ACILHIDRAZONAS FOR THE TREATMENT OF NEUROLOGICAL DISEASES (Machine-translation by Google Translate,

专利摘要:
Acylhydrazones for the treatment of neurological diseases. The present invention relates to a group of compounds with a structural nucleus of acylhydrazone that have a modulating capacity for the interaction between NCS-1 and Ric8a proteins, involved in the process of regulating the number of synapses and the probability of neurotransmitter release. These compounds are therefore useful for the treatment of neurological diseases in which the number of synapses is affected, such as Alzheimer's disease, Huntington's disease or Parkinson's disease. (Machine-translation by Google Translate, not legally binding)
公开号:ES2750924A1
申请号:ES201830933
申请日:2018-09-27
公开日:2020-03-27
发明作者:Fernandez Ruth Perez；Martin Andrea Canal；Barrena Maria Jose Sanchez；Aparicio Alicia Mansilla
申请人:Consejo Superior de Investigaciones Cientificas CSIC；Fundacion para la Investigacion Biomedica del Hospital Universitario Ramon Y Cajal；
IPC主号:

专利说明:

[0001]
[0002] Acylhydrazones for the treatment of neurological diseases
[0003]
[0004] The present invention refers to a group of compounds with a structural nucleus of acylhydrazones that have a modulating activity of the interaction between the NCS-1 and Ric8a proteins, involved in the process of regulation of the number of synapses and the probability of neurotransmitter release . Because of this, the compounds of the invention are useful for the treatment of neurological diseases in which the number of synapses is affected, such as Alzheimer's disease, Huntington's disease or Parkinson's disease.
[0005]
[0006] BACKGROUND OF THE INVENTION
[0007]
[0008] In order to transmit nerve impulses to coordinate each function in the body, neurons communicate through synapses. These require the formation of a neurotransmitter-releasing locus in the pre-synaptic neuron and an associated receptor field in the post-synaptic neuron. For the proper functioning of nerve transmission, an adequate balance between the number of releasing sites and the probability of neurotransmitter release is necessary. Sometimes imbalances in this balance occur, negatively affecting the functioning of the nervous system. When the imbalance is due to excess synapse, neurodevelopment is affected, causing disorders such as Angelman syndrome, fragile X chromosome syndrome and Rett syndrome, other autism spectrum disorders, epilepsy, bipolar disorder, among others. On other occasions, the problem is synaptic loss increasingly frequent as a consequence of the aging of the world population, and which has as a consequence the development of neurodegenerative diseases such as Alzheimer's disease, Huntington's, Parkinson's, etc.
[0009]
[0010] Synaptic restoration using drugs has great therapeutic value. By inhibiting the interaction between neuronal calcium sensor 1 (NCS-1) and guanylyl exchange factor (Ric8a), it has been shown that the normal number of synapses is restored in an animal model of fragile X syndrome (Mansilla, A. et. Al. PNAS 2017, 114, E999-E1008). However, currently they have not been published. compounds that stabilize the interaction of the protein complex (NCS-1: Ric8a) and restore the lost synapses, which would contribute to an improvement of the cognitive functions and of the memory altered in neurodegenerative diseases such as Alzheimer's.
[0011]
[0012] Compounds with a structural nucleus of acylhydrazone have been described that are useful for the treatment of neurological diseases such as epilepsy (Angelova et al., J. Drug Dev Res. 2016 Nov, 77 (7): 379-392) or as the disease Alzheimer's (Dias Viegas FP et al. Eur J Med Chem. 2018 Mar 10, 147: 48-65), although no compounds of this type have been described that efficiently treat neurological diseases by improving the number of synapses by modulating the interaction of the NCS-1 and Ric8a proteins.
[0013]
[0014] DESCRIPTION OF THE INVENTION
[0015]
[0016] The protein complex formed by NCS-1 and Ric8 regulates the number of synapses and the probability of neurotransmitter release, making it a good therapeutic target for the treatment of diseases in which the synapse is altered. The affinity of the compounds for dNCS-1 ( Drosophila neuronal calcium sensor ) has been verified from fluorescence spectroscopy studies . The Drosophila neuronal calcium sensor (dNCS-1) is conserved from yeast to humans.
[0017]
[0018] On the other hand, through immunoprecipitation techniques with human proteins, the ability to promote (activate) the binding of both proteins in the form of the NCS-1 / Ric8a complex has been demonstrated.
[0019]
[0020] In a first aspect, the present invention relates to a compound of formula (I):
[0021]
[0022]
[0023]
[0024] where
[0025] R1 is selected from OH, NO2, or halogen;
[0026] R2 and R3 are independently selected from H, NO2, or halogen;
[0027] R4 is selected from:
[0028] - aryl optionally substituted by an OH group or by a C1-C6 alkyl,
[0029] - heteroaryl optionally substituted by halogen, or
[0030] - a CHR5R6 group, where R5 is selected from OH or H and R6 is selected from aryl, heteroaryl or a [CH2] n-O- [C1-C4 alkyl] group, where n is a value between 1 and 4;
[0031] or any of its isomers or salts, for use in the treatment of a neurological disease that is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease and other neurodegenerative diseases, as well as Angelman syndrome, X chromosome syndrome brittle, Rett syndrome, autism spectrum syndromes, epilepsy, Rett syndrome, schizophrenia, bipolar disorder, amyotrophic lateral sclerosis, Friederich's ataxia, progressive supranuclear palsy, frontotemporal dementia, Lewy body dementia, Creutzfeldt-Jakob disease;
[0032]
[0033] with the proviso that the compound of formula (I) is not one of the following compounds:
[0034]
[0035]
[0036]
[0037]
[0038] In a preferred embodiment, R1 is OH.
[0039]
[0040] In another preferred embodiment, R2 is NO2.
[0041]
[0042] In another more preferred embodiment when R1 is OH, R2 is NO2.
[0043]
[0044] In another preferred embodiment, R3 is H.
[0045]
[0046] In another even more preferred embodiment when R1 is OH and R2 is NO2, R3 is H.
[0047] In another preferred embodiment, R1 is NO2.
[0048]
[0049] In another preferred embodiment, R2 is halogen, preferably R2 is chlorine.
[0050]
[0051] In another more preferred embodiment when R1 is NO2, R2 is halogen, preferably R2 is chlorine.
[0052]
[0053] In another even more preferred embodiment when R1 is NO2 and R2 is halogen, preferably R2 is chlorine, R3 is H.
[0054]
[0055] In another more preferred embodiment when R1 is NO2, R2 is H.
[0056]
[0057] In another even more preferred embodiment when R1 is NO2 and R2 is H, R3 is halogen, preferably R3 is fluoro.
[0058]
[0059] In another preferred embodiment, R1 is halogen, preferably R1 is fluoro.
[0060]
[0061] In another preferred embodiment, R2 is H.
[0062]
[0063] In another more preferred embodiment when R1 is halogen, preferably fluorine, R2 is H.
[0064]
[0065] In another even more preferred embodiment when R1 is halogen, preferably R1 is fluoro, and R2 is H, R3 is NO2.
[0066]
[0067] In another preferred embodiment, R4 is a heteroaryl that is selected from thiophenyl or furanyl, where said groups can be optionally substituted by halogen.
[0068]
[0069] In another preferred embodiment, R4 is an aryl that is selected from phenyl or naphthyl, where phenyl can be optionally substituted by C1-C4 alkyl and naphthyl can be optionally substituted by OH.
[0070]
[0071] In another preferred embodiment, the compound of formula (I) for use as described above is selected from the following list:
[0072] • (E) -W '- (3-hydroxy-4-nitrobenziliden) furan-2-carbahydrazide (A3H2)
[0073] • (E / Z) -W '- (3-hydroxy-4-nitrobenzyliden) -2- (1H-indole-3-yl) acetohydrazide (A3H3) • (E) -W' - (3-hydroxy-4- nitrobenziliden) -3-methoxypropanhydrazide (A3H4)
[0074] • (±) - (E / Z) -2-hydroxy-W '- (3-hydroxy-4-nitrobenziliden) -2-phenylacetohydrazide (A3H6)
[0075] • (E / Z) -W '- (3-hydroxy-4-nitrobenziliden) thiophene-2-carbohydrazide (A3H8)
[0076] • (E) -4- (tert-butyl) -W- (3-hydroxy-4-nitrobenziliden) benzohydrazide (A3H17) • (Z) -5-chloro-W- (3-hydroxy-4-nitrobenziliden) thiophene- 2-carbohydrazide (A3H18) • (E / Z) -3-hydroxy-W- (3-hydroxy-4-nitrobenziliden) -2-naphthohydrazide (A3H19) • (Z) -W- (4-chloro-3-nitrobenziliden ) furan-2-carbohydrazide (A5H2)
[0077] • (±) - (E / Z) -W- (4-chloro-3-nitrobenziliden) -2-hydroxy-2-phenylacetohydrazide (A5H6)
[0078] • (E / Z) -W- (4-chloro-3-nitrobenziliden) thiophene-2-carbohydrazide (A5H8)
[0079] • (E) -W- (5-fluoro-2-nitrobenziliden) furan-2-carbohydrazide (A7H2)
[0080] • (E / Z} -W- (5-fluoro-2-nitrobenziliden) thiophene-2-carbohydrazide (A7H8)
[0081] • (E) -W- (2-fluoro-5-nitrobenziliden) furan-2-carbohydrazide (A8H2)
[0082] • (E) - (W- (2-fluoro-5-nitrobenziliden) -2-hydroxy-2-phenylacetohydrazide (A8H8)
[0083]
[0084] A second aspect of the invention relates to a compound that is selected from:
[0085] • (E) -W '- (3-hydroxy-4-nitrobenziliden) furan-2-carbahydrazide (A3H2)
[0086] • (E / Z) -W '- (3-hydroxy-4-nitrobenzyliden) -2- (1H-indole-3-yl) acetohydrazide (A3H3) • (E) -W' - (3-hydroxy-4- nitrobenziliden) -3-methoxypropanhydrazide (A3H4)
[0087] • (E) -4- (tert-butyl) -W- (3-hydroxy-4-nitrobenziliden) benzohydrazide (A3H17) • (Z) -5-chloro-W- (3-hydroxy-4-nitrobenziliden) thiophene- 2-carbohydrazide (A3H18) • (E / Z) -3-hydroxy-W- (3-hydroxy-4-nitrobenziliden) -2-naphthohydrazide (A3H19) • (E) -W- (5-fluoro-2-nitrobenziliden ) furan-2-carbohydrazide (A7H2)
[0088] • (E / Z} -W- (5-fluoro-2-nitrobenziliden) thiophene-2-carbohydrazide (A7H8)
[0089] • (E) -W- (2-fluoro-5-nitrobenziliden) furan-2-carbohydrazide (A8H2)
[0090] • (E) - (W- (2-fluoro-5-nitrobenziliden) -2-hydroxy-2-phenylacetohydrazide (A8H8)
[0091]
[0092] A third aspect of the invention relates to a compound of the second aspect of the invention for use as a medicine.
[0093]
[0094] A fourth aspect of the invention relates to a pharmaceutical composition comprising at least one compound of the second aspect of the invention and at least a pharmaceutically acceptable vehicle.
[0095]
[0096] The term "aryl" refers in the present invention to a phenyl, naphthyl, indenyl, phenanthril or anthracil radical, preferably phenyl and naphthyl. The aryl radical can be optionally substituted by one or more substituents such as alkyl, haloalkyl, aminoalkyl, dialkylamino, hydroxyl, alkoxy, phenyl, mercapto, halogen, nitro, cyano and alkoxycarbonyl.
[0097]
[0098] The term "heteroaryl" refers to an aryl, as defined above, that contains at least one non-carbon atom, such as S, N, or O, forming part of the aromatic ring. Examples of heteroaryl radicals include, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrazinyl, pyridazinyl, pirimidininilo, triazinyl, tienofuranilo, tienopirrolilo, pirrolopirazolilo, benzothiophenyl, benzofuranyl, indolyl, isoindolyl, benzimidazolyl, benzothiazolyl, imidazopyridinyl, pyrazolopyridinyl, isoquinolinyl, quinolinyl, quinolizinyl, naphthyridinyl, quinazolinyl, quinoxalinyl. Heteroaryl groups may be optionally substituted at any of their positions by one or more substituents or by two substituents forming a fused ring to heteroaryl and said substituents are independently selected from such as CF3, C1-C6 alkyl, S-C1-C6 alkyl, halogen, CN, O-C1-C6-alkyl, NO2, COO-C1-C6-alkyl, NHCO-C1-C6-alkyl, NH2 and NH-C1-C6-alkyl, and more preferably between CF3, C1-C6-alkyl, halogen, CN and NO2.
[0099]
[0100] The term "alkyl" refers, in the present invention, to linear or branched hydrocarbon chain radicals, having from 1 to 10 carbon atoms, preferably from 1 to 6, and more preferably from 1 to 4, and which are bind to the rest of the molecule via a single bond, for example methyl, ethyl, n-propyl, / -propyl, n-butyl, tere-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl groups can be optionally substituted by one or more substituents such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.
[0101]
[0102] The term "halogen" refers, in the present invention, to fluorine, chlorine, bromine or iodine.
[0103]
[0104] The compounds of the invention, in their therapeutic use or forming part of a pharmaceutical composition, they can be in crystalline form as free compounds or as solvates and both forms are intended to be within the scope of the present invention. In this sense, the term "solvate", as used herein, includes both pharmaceutically acceptable solvates that can be used in the manufacture of a medicine, and pharmaceutically unacceptable solvates, which can be useful in the preparation of solvates or salts pharmaceutically acceptable. The nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. Solvates can be obtained by conventional solvation methods well known to those skilled in the art.
[0105]
[0106] Compounds of the invention for therapeutic use or as part of a pharmaceutical composition are prepared in solid or aqueous suspension, in a pharmaceutically acceptable diluent. These preparations can be administered by any appropriate route of administration, for which said preparation will be formulated in the pharmaceutical form appropriate to the chosen route of administration. In a particular embodiment, the administration of the compound of formula (I) provided by this invention is carried out orally, topically, rectally or parenterally (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, etc.). The preparation of the different pharmaceutical forms of drug administration and of the excipients necessary to obtain them can be found in different manuals handled by experts in the field.
[0107]
[0108] The compounds described in the present invention, their pharmaceutically acceptable salts, solvates as well as the pharmaceutical compositions containing them can be used together with other additional drugs to provide a combination therapy. Said additional drugs may be part of the same pharmaceutical composition or, alternatively, may be provided in the form of a separate composition for simultaneous or non-simultaneous administration of the pharmaceutical composition comprising a compound of the invention, or a salt, stereoisomer or pharmaceutically acceptable solvate thereof.
[0109]
[0110] Unless otherwise indicated, the compounds of the invention also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds that have such a structure, except for the replacement of a hydrogen by a deuterium or by tritium, or the replacement of a carbon by a carbon enriched in 13C or 14C or a nitrogen enriched in 15N, is within the scope of this invention.
[0111]
[0112] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
[0113]
[0114] BRIEF DESCRIPTION OF THE FIGURES
[0115]
[0116] FIG. 1 . It shows the results of immunoprecipitation assays in HEK293 cells transfected with human V5-Ric8a and NCS-1 and in the presence or not of the compounds. The DMSO lane represents a positive control of the interaction.
[0117]
[0118] FIG. 2. Shows the representation of the fluorescence emission of the dNCS-1-ligand complex in the presence of Calcium, in increasing concentrations of ligand. The adjustment of the curves has been made considering a 1: 1 stoichiometry. For the correct comparison of the curves the intensities have been normalized and represented as (I0 - I) / I0. X. The value of r2 is 0.998.
[0119]
[0120] EXAMPLES
[0121]
[0122] Next, the invention will be illustrated by tests carried out by the inventors, which shows the effectiveness of the product of the invention.
[0123]
[0124] Example 1. Synthesis of the compounds of the invention.
[0125]
[0126] Preparation and preparation of (E) -W '- (3-hydroxy-4-mtrobenzMiden) furan-2-carbahydrazide (A3H2).
[0127]
[0128]
[0129]
[0130] 3-hydroxy-4-nitrobenzaldehyde (319 mg, 1.91 mmol), 2-furoic acid hydrazide (200 mg, 1.59 mmol), MeOH (60 mL). The product is filtered and washed with MeOH at rt to obtain the compound as a yellow solid (0.30 g, 70%) (E> 99). Mp : 256 - 257 ° C. 1 H-NMR (300 MHz, DMSO-d6) 512.07 (s, 1H), 11.18 (s, 1H), 8.40 (s, 1H), 7.98 (m, 2H), 7.49 ( d, J = 1.7 Hz, 1H), 7.40 - 7.26 (m, 2H), 6.72 (d, 1H, J = 1.7 Hz). 13 C-NMR (75 MHz, DMSO-cfe): 5 164.5, 152.3, 146.7, 145.2, 140.5, 140.0, 137.2, 125.9, 118.3, 118.5, 116.8, 112.6. Anal. Caled , for C12H9N3O5: C, 52.37%; H, 3.30%; N, 15.27%.
[0131] Found: C, 52.43%; H, 3.30%; N, 15.11%. HPLC-MS : tR: 7.02 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 276 m / z.
[0132]
[0133] Preparation and preparation of (£ / Z) -W, - (3-hydroxy-4-mtrobenzMiden) -2- (1H-mdol-3-yl) acetohydrazide (A3H3).
[0134]
[0135]
[0136]
[0137] 3-hydroxy-4-nitrobenzaldehyde (330 mg, 1.97 mmol), indole-3-acetic hydrazide hydrazide (300 mg, 1.58 mmol), MeOH (60 mL). Filter and wash the product with MeOH to obtain it as an orange solid (0.30 g, 70%) (E: Z = 40:60). M.p .: 216 - 217 ° C. 1H-NMR (300 MHz, DMSO-d6) 511.79 (s, 1H, E), 11.54 (s, 1H, Z), 11.19 (s, 1H), 7.88 (s, 1H), 7.70-7.65 (m, 1H ), 7.39-7.36 (m, 1H), 7.27-7.25 (m, 1H), 7.15 (s, 1H), 7.02-6.98 (m, 1H), 6.92-6.88 (m, 1H), 6.78 (s, 1H) ), 6.72-6.68 (m, 1H), 5.89 (s, 1H), 3.68 (s, 2H).
[0138] 13C-NMR (75 MHz, DMSO-d6): 5 172.1, 150.8, 148.1, 142.8, 137.4, 136.4, 130.5, 125.9, 123.9, 122.6, 121.0, 120.2, 119.2, 116.7, 111.9, 110.6, 42.5. Anal. Calcd, for C17H14N4O4: C, 60.35%; H, 4.17%; N, 16.56%. Found: C, 60.13%; H, 4.20%; N, 16.56%. HPLC-MS: tR: 8.45 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 339 m / z.
[0139]
[0140] Preparation and preparation of (Ej-W '- (3-hydroxy-4-nitrobenziliden) -3-methoxypropanhydrazide (A3H4).
[0141]
[0142] 3-hydroxy-4-nitrobenzaldehide (339 mg, 2.0 mmol), 3-methoxypropionic acid hydrazide (180 pL, 1.69 mmol), MeOH (60 mL). It has been purified by automatic column chromatography by Biotage system using DCM / MeOH (0-6%) as eluents obtaining a yellow solid (437 mg, 97%) (E> 99). Mp: 163 -164 ° C. 1 H-NMR (300 MHz, DMSO-d6): 5 11.61 (s, 1H), 11.13 (s, 2H), 8.13 (s, 1H), 7.95-7.92 (m, 3H), 3.66-3.58 (m, 2H), 3.24 (s, 3H), 2.47 (t, 2H, J = 6.8 Hz). 13 C-NMR (75 MHz, DMSO-d6): 5 172.1, 151.8, 143.1, 140.2, 139.8, 136.6, 125.3, 117.4, 67.4, 57.4, 34.4. Anal. Caled , for C11H13N3O5: C, 49.44%; H, 4.90%; N, 15.72%.
[0143] Found: C, 49.51%; H, 4.82%; N, 15.61%. HPLC-MS : tR: 6.47 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 268 m / z.
[0144]
[0145] Preparation and preparation of (E) -4- (fert-butyl) -W '- (3-hydroxy-4-nitrobenziliden) benzohydrazide (A3H17).
[0146]
[0147]
[0148]
[0149] 3-hydroxy-4-nitrobenzaldehyde (267 mg, 1.6 mmol), 4-tert-butylbenzoic acid hydrazide (255.5 mg, 1.33 mmol), EtOH (60 mL). Wash and filter with EtOH to obtain a yellow solid (430 mg, 95%) (E> 99). Mp: 220-221 ° C. 1 H-NMR (300 MHz, DMSO-d6): 512.01 (s, 2H), 11.19 (s, 2H), 8.42 (s, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.2 Hz, 5H), 7.61 - 7.49 (m, 7H), 7.31 (d, J = 8.6 Hz, 2H), 3.34 (s, 1H), 3.18 (s, 6H), 1.32 (s, 9H). 13 C-NMR (75 MHz, DMSO-d6): 5 155.4 (C-1), 152.8 (C-1), 145.5 (C-1), 141.4 (C-1), 137.6 (C-1), 130.8 (C-1), 128.1 (C-1), 126.4 (C-1), 125.7 (C-1), 118.5 (C-1), 116.8 (C-1), 49.1 (C-1), 39.9 ( C-1), 35.2 (C-1), 31.4 (C-1). Anal. Calcd , for C18H19N3O4: C, 63.33%; H, 5.61%; N, 12.31%; Found: C, 63.10%; H, 6.08%; N, 12.25%. HPLC-MS : tR: 9.72 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 342 m / z.
[0150]
[0151] Preparation and obtaining of (Z) -5-chloro-W '- (3-hydroxy-4-mtrobenziliden) thiophene-2- carbohydrazide (A3H18).
[0152]
[0153]
[0154]
[0155] 3-hydroxy-4-nitrobenzaldehyde (279 mg, 1.71 mmol), 5-chlorothiopfen-2-carboxylic acid hydrazide (249.7 mg, 1.41 mmol), EtOH (60 mL). Wash and filter with EtOH to obtain a yellow solid (404 mg, 90%) (Z> 99) mp: 255-256 ° C. 1 H-NMR (300 MHz, DMSO-cfe): 5 12.17 (s, 1H, H-2), 11.28 (s, 1H, H-8), 8.12 - 7.94 (m, 2H, H-10, 14) , 7.94 - 7.79 (m, 1H, H-11), 7.51 (m, 1H, H-4), 7.33-7.31 (m, 2H, H-13, 6). 13 C-NMR (75 MHz, DMSO-cfe): 5 161.2 (C-1), 152.8 (C-7), 143.3 (C-15), 140.3 (C-9), 138.2 (C-12), 137.9 (C-11), 135.2 (C-10), 130.3 (C-5), 127.2 (C-4), 126.6 (C-6), 118.6 (C-13), 117.4 (C-14). Anal. Calcd , for C12H8ClN3O4S: C, 44.25%; H, 2.48%; N, 12.90%; S, 9.84%. Found: C, 44.16%; H, 2.58%; N, 12.85%; S, 9.87%. HPLC-MS : tR: 9.48 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 326 m / z.
[0156]
[0157] Preparation and preparation of (£ / Z) -3-hydroxy-W- (3-hydroxy-4-nitrobenziliden) -2-naphthohydrazide (A3H19).
[0158]
[0159]
[0160]
[0161] 3-hydroxy-4-nitrobenzaldehyde (198mg, 1.18mmol), 3-hydroxy-2-naphthoic acid hydrazide (206mg, 0.98mmol), EtOH (60mL). The product is filtered and washed with EtOH to obtain a yellow solid (287 mg, 83%) (E: Z = 88:12). PS: 264-265 ° C. 1 H-NMR (300 MHz, DMSO-cfe): 5 12.13 (s, 1H, H-2, £), 12.05 (s, 1H, H-2, Z), 11.21 (s, 2H, H-10, 21), 8.44 (m, 2H, H-4, 7), 7.96 (d, J = 8.4 Hz, 2H, H-14, 17), 7.78 (d, J = 8.3 Hz, 1H, H-6), 7.60 - 7.43 (m, 2H, H-15, 16), 7.43 - 7.30 (m, 3H, H-11, 12, 19) .13 C-NMR (75 MHz, DMSO-d6): 5 164.2 (C- 1), 154.2 (C-20), 152.8 (C-9), 146.4 (C-4), 140.9 (C-8), 137.8 (C-5), 136.3 (C-22), 131.1 (C-18 ), 129.1 (C-13), 128.7 (C-7), 127.3 (C-12), 126.4 (C-6), 126.3 (C-17), 124.3 (C-14), 121.2 (C-16) , 118.6 (C-15), 117.0 (C-11), 111.0 (C-19). Anal. Calcd , for C18H13N3O5:: C, 61.54%; H, 3.73%; N, 11.96%. Found: C, 61.51%; H, 3.76%; N, 11.97%. HPLC-MS : tR: 9.38 min (Column ACE-Excel 3 C18PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 353 m / z.
[0162]
[0163] Preparation and preparation of £) -W '- (5-fluoro-2-nitrobenziliden) furan-2-carbohydrazide (A7H2).
[0164]
[0165]
[0166]
[0167] 5-fluoro-2-nitrobenzaldehyde (250mg, 1.5mmol), 2-furoic acid hydrazide (150mg, 1.2mmol), MeOH (50mL). It is purified by automatic column chromatography on silica Biotage system, DCM / MeOH (0 - 3%) obtaining a yellow solid (0.330 g, 99%) (E> 99). Mp: 194 - 195 ° C. 1 H-NMR (300 MHz, DMSO-d6): 512.33 (s, 1H, H-2), 8.91 (s, 1H, H-4), 8.23 (dd, J = 9.2 Hz, 1H, H-14), 8.00 (d, J = 1.8 Hz, 1H, H-7), 7.81 (dd, J = 9.6, 2.9 Hz, 1H, H-12), 7.56 (dd, J = 9.0 , 2.9 Hz, 1H, H-13), 7.40 (d, J = 3.4 Hz, 1H, H-10), 6.75 (dd, J = 3.4, 1.8 Hz, 1H, H-8). 13 C-NMR (75 MHz, DMSO- d 6): 5 165.66 (C-1), 162.30 (C-9) 146.31 (C-7), 144.49 (C-4), 132.26 (C-6), 132.19 (C-11), 128.30 (C-14), 128.25 (C-5), 117.58 (C-13), 117.50 (C-10), 113.96 (C-12), 112.16 (C-8). Anal. Calcd , for C12H8FN3O4: C, 51.99%; H, 2.91%; N, 15.16%. Found: C, 51.94%; H, 2.91%; N, 15.00%.%. HPLC-MS : tR: 7.00 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 278 m / z.
[0168]
[0169] Preparation and preparation of (£ / Z) -W '- (5-fluoro-2-nitrobenziliden) thiophene-2-carbohydrazide (A7H8).
[0170]
[0171]
[0172]
[0173] 5-fluoro-2-nitrobenzaldehyde (220 mg, 1.3 mmol), 2-thiophenecarboxylic acid hydrazide (160 mg, 1.1 mmol), MeOH (50 mL). The solid obtained by crystallization is filtered and washed as an orange solid (0.270 g, 82%) (E: Z = 26:74). Mp: 218 - 219 ° C. 1 H-NMR (300 MHz, DMSO-d6): 512.33 (s, 1H, H-2, E), 12.32 (s, 1H, H-2, Z), 8.89 (s , 1H, H-7), 8.25 (dd, J = 9.1, 5.0 Hz, 1H, H-14), 8.00 (s, 2H, H-8, 10), 7.86 (s, 1H, H-4), 7.57 (d, J = 9.1 Hz, 1H, H-12), 7.27 (d, J = 5.0 Hz, 1H, H-13). 13 C-NMR (75 MHz, DMSO- d6): 5 165.66 (C-1), 162.31 (C-9), 147.16 (C-7), 144.55 (C-4), 133.05 (C-11), 132.65 (C-6), 128.23 (C-14), 127.98 (C-5), 117.59 (C-13), 117.54 (C-10), 114.84 (C12), 114.21 (C-8). Anal. Caled , for C12H8FN3O3S: C, 49.15%; H, 2.75%; N, 14.33%; S, 10.93%. Found : C, 49.29%; H, 2.82%; N, 14.21%; S, 10.92%. HPLC-MS : tR: 8.25 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 294 m / z.
[0174]
[0175] Preparation and preparation of (£) -W '- (2-fluoro-5-nitrobenziliden) furan-2-earbohydrazide (A8H2).
[0176]
[0177]
[0178]
[0179] 2-fluoro-5-nitrobenzaldehyde (240mg, 1.44mmol), 2-furoic acid hydrazide (150mg, 1.2mmol), MeOH (50mL). It is purified by automatic column chromatography on silica Biotage system using DCM / MeOH (9-10%) obtaining an orange solid (0.26 g, 75%) (E> 99). Mp: 187-188 ° C. 1 H-NMR (300 MHz, DMSO- d6): 512.23 (s, 1H, H-2), 8.68 (m, 2H, H-9, 10), 8.35 (m, 1H , H-14), 8.00 (m, 1H, H-4), 7.63 (m, 1H, H-12), 7.38 (s, 1H, H-7), 6.75 (m, 1H, H-13). 13 C-NMR (75 MHz, DMSO-d6): 5165.47 (C-1), 162.31 (C-6), 153.61 (C-9), 146.40 (C-4), 143.84 (C-10), 138.59 ( C-8), 126.79 (C-7), 126.64 (C-14), 123.40 (C-5), 123.24 (C-13), 118.00 (C-10), 117.65 (C-12), 112.20 (C -8). Anal. Caled , for C12H8FN3O4: C, 51.99%; H, 2.91%; N, 15.16%. Found : C, 51.90%; H, 2.91%; N, 15.05%. HPLC-MS : tR: 6.97 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 278 m / z.
[0180]
[0181] Preparation and obtaining of (E) - (W '- (2-fluoro-5-nitrobenziliden) -2-hydroxy-2-phenylaeetohydrazide (A8H8).
[0182]
[0183]
[0184]
[0185] 2-fluoro-5-nitrobenzaldehyde (220 mg, 1.3 mmol), mandelic acid hydrazide (150 mg, 1.1 mmol), MeOH (50 mL). It is purified by automatic column chromatography on silica Biotage system using DCM / MeOH (0 - 4%) obtaining a white solid (0.16 g, 50%) (E> 99). Mp: 237-238 ° C. 1 H-NMR (300 MHz, DMSO-d6): 512.20 (s, 1H, H-2), 8.82 (s, 1H, H-10), 8.69 (s, 1H, H -8), 8.35 (m, 1H, H-14), 8.06 (s, 1H, H-7), 7.96 (s, 1H, H-4), 7.64 (m, 1H, H-12), 7.27 ( m, 1H, H-13). 13 C-NMR (75 MHz, DMSO d 6): 5 165.57 (C-1), 163.35 (C-6), 161.49 (C-9), 144.43 (C-4), 144.27 (2C, C-7, 14), 132.64 (C-11 ), 126.76 (2C, C-8, 10), 126.6 (C-5), 118.06 (C-13), 117.78 (C-12). Anal. Caled , for C12H8FN3O3S: C, 49.15%; H, 2.75%; N, 14.33%; S, 10.93%. Found : C, 49.36%; H, 2.79%; N, 14.24%; S, 10.95%. HPLC-MS : tR: 7.85 min (Column ACE-Excel 3 C18-PFP, gradient 15-85% H2O: CH3CN 0.1% formic), [M + H] + = 318 m / z.
[0186]
[0187] Example 2. Modulation tests of the NCS1 / Ric8a interaction in vitro.
[0188]
[0189] In order to assess the ability of the compounds of the invention A3H3, A5H2 and A3H4 to modulate the interaction between human NCS-1 and Ric8a, HEK293 cells were transfected with a version of Ric8a labeled with the V5 epitope (Ric8a-V5) and NCS-1, incubating the cells and the subsequent cell lysate with the product to be tested. Subsequently, by means of the immunoprecipitation technique, the amount of Ric8a-V5 bound to NCS-1 was assessed. The data (Fig. 1) indicate that the presence of the compounds allows interaction with Ric8-a and demonstrate that the presence of the A3H3 product increases the binding of NCS-1 with Ric8a by more than double.
[0190]
[0191] Example 3. Measurement of the affinity of the compounds for the NCS-1 protein.
[0192]
[0193] The affinity of the compounds for dNCS-1 was measured using the fluorescence spectroscopy technique. The emission intensity of the complex is analyzed where the signal is monitored at increasing concentrations of ligand (1: 0 to 1:30 equivalents) keeping the protein concentration constant 7.8 pL, 30.8 pM, 1 eq.). With this, its affinity constant is obtained through the apparent dissociation constant. In Fig. 2 the graph of A3H3, A3H2 and A3H4 is shown as an example. Experiments are performed at 5 ° C with dNCS-1 in 50 mM Tris buffer, pH 7.9, 125 mM NaCl, 0.5 mM CaCl2 with 5% DMSO in a total volume of 300 pL. The experiment is performed at the excitation wavelength of 295 nm, and at the emission wavelength of 345 nm (maximum emission) depending on the compound in a Jobin Yvon Floromax4 Spectrofluorimeter equipped with a Peltier Thermostat, the values obtained are the result of the normalized average of the three repetitions for each measurement. The studied compounds do not present emission in the measured fluorescence range. The affinity constants found for the tested compounds are as follows:
[0194]

权利要求:
Claims (21)
[1]
1. Compound of formula (I):

[2]
2. Compound for use according to claim 1, wherein R1 is OH.
[3]
3. Compound for use according to claim 2, wherein R2 is NO2.
[4]
4. Compound for use according to claim 3, wherein R3 is H.
[5]
5. Compound for use according to claim 1, wherein R1 is NO2.
[6]
6. Compound for use according to claim 5, wherein R2 is halogen.
[7]
7. Compound for use according to claim 6, wherein R2 is chlorine.
[8]
8. Compound for use according to any of claims 6 or 7, wherein R3 is H.
[9]
9. Compound for use according to claim 5, wherein R2 is H.
[10]
10. Compound for use according to claim 9, wherein R3 is halogen.
[11]
11. Compound for use according to claim 10, wherein R3 is fluoro.
[12]
12. Compound for use according to claim 1, wherein R1 is halogen.
[13]
13. Compound for use according to claim 12, wherein R1 is fluoro.
[14]
14. Compound for use according to any of claims 12 or 13, wherein R2 is H.
[15]
15. Compound for use according to claim 14, wherein R3 is NO2.
[16]
16. Compound for use according to any of claims 1 to 15, wherein R4 is a heteroaryl that is selected from thiophenyl or furanyl, where said groups can be optionally substituted by halogen.
[17]
17. Compound for use according to any one of claims 1 to 15, wherein R4 is an aryl that is selected from phenyl or naphthyl, where the phenyl can be optionally substituted by C1-C4 alkyl and the naphthyl can be optionally substituted by OH.
[18]
18. Compound of formula (I) for use according to claim 1 which is selected from the following list:
• (E) -W '- (3-hydroxy-4-nitrobenziliden) furan-2-carbahydrazide
• (E / Z) -W '- (3-hydroxy-4-nitrobenzyliden) -2- (1H-indole-3-yl) acetohydrazide
• (E) -W '- (3-hydroxy-4-nitrobenziliden) -3-methoxypropanhydrazide
• (±) - (E / Z) -2-hydroxy-W '- (3-hydroxy-4-nitrobenziliden) -2-phenylacetohydrazide • (E) -W' - (3-hydroxy-4-nitrobenziliden) thiophene- 2-carbohydrazide
• (E) -4- (tert-butyl) -W- (3-hydroxy-4-nitrobenziliden) benzohydrazide
• (Z) -5-chloro-W- (3-hydroxy-4-nitrobenziliden) thiophene-2-carbohydrazide
• (E / Z) -3-hydroxy-W- (3-hydroxy-4-nitrobenziliden) -2-naphthohydrazide
• (Z) -W- (4-chloro-3-nitrobenziliden) furan-2-carbohydrazide
• (±) - (E / Z) -W- (4-chloro-3-nitrobenziliden) -2-hydroxy-2-phenylacetohydrazide
• (E / Z) -W- (4-chloro-3-nitrobenziliden) thiophene-2-carbohydrazide
• (E) -W- (5-fluoro-2-nitrobenziliden) furan-2-carbohydrazide
• (E / Z) -W- (5-fluoro-2-nitrobenziliden) thiophene-2-carbohydrazide
• (E) -W- (2-fluoro-5-nitrobenziliden) furan-2-carbohydrazide
• (E) - (W- (2-fluoro-5-nitrobenziliden) -2-hydroxy-2-phenylacetohydrazide.
[19]
19. Compound selected from:
• (E) -W '- (3-hydroxy-4-nitrobenziliden) furan-2-carbahydrazide
• (E / Z) -W '- (3-hydroxy-4-nitrobenzyliden) -2- (1H-indole-3-yl) acetohydrazide
• (E) -W '- (3-hydroxy-4-nitrobenziliden) -3-methoxypropanhydrazide
• (E) -4- (tert-butyl) -W- (3-hydroxy-4-nitrobenziliden) benzohydrazide
• (Z) -5-chloro-W- (3-hydroxy-4-nitrobenziliden) thiophene-2-carbohydrazide
• (E / Z) -3-hydroxy-W- (3-hydroxy-4-nitrobenziliden) -2-naphthohydrazide
• (E) -W- (5-fluoro-2-nitrobenziliden) furan-2-carbohydrazide
• (E / Z) -W- (5-fluoro-2-nitrobenziliden) thiophene-2-carbohydrazide
• (E) -W- (2-fluoro-5-nitrobenziliden) furan-2-carbohydrazide
• (E) - (W- (2-fluoro-5-nitrobenziliden) -2-hydroxy-2-phenylacetohydrazide.
[20]
20. Compound according to claim 19 for use as a medicine.
[21]
21. Pharmaceutical composition comprising at least one compound according to claim 19 and at least one pharmaceutically acceptable carrier.

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