method of Rasmussen et al in Synthesis (1988) 456-459. The compounds of the formula III can therefore be prepared by reacting a compound of the formula VI wherein T is as defined above, with benzoyl isothiocyanate, followed by the alkaline-aqueous cleavage of the resulting benzoyl thiourea derivative.

The compounds of the formula VI can be prepared by reduction of a corresponding compound of the formula VII, where T is as defined above. The reduction reaction can be carried out under a number of conditions, for example those described in J. March "Advanced Organic Chemistry" 3 / a. Edition (1985) on pages 1103-1104. These include catalytic hydrogenation, the use of Zn, Sn, or a Fe metal, A1H3-A1C13, sulfides and others. It is preferred to carry out the reaction by hydrogenation at atmospheric pressure in the presence of a palladium and carbon catalyst for typically 1-4 hours, or until the reduction is complete. The compounds of the formula VII in which T is as defined above and X or at least one of X and Y represent alkyl with Cl a 6 or the group - (CH 2) n Q, can be prepared by the reaction of a corresponding compound of the formula VII in which X and / or Y represent hydrogen with a compound of the formula II.

The reaction can be carried out under conditions analogous to those described above in process (a). The compounds of the formula VII in which T represents a saturated or unsaturated alkylene chain with C3_5 substituted by - (CH2) m-NXY or -0- (CH2) 2-NH-substituted by - (CH2) m-NXY can to be prepared by the reaction of a corresponding compound in which T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) mL or -0- (CH2) 2-NH-substituted by - (CH2) mL and L represents a separation group, with a compound of formula III. This reaction can be carried out under conditions analogous to those described above in process (b). The compounds of the formula VII in which X represents hydrogen are either known or can be prepared by known methods. For example, they can be prepared by nitration of the non-nitrated derivative. This nitration reaction is carried out conventionally by reacting the un-nitrated aromatic compound with nitric acid either alone or in water, acetic acid, acetic anhydride or sulfuric acid. Additional details of these reactions and additional alternative reagents are described in J.

March "Advanced Organic Chemistry" 3 / a. Edition (1985) on pages 468-470. The compounds of the formulas II, III and V are either known or can be prepared by conventional methods known per se. The compounds of formula I can be prepared as such, or as acid addition salts of the type described above. Alternatively, they may be prepared as a pharmaceutically unacceptable addition salt, for example an oxalic acid salt, or any product thereof may be subsequently converted to a pharmaceutically acceptable salt by conventional means. The salts of the compounds of the formula I can be formed by reacting the free acid, the base or a salt thereof, with one or more equivalents of the appropriate base or acid. The reaction can be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, for example water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents , which can be removed in vacuo or by drying with freezing. The reaction can be an exchange process or it can be carried out on an ion exchange resin.

Where necessary, the amine or other reactive groups can be protected using a protecting group as described in the standard text "Protecting Groups in Organic Synthesis", 2 / a. Edition (1991) by Greene and Wuts. Amine protecting groups which can be mentioned in particular include alkyloxycarbonyl with C2 to 7, for example t-butyloxycarbonyl, phenylalkyloxycarbonyl with C8 to 13, for example benzyloxycarbonyl. However, it is preferred to protect the amine groups by treatment with trifluoroacetic anhydride in a suitable solvent (for example methylene chloride, methanol) at room temperature. Deprotection can be achieved by hydrolysis in water. The compounds of the invention and the intermediates can be isolated from their reaction mixtures by standard techniques. The term "alkyl with Cl to 6" includes aliphatic and cyclic alkyl, straight chain, branched, saturated, unsaturated, containing 1 to 6 carbon atoms. "Alkyl with Cl a 3" can be interpreted in a similar way. The compounds of the formula I can exist in enantiomeric forms. The various optical isomers can be isolated by the separation of a racemic mixture of the compounds using conventional techniques, for example fractional crystallization, or HPLC. Alternatively, the individual enantiomers can be made by the reaction of the appropriate optically active starting materials, under reaction conditions which do not cause racemization. Intermediate compounds can also exist in enantiomeric forms and can be used as enantiomers, diastereomers, racemates or purified mixtures. The compounds of the general formula I possess a useful pharmacological activity in animals. In particular, they possess a useful nitric oxide synthase inhibitory activity, and are expected to be useful in the treatment or prophylaxis of human diseases or conditions in which the synthesis or over-synthesis of nitric oxide forms a contributing part; for example, hypoxia, for example in cases of cardiac arrest, attacks and neonatal hypoxia, neurodegenerative conditions that include nerve degeneration and / or necrosis of the nerves in disorders such as ischemia, hypoxia, hypoglycemia, epilepsy , and in external wounds (such as damage to the spinal cord or head), hyperbaric oxygen seizures and toxicity, dementia such as presenile dementia, Alzheimer's disease and AIDS-related dementia, Sydenham's chorea, Parson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Korsakoff's disease, imbecility related to a disorder of brain vessels, sleep disorders, schizophrenia, anxiety, depression, affective disorder of the seasons, delayed departure from airplanes, depression or other symptoms associated with premenstrual syndrome (PMS), anxiety and shock septic. The compounds of the formula I can also be expected to show activity in the prevention and reversal of tolerance to opiates and diazepines, the treatment of drug addiction, pain relief and the treatment of migraine and other migraine headaches. Vascular head. The compounds of the present invention may also show useful immunosuppressive activity, may be useful in the treatment or prophylaxis of inflammation, in the treatment of gastrointestinal motility disorders and in the induction of labor. The compounds may also be useful in the treatment of cancers that express nitric oxide synthase. The compounds of formula I are expected to be particularly useful in the treatment or prophylaxis of neurodegenerative or migraine conditions or for the prevention and reversal of tolerance to opiates and diazepines or for the treatment of drug addiction and especially in the treatment or prophylaxis of neurodegenerative disorders. Of particular interest are the conditions selected from the group consisting of hypoxia, ischemia, attacks and Amyotrophic Lateral Sclerosis. Accordingly, according to a further aspect of the invention there is provided the use of a compound of the formula I or a pharmaceutically acceptable salt thereof as a pharmaceutical substance. According to another feature of the invention, the use of a compound of the formula I or a pharmaceutically acceptable salt thereof as a pharmaceutical substance is provided. According to another feature of the invention, there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of the diseases or conditions mentioned above.; and a method of treatment or prophylaxis of one of the diseases or conditions mentioned above, which comprises administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, to a person suffering from or being susceptible to such a disease or condition.

For the therapeutic indications mentioned above, the dosage administered, of course, will vary with the compound employed, the mode of administration and the desired treatment. However, in general, satisfactory results are obtained when the compounds are administered to a human at a daily dose of the solid form between 1 mg and 2000 mg per day. The compounds of the formula I, and the pharmaceutically acceptable derivatives thereof, can be used by themselves, or in the form of medicinal preparations suitable for enteral or parenteral administration.

Detailed description of the invention According to the invention, there is provided a pharmaceutical composition preferably comprising less than 80% and more preferably less than 50% of a compound of the formula I, or a pharmaceutically acceptable salt thereof, mixed with a pharmaceutically acceptable diluent or carrier. A method of preparing such a pharmaceutical formulation comprising mixing the ingredients is also provided.

Examples of such diluents and carriers are: for tablets and dragees: lactose, starch, talc, stearic acid; for capsules: tartaric acid or lactose; for injectable solutions: water, alcohols, glycerin, vegetable oils; for suppositories: natural or hardened oils or waxes. The compositions in a form suitable for oral administration, ie the esophageal administration include: tablets, capsules and lozenges; Sustained or prolonged release compositions include those in which the active ingredient is bound to an ion exchange resin which is optionally coated with a diffusion barrier to modify the release properties of the resin. It is preferred that the composition contain up to 50% and more preferably up to 25% by weight of the compound of the formula I, or a pharmaceutically acceptable derivative thereof. Enzymatic nitric oxide synthase has a number of isoforms and compounds of formula I, and pharmaceutically acceptable salts thereof, can be selected to inhibit the activity of nitric oxide synthase by methods based on those of Bredt and Snyder in Proc. . Nati Acad. Sci. (1990) 87, 682-685 and Forstermann et al., Eur. J. Pharm. (1992) 225, 161-165 as follows. Nitric oxide synthase converts H-L-arginine to H-L-citrulline which can be separated by cation exchange chromatography and quantified by scintillation counting.

Choose (A) Selection to inhibit the activity of neuronal nitric oxide synthase The enzyme was isolated from the hippocampus or rat cerebellum. The cerebellum or hippocampus of a male Sprague-Dawley rat (250-275 g) is removed following anesthesia with C02 and decapitation of the animal. The cerebellar or hippocampal supernatant is prepared by homogenization in 50 mM Tris-HCl with a buffer solution of 1 mM EDTA (pH 7.2 at 25 ° C) and centrifugation for 15 minutes at 20,000 gravities. The residual L-arginine was removed from the supernatant by chromatography through columns filled with sodium and Dowex AG-50W-X8 hydrogen successively, and further centrifugation at 1000 gravities for 30 seconds. For the assay, 25 μl of the final supernatant is added to each of the 96 wells (of a 96-well filter plate) containing 25 μl of a solution of L-arginine (18 μM concentration of 1 H-L-arginine , 96 nM 3H-L-arginine) and either 25 μl of a test buffer or assay (50 mM HEPES, 1 M EDTA, 1.5 mM CaCl 2, pH 7.4) or 25 μl of the test compound in the buffer solution at 22 ° C. 25 μl of the complete test buffer (50 M HEPES, 1 mM EDTA, 1.5 mM CaCl 2, 1 mM DTT, 100 μM NADPH, 10 μg / ml calmodulin, pH 7.4) are added to each test tube. the reaction and the reaction is stopped after 10 minutes by the addition of 200 ul of a suspension of the buffer solution of termination (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 200-400 mesh. The labeled L-citrulline is separated from the labeled L-arginine by filtering each filter plate and 75 ul of each finished reaction is added to 3 ml of a cocktail (multi-component substance) of scintillations. The L-citrulline is then quantified by scintillation counting. In a typical experiment using the cerebellar supernatant, the basal activity is increased by 20,000 dpm / ml of the sample above a blank of the reagent which has an activity of 7,000 dpm / ml. The N-nitro-L-arginine, which gives 80% inhibition of nitric oxide synthase at a concentration of 1 μM, is tested in the assay to verify the procedure.

Selection B (B) Selection for the inhibitory activity of inducible nitric oxide synthase The enzyme is prepared, after induction, from the cell line of the cultured human colorectal carcinoma, DLD-1 (obtained from the European Collection of Animal Cell Cultures), the DLD-1 cells are cultured in an RPMI 1640 medium supplemented with 10% of 10% fetal bovine serum, 4 mM of L-glutamine and antibiotics (100 units / ml of penicillin G, 100 μg / ml of streptomycin and 0.25 μg / ml of amphotericin B) and 100 μg / ml of kanamycin. The cells were routinely grown in 225 cm3 containers containing 35 ml of the medium maintained at 37 ° C and in a humidified atmosphere containing 5% C02. Nitric oxide synthase is produced by cells in response to interferon-approximately 250 U / ml IL-1, 1000 U / ml IFN ?, 200 U / ml IL-6, and 200 U / ml TNF-alpha. After a period of 17-20 hours in the culture, the collection of the cells is effected by scraping the cell sheet from the surface of the container in the culture medium. The cells are collected by centrifugation (1000 gravities for 10 minutes) and the lysate prepared by adding a solution containing 50 mM Tris-HCl (pH 7.5 at 20 ° C) to the cell microsphere, 10% (v / v) of glycerol, 0.1% (v / v) of Triton-X-100, 0.1 μM of dithiothreitol and a cocktail or mixture of protease inhibitors comprising leupeptin (2 μg / ml), a trypsin inhibitor of the soybean seed (10 μg / ml), aprotinin (5 μg / ml) and phenylmethylsulfonyl fluoride (50 μg / ml). For the test or assay, 25 μl of the cocktail or mixture of the substrate (50 mM Tris-HCl (pH 7.5 at 20 ° C), 400 μM of NADPH, 20 μM of the flavin adenine dinucleotide, 20 μM of the flavin mononucleotide, μM of tetra idrobiopterin, 12 μM of L-arginine and 0.025 μCi of L- [3 H] arginine) are added to 96-well filter pockets (0.45 μM pore size) containing 25 μl of a solution of the compound test in 50 mM Tris-HCl. The reaction is initiated by adding 50 μl of the lysate of the cells (prepared as above) and after incubation for 1 hour at room temperature is terminated by the addition of 50 μl of a 3 M aqueous solution of nitroarginine and 21 mM EDTA . The labeled L-citrulline is separated from the labeled L-arginine using Dowex AG-50W. 150 μl of a 25% aqueous suspension of Dowex 50W (Na + form) are added to the test or assay after which the complete assembly is filtered on the 96-well plates. 70 μl of the filtrate are sampled and added to the cavities of the 96-well plates containing the solid scintillation compound. After allowing the samples to dry, L-citrulline is quantified by scintillation counting. In a typical experiment the basal activity is 300 dpm per 70 μl of the sample, which is increased to 1900 dpm in the reagent controls. The aminoguanidine, which gives an IC 50 (50% inhibitory concentration) of 10 μM, is tested as a standard to verify the procedure.

Selection C (C) Selection for the inhibitory activity of endothelial nitric oxide synthase The enzyme can be isolated from endothelial cells of the human umbilical vein (HUVECs) by a method based on that of Pollock et al (1991) Proc. Nat. Acad. Sci., 88, 10480-10484. The HUVECs were purchased from Clonetics Corp (San Diego, CA, USA) and cultivated until confluence. Cells can be maintained until they pass 35-40 without significant loss of nitric oxide synthase yield. When the cells reached confluence, they were resuspended in a buffered saline Dulbecco's phosphate solution, centrifuged at 800 rpm for 10 minutes, the cell pellets were homogenized in 50 mM ice-cold Tris-HCl, 1 M EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 μM leupeptir.aa pH 4.2. Following centrifugation at 34, 000 rpm for 60 minutes, the pill was solubilized in the homogenization buffer which also contains 20 M of CHAPS. After about 30 minutes of incubation on ice, the suspension is centrifuged at 34,000 rpm for 30 minutes. The resulting supernatant is stored at -80 ° C until used. For the test or assay, 25 μl of the final supernatant is added to each well of a 96-well filter plate containing 25 μl of the L-arginine solution (of a concentration of 12 μM of 1H-L-arginine, 64 nM of 3 H-L-arginine) and either 25 μl of a test buffer solution (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2, pH 7.4) or 25 μl of the test compound in the buffer at 22 ° C. 25 μl of the complete test solution (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2, 1 mM DDT, 100 μm NADPH, 10 μg / ml calmodulin, 12 μM tetrahydrobiopterin, pH 7.4) are added to each well. start the reaction and the reaction is stopped after 30 minutes by the addition of 200 ul of a 50% suspension of the buffer buffer (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 mesh 200-400. The labeled L-citrulline is separated from the labeled L-arginine by filtration to another 96-well plate and 75 ul of each finished reaction is added to 3 ml of the scintillation cocktail. The L-citrulline is then quantified by the count of the scintillations. In a typical experiment, the basal activity is increased by 5,000 dpm / ml of the sample above a reagent blank which has an activity of 1500 dpm / ml. A reference standard, N-nitro-L-arginine, which gives 70-90% inhibition of nitric oxide synthase at a concentration of 1 μM, is tested in the assay to verify the procedure. The compounds can also be tested in an ex-vivo assay to determine the extent of brain penetration.

Selection D (D) Ex vivo assay for the inhibition activity of neuronal nitric oxide synthase Male Sprague-Dawley rats (250-275 g) were dosed intravenously at 10 mg / kg with the test compound dissolved in 0.9% salted solution or with brine alone as control. At a predetermined time interval (typically 2-24 hours) after treatment, the animals were sacrificed, the cerebellum removed and the supernatant prepared and evaluated to verify the activity of the nitric oxide synthase as described in Selection A. As a Additional confirmatory test, a fraction of the cerebellar supernatant was applied to a column of 2'-5'-ADP Sepharose (which binds to nitric oxide synthase) and subsequently eluted with NADPH. The eluent was tested to verify the activity of nitric oxide synthase following the procedure of Screening A. Compounds that penetrate the brain of the rat and inhibit neuronal nitric oxide synthase, led to reduced activity of nitric oxide synthase both in the preparation of the supernatant and in the eluent from the column of 2'-5'-ADP Sepharose. In the selections for nitric oxide synthase inhibition activity, the activity of the compound is expressed as IC5o (the concentration of the drug substance which gives 50% inhibition of the enzyme in the assay). The IC 50 values for the test compounds were initially estimated from the inhibitory activity of the 1, 10 and 100 μM solutions of the compounds. Compounds that inhibited the enzyme by at least 50% at 10 μM were retested using more appropriate concentrations so that an IC50 could be determined. In Selection A above (a selection for activity against the neuronal isoform of nitric oxide synthase), the compound of Example 1 below gave an IC 50 of less than 10 μM which indicates that it is expected to show useful therapeutic activity. In Selections B and C (the selections for activity against the macrophage and the endothelial isoforms of nitric oxide synthase) the compound of Example 1 gave IC 50 values greater than 10 times those obtained in Section A, indicating that the same sample a desirable selectivity. The compounds of Example 2 were also tested in Selection A and also gave an 'IC50 value of less than 10 μM. Therefore, this compound is also expected to show useful therapeutic activity. When compared with the compounds of the prior art, the compounds of the formula I and the pharmaceutically acceptable salts thereof have the advantage that they may be less toxic, more effective, longer in activity, may have a longer range. broad spectrum of activity, they can be more potent, more selective for the neuronal isoform of the nitric oxide synthase enzyme, to produce a lower level of side effects, they can be absorbed more easily or have other useful pharmacological properties. The invention is illustrated, but not in a limiting manner, by the following examples: Example 1 N- (1, 2, 3, 4-tetrahydroisoquinolin-7-i1) carbamimidothioic acid ethyl ester (a) 2- (7-amino-1,2,3,4-tetrahydroisoquinoline) 2,2,2-trifluoroacetamide To a solution of 4.21 g (23.6 mmol) of 7-nitro-1,2,3,4-tetrahydroisoquinoline and 3.6 ml, 926 mmol) of triethylamine in 100 ml of methylene chloride at 0 ° C is added 3.5 ml ( 25 mmoles) of trifluoroacetic anhydride, the reaction mixture is stirred overnight. The reaction mixture is extracted with dilute hydrochloric acid. The aqueous phase is basified and extracted with methylene chloride. The organic phase (magnesium sulfate) was dried to give N- (7-nitro (1,2,3,4-tetrahydroisoquinoline) trifluoroacetamide as a yellow solid.This compound was immediately received in 200 ml of ethanol, 0.50 g of 5% palladium on carbon are added, and the mixture is hydrogenated over a Parr hydrogenator at 3.17 kg / cm2 (45 psi) for 1.5 hours.The catalyst was removed by filtration and the solvent was evaporated. ml of petroleum ether to give 5.45 g (95%) of the title compound as a gray solid, mp 61-3 ° C. (b) 1,2,3,4-tetrahydroisoquinolin-7-thiourea To a solution of 1.3 ml (9.7 mmol) of benzoyl isothiocyanate in 13 ml of refluxing acetone 1.25 g (5.12 mmol) of 2- (7-amino-1,2,3,4-tetrahydroisoquinoline) trifluoroacetamide are added rapidly to a speed such that reflux is controlled. After the addition is complete, the reaction mixture is stirred for 3 h. After cooling, the solid is collected and washed with 30 ml of acetone to give 1.74 g (83%) of the intermediate compound of l-benzoyl-3- [2- (2-2-2-trifluoroacetamide) 1,2, 3,4-tetrahydroisoquinolin-7-thiourea as a colorless white solid. This compound was immediately added to 20 ml of a 5% sodium hydroxide solution and the resulting solution is heated at 80 ° C for 1 hour. During cooling to room temperature, the solution is filtered to give 0.78 g (74%) of the title compound, m.p. 198-203 ° C.

(C) N- (1, 2,3,4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid ethyl ester To a suspension of 0.75 g (3.61 mmol) of 1, 2, 3, 4-tetrahydroisoquinolin-7-thiourea in 10 ml of isopropanol is added 0.35 g (3.7 mmol) of methanesulfonic acid in 2 ml of isopropanol. The reaction mixture is stirred for 0.25 h before 0.85 ml (8.4 mmol) of ethyl methanesulfonate is added. The reaction mixture is refluxed for 4 hours. The solvent is removed by vacuum distillation to give an oil which is dissolved in 100 ml of water. The aqueous phase is made basic with saturated sodium bicarbonate and the aqueous phase is extracted 8 times with 100 ml of methylene chloride. The combined extracts are dried over magnesium sulfate and concentrated to give 0.61 g of an oil which solidifies during rest. Column chromatography on silica gel using 10% methanol in chloroform saturated with ammonia afforded 0.45 g (53%) of the title compound as a white solid, EM 236 (M + H).

Example 2 N-5- (2- (((3-chlorophenyl) methyl) (methyl) amino) indanyl) carbamimidothioic acid ethyl ester (a) 2- ((3-Chlorophenyl) carbonyl) amino-5-nitroindane To the 2-amino-5-nitroindane hydrochloride (1.5 g, 7.0 mmol) in methylene chloride (50 ml) at 0 ° C is added triethylamine (2.1 ml, 15.0 mmol) followed by 3-chlorobenzoyl chloride (1.0 ml, 7.5 mmoles). The mixture is immediately emptied in water and the layers are separated. The aqueous layer is extracted with methylene chloride (2 X 20 mL) and the combined extracts are washed with water, dried over MgSO 4, filtered, and concentrated to an oil which was homogeneous by CCD and used immediately in the next step: E.M. (M + H) + = 317. [b) 2- ((3-Chlorophenyl) methyl) amino-5-nitroindane 2- ((3-chlorophenyl) carbonyl) amino-5-nitroindane (2.2 g, 7.0 mmol) in THF (75 mL) is added BH3"THF (1.0 M, 35 mL, 35 mmol) per drop. The mixture is refluxed for 12 hours, cooled to 0 ° C, quenched with 4N HCl (60 mL), and refluxed for 1 hour. The resulting solution is evaporated to an oil, made basic with 50% NaOH, and extracted with methylene chloride (3 X 20 mL). The combined extracts are washed with water, dried over MgSO, filtered and concentrated to an oil. Treatment with IPA / HC1 yielded 2- ((3-chlorophenyl) methyl) amino-6-nitroindane: (2.1 g, 88% two steps); p.f. 234-237 ° C. (c) 2- ((3-chlorophenyl) methyl) (methyl) amino-5-nitroindane Al 2- ((3-chlorophenyl) methyl) amino-5-nitroindane (4.4 g, 14.5 mmol) in formic acid (5.5 mL) is added formaldehyde (12 mL). The mixture is refluxed for 30 minutes, cooled, neutralized with 2N NaOH, and extracted with ethyl acetate (3 X 70 mL). The combined extracts are washed with water, dried over MgSO4, filtered and concentrated to an oil: (4.2 g, 91%); E.M. (M + H) + = 317. (d) 2- ((3-chlorophenyl) methyl) (methyl) amino-5-aminobenzene 2- ((3-Chlorophenyl) methyl) (methyl) amino-5-nitroindane (4.3 g, 13.6 mmol) in 85% AcOH / H20 (100 mL) is added zinc metal (7.1 g, 109.0 mmol). The mixture is stirred for 5 minutes, filtered through celite, and evaporated to an oil. The oil is poured into basic water and extracted with ethyl acetate (3 X 100 mL). The combined extracts are washed with water, dried over MgSO4, filtered, and concentrated to an oil: (3.6 g, 92%); E.M. (M + H) + = 287. (e) 5- (2- (((3-chlorophenyl) methyl) (methyl) amino) indanyl) -1-benzoy1-2-thiourea To a solution of benzoyl isothiocyanate (2.7 g, 16.5 mmol) in 15 ml of dry acetone, preheated to very mild reflux, is added quickly, at a rate such as to control a vigorous reflux, 2- ((3-chlorophenyl) methyl) (methyl) amino-5-aminobenzene (3.6 g, 12.4 mmoles) dissolved in 10 ml of dry acetone. The reaction mixture is refluxed for 30 minutes, poured onto ice with vigorous stirring, and extracted with ethyl acetate (3 X 100 mL). The combined extracts were washed with water, dried over MgSO4, filtered, and concentrated to a solid which was recrystallized from IPA: (3.12 g, 58%); p.f. 128-130 ° C. (f) 5- (2- (((3-chlorophenyl) methyl) (methyl) amino) indanyl) -2-thiourea A mixture of 5- (2- (((3-chlorophenyl) methyl) (methyl) amino) indanyl) -l-benzoyl-2-thiourea (3.1 g, 7.12 mmol) and 40 ml of 2.5 N aqueous sodium hydroxide are added. heat at 90 ° C for 35 minutes with shaking. The hot reaction mixture is poured into 60 ml of water with stirring. The product is extracted in three portions of methylene chloride. The combined extracts are washed with water, dried over magnesium sulfate and concentrated to dryness. The residue is chromatographed on silica gel (ethyl acetate / hexane 8: 1) and concentrated to an oil: (2.2 g, 93%); E.M. (M + H) + = 246. (g) Ethyl N-5- (2 - (((3-chlorophenyl) methyl) (methyl) amino) indanyl) carba imidothioic acid ester - (2- (((3-chlorophenyl) methyl) (methyl) amino) -indanyl) -2-thiourea (2.2 g, 6.33 mmol) was suspended in 20 ml of ethanol 200, and the mixture is treated with 0.41 ml of methanesulfonic acid, and 1.35 ml of ethyl methanesulfonate. The mixture is refluxed for 4 hours, evaporated, made basic with saturated bicarbonate, and extracted with methylene chloride (3 X 30 mL). The combined extracts are washed with water, dried over MgSO, filtered, and concentrated to an oil which is dissolved in ethyl acetate and treated with IPA / HC1. The solids are filtered and washed with IPA: (2.40 g, 83%); p.f. decomposition > 150 ° C.

Example 3 N- (2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid ethyl ester dihydrochloride (a) 7-Nitro-2-methyl-1,2,3,4-tetrahydroisoquinoline hydrochloride A solution of 4.00 g (18.7 mmol) of 7-nitroisoquinoline in 10 ml of formic acid and 17 ml of 38% aqueous formaldehyde is heated at reflux for 1 hour. The reaction mixture is cooled, poured on ice and basified with aqueous ammonia. The gummy residue which was precipitated is extracted twice with methylene chloride. The dried organic phase (magnesium sulfate) is concentrated to give 7-nitro-2-methyl-1,2,3,4-tetrahydroisoquinoline as a thick oil. This oil was immediately received in ethanol (50 ml) and a solution of the hydrochloric acid in ethanol was added until the solution was distinctly acidic until it became litmus. The ether was added to induce precipitation and the resulting solid was collected to give 3.99 g (93%) of the title compound as a yellow solid, m.p. 236-8 ° C (decomposition). (b) Hydrochloride of 7-amino-2-methyl-1,2,3,4-tetrahydroisoquinoline A suspension of 3.98 g (17.5 mmol) of the hydrochloride of 7-nitro-2-methyl-1,2,3,4-tetrahydroisoquinoline and 0.4 g of 10% palladium on carbon in 200 ml of ethanol is hydrogenated at 3.52 kg. / cm2 (50 psi) for 2 hours. The catalyst is filtered and washed with a small amount of water. The filtrate is concentrated producing an aqueous solution. Absolute ethanol was added and evaporated by removing excess water until a solid was produced. This solid was dissolved in hot ethanol (60 mL) and the ether added slowly to induce crystallization. The product is collected to give 3.38 g (97%) of the title compound as a colorless white solid, m.p. 114-9 ° C.

(C) 2-Methyl-l, 2,3, 4-tetrahydroisoquinolin-7-thiourea A solution of 3.88 g (19.5 mmol) of the hydrochloride of 7-amino-2-methyl-1,2,3,4-tetrahydroisoquinoline in 100 ml of water is made basic with a solution of potassium carbonate and extracted twice. with methylene chloride. The dry organic phase (magnesium sulfate) is concentrated to give 3.13 g (99%) of the free base as an oil. This oil is taken up in acetone (75 ml) and 2.21 g (19.4 mmol) of trifluoroacetic acid in 100 ml of acetone are added. The solution is heated to reflux where 5.2 ml (39 ml) of benzyl isothiocyanate are added dropwise. The reaction mixture is heated for 1 hour before cooling to room temperature. The solvent was removed in vacuo and the resulting oil is taken up in methanol (150 ml) and 2.5 M sodium hydroxide (50 ml). This solution is heated at 65 ° C for 1 hour before cooling to room temperature. The methanol is removed by vacuum distillation and the aqueous solution is cooled to precipitate the product. The solid is collected to give 2.22 g of the title compound as a faint yellow solid, m.p. 184-6 ° C. A second crop of the title compound (0.79 g, 69% total yield) was also obtained. (d) N- (2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid ethyl ester dihydrochloride To a suspension of 0.88 g (4.0 mmol) of the 2-Methyl-1,2,4,4-tetrahydroisoquinoline-7-thiourea in 8 ml of isopropanol is added 0.39 g (3.9 mmol) of methanesulfonic acid. The solution is refluxed for 0.5 hours to ensure the formation of the methanesulfonate salt, both the salt and the free base were insoluble in isopropanol. To this solution 1.5 ml (14 mmol) of ethyl methanesulfonate are added and the heating is continued overnight to produce a clear solution. The solvent is removed in vacuo and the resulting oil is taken up in water, made basic with potassium carbonate and extracted twice with methylene chloride. The dry organic phase (magnesium sulfate) is concentrated to give an oil. This oil is received in ethanol until it becomes distinctly acidic and becomes litmus. The ether is added and the salt became a viscous oil. The solvent is decanted and the oil is washed several times with ether. The oil is received in water (250 ml) and the solution is treated with decolorizing carbon. The solution is filtered and the filtrate is diluted to 500 ml with water. This solution is dried by freezing to give 1.06 g (78%) of the title compound as a monohydrate. MS (CI) 250 (M + H); NMR (DMS0 / D20) 7.33 (d, 1H), 7.21 (d, 1H), 7.17 (s, 1H), 4.36 (s amp., 2H), 3.0-3.6 (m, 6H), 3.17 (s, 3H) ), 1.30 (t, 3H).

Example 4 N- (2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid methyl ester dihydrochloride To a suspension of 1.00 g (4.52 mmol) of the 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-thiourea (Example 3, step (c)) in 10 ml of isopropanol 0.44 g (4.5 mmol) are added. ) of methanesulfonic acid. The solution is stirred at room temperature for 2 hours to ensure the formation of the methanesulfonate salt, as both the salt and the free base were insoluble in isopropanol. To this solution is added 6.7 g (47 mmol) of methyl iodide and the reaction mixture is stirred overnight. The solvent is removed by distillation under vacuum and the residue is dissolved in water, treated with decolorizing carbon and filtered to give a clear, colorless, aqueous solution. This solution is made basic with potassium carbonate and extracted twice with methylene chloride. The organic phases were combined, dried (magnesium sulfate), and concentrated in vacuo to give 1.02 g (96%) of the product as a free base. This oil is received in ethanol and is made distinctly acid by the addition of hydrochloric acid in ethanol. The addition of excess ether caused the salt to separate as an oil. The solvent was decanted and this oil was washed several times with ether. This oil is received in 250 ml of water and again treated with decolorizing carbon. The solution is filtered and the filtrate is diluted to 500 ml with water. This solution is dried by freezing to give g of the title compound as a white solid. MS (Cl) 236 (M + H). NMR (DMSO / D20) 11.6-11.9 (broad, 1H), 9.4-9.7 (broad, 1H), 7.36 (d, 1H), 7.24 (d, 1H), 7.18 (s, 1H), 4.2-4.6 (broad m, 2H), 3.0-3.7 (broad m , 4H), 2.87 (s, 3H), 2.70 (s, 3H).

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, priority is claimed as contained in the following

Claims (10)

1. A compound of the formula I characterized in that D represents alkyl with Cl at 6 T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) m-NXY; -0- (CH2) 2-NH- substituted by - (CH2) m-NXY; or -U- (CH2) a-N (X) - (CH2) b-; U represents NH, 0 or CH2; a and b, which may be the same or different, represent an integer from 0 to 3, provided that a + b is in the range of 1 to 3; X and Y, which may be the same or different, represent hydrogen, alkyl with Cl to 6, or a group ~ (CH2) nQ, or NXY together represent piperidinyl, pyrrolidinyl, morpholinyl, or tetra idioisoquinolinyl; Q represents phenyl optionally substituted by alkyl with Cl a 6, alkoxy with Cl a 6, trifluoromethyl, halogen, nitro or cyano; m and n independently represent a whole number of 0 to 5; and pharmaceutically acceptable salts thereof.

2. A compound of the formula I, according to claim 1, characterized in that T represents -U- (CH 2) a-N (X) - (CH 2) b-.

3. A compound of the formula I, according to claim 1 or claim 2, characterized in that T represents -U- (CH 2) a-N (X) - (CH 2) b- and U represents CH 2.

4. A compound of formula I, according to any of claims 1 to 3, characterized in that T represents -U- (CH 2) a-N (X) - (CH 2) -, U represents CH 2 and a and b each represent 1.

5. A compound of the formula I, according to any of claims 1 to 4, characterized in that D represents ethyl.

6. A compound of formula I, according to claim 1, characterized in that it is: N- (1, 2, 3, 4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid ethyl ester; N-6- (2 - (((3-chlorophenyl) methyl) (methyl) amino) indanyl) carbamimidothioic acid ethyl ester; N- (2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid ethyl ester; N- (2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl) carbamimidothioic acid methyl ester; or a pharmaceutically acceptable salt of any of them.

7. A compound according to any of claims 1 to 6, characterized in that it is used as a pharmaceutical substance.

8. A pharmaceutical composition, characterized in that it comprises a compound according to any of claims 1 to 6, mixed with a pharmaceutically acceptable diluent or carrier.

9. The use of a compound according to any of claims 1 to 6, in the manufacture of a medicament for the treatment or prophylaxis of neurodegenerative disorders, or for the prevention and reversal of tolerance to opiates and diazepines or for the treatment of drug addiction.

10. A process for the preparation of the compounds of the formula I, as defined in any of claims 1 to 6, and the pharmaceutically acceptable salts thereof, characterized in that it comprises: (a) preparing a compound of the formula I in the wherein X or at least one of X and Y represent alkyl with Cl to 6 or the group - (CH2) nQ, by reacting a corresponding compound of formula I in which X or one or both of X and Y represent hydrogen, with a compound of formula II Rx-L II wherein R 1 represents alkyl with Cl a 6 or the group - (CH 2) n Q and L is a separation group; or (b) preparing a compound of the formula I in which T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) m-NXY or -0- (CH2) 2-NH- substituted by - (CH2) m-NXY by the reaction of a corresponding compound in which T represents a saturated or unsaturated alkylene chain with C3-5 substituted by - (CH2) mL or -0- (CH2) 2-NH- substituted by - (CH2) mL and L represents a separation group, with a compound of formula III XYNH III wherein X and Y are as defined in claim 1; or (c) reacting a compound of formula IV wherein T is as defined in claim 1, with a compound of the formula V D - L V wherein D is as defined in claim 1 and L is a separation group, and wherein it is necessary or desirable to convert the resulting compound to a pharmaceutically acceptable salt thereof or vice versa.