MXPA97004560A - Noved receiver - Google Patents

Abstract

A receptor in substantially pure form obtained from rat forebrain tissue characterized in that a) Compound A binds to it with a Kd of 48 mM for rat forebrain tissue, b) Compound A binds to it with a Bmax 220 pmol / g protein for rat forebrain tissue c) Compound D binds to it with a Kd of 2nM for rat forebrain tissue d) Compound B binds to it with a Bmax of 220 pmol / g protein for rat forebrain tissue, and homologous receptors from other sources that share at least 85% homology with the prosencephalon tissue of ra

Description

NOVEDOUS RECEIVER FIELD OF THE INVENTION The present invention relates to a novel receptor in substantially pure form, to a soluble form of the receptor and to its use as a therapeutic agent; to a method for selecting compounds useful in the treatment of disorders by interaction with the receptor, to novel compounds discovered to carry out the selection method; to the recombinant receptor and to its use in said selection method; to the preparation of onoclonal and polyclonal antibodies that bind to the receptor; to the use of said antibodies as therapeutic agents, and to a method of determining the effectiveness of the therapeutic agents that bind to the receptor.

BACKGROUND OF THE INVENTION UIO 92/22293 (SmithKIine Beecham) describes a class of compounds that have shown, through behavioral models, to possess certain activities on the CNS, in particular, in the treatment and / or prevention of epilepsy. An example of said compound described in the above patent application is trans- (+) - B-acetyl-4S- (4-fluorobenzolamino) -3,4 dihydro-2,2-dimethyl-2H-1-benzoyl? iran-3R-ol (hereinafter referred to as the compound fi). UO / 94/13656, UO / 94/13657, UO / 94/13292, UIO / 94/13297, PCT / EP / 95/02076, PCT / EP / 95/02249 and PCT / EP / 95/02246, describe other compounds that possess certain activities on CNS, in particular PCT / EP / 95/02076, describe the "cold" compound of Example 4 in the present application, that is, compound B. The compounds described in the aforementioned patents do not bind to any known receptor and now a novel receptor has been identified to which said compounds are bound. Therefore, the present invention provides a receptor in substantially pure form which is obtained from rat forebrain tissue which is characterized in that: a) The compound fl binds with a Kd of 40nM for rat forebrain tissue, b) Compound fl binds to it with a Bm «x of 220 pmol / g protein for rat forebrain tissue; c) Compound B is bound to it with a Kd of 2nM for rat forebrain tissue; d) Compound B is bound to it with a Brnax of 220pmol / g protein for rat forebrain tissue; And homologous receptors from other sources that share at least 85% homology with the rat forebrain tissue. Other known anticonvulsant compounds include diazepam, phenytoin, pentobarbitone, valproate, carbarnazepine, vigrabatrin, lamotrigine, ethosuximide and gabapentin, which do not bind to the novel receptor. In addition, the novel receptor can be found in human or rodent neuroblast a and glioma cell cultures. These can be used without preparation or they can be prepared with the same method as for brain tissue. In human neuroblastoma cell lines, for example SH5Y5Y or I R 32, compound B binds in the novel receptor with a Kd of 2nM and a Brnaz of 150 pmol / g of protein. The novel receptor is isolated in substantially pure form by conventional techniques. For example, an aliquot (for example containing 1 to 10 mg of protein / ml) of the tissue containing the novel receptor (for example the one described above) is mixed with radiolabeled compound (for example 125 I) with photoaffinity mark (for example compound C -see example 6). Preferably, the final concentration of the photoaffinity label compound in the mixture is 0.1 to 1000 pM. The mixture can be suitably incubated for about 1 hour at room temperature. The mixture is then exposed to UV light (for example 366 nm from a 6W lamp) for about 30 minutes. The tissue is then washed by centrifugation to remove unbound photoaffinity label compound. The photoaffinity-labeled receptor can be initially separated from the other proteins by means of gel permeation chromatography (for example with Super6se) under non-reducing conditions. The protein fractions that contain the receptor can then be precipitated with tpcloroacetic acid, as described by Bensadoun and Ueinstein (Bensadoun fl., and Ueinstein D. (1976) Anal. Biochem. 70, 241-250). The proteins are then separated by means of electrophoresis with Dodecyl Sodium Sulfate-Polyacrylamide Gel (SDS-PAGE) under red? Ctorae conditions. 6% (w / v) bar gels covered with a gel superimposed on 5% (w / v), or vertical plate gel at 10% (w / v), or gradient 4-20% (p / v), based on the Laernrnli method (see Lae mli UK (1970) Nature, 227,680-685). Additional purification of the receptor can be achieved by means of Isoelectric Focusing (IEF) in polyacrylamide gels with immobilized pH gradient of material prepared by preparative SDS-PAGE. The molecular weight of the receptor is on the scale of 130 kiloDaltons when analyzed by gel electrophoresis (SDS-PAGE). A second aspect of the invention provides a soluble form of the above receptor. Soluble forms of said receptor can be prepared in accordance with conventional techniques. Said soluble forms of said receptor are believed to possess therapeutic utility and therefore the present invention extends to the use of a soluble form of said receptor as a therapeutic agent.

The present invention also extends to the use of a soluble form of said receptor in the manufacture of a medicament for the treatment and / or prevention of anxiety, mania, depression, disorders associated with a szaramoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepinae, disorders that can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia. The present invention also extends to a method of treatment and / or prevention of anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkineon disease, psychosis, migraine and / or cerebral ischemia, which comprises administering an effective or prophylactic amount of the soluble receptor to a patient in need thereof . The present invention also extends to a pharmaceutical composition for use in the treatment or prevention of anxiety, mania, depression, disorders associated with sterobacillic hemorrhage or neural shock, effects associated with abstinence from substances of abuse such as cocaine, nicotine , alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia, which comprises the soluble receptor in admixture with pharmaceutically acceptable vehicles. Said pharmaceutical compositions and vehicles are well known in the art and can be prepared by conventional techniques. In a third aspect, the present invention provides a method of selecting compounds having therapeutic activity associated with binding to said receptor, which comprises: contacting a test compound with a substrate in which the novel receptor is present and measure the degree of union. Substrates in which the novel receptor can be found include, but are not limited to, brain tissue from rats, humans, marmosets, dogs, cats and mice. Said brain tissue can be suitably homogenized in an aqueous medium regulated in its pH such as HEPES 5 to SOmtl, pH 7.4 or pH regulator Tris / HCl. The homogenized tissue can be washed by centrifugation and resuspension. Subcellular fractions prepared from this fabric can also be used. The contact of the test compound with a substrate in which the novel receptor is present can be carried out by mixing an aliquot (containing for example 1 to 10 mg of protein / ml) with a radiolabeled test compound (for example 3H). or 125?). Preferably, the final concentration of the test compound in the mixture is 0.1 to lOOOntl, most preferably 1 to 50 nt. The mixture can be suitably incubated for 1 hour at about room temperature. The unbound test compound is then separated by filtration of the bound test compound. This can be done using Uhat an glass fiber filters, preferably of the GF / B or 6F / C type. The filters can be washed appropriately with ice-regulated medium at their pH (preferably of the type used in the preparation of the fabric). The amount of radioactivity bound to the tissue trapped in the filters can be measured by the addition of liquid scintillation mixture to the filters, followed by counting in a liquid scintillation counter (for 3H), or by direct counting of the filters in a range counter (for 125?). Alternatively, when intact adherent cells are used in a culture dish, the unbound test compound can be separated from the bound test compound by washing the cells with pH regulated medium., cooled with ice, followed by dissolution of the cells in sodium hydroxide solution and counting in a liquid scintillation counter or gamma counter as before. The radiolabelling of the test compound is carried out using conventional techniques. Alternatively, the binding affinities of the non-radiolabeled test compounds can be established by measuring the amount of displacement of a radiolabeled compound that is known to bind to the receptor, such as compound A and B and other compounds mentioned or covered by the patents or applications mentioned above, using conventional techniques. It should be appreciated that the radio-labeled compounds that bind to the receptor and can be replaced using this screening method are novel and form a further aspect of the present invention. It can be advantageous to use recombinant.es receptors in the selection method that can be prepared by conventional techniques. For example, a means for isolating a nucleic acid encoding a novel human receptor is to probe a cDNA or human genomic library with a naturally-designed or artificially designed probe using recognized methods (see for example "Current Protocols in Molecular Biology", Aus. ? bel, FM, et al., (eds.) Greene Publishing Assoc. and Oohn Wiley Interscience, New York, 1989, 1992). The isolated nucleic acid molecules obtained herein can be used to obtain complementary copies of genomic DNA, cDNA or RNA, from human, mammalian or other animal sources, or to select said sources and look for related sequences that include regulatory and regulatory elements. transcription control defined above, as well as other regions that determine stability, processing, translation and tissue specificity of the 5 'and / or 3' regions in relation to the coding sequences.

The proteins of this invention are preferably made by recombinant genetic engineering techniques. The isolated nucleic acids, particularly the DNAs can be introduced into expression vectors by operably linking the DNA to the necessary expression control regions (eg, regulatory regions) required for the expression of the gene. The vectors can be introduced into the appropriate host cells such as prokaryotic (for example bacterial) or eukaryotic cells (for example yeast, insect or mammal) by methods well known in the art (flusubel et al., Supra). The coding sequences of the desired proteins that have been prepared or isolated can be cloned into a suitable vector or replicon. Numerous cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells that can be transformed include bacteriophage lambda (E. coli), pBR322 (E.coli), pflCY.177 (E.coli), pKT230 (gram-negative bacteria), pGV1106 ( gram-negative bacteria), pLOFRl (gram-negative bacteria), pME290 (gram-negative bacteria, not E. coli), pHV14 (E. coli and Bacillus s? btilis), pBD9 (Bacillus), pIJBl (Streptomyces), pUC6 (streptomyces), YIp5 (Saccharomyces, a cell system of insect baculovirus, YCpl9 (Saccharomyces) See, generally, "DNA Cloning": Vols. I and II, Glover et al., ed.

IRL Press Oxford (1985) (1987) and; T. Maniatis and others.

("Molecular Cloning" Cold Spring Harbor Laboratory (1982) .The gene can be placed under the control of a promoter, ribose a binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as elements of "control"), since the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construct.The coding sequence may or may not contain a signal peptide or sequence The antigens of the subunit of the present invention can be expressed using for example the tac promoter of E. coli or the promoter of the protein A gene. (spa) and signal sequence. The guide sequences can be removed by the bacterial host in post-translational processing. See, e.g., Patents of E.U.A. Nos. 4,431,739; 4,425,437; 4,338,397. In addition to control sequences, it may be convenient to add regulatory sequences that allow the regulation of the expression of protein sequences related to the growth of the host cell. Regulatory sequences are known to those skilled in the art, and examples include those which cause the expression of a gene to be activated or deactivated in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements, for example enhancement sequences, may also be present in the vector.

An expression vector is constructed in such a way that the particular coding sequence is located in the vector with appropriate regulatory sequences, the location and orientation of the coding sequence with respect to the control sequences is such that the coding sequence is transcribed under the "control * 1" of the control sequences (ie, polymerase RNA that binds to the DNA molecule in the control sequences that transcribe the coding sequences) It may be desirable to modify the sequences encoding the particular antigen of interest For example, in some cases it may be necessary to code the sequence so that it can be linked to the control sequences in the proper orientation, that is, to maintain the reading frame.Lae control sequences and other regulatory sequences can ligate to the coding sequence before insertion into a vector, such as the cloning vectors described above Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. In some cases, it may be convenient to add sequences that cause the secretion of the peptide from the host organism, with subsequent excision of the s <.;., »The secreter. It may also be convenient to produce mutants or analogs of the receptors of interest. Mutants or analogs can be prepared by deleting a portion of the protein coding sequence, by inserting a sequence, and / or by substituting one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed utagénesis, are well known to those skilled in the art. See, v. gr. , T. Maniatis et al., Supra DNfl Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra. A number of prokaryotic expression vectors are known in the art. See, for example, U.S. Patent Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366, 246; 4,342,832; see also British patent applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and the European Patent Application 103,395. Yeast expression vectors are also known in the art. See for example the Patents of E.U.fl. Nos. 4,446,235; 4,443,539; 4,430,428; see also European patent application 103,409; 100.561; 96,491. pSV2neo (as described in 3. Mol. flppl.Genet.1: 327-341), which uses the last SV40 promoter to handle expression in mammalian cells, or pCDNAlneo, a vector derived from pCDNAl (Mol. .: 125-29) which uses the CMV promoter to handle the expression. These latter two vectors can be employed for transient or stable expression (using resistance G418) in mammalian cells. Also useful are expression systems in insect cells, for example Drosophila, see for example PCT applications US 89/05155 and US 91/06838, as well as application EP88 / 304093.3 Depending on the expression system and the selected host, proteins of the present invention are produced by the development of transformed host cells by an expression vector described above under the conditions in which the protein of interest is expressed. The protein is isolated after the host cells and purified. If the expression system secretes the protein into the culture medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from the cellular ones or recovered from the membrane fraction of the cell. In the case, as in the present, wherein the protein is located on the cell surface, intact cells or isolated membranes can be used as a readable source of the desired genetic product. The selection of appropriate growth conditions and recovery methods are within the practice of the art. An alternative method for identifying proteins in the present invention is to construct libraries, use the resulting clones to transform E. coli and collect and select individual colonies using polyclonal serum or monoclonal antibodies to the desired receptor. The proteins of the present invention can also be produced by means of chemical synthesis such as or peptide synthesis in solid phase, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. The chemical synthesis of peptides is not particularly preferred. The proteins of the present invention or their fragments comprising at least one epitope can be used to produce antibodies, both polyclonal and onoclonal. If polyclonal antibodies are desired, a selected mammal (for example mouse, rabbit, goat, horse, etc.) is immunized with a receptor of the present invention or s? fragment, or? mutated receptor. The serum of the immunized animal is collected and treated in accordance with known procedures. If sera containing polyclonal antibodies are used, the polyclonal antibodies can be purified by immunoaffinity chromatography or other known methods. The monoclonal antibodies to the protein of the present invention and to fragments thereof, they can also be easily produced by an expert in the field. The general methodology for making monoclonal antibodies using hybridization technology is well known. Immortal antibody cell lines can be created by cell fusion, and also by other techniques such as direct transformation of oncogenic DNA lymphocytes, or transfection with Epstein-Barr virus. See, v. gr. , M. Schreier and others, "Hibridorna Techniquee" (1980); Ha merling et al., "" Monoclonal Antibodies and T-cell Hybridomas "(1981), Kennett et al.," Monoclonal Antibodies "(1980), see also US Patent Nos. 4,341,761, 4,399,121, 4,427,783, 4,444,887, 4,452,570, 4,466,917; 4,472,500, 4,491,632, and 4,493,890 Panels of monoclonal antibodies raised against the antigen of interest or fragments thereof can be selected for different properties, ie, by isotype, epitope, affinity, etc. Monoclonal antibodies are useful in purification, using in unaafinity techniques, of the individual antigens against which they are directed Alternatively, genes encoding the monoclonal of interest can be isolated from the hybridomas by means of PCR techniques known in the art and cloned and expressed in the appropriate vectors The antibodies of this invention, whether polyclonal or monoclonal, have additional utility since they can be used as reagents in immunoassays, RIA, ELISA, and the like. In other embodiments, the cell membrane fractions comprising the isolated receptor or receptors isolated or immobilized on solid supports can be used to measure the binding of the ligand to be tested. When recombinant cells are used for the purposes of receptor expression, it is preferred to use cells with little or no endogenous receptor activity so that if there is any binding, this is due to the presence of the expressed receptor of interest. Preferred cells include embryonic kidney cells from human, monkey kidney (HEK-293cell), fibroblast cells (COS), Chinese hamster ovary (CHO) cells, rosophil L cells, or murine. It is also preferred to use as a host cell, one in which there is a second messenger system sensitive to the receptor. Second known messenger systems include, but are not limited to, increases or decreases in hydrolysis of phosphoinositide, adenylate cyclase, guanylate cyclase or ion channel activity in response to ligand binding to the extracellular receptor domains. In a further embodiment, a specifically designed receiver union indicator may be constructed. For example, a fusion protein can be made by fusing the receptor of this invention with a protein domain that is sensitive to ligand receptor binding. Such domain, referred to herein as an indicator domain, is capable by itself or in association with accessory molecules, of generating an analytically detectable signal that is indicative of ligand receptor binding. Alternatively, cell membrane preparations can be employed from transfected or transformed cells. In such a case the binding of an analytically detectable ligand is measured. The use of radioactively and non-radioactively labeled ligands is contemplated in this invention. All of the above techniques that are useful for ligand identification are also useful in the selection of drugs and drug development protocols. A fourth aspect provides novel compounds (hereinafter referred to as compounds of formula X) and pharmaceutically acceptable salts, hydrates or solvates thereof, identified in the above selection method. The pharmaceutically acceptable salts, hydrates and saturates thereof can be prepared in conventional manner. The invention also extends to the use of a compound of formula X or a pharmaceutically acceptable salt, hydrate or saturated thereof as a therapeutic agent. The invention also extends to a method of treating or preventing disorders comprising administering an effective and / or prophylactic model amount of a compound of formula X or a pharmaceutically acceptable salt, hydrate or solvate thereof to a patient in need of same. The invention also extends to the use of a compound of formula X in the manufacture of a medicament for the treatment or prevention of anxiety, mania, depression, disorders associated with a sassonocele or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with agents anticonvulsants such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia. The invention also extends to a pharmaceutical composition for use in the treatment or prevention of anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from subetancies of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia, which comprises mixing a compound of formula X with a pharmaceutically acceptable carrier. Said novel compounds identified by the aforementioned selection method can be prepared using techniques known in the field of organic chemistry. A fifth aspect of the present invention provides a monoclonal or polyclonal antibody that binds to the novel receptor. Said monoclonal and polyclonal antibodies can be recognized and prepared by conventional techniques. Therefore, the present invention also provides the use of a monoclonal or polyclonal antibody that binds to the novel receptor as a therapeutic agent. The invention also provides the use of a monoclonal or polyclonal antibody that binds to the novel receptor in the manufacture of a medicament for the treatment and / or prevention of anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the associated effects with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia. The invention also provides a method of treatment and / or prevention of anxiety, mania, depression, disorders associated with subarachnoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepine, disorders that They can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkineon's disease, sycosis, migraine and / or cerebral ischemia, which comprises administering an effective and / or prophylactic amount of a monoclonal or polyclonal antibody that binds to the novel receiver. The present invention also extends to a pharmaceutical composition for use in the treatment and / or prevention of anxiety, mania, depression, disorders associated with subarachnoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine , nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia; which comprises the mixture of a monoclonal or polyclonal antibody with a pharmaceutically acceptable carrier.

A sixth aspect of the present invention provides radiolabeled compounds that bind to the novel receptor. Said radio-zero compounds can be prepared by conventional techniques. Particular examples of said compounds are described in the examples. Therefore, the present invention also provides the use of radio-labeled compounds that bind to the novel receptor or diagnostic tools to detect changes or abnormalities in the novel receptor. These changes may be present in diseases associated with binding at the receptor mentioned in the present invention. Such radio-annonated compounds can also be used as research tools to study the properties of the novel receptor. Preferred radiolabelled compounds include those presented in Examples 2 to 5. The following examples illustrate the present invention.

E3EMPL0 1 Adult male Mistar rats were sacrificed and the brains were removed by dissection. All the forebrain tissue was dissected and homogenized in an aqueous medium regulated in its pH. The homogenized tissue was washed by centrifugation and re-suspended in the same pH regulator. After centrifugation, the resuspended tissue was used freshly prepared or stored in freezing for periods of up to 3 rnesee or before use. Aliquots of tissue prepared as above were mixed at a concentration of 1-10 mg protein / ml, with aliquots of 1"3 H] -compound A, dissolved in regulated medium (50nM HEPES, pH7.4). At the end of [3H3 ~ Compound A in the mixture was on the scale of 20-50nM, the mixture was incubated at room temperature for 1 hour, then C3H3-Compound A bound to the tissue of C3H] -compound A unbound, was separated by filtration medium This filtration was through What an glass fiber filters (GF / B or GF / C), then the filters were quickly washed with medium regulated in their pH in ice water (50mM HEPES, pH7. 4) The amount of radioactivity bound to the tissue trapped in the filters was measured by the addition of liquid scintillation combination filters, followed by counting in a liquid scintillation counter.To determine the "specific" binding amount of [3H3- compound fl (that is, site-specific binding novel) parallel tests were carried out as above in which C3H.I-compound fl and tissue were incubated together, in preedence of a high concentration (l-10uM) of an unlabeled compound that also binds to the novel site, avoiding thus the binding of C3H3-compound A to this tissue. The same unlabeled compound A was used, but other compounds that bind to the novel site can also be used. The amount of C3H3-7? Compound A remaining in the presence of this unlabelled compound is defined as "non-specific" binding. This amount is subtracted from the total amount of C3H3-Compound A, which binds (i.e., the one present in the presence of unlabeled compounds) to obtain the "specific" binding amount of C3H3-Compound A to a novel site. The estimates of the density of the novel binding site in the tissues and their affinity for the C3H-compound A were obtained by incubating tissue together with C3H3-compound in a scale of concentrations. The specific binding levels (as defined above) were then used at different concentrations of C3H3-Compound A to calculate the dissociation constant (* D) of the C3H3-compound fl for the novel site and the density (Bma ") of this site in the tissue.

RESULTS The calculated values for compound fl are KD 40nM and B "? 220 pmol / g protein for rat forebrain tissue. Using a method similar to that described, except that the concentration of compound B was approximately 10 times lower, the values calculated for compound B are KD 2nM and Bmax 220 pmol / g protein), for rat forebrain tissue.

EXAMPLE 2 Synthesis of Ccarboxyl-i * C3-Coropuesto fl y [carboxyl-14c3- Compound fl 1- CCARboxyl-1 * C34-fluorobenzoic acid To a stirred suspension of D4C3 potassium cyanide (100 Ci, 60 mCi / mrnoles-i) in anhydrous dirnethyphrhamide was added copper (I) iodide (158 mg, 0.83 mmole), and -fluoro-iodobenzene (433 mg, 1.95 mmol). The mixture was heated to reflux, under nitrogen, for 22 hours. The solution was made alkaline with the addition of 5N sodium hydroxide (2 rnl) and diluted with water (20 ml). The mixture was completely extracted with diethyl ether; the combined extracts were washed successively with saturated brine (2x30 mL), water (2x30 mL), dried over magnesium sulfate; it was filtered and the ether was separated by distillation. The residue was dissolved in ethanol (15 ml), potassium hydroxide (1.34 g) in water (8 ml) was added, and the resulting solution was heated under reflux for 15 hours. The cooled reaction mixture was diluted with water (20 ml), the pH was adjusted to 1-2 with 1 N hydrochloric acid, and the mixture was completely extracted with ethyl acetate. The combined extracts were washed with water (1x50 ml), dried over magnesium sulfate, filtered and evaporated to dryness, yielding Ccarboxyl-i * C34-fluorobenzoic acid (196 mg, 1.38 mmol, 68.4 mCi, 68.4%). 2. - CCarboxil-i * C3 Compound fl They were dissolved, Ecarboxyl-1,, iC3-4-fluorobenzoic acid (196rng, 68.4mC.i, 1.38mmolee), 1-hydroxybenzotriazole hydrated (191.4mg, 1, 42mmol) and l- (3-dimethylaninopropyl) -3-ethylcarbodiimicla hydrochloride (265mg, 1.38 rnols), in anhydrous dichloromethane (6 ml). To this was added a solution of (3R, 4S) -4-amino-3,4-dihydro-2,2-dimethyl-2H-benzo [b3pyran-3-ol (258 mg, 1.38 mmol, contains 95 p / p of methanol) and triethylamine (0.190 rnl) in dichloromethane (2.0 ml). The mixture was filtered at room temperature for 19.5 hours and the solvent was evaporated in vacuo. The residue was taken up in ethyl acetate (25 ml), washed successively with dilute HCl (2x25 nl), water (25 ml), saturated aqueous sodium bicarbonate (25 ml) and water (25 ml); dried over magnesium sulfate, filtered and evaporated to dryness. Ccarboxyl-1 ** »C3-Fl-Compound was purified by column chromatography (40 gillum, eluted with ethyl acetate / hexane 1: 3 v / v to remove non-polar impurities and 100% ethyl acetate elute p * C3-Compound fl, yielding [14C-Compound fl (179.7mg, 0.50 mmoles) A portion (130 mg) of this material was further purified by means of reparative HPLC ee i (column of Spherisorb 5U, silica 22.5 x 250 rnm, eluted at lOml / min with chloroform / methanol 95: 5 v / v) to produce C1 * C3-Compound A (107rng). This batch was left to equilibrate over water for 6 hours, then completely dried under vacuum. it had a radiochemical purity of 99.7%, an optical purity> 99%, a chemical purity of 95.5% ("as such") and a specific activity of 59.6Ci.mmoles-i.The spectrum * HRMN was consistent with the eetruct It is identical to the unlabeled Comet A prepared in W092 / 22293. The analytical systems are detailed below.

SCHEME 1 SYNTHESIS OF C1 «C] C0 POSITION [1 C] Compound A *? Radiocarbon tail of carbon-14 EXAMPLE 3 Synthesis of [carboxyl-i * C3. Synthesis of f3S, 4S) -6-acetyl-3, 4-dihydro-2, 2-diroetí 1-4- (3-chloro-4-fluorophenyl L "** C3ca rbonilaroino) -2H-benzole" b3 pyran 3-ol 3-Chloro-4-flurobenzoC1 * C3niile 3-Chloro-4-fluoroiodobenzene (472.8 mg, 1.84 mrnole), Ci * C3cy. Potassium hydrogen (IDOrnCi at a specific activity of 60mC.i.m.mole- *; 1.67 rnmolee , 108.8mg) and copper iodide (I) (173.9mg, 0.91 mmoles) in N-rnetylpyrrolidinone (4.5 rnl). Afterwards, the mixture was heated under nitrogen to 150 ° C with stirring, for a total of 19.25 hours and allowed to stand at room temperature for 49 hours). Examination by CCD (silica, eluent ethyl acetate / n-hexane 1: 5 v / v) showed that the reaction had proceeded to approximately 73% conversion. The reaction mixture was then partitioned between water (100 ml) and ethyl acetate (ICIO ml). The organic layer was washed successively with 2% w / v ferric chloride solution (100 ml), water (100 ml), sodium ethabisulfite solution 2% w / v (100 ml), water (100 ml) and brine (100 ml). Then the organic layer was dried over magnesium sulfate, filtered to remove the desiccant, evaporated to dryness under reduced pressure and the residues were subjected to column chromatography on silica (Merck Art. 9385) eluting with ethyl acetate / n- Hexane 1: 8 The relevant fractions were collected and combined to produce a solution containing approximately 72.7rnCi, corresponding to 1.21 mmole, 188rng; (72.7% yield) of 3-chloro-4-fluorobenzo [i * C3nitrile.

Synthesis of 3-chloro-4-fluorobenzoCi * C3-carboxylic acid A solution of 3-chloro-4-fluorobenzoCl4C3nitrile (36 Ci at specific activity of BO Ci.mmoles- *; 0.6 mmol, 94.5rng) in ethyl acetate / n -hexane 1: 8 v / v (85 rnl) was reduced to dryness under reduced pressure. The residues were suspended in concentrated hydrochloric acid (8.0 ml) and heated to 100 ° C, with stirring, under nitrogen, for 6 hours. At approximately one hour intervals the mixture was stirred to again wash sublimated material in the mixture. The mixture was allowed to cool and stand at room temperature overnight. The mixture was then partitioned between water (30 ml) and ethyl acetate (40 ml). The aqueous layer was extracted again with ethyl acetate (40 ml); the organic layers were combined and extracted in 2M sodium hydroxide solution (40 rnl). The organic layer was extracted 2 more times in 2M sodium hydroxide solution (2x20 ml). The combined alkaline aqueous layers were carefully acidified to pH 1 with the addition of concentrated hydrochloric acid. The aqueous solution was then extracted 2 times in ethyl acetate (2x80 ml); the organic layers were combined, dried over magnesium sulfate and filtered to remove the desiccant. The desiccant was washed several times with small portions of ethyl acetate (total volume 50 ml) and the washings were combined with the filtrate. Then all of the solvent was evaporated under reduced pressure to yield a solid (75 rng, 0.43 mmol, 71.7% yield) of 3-chloro-4-fluorobenzene C14C3carboxylic acid.

Synthesis of C3S, 4S) -B-acetyl-3,4-dihydro-2,2-diroethyl-4- (3-chloro-4-fluoro-phenylCi * C3carbonylamino) -2H-benzoL'-3-pyran-3-Ql It was dissolved 3-chloro-4-fluorobenzene D4C3carboxylic acid (75 mg, 0.43 mmol) in DMF (2.5 rnL). To this solution was added hydroxybenzitriazole (82.6 mg, 0.61 mmol, 1.42 eq) [dried at room temperature under vacuum for 24 hours3 and l- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (113.8 mg, 0.59 mmol, 1.37 eq ) [dried at room temperature under vacuum for 24 hours3 and the mixture was stirred at room temperature for 30 minutes. A solution of (3S, 4S) -6 ~ acetyl-3,4-dihydro-2,2-dimethyl-4- (3-chloro-4-fluoro-phenyl [14C3carbonylamino) -2H-benzoCb pyran-3 was added. -ol (150 mg, 0.64 mmol, 1.49 eq) in DMF (2.5 mL) and the mixture was stirred at room temperature overnight. Then the mixture was partitioned between water (50 mL) and ethyl acetate (100 mL); the aqueous layer was extracted again in ethyl acetate (80 L). The organic layers were combined, washed with water (50 mL), dried over magnesium sulfate and filtered to remove the desiccant. The desiccant was washed on the filter with ethyl acetate (50 rnL); the filtrate and the washings were combined and evaporated to dryness under reduced pressure. The residues were subjected to column chromatography on silica eluting with ethyl acetate / n-hexane 1: 1 v / v. The relevant fractions of the column were combined, evaporated to dryness under reduced pressure and the foamy residues obtained in this way were recrystallized from acetone / n-hexane to produce a white solid product which was dried under vacuum to give (3S, 4S) -6-acetyl-3,4-dihydro-2,2-dimethyl-4- (3-chloro-4-fluoro-phenyl [14C3carbonylamino) -2H-benzoCb3pyrano-3-ol. The product had a radiochemical purity of 98.7%, a chemical purity of 99.4% ("as such"), a chiral purity of 99.9% and a specific activity of 59.4m Ci.mmoles-i. The H NMR spectrum was consistent with the structure and identical to the unlabeled compound prepared as described in the aforementioned patents.

EXAMPLE 4 1251] Compound B The p > Aladio (II) catalyzed the coupling of the compound B unlabelled with bis (tributyltin) to give the detribethytannate derivative. The white compound labeled with iodo-125, compound B- [i25ii / was then obtained by staging radioiodine on a 100-acre portion of the tributylstannane derivative with 5.0 mCi of iodide-D25 i] t in the presence of 1.0-2.0 micrograms of Chlorunide-T as an oxidant in 3% acetic acid in ethanol. This procedure gave 3.4-3.9 mCi (68-78% radiochemical yield) of [125 i3Com? Set B], at a radiochemical purity of at least 99% after purification by HPLC (Baker silica gel column, 4.6 rnm ID x25 c, 98.2 hexane / isopropanol, eluted at 1.0 mL / min with UV measured at 230 nm). It was determined that the specific activity (derived from measurements of mass concentrations and radioactivity) was 1775-1800 Ci / mmoles.

EXAMPLE 5 C3H3 Compound A Compound D (1.0-1.2 rng, 1.9-2-4 umoles) was dissolved in 1.0 rnL of 9: 1 (v / v) DMF (Baker) / triethylanine (Aldrich). This solution was added 1.0-1.2 mg (100% p) of palladium on carbon at .1.0% (Aldrich). The reaction mixture was stirred under a range of 3.6-4.9 Ci of tritium gas for 16-17 hours at room temperature. Removal of volatile components by addition of rnetanol (3 x 2 L) followed by vacuum transfer left 108-146 mCi of crude product. Radio-HPLC analysis showed that this material contained 61-66% of [3H3 Compound fl. This material was purified by means of reverse phase HPLC (Beckman Octil column, 4.6 x 250 mm, acetonitrile / water / trifluoroacetic acid (40: 60: 0.1), UV detection at 220-240 nm, flow rate lmL / min. ) in four to five injections. The eluate corresponding to the product was lyophilized and then dissolved in ethanol to provide 2425 rnCi of [H3 Compoteto fl. The analysis gave a specific activity of 21.5-29 Ci / moles (abundance of isotope by chemical ionization-mass ethano spectrum, NH3 gas reagent) and a radiochemical purity of at least 99% (column of Ultraphere ODS 5 um, 4.6x 250 mm, linear gradient of acetonitrile / water / trifluoroacetic acid from 34: 66: 0.1 to 90:10:01 for 10 min at 1.0 L / min, UV detection at 230 nm and detection of radioactivity by means of a flow-titration monitor of online radioactivity).

Compound D H Compound A EXAMPLE 6 Compound C The photoafin mark of 1-5 -3-acetyl-3,4-dihydro-2,2-dimethyl-8- [i25i3-iodo-4S- [3-ttrifluoromethyl-3H-diazirin-3-yl} -benzoylamino3-2H-benzoCb3pirano-3R-ol was prepared by introducing the radiolabel by ipso substitution of 8-trib? leetannane under normal conditions. { Na [1251] (without added vehicle / chloramine-T.) And [125 i3-6-acetyl-3,4-dihydro-2? 2-dirnethyl-8-Ci Si3yodo-4S- [3- { 3-trifluorornethyl-3H-diazirin-3-yl.} - benzoylamino-3H-benzo [bliropr-3R-ol, [125?] SB-224172 crude, purified by HPLC fJNovapak C18 eluted with 0.1% aqueous TFO / acetonitrile (1.1 v v).) The stannane was prepared by reaction of 6-flcetyl-3,4-dihydro-2,2-dimethyl-8-iodo-4S-amino-2H-benzo [b] pyrano-3R-ol with hexabutyl diamine and bis (triphenylphosphine) of dibro-opaladium and subsequent condensation with 3-C3- (trifluoromethyl) -3H-diazirin-3-yl-3-benzoic acid.

Flowchart EXAMPLE 7 a) 3-ClJl-Diazirino-2-trifluoroethyl) benzoic acid To a stirred solution of 3- (1, 1-Diazirino-2-trifluoroethyl) benzyl alcohol (2.7 g, prepared according to M. Ceruso and GD Preetwich , Bioorg and Med Chem Letts, 1994 4, 2179-2184) in dioxane 810 mL) and 0.2M KOH solution (63 mL) were added KMN04 (2.45 g) in portions over 2.5 hours. The mixture was then filtered through a pad of Celite to remove excess Mn02 and the filtrate was extracted with ether. The aqueous layer was acidified with 1M H2SO4 (25 mL) and extracted with ether. The organic layer was washed with water, brine and dried over anhydrous Na 2 SO 4. Filtration and evaporation in vacuo gave the compound of the description as a light yellow solid (2.45 g). b) trans-6-flcetyl-4S- (3-tl, l-diazirino-2-trifluoro-roethyl) -benzoylamino) -3,4-dihydro-2,2-dimethyl-8-iodo-2H-l- benzopyran-3R-ol (Compound C) To a solution of diazirinylbenzoic acid from the previous description (0.124 g) in DMF (2 ml) was added dinethylaminopropylcarbodiirnide deethyl hydrochloride (0.095 g) and 1-hydroxybenzotriazole (0.067 g). This solution was stirred at room temperature for 10 minutes. trans-6-flcetyl-4R-amino) -3,4-dihydro-2, 2-dirnethyl-8-iodo-2H-l-benzo? iran-3R-ol (0.015 g, prepared as in description 1 and example 8 of PCT / EP / 95/02249) was added to the solution and stirring was continued for 3 hours. The mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with dilute HCl, water, saturated NaHCO 3 solution and brine and dried over anhydrous Na 2 SO 4. Filtration and vacuum evaporation gave solid crude (0.37 g) which was recrietalized from ethyl acetate: n-hexane to give the compound of Example 7 as crystals. P.f. 109-100oCia3D25 + 55.3 ° (MeOH, c = .l..1)

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. - A receptor in substantially pure form obtained from rat procencephalic tissue, characterized in that; a) Comet A is bound to it with a Kd of 0nM for rat procencephalic tissue, b) Compound A binds to it with a Brnax of 220 pmoles / g protein for rat procencephalic tissue; c) Compound B is bound to it with a Kd of 2nM of rat procencephalic tissue; d) Compound B is bound to it with a Bmax of 220 pmol / g protein for rat procencephalic tissue; and receive homologs from other sources that share at least 85% homology with rat procencephalic tissue.

2. A soluble form of the receptor according to claim 1.

3. A receiver according to claim 1, which has a molecular weight in the region of 130 kiloDaltons, when analyzed by means of electoforesis gel (SDS) -PAGE).

4. The use of a soluble form of the receptor according to claim 2, in the manufacture of a medicament for treating and / or preventing anxiety, mania, depression, disorders associated with an arachnoid hemorrhage or neural shock, the effects associated with abstinence from abuses such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents, such as epilepsy; Parkinson's disease, psychosis, migraine and / or cerebral ischemia.

5. A pharmaceutical composition for use in the treatment or prevention of anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents, such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia, comprising the soluble receptor according to claim 2 in admixture with pharmaceutically acceptable carriers. 6. A method of selecting compounds that have therapeutic activity associated with receptor binding according to claim 1, which comprises contacting a test compound with a substrate in which the novel receptor is present and measuring the degree of Union. 7. A radiolabelled compound which binds to the receptor according to claim 1, and can be displaced using the selection method according to claim 6. 8.- A novel compound and pharmaceutically acceptable salts, hydrates or solvates. of the same identified in the selection method in accordance with the claim

6. 9. The use of a kit according to claim 8 in the manufacture of a medicament for the treatment or prevention of anemia, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents, such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia. 10. A pharmaceutical composition for use in the treatment or prevention of anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from subetanciae of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders which can be treated and / or prevented with anticonvulsant agents, such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia, which comprises the mixture of a compound according to claim 8, with a pharmaceutically acceptable carrier . 11. A monoclonal or polyclonal antibody that binds to the receptor according to claim 1. 12. The use of a monoclonal or polyclonal antibody according to claim 11 that binds to the novel receptor., in the manufacture of a medicament for treating and / or preventing anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from sub-levels of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and / or prevented with anticonvulsant agents, such as epilepsy, Parkinson's disease, psychoeisis, migraine and / or cerebral ischemia. 13. A pharmaceutical composition for use in the treatment and / or prevention of anxiety, mania, depression, disorders associated with a subarachnoid hemorrhage or neural shock, the effects associated with abstinence from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines , disorders that can be treated and / or prevented with anticonvulsant agents, such as epilepsy, Parkinson's disease, psychosis, migraine and / or cerebral ischemia, comprising the mixture of a monoclonal or polyclonal antibody according to claim 15 with a vehicle pharmaceutically acceptable. 14. A radio-labeled component that connects the novel receiver according to claim 1. 15. The use of radio-controlled compounds according to claim 14, which bind to the novel receiver as diagnostic tools for detecting changes or abnormalities in the novel receiver according to claim 1.