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    • 专利摘要:
    New pyrazolone-like compounds, their use for the diagnosis of allergies to beta-lactam antibiotics, preferably aminocephalosporins and/or aminopenicillins, and preparation procedure. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2710544A1
    申请号:ES201731240
    申请日:2017-10-20
    公开日:2019-04-25
    发明作者:Jaen María José Torres；Vega María Isabel Montanez；Mayorga Cristobalina Mayorga；Ortiz Angela Martin-Serrano；Diaz Inmaculada Dona；Villatoro Ezequiel Perez-Inestrosa
    申请人:Universidad de Malaga；Servicio Andaluz de Salud；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] FIELD OF THE TECHNIQUE
    [0005]
    [0006] The present invention belongs to the field of medicine, pharmacy and biological chemistry, and refers, fundamentally, to the use and preparation of new pyrazolone-type compounds for the diagnosis of allergies to beta-lactam antibiotics, preferably aminocephalosporins and / or aminopenicillins.
    [0007]
    [0008] BACKGROUND OF THE INVENTION
    [0009]
    [0010] Cephalosporins, a class of beta-lactam antibiotics, derivatives of cephalosporanic acid are, after penicillins, the most widely used antibacterial agents for the treatment and prophylaxis of infectious diseases, but also one of the main causes of adverse immune reactions.
    [0011]
    [0012]
    [0013]
    [0014]
    [0015] However, unlike penicillins, for which the structures responsible for the allergy have been identified, the metabolites of the cephalosporins that present antigenic character, and therefore responsible for the immunological responses to cephalosporins, are not yet fully characterized.
    [0016]
    [0017] Although it is not known with certainty what this structure consists of, if there is a clinical and immunochemical evidence that the side chain R can contribute to the induction of specific Immunoglobulin E (IgE) and to the development of cross-reactivity responses between different cephalosporins . In fact, some patients can react selectively against the cephalosporin involved in the reaction, others against several cephalosporins with the same or similar R side chain (Antunez et al., J. Allergy Clin. Immunol.
    [0018] 117, 404-410, 2006; Romano, A. et al. J. Allergy Clin. Immunol. 106, 1177-1183, 2000; Saenz from San Pedro, B. et al. Ann. Allergy, Asthma Immunol. 89, 101-103, 2002) and a third group of patients may present cross-reactivity with other beta-lactams, especially penicillins, with the same or similar R (Romano, A. et al., J. Allergy Clin. Immunol. 1177 1183, 2000; Kelkar, PS et al. N. Engl. J. Med. 345, 804-809, 2001; Miranda, A. et al. J. Allergy Clin. Immunol. 98, 671-677, 1996; Novalbos, A. et al. Clin. Exp. Allergy 31,438-443 2001).
    [0019]
    [0020] Antunez et al. prepared a series of monomeric conjugates of cephalosporins to butylamine, and evaluated their molecular recognition by IgE in patients allergic to different cephalosporins (Antunez et al., J. Allergy Clin. Immunol., 117, 404-410, 2006). As a consequence of this conjugation to butylamine, a series of different structures are formed, difficult to characterize, but it is assumed that most of the conjugates formed include the R side chain in their structure. In patients allergic to cephalosporins, a recognition was observed selective of the side chain R in 67% of the cases, with some degree of cross-reactivity to cephalosporins with identical R side chain or similar. This study was very useful to confirm the importance of the side chain in recognition of IgE specific to cephalosporins, but does not give more information about the specific structure that forms the epitope or antigenic determinant.
    [0021]
    [0022] There are 3 studies that have addressed this problem through the synthesis of perfectly defined structures, as possible antigenic determinants of cephalosporins, which have been evaluated immunochemically.
    [0023]
    [0024] Sanchez-Sancho et al have identified a possible basic structure of the antigenic determinant, which remains bound to the carrier protein in the process of conjugation of the cephalosporins (Sanchez-Sancho, F. et al., J. Mol. Recognit., 16, 148- 156, 2003). These researchers postulated the formation of a cephalosporoyl conjugate that underwent a fragmentation of the dihydrothiazine portion as soon as it formed, giving rise to a complex mixture of unstable compounds difficult to isolate and analyze. The structure proposed by these researchers was an aN-acyl-L-alanylbutanamide, which comprises the acyl R side chain of the cephalosporins and the aminoaddic residue included in the betalactamic structure of the cephalosporins, losing the R 'substituent. In this proposed structure, the N-butyl chain aims to emulate the alkyl chain of the lysine residues present in the proteins. However, the functional group present in Carbon 3 of said derivatives (called group G = CH3) was not totally determined, since this carbon could present diverse functionalizations. Subsequently, a series of derived molecules with different functional groups G (CH2OH, C (OCH3) 2) were developed, which were shown to refine the molecular recognition by the IgE of patients allergic to cephalosporins (Montanez, MI et al., Chem. Res. Toxicol. 24, 706-717, 2011). These results were satisfactory for a series of cephalosporins, but could not be demonstrated with the a-amino-cephalosporins.
    [0025]
    [0026] In the case of a-amino-cephalosporins, it is hypothesized that the antigenic determinants could have a pyrazinone structure, derived from an intramolecular cyclization of the amino group of the side chain to the carbonyl group, which is proposed to be formed in the carbon 6 of the intermediate product of aminolysis of the cephalosporin with proteins. In the case of the aminocephalosporins cefaclor and cephalexin, the formation of a pyrazinone-type degradation product (1,6-dihydro-5-phenyl-6-oxo-N-propylpyrazinecarboxamide) has been described by reacting them in the laboratory with propylamine in aqueous medium (Venemalm, L. Bioorg, Med. Chem. Lett., 11, 1869-1870, 2001). However, the immunochemical evaluation of this pyrazinone could not be carried out due to the low yield obtained and the difficulty involved in its chemical synthesis by other procedures (total synthesis), resorting to the synthesis of a similar compound (1,6-dihydro-5-acid). phenyl-6-oxo-pyrazine-acetic acid) that was conjugated to carrier molecules for their immunological evaluation. The results indicate that 60% of patients with positive IgE, through ImmunoCAP, to cefaclor presented IgE directed to this structure. However, the compound synthesized has a carboxymethyl radical as a substituent in the pyrazinone ring, which implies, in relation to the in vivo degradation hypothesis, the introduction of an additional methylene group (between the pyrazinone ring and the carbonyl group) into a fragment. of the molecule with notable influence on molecular recognition.
    [0027]
    [0028] Therefore, the fact that the chemical structure of the antigenic determinant of cephalosporins has not been fully characterized, is hindering the development of diagnostic tools that allow an adequate evaluation of the existence of an allergic reaction to this group of antibiotics. Currently, there is only one commercial method, immunoCAP-FEIA, for detecting IgE specific to cefaclor (Ariza, A. et al., J. Investig Allergol, Clin. Immunol. 25, 12-25, 2015), which does not have the adequate sensitivity.
    [0029]
    [0030] BRIEF DESCRIPTION OF THE INVENTION
    [0031]
    [0032] The present invention faces the problem of providing a method that allows the evaluation of the allergic reaction to cephalosporins and that presents an adequate sensitivity. Thus, the present inventors have discovered a compound of formula (I) that recreates the antigenic determinant of aminocephalosporins, as well as a method for its synthesis, which is useful for the detection of IgE antibodies generated by hypersensitivity to betalactams, preferably aminocephalosporins and / or aminopenicillins.
    [0033]
    [0034] Thus, a first aspect of the present invention relates to a compound of general formula (I):
    [0035]
    [0036]
    [0037]
    [0038]
    [0039] or any of its salts, tautomers or hydrates, where:
    [0040]
    [0041] R1 is selected from linear or branched, substituted or unsubstituted C1-C10 alkyl, straight or branched (C2-C10) alkenyl, substituted or unsubstituted, and straight or branched, substituted or unsubstituted alkynyl (C2-C10).
    [0042]
    [0043] R2 is selected from hydrogen and hydroxyl
    [0044]
    [0045] With the condition that the compound is excluded:
    [0046]
    [0047] W-propyl-6-oxo-5-phenyl-1,6-dihydropyrazine-2-carboxamide
    [0048]
    [0049] A second aspect of the invention relates to a composition comprising or consisting of at least one compound of the present invention, or any combination thereof, hereinafter the composition of the invention.
    [0050]
    [0051] Another aspect of the invention relates to a compound of the invention, or a composition of the invention, for use in medicine.
    [0052]
    [0053] Another aspect of the invention relates to a compound of the invention, or a composition of the invention, for the detection of IgE antibodies generated by hypersensitivity to betalactams.
    [0054]
    [0055] Another aspect of the invention relates to a method of obtaining the compounds of the invention of formula (I) as described below.
    [0056]
    [0057] Another aspect of the invention relates to an in vitro method of obtaining useful data for the prevention, identification, screening and / or diagnosis of the hypersensitivity to the aminocephalosporins of an individual.
    [0058] Another aspect of the invention relates to an in vitro method for the prevention, identification, screening and / or diagnosis of hypersensitivity to the aminocephalosporins of an individual.
    [0059]
    [0060] Another aspect of the invention relates to a kit or device that comprises or consists of at least one compound of the invention or in the composition of the invention.
    [0061]
    [0062] BRIEF DESCRIPTION OF THE FIGURES
    [0063]
    [0064] Figure 1. Results of inhibition of RAST, using as inhibitors compound 1, compound 3 and the conjugate cefaclor-butylamine, at concentrations 10 and 100 mM, in 10 cases of patients allergic to cefaclor.
    [0065]
    [0066] Figure 2. RAST inhibition results, using as inhibitors compound 2, compound 4 and the cefadroxil-butylamine conjugate, at concentrations 10 and 100 mM, in 11 cases of patients allergic to amoxicillin. The cases of group A present cross-reactivity with benzylpenicillin while those of group B have selective hypersensitivity to amoxicillin.
    [0067]
    [0068] DETAILED DESCRIPTION OF THE INVENTION
    [0069]
    [0070] The fact that the chemical structure of the antigenic determinant of cephalosporins has not been fully characterized, is hindering the development of diagnostic tools that allow an adequate evaluation of the existence of an allergic reaction to this group of antibiotics.
    [0071]
    [0072] The authors of the present invention have identified a family of compounds, with structure of pyrazolone, derivatives of alpha-aminocephalosporins that allows the detection of IgE antibodies generated in allergic reactions to betalactams, preferably aminocephalosporins and / or aminopenicillins, and which elevates sensitivity of the immunoassay compared to the synthetic determinants proposed to date.
    [0073]
    [0074] COMPOUNDS OF THE INVENTION
    [0075]
    [0076] Thus, a first aspect of the present invention relates to a compound of general formula (I):
    [0077]
    [0078]
    [0079] or any of its salts, tautomers or hydrates, where:
    [0080]
    [0081] R1 is selected from linear or branched, unsubstituted or substituted Cr C10 alkyl, linear or branched (substituted or unsubstituted) C2-Ci0 alkenyl, and straight or branched, substituted or unsubstituted alkynyl (C2-C10);
    [0082]
    [0083] R 2 is selected from hydrogen, hydroxyl and alkyloxy, wherein the hydrocarbon chain of the ether function is a linear or branched alkyl of 1 to 5 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n -butyl, tert-butyl, etc.
    [0084]
    [0085] With the proviso that the compound is excluded:
    [0086]
    [0087] W-propyl-6-oxo-5-phenyl-1,6-dihydropyrazine-2-carboxamide
    [0088]
    [0089] The term "alkyl" refers in the present invention, unless otherwise indicated, to aliphatic, linear or branched chains, having from 1 to 10 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, npentyl, n-hexyl, etc. Preferably the saturated hydrocarbon chain has between 1 and 6 carbon atoms. More preferably it is n-butyl. The alkyl radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a group substituted or not selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide; preferably the substituent is hydroxyl or azide.
    [0090]
    [0091] The term "alkenyl" refers in the present invention, and unless otherwise indicated, to aliphatic chains unsaturated with at least one double bond, linear or branched, having from 2 to 10 carbon atoms.
    [0092]
    [0093] The term "alkynyl" refers in the present invention, and unless otherwise indicated, to aliphatic chains unsaturated with at least one triple bond, linear or branched, having from 2 to 10 carbon atoms.
    [0094]
    [0095] In the present invention, the chains unsaturated hydrocarbon chains, alkenyl and alkenyl, may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide; preferably the substituent is hydroxyl or azide.
    [0096]
    [0097] In a preferred embodiment of this aspect of the invention, R 1 is a linear or branched (C 1 -C 10) alkyl, substituted or unsubstituted; more preferably it is an n-butyl.
    [0098]
    [0099] In another preferred embodiment of this aspect, R1 is substituted by an azide group or an alcohol group; preferably R1 is a substituted alkyl; and even more preferably R1 is a substituted n-butyl.
    [0100]
    [0101] In another preferred embodiment of this aspect, R 2 is hydrogen or hydroxyl.
    [0102]
    [0103] In another preferred embodiment of this aspect, the compound is selected from the list consisting of N-butyl-6-oxo-5-phenyl-1,6-dihydropyrazine-2-carboxamide and / or N-butyl-5- (4 -hydroxyphenyl) -6-oxo-1,6-dihydropyrazine-2-carboxamide or any of its salts, tautomers or hydrates.
    [0104]
    [0105] In another preferred embodiment of this aspect, the compounds of formula (I) of the present invention are attached to carrier molecules or solid support. Illustrative, non-limiting examples of carrier molecules are proteins, synthetic pho- lymers (such as poly-L-Lysine (PLL)), and dendrimers. Illustrative, non-limiting, solid support examples are nanoparticles, microparticles, spheres / sheets of silica, cellulose, zeolites, plastic, latex, glass, Sepharose (polymeric polysaccharide material) and phenyl-octyl Sepharose CL4B; more preferably the compounds of formula (I) of the present invention are attached to dendrimers.
    [0106]
    [0107] The binding to carrier molecules or solid support can be carried out, but without limitation, through at least one substituent present in the hydrocarbon chain R1, based on the general reactivity of such substituent, and following the methodologies usually described in the art. Preferably such a substituent is hydroxyl or azide.
    [0108]
    [0109] COMPOSITION OF THE INVENTION
    [0110]
    [0111] Another aspect of the invention relates to a composition comprising or consisting of at least one compound of the present invention, or any combination thereof, and to which we refer hereinafter as the composition of the invention.
    [0112] In a preferred embodiment of this aspect of the invention, the concentration of the compound of the invention is between 0.5 mM and 500 mM, more preferably between 10 mM and 200 mM, and even more preferably is 100 mM.
    [0113]
    [0114] In another preferred embodiment of this aspect, the composition comprises at least one other active ingredient.
    [0115]
    [0116] As used in this invention the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological or other different effect in the diagnosis , cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the elaboration of the drug and are present therein in a planned modified form that provides the specific activity or effect.
    [0117]
    [0118] In another preferred embodiment of this aspect, the composition further comprises at least one excipient and / or a pharmaceutically acceptable veticule.
    [0119]
    [0120] In another alternative embodiment of this aspect, the composition consists of at least one compound of the present invention, or any of its combinations as single or only active ingredients, although it may comprise excipients and / or pharmaceutically acceptable vehicles.
    [0121]
    [0122] In another preferred embodiment, the composition of the invention is a pharmaceutical composition.
    [0123]
    [0124] Also, within the scope of the present invention are the prodrugs of the pharmaceutical compositions of the invention. The term "prodrug" or "prodrug" as used herein includes any derivative of a compound of the invention, such as a compound of formula (I), which when administered to an individual or added to an isolated biological sample. of an individual can be transformed directly or indirectly into said compound, such as the compound of formula (I), in said individual. Advantageously, said derivative is a compound that increases the bioavailability of the compound when it is administered to an individual or added to a biological sample isolated from an individual or that enhances the release of the compound in a biological compartment. The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
    [0125] Within the scope of the present invention are "derivatives" which include both pharmaceutically acceptable compounds, ie, derivatives of the compound of formula (I) that can be used in the preparation of a medicament or food compositions, and derivatives pharmaceutically not acceptable, since these may be useful in the preparation of pharmaceutically acceptable derivatives. The derivatives may include stereoisomers, depending on the presence of multiple bonds, or chiral centers. Stereoisomers (enantiomers, diastereoisomers, meso forms) and mixtures thereof fall within the scope of the present invention, that is, the term stereoisomer also refers to any mixture of stereoisomers (racemic or mixtures in different proportions of the different stereoisomers) ). The diastereoisomers, as well as their mixtures, can be separated by conventional techniques.
    [0126]
    [0127] Any of the compounds of the invention may be in crystalline form, or in liquid form, for example oils, as free compounds or as solvates. In this sense, the term "solvate", as used herein, includes both pharmaceutically acceptable solvates, that is, solvates of the compound that can be used in the manufacture of a medicament, and pharmaceutically unavailable solvates, which can be useful in the preparation of solvates or pharmaceutically acceptable salts. The nature of the pharmaceutically acceptable solvate is not critical, so long as it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. The solvates can be obtained by conventional solvation methods known to those skilled in the art.
    [0128]
    [0129] For their application in diagnosis or therapy as pharmaceutical compositions, the compounds of the invention, their salts, prodrugs or solvates, will preferably be in a pharmaceutically acceptable or substantially pure form, that is, having a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels. The purity levels for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and still more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or of its salts, solvates or prodrugs.
    [0130] On the other hand, we note that the adjuvants and pharmaceutically acceptable vehicles that can be used in the pharmaceutical compositions of the invention are the adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of therapeutic compositions.
    [0131]
    [0132] The compounds described in the present invention, their salts, prodrugs and / or solvates as well as the pharmaceutical compositions containing them can be used together with other drugs or additional active ingredients to provide combination therapy. Said additional drugs can be part of the same pharmaceutical composition or, alternatively, can be provided in the form of a separate composition for simultaneous administration, or not, to that of the pharmaceutical composition comprising a compound of formula (I), or a salt, prodrug or solvate thereof.
    [0133]
    [0134] MEDICAL USES OF THE INVENTION
    [0135]
    [0136] Another aspect of the invention relates to a compound of the invention, or a composition of the invention, for use in medicine, or alternatively to the use of the compound of the invention or a composition of the invention in the manufacture of a medicament.
    [0137]
    [0138] The compounds of the present invention can be used for the diagnosis of allergies to antibiotics with structures of betalactam type.
    [0139]
    [0140] Therefore, another aspect of the invention relates to a compound of the invention, or a composition of the invention, for the detection of IgE antibodies generated by hypersensitivity to betalactams, or alternatively, to the use of at least one compound of the invention. , or a composition of the invention, in the manufacture of a medicament for the detection of IgE antibodies generated by hypersensitivity to betalactams.
    [0141]
    [0142] In a preferred embodiment of this aspect, betalactams are aminopenicillins and / or aminocephalosporins; more preferably the betalactams are aminocephalosporins, and even more preferably cefaclor and cefadroxil.
    [0143]
    [0144] In another preferred embodiment of the invention, the compound for use in medicine or for the detection of IgE antibodies generated by hypersensitivity to betalactams is selected from the list consisting of N-butyl-6-oxo-5-phenyl-1,6 -dihydropyrazine-2-carboxamide and / or N-butyl-5- (4-hydroxyphenyl) -6-oxo-1,6-dihydropyrazine-2-carboxamide or any of its salts, tautomers or hydrates.
    [0145]
    [0146] The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, compounds with structures of beta-lactam type are used for the diagnosis of allergies.
    [0147]
    [0148] METHOD OF OBTAINING THE COMPOUNDS
    [0149]
    [0150] Another aspect of the invention relates to a method for obtaining the compounds of the invention of formula (I), hereinafter the first method of the invention, characterized in that it comprises the following steps:
    [0151]
    [0152] (1) Obtaining a compound of formula (VI)
    [0153]
    [0154]
    [0155]
    [0156]
    [0157] by reacting the compounds of formula (II), (III), (IV) and (V) in an organic solvent
    [0158]
    [0159]
    [0160]
    [0161]
    [0162] where R3 and R4 are amine protecting groups that are selected independently; wherein the compound of formula (IV) presents in alpha position to the aldehyde group an acetal group as protecting group of a carbonyl group, so that R5 and R6 are independently selected from the group consisting of linear, branched, organic carbon chain, heterodclic, saturated and unsaturated, for example methyl, ethyl, npropyl, i-propyl, etc .; and wherein R5 and R6 may also be linked by a carbon-carbon covalent bond forming a cyclic acetal, for example of the 1,3-dioxolane or 1,3-dioxane type; and wherein R1 and R2 are defined as defined by the compounds of formula (I) of the present invention.
    [0163]
    [0164] (2) obtaining a compound formula (VII) from the compound of formula (VI) by eliminating the amine protecting groups R3 and R4 and breaking up the acetal formed by -OR5 and -OR6.
    [0165]
    [0166]
    [0167] (3) obtaining a compound of formula (I) by intramolecular cyclization of the compound of formula (VII), through a nucleophilic attack of the primary amino group on the aldeddo group, and subsequent aromatization.
    [0168]
    [0169] Said method also serves to obtain the compound N-propyl-6-oxo-5-phenyl-1,6-dihydropyrazine-2-carboxamide.
    [0170]
    [0171] As used in the present invention, the term "amine protecting group" is understood, unless expressly indicated otherwise, to be a group of those commonly used in organic chemistry that may be attached to a nitrogen atom to protect said atom from Nitrogen to participate in an unwanted reaction and that can be easily removed after the reaction Illustrative examples of these protecting groups, but not limited to, are a t-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (CBz) or , 5-dimethoxybenzyl Other suitable protective groups can be found in such texts as Greene's Protective Groups in Organic Synthesis, 4th ed., Wuts, PGM, Greene, TW, John Wiley & Sons: Hoboken, New Jersey, 2007.
    [0172]
    [0173] The removal of the protective groups and acetal cleavage of step (2) takes place according to methods known in the literature and which can be found in texts such as the previous one. For example, the Boc group can be cleaved with acid medium, the CBz group with acid medium or with H2 / Pd, etc.
    [0174]
    [0175] In a preferred embodiment of this aspect of the invention, R3 and R4 are independently selected from tert-butoxycarbonyl (Boc), 2,5-dimethoxybenzyl, trityl, fluorenylmethyloxycarbonyl (Fmoc) and benzyloxycarbonyl (CBz); more preferably R3 is tert-butoxycarbonyl (Boc) and R4 is 2,5-dimethoxybenzyl.
    [0176]
    [0177] In another preferred embodiment of this aspect, R5 and R6 are lmeal or branched (C1-C5) carbon chains that are selected independently; more preferably they are methyl.
    [0178]
    [0179] In another preferred embodiment of this aspect, R5 and R6 are joined by a carbon-carbon bond forming a 5- or 6-membered cyclic acetal; more preferably a 5-membered dicalic acetal.
    [0180] In another preferred embodiment of this aspect, R3 is tert-butoxycarbonyl (Boc), R4 is 2,5-dimethoxybenzyl, and R5 and R6 are methyl.
    [0181]
    [0182] In another preferred embodiment of this aspect, deprotection step (2) is carried out by treatment of the compound of formula (VI) with an acidic medium, and optionally with a previous or subsequent treatment with a mild basic medium.
    [0183]
    [0184] In another preferred embodiment of this aspect, step (2) is carried out by treating the compound of formula (VI) with an acidic medium; more preferably the acid medium is a trifluoroacetic acid (TFA) solution.
    [0185]
    [0186] In another preferred embodiment of this aspect, steps (2) and (3) of the method are carried out in a single reaction step by treatment of the compound of formula (VI) with an acidic medium; more preferably the acidic medium is a trifluoroacetic acid (TFA) solution; even more preferably it is a solution of trifluoroacetic acid (TFA) in a chlorinated organic solvent; and even more preferably is 30% trifluoroacetic acid (TFA) in dichloroethane.
    [0187]
    [0188] METHOD OF THE INVENTION
    [0189]
    [0190] Another aspect of the invention relates to an in vitro method of obtaining useful data for the prevention, identification, screening and / or diagnosis of the hypersensitivity to the aminocephalosporins of an individual, hereinafter second method of the invention, comprising or consists of the following steps:
    [0191]
    [0192] to. Collecting a blood sample from a subject, and incubating its serum with a compound or composition as defined in the present invention;
    [0193] b. Quantify the amount of specific IgE antibodies that are generated against the compound of section a); Y
    [0194]
    [0195] c. compare the amount obtained in step b) with the amount obtained in a negative control sample.
    [0196]
    [0197] In a preferred embodiment of this aspect of the invention, the quantification of step b) is carried out by a technique selected from the group consisting of: RAST (radioalergoadsorption test), ELISA (enzyme-linked immunosorbent assay) , FAST (fluorescence allergy-adsorption test), MAST (multiple chemiluminescence-allergy test), LAST (latex-allergy-adsorption test), and immunoassay performed in microarray support with fluorescent detection; more preferably it is carried out by RAST.
    [0198]
    [0199] The control sample can be analyzed, for example, simultaneously or consecutively, together with the biological sample problem. The comparisons described in the methods of the present invention can be performed manually or computer-assisted.
    [0200]
    [0201] Steps (a), (b) and / or (c) of the method described above can be fully or partially automated, for example, by means of a sensor robotics equipment for quantification in step (a) and (b) or the comparison in step (c).
    [0202]
    [0203] Another aspect of the invention relates to an in vitro method for the prevention, identification, screening and / or diagnosis of hypersensitivity to aminocephalosporins of an individual, hereinafter referred to as the third method of the invention, comprising steps (a) - (c) as described in the second method of the invention, and further comprises:
    [0204]
    [0205] d. diagnose the individual as allergic when the value obtained in stage c) is superior to the negative control.
    [0206]
    [0207] An "individual" or "subject", as used herein, refers to a mammal, human or non-human, in observation, and more preferably a human being.The individual can be anyone, an individual predisposed to allergy to aminocephalosporins or an individual suffering from allergy.
    [0208]
    [0209] KIT OR DEVICE OF THE INVENTION
    [0210]
    [0211] Another aspect of the invention relates to a kit or device that comprises or consists of at least one compound of the invention or in the composition of the invention.
    [0212]
    [0213] In a preferred embodiment of this aspect of the invention, the kit or device of the invention further comprises the elements necessary for the performance of an immunoglobulin E (IgE) antibody detection test generated in allergic reactions to aminocephalosporins.
    [0214]
    [0215] In another alternative embodiment of this aspect, the kit or device consists of at least one compound of the invention or in the composition of the invention, although it may also comprise the elements necessary for the performance of a detection test of the invention. Immunoglobulin E (IgE) antibodies generated in allergic reactions to aminocephalosporins.
    [0216]
    [0217] The kit or device of the invention can include another additional device for taking the isolated biological sample, preferably blood serum.
    [0218]
    [0219] EXAMPLE OF THE INVENTION
    [0220]
    [0221] In the following the invention is illustrated by the synthesis of some chemical compounds that respond to the general formula (I), as defined in the present invention, together with immunochemical tests carried out by the inventors with these synthetic compounds and which show its usefulness in the field of medicine and biological chemistry, and in particular, for the detection of IgE antibodies generated in allergic reactions to aminocephalosporins.
    [0222]
    [0223]
    [0224]
    [0225] Synthesis and characterization of synthesized compounds
    [0226]
    [0227] Equimolar amounts of all reagents are used: 60% aqueous solution of 2,2-dimethoxyacetaldehyde, butyl isocyanide, 2,5-dimethoxybenzylamine and N-Boc-phenylglycine (for the synthesis of compound 1) or N-Boc-hydroxyphenylglycine ( for the synthesis of compound 2), and allowed to react in methanol at room temperature for a minimum of 48 hours. When the reaction is finished, dichloromethane (CH2Cl2) and two equivalents of para-toluenesulfonic acid supported on a polystyrene resin (PS-p-TsOH) are added and maintained in agitation at room temperature for a minimum of one hour for the elimination of possible remains of the isocyanide. After that time, it is filtered to remove PS-p-TsOH and this is washed three times with methanol (MeOH), ethyl acetate (AcOEt) and dichloromethane (CH2Cl2). The organic fractions are united and the solvents are removed to obtain a residue corresponding to the UGI adduct. For the deprotection step, 30% trifluoroacetic acid (TFA) in dichloroethane (DCE) is added and keep at reflux (80 ° C) for two hours, after which time it is allowed to cool, basify with saturated solution of NaHCO 3 until pH 8 and extract with AcOEt. The organic phase is dried over anhydrous MgSO 4 and, after removing the solvent, a yellow syrup is obtained which is purified by chromatography on silica gel with CH 2 Cl 2 / MeOH mixtures to obtain the final compound (compound 1 or composite case 2) in the form of solid yellow. The structures were characterized by nuclear magnetic resonance (NMR-)
    [0228] 1 H, 13 C-NMR) and mass spectrometry (MS).
    [0229]
    [0230] Compound 1: M-butyl-6-oxo-5-phenyl-1,6-dihydropyrazine-2-carboxamide
    [0231]
    [0232] 1 H-NMR (400 MHz, MeOD): 5 (ppm) 8.52 (s, 1H; N-CH = CCO), 8.19 (m, 2H; Ar-ortho), 7.45 (m, 3H; Ar-meta + for ), 3.41 (t, 3 J (H, H) = 7.2 Hz, 2H; -CONH-CH 2 -), 1.62 (m, 2H; -CH 2 -CH 2 -CH 2 -), 1.43 (m, 2H ; -CH 2 -CH 2 -CH 3 ), 0.98 (t, 3 J (H, H) = 7.6 Hz, 3H; -CH 2 -CH 3 ).
    [0233]
    [0234] 13 C NMR (100 MHz, MeOD): 5 (ppm) 164.3, 157.7, 149.8, 136.9, 133.5, 131.0, 130.29, 129.1 (x2), 40.41, 32.61, 21.14, 14.08.
    [0235]
    [0236] ESI MS calculated for C 15 H 17 N 3 O 2 (MH) + 271.13, found: 272.1393.
    [0237]
    [0238] Compound 2: M-butyl-5- (4-hydroxyphenyl) -6-oxo-1,6-dihydropyrazine-2-carboxamide
    [0239]
    [0240] 1 H NMR (400 MHz, MeOD): 5 (ppm) 8.39 (s, 1H; N-CH = CCO), 8.17 (d, 3 J (H, H) = 9.5 Hz, 2H; Ar-ortho), 6.86 (d, 3 J (H, H) = 9.5 Hz, 2H; Ar-meta), 3.39 (t, 2H; -CONH-CH 2 -), 1.60 (m, 2H; -CH 2 -CH 2 -CH 2 -), 1.40 (m, 2H; -CH 2 -CH 2 -CH 3 ), 0.97 (t, 3 J (H, H) = 7.6Hz, 3H; -CH 2 -CH 3 ).
    [0241] 13 C NMR (150 MHz, MeOD): 5 (ppm) 164.1, 160.9, 157.1, 150.5, 135.6, 131.7, 130.4, 128.0, 115.9, 40.4, 32.6, 21.1, 14.1.
    [0242]
    [0243] ESI MS calculated for C 15 H 17 N 3 O 3 (MH) + 287.13, found: 288.1342.
    [0244]
    [0245] Study of the recognition of the synthetic structures by specific IgE antibodies
    [0246]
    [0247] The study of the recognition of type (I) synthetic structures by specific IgE antibodies has been carried out by competitive inhibition studies of RAST immunoassay, using human serum from patients who have been suffering from allergic reactions to aminocephalosporins (specifically cefaclor) or penicillins that possess the same side chains (specifically amoxicillin). In these inhibition studies, disks activated with BrCN, coupled to PLL and conjugated to the beta-lactam that caused the allergic episode of each patient, have been used as solid phase. As products in fluid phase the synthetic structures type (I) have been used.
    [0248] Before carrying out the inhibition study with the new synthetic structures, it is necessary to do the direct RAST to be able to know what each patient is allergic to.
    [0249]
    [0250] Radioimmunoassay for the determination of IgE: RAST
    [0251]
    [0252] The in vitro determination of the presence of IgE antibodies was carried out by RAST to cefaclor, amoxicillin and benzylpenicillin, using solid phase functionalized cellulose discs with each of the drugs conjugated to poly-L-Lysine.
    [0253]
    [0254] Serum was obtained from peripheral blood, which was kept at -20 ° C until its inclusion in the trials. Briefly, the assays are performed in duplicate, incubating 30 ^ l of serum with the solid phase previously functionalized with the beta-lactam-PLL conjugates for 3 hours. After three washes with PBS-tween buffer, radioactive labeled anti-IgE is added and left to incubate overnight. The discs are washed 3 times with PBS-tween buffer and their radioactivity is measured with a gamma counter. The results are calculated as a percentage with respect to a maximum and were considered positive if they were superior to 2%, result value of the mean (M) plus two standard deviations (SD) of a negative control group.
    [0255]
    [0256] Immunochemical recognition study: RAST inhibition
    [0257]
    [0258] In order to determine the recognition of the IgE antibodies to the different synthetic structures, competitive immunoassays were performed in those sera that showed high levels of RAST (> 7%). For this, the betalactam involved in the allergic reaction conjugated to PLL was used in the solid phase. The RAST inhibition test consists of a competition study between the conjugates in fluid phase and a conjugate in solid phase, by capturing the specific IgE available in the fluid phase. The results were calculated through the following formula:% Inhibition = (% RAST serum not inhibited -% RAST serum inhibited) x 100 /% RAST serum not inhibited.
    [0259]
    [0260] In particular, the RAST inhibition test was carried out by incubating in the fluid phase the serum of the patients at different concentrations (100 mM and 10 mM) of the compounds of formula (I) overnight (16 h). Later the solid phase is added for its incubation and the same protocol used for the RAST is followed. The inhibition test of the RAST is considered significant, that is, that the IgE show a specific recognition, when the inhibition of the RAST is equal to or greater than 50%.
    [0261] Results:
    [0262] Molecules derived from cefaclor and cefadroxil have been synthesized, corresponding to antigenic determinants of formula (I) (Compound 1 and Compound 2) according to the degradation of aminocephalosporins. The synthetic strategy employed has consisted in the reaction of Ugi / Deprotection / Cycling (UDC). This strategy has employed the multicomponent reaction of Ugi (U-4CR), in which an aldehyde or ketone, an isocyanide, an amine and a carboxylic acid are simultaneously used to give a bisamide (Ugi adduct). In said adduct, the aldehyde and amino groups are suitably protected. The subsequent elimination of the protective groups and aromatization gives rise to pyrazinone structures. It is a fairly efficient method in which all the starting products are commercial and the target molecule is achieved after only two synthetic steps and one purification step.
    [0263]
    [0264] The immunological recognition of these new compounds 1 and 2 (pyrazinone-like structures) was evaluated, together with previously described synthetic determinants (Compound 3 and Compound 4 - structures in Figure 1 and Figure 2. Montanez, MI et al., Chem. Res. Toxicol .
    [0265] 24, 706-717, 2011) and the monomeric conjugates with butylamine (Antunez et al., J. Allergy Clin, Immunol., 117, 404-410, 2006) by radioallergosorbent inhibition (RAST) inhibition studies using serum from allergic patients. betalactams. In this way we compared the recognition of the perfectly defined synthetic structures, compound 1 and compound 2, with the mixture of compounds resulting from the cephalosporin-butylamine conjugation.
    [0266]
    [0267] The immunological evaluation of structures derived from cefaclor (1, 3 and cefaclorbutylamine) was carried out at two concentrations (100 and 10 mM) with 10 sera from patients allergic to cefaclor. The test consists of a competition between the recognition of IgE by the solid phase (poly-L-lysine-cefaclor) vs the inhibitors (compound 1, compound 3 and cefaclor-butylamine) in the fluid phase. The inhibition results of RAST are shown in Figure 1.
    [0268]
    [0269] The results comparing both synthetic structures, at 100 mM concentration, showed that in 70% of cases compound 1 presented a higher percentage of inhibition, ie greater recognition, than compound 3. In general, at a concentration of 10 mM, the Decay recognition being similar for both structures. Considering the percentage of significant inhibition when it is greater than 50%, the recognition at the highest concentration was significant in 50% of the cases for compound 1, in 30% for compound 3 and in 60% of the cases. cases for the conjugate cefaclor-butilamina.
    [0270] The immunological evaluation of structures derived from cefadroxil (2, 4 and cefadroxilobutilamina) was carried out at two concentrations (100 and 10 mM) with a group of patients with cross-reactivity amoxicillin / benzylpenicillin (6 cases) and another group with selective hypersensitivity to amoxicillin (5 cases), all with positive direct RAST to cefadroxil. The test consists of a competition between the recognition of IgE by the solid phase (poly-L-lysine-amoxicillin) vs the inhibitors (compound 2, compound 4 and cefadroxil-butylamine) in the fluid phase. The results of inhibition of RAST are shown in Figure 2.
    [0271]
    [0272] The results comparing both synthetic structures in 6 patients with cross-reactivity, at 100 mM concentration, considering the percentage of significant inhibition when it is greater than 50%, showed that the recognition was significant in 50% of the cases for compound 2, in 83% of cases for compound 4 and in 100% of cases for the conjugate cefadroxil-butylamine.
    [0273]
    [0274] The results comparing both synthetic structures in 5 patients with selective hypersensitivity to amoxicillin, at 100 mM concentration, showed that the recognition was significant in 40% of the cases for both compound 2 and compound 4, and in 80% of the cases for the cefadroxil-butylamine conjugate.
    [0275] These results confirm that the recognition in the case of patients with cross reactivity to other betalactams is directed to the nuclear region thereof (betalactamic ring), present in structure 4 and modified in structure 2. On the contrary, in patients With selective hypersensitivity, the recognition of both synthetic structures is very similar, as expected because the recognition is directed to the side chain R1, present in both structures.
    [0276]
    [0277] The results encompassing the 11 cases of patients allergic to amoxicillin show 54% of cases in which compound 2 inhibits more than 4. However, considering the percentage of significant inhibition when it is greater than 50%, the recognition was significant in 45% of the cases for structure 2, 64% for structure 4 and 91% for the conjugate cefadroxil-butylamine.
    [0278]
    [0279] Conclusion: Well-defined structures have been successfully prepared, corresponding to synthetic determinants of formula (I) derived from aminocephalosporins (with the side chains of cefaclor and cefadroxil). These compounds are recognized by the IgE of patients allergic to aminocephalosporins and / or penicillins to a greater extent than the synthetic structures previously described. Its use as inhibitors in inhibition of the RAST is useful to determine the degree of recognition and if there are crossed reactivities. The use of these solid phase anchored structures in the quantification of specific IgE by RAST may increase the sensitivity of the assay.
    [0280]
    [0281] These structures of formula (I) also include the corresponding antigenic determinants of other aminocephalosporins with the same R1 side chains, such as cefprozil, cephaloglycine and cephalexin.
    权利要求:
    Claims (24)
    [1]
    1. A compound of general formula (I):

    [2]
    2. The compound according to claim 1, wherein R1 is a linear or branched (C1-C10) alkyl, substituted or unsubstituted.
    [3]
    3. The compound according to claims 1-2, wherein R1 is substituted by an azide group or an alcohol group
    [4]
    4. The compound according to claims 1-2, wherein R1 is butyl
    [5]
    5. The compound according to claims 1 to 4, wherein R2 is hydrogen
    [6]
    6. - The compound according to claims 1 to 4, wherein R2 is hydroxyl
    [7]
    7. - A composition comprising at least one compound according to any of claims 1-6, or any combination thereof.
    [8]
    8. The composition according to the preceding claim, which also comprises at least one excipient and / or a pharmaceutically acceptable vehicle.
    [9]
    9. A compound according to any of claims 1-6, or a composition according to any of claims 7-8, for use in medicine.
    [10]
    10. A compound according to any of claims 1-6 or a composition according to any of claims 7-8 for the detection of IgE antibodies generated by hypersensitivity to beta-lactams.
    [11]
    11. - The compound or composition according to the previous revindication where betalactams are aminopenicillins and / or aminocephalosporins.
    [12]
    12. The compound or composition according to any of claims 10-11, wherein the betalactams are aminocephalosporins, preferably cefaclor and cefadroxil.
    [13]
    13. The compound according to any of claims 9-12, selected from the list consisting of N-butyl-6-oxo-5-phenyl-1,6-dihydropyrazine-2-carboxamide and / or N-butyl-5. - (4-hydroxyphenyl) -6-oxo-1,6-dihydropyrazine-2-carboxamide or any of its salts, tautomers or hydrates
    [14]
    14. A method for obtaining a compound of formula (I) as described in any one of the preceding claims, characterized in that it comprises the following steps:
    (1) Obtaining a compound of formula (VI)

    [15]
    15. The method according to the preceding claim, wherein R3 and R4 are independently selected from tert-butoxycarbonyl (Boc), 2,5-dimethoxybenzyl, trityl, fluorenylmethyloxycarbonyl (Fmoc) and benzyloxycarbonyl (CBz).
    [16]
    16. The method according to any of claims 14-15, wherein R4 is tert-butoxycarbonyl (Boc), R5 is 2,5-dimethoxybenzyl and R5 and R6 are methyl.
    [17]
    17. - The method according to any of claims 14-16, wherein step (2) is carried out by treatment of the compound of formula (VI) with an acid medium, and optionally with a previous or subsequent treatment with a basic medium soft.
    [18]
    18. - The method according to any of claims 14-16, wherein step (2) is carried out by treating the compound of formula (VI) with an acid medium.
    [19]
    19. - The method according to the preceding claim wherein steps (2) and (3) are performed in a single reaction step by treatment of the compound of formula (VI) with an acid medium.
    [20]
    20. An in vitro method for obtaining useful data to diagnose hypersensitivity to aminocephalosporins, which includes the following steps:
    to. Collecting a blood sample from a subject, and incubating its serum with a compound, as defined in any one of claims 1-6, or with a composition, as defined in any of claims 7- 8;
    b. Quantify the amount of specific IgE antibodies that are generated against the compound of section a);
    c. comparing the amount obtained in step b) with the amount obtained in a negative control sample; Y
    [21]
    21. - An in vitro method for diagnosing hypersensitivity to aminocephalosporins comprising steps (a), (b) and (c) according to the preceding claim, and further comprises:
    d. diagnose the individual as allergic when the value obtained in stage c) is superior to the negative control.
    [22]
    22. The method according to any of claims 20-21, wherein step (b) is carried out by a technique selected from the group consisting of: RAST (radioalergoadsorption test), ELISA (bound immunosorbent assay) to enzymes), FAST (fluorescence allergy-adsorption test), MAST (multiple chemiluminescence-allergy test), LAST (latex-allergy-adsorption test), and immunoassay performed in microarray support with fluorescent detection.
    [23]
    23. A kit or device comprising at least one compound according to any of claims 1-6 or a composition according to any of claims 7-8.
    [24]
    24. - The kit or device according to the preceding claim which also comprises the elements necessary for the performance of a test for the detection of antibodies immunoglobulin E (IgE) generated in allergic reactions to aminocephalosporins.
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