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    • 专利摘要:
    Derived from Purina CDC7 inhibitors and their use for the treatment of neurological pathologies. The present invention relates to the use of a series of purine derivatives that have the following general formula: {IMAGE-01} And that they are capable of inhibiting CDC7 kinase activity, making them useful for the treatment of neurological diseases such as Alzheimer's disease, amyotrophic lateral sclerosis or frontotemporal dementia, among others, where TDP hyperphosphorylation occurs. -43 and subsequent CDC7-induced agglomerate formation. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2749743A1
    申请号:ES201830914
    申请日:2018-09-21
    公开日:2020-03-23
    发明作者:Gil Ana Martínez；Ayuso-Gontan Carmen Gil；Requero Angeles Martín；Prats Elisa Rojas；Loreto Martinez-Gonzalez
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] The present invention relates to the use of a series of purine derivatives that are capable of inhibiting CDC7 kinase activity, making them useful for the treatment and / or prevention of neurological diseases such as amyotrophic lateral sclerosis, the disease Alzheimer's disease or frontotemporal dementia, where hyperphosphorylation of TDP-43 and subsequent CDC7-induced agglomerate formation occur.
    [0005]
    [0006] STATE OF THE ART
    [0007]
    [0008] Kinase hyperactivity occurs in many types of diseases and particularly in neurodegenerative diseases and cancer. The hyperphosphorylation of the TDP-43 protein induces the formation of aggregates that have been detected in patients with amyotrophic lateral sclerosis or frontotemporal lobar degeneration. CDC7 kinase has been found to be responsible for the dual hyperphosphorylation of TDP-43 in serine 409/410 in certain models, so inhibition of this CDC7 would be an interesting strategy to develop drugs for neurodegerative diseases, such as amyotrophic lateral sclerosis (ALS) or with frontotemporal lobar degeneration (Lianchko, NF et al., Ann Neurol. 2013 74 (1): 39-52). There are other neurological diseases also mediated by TDP-43, such as chronic traumatic encephalopathy and age-related cognitive decline (Iverson GL, et al., Neurosci Biobehav Rev. 2015 Sep; 56: 276-293; Nag S, et al ., Neurology. 2017 Feb 14; 88 (7): 653-660; Wilson RS, et al., JAMA Neurol. 2013 Nov; 70 (11): 1418-1424).
    [0009]
    [0010] US2013 / 0072506A1 describes 6,8-disubstituted purine derivatives that are useful for a number of therapeutic and cosmetic uses. Among the possible therapeutic uses, mention is made of the treatment of multiple sclerosis or as antineurodegenerative drugs.
    [0011]
    [0012] WO2007 / 124288A1 describes a series of compounds with an indazole structural core that have the ability to inhibit CDC-7 and that are useful for the treatment of a disease in which this kinase is involved, such as cancer.
    [0013]
    [0014] In ACS Med. Chem. Lett. 2013, 4, 211-215, Penning et al. describe a study on the interaction of azaindole-derived compounds with CDC-7 and the possibility of using these compounds in cancer therapy.
    [0015]
    [0016] US2011 / 015172A1 describes a family of pyrrolpyrazines that are kinase inhibitors such as CDC-7 and their use for the treatment of diseases associated with this kinase such as cancer.
    [0017]
    [0018] DESCRIPTION OF THE INVENTION
    [0019]
    [0020] The present invention provides a series of purine-derived compounds that are CDC-7 inhibitors and useful as potential drugs for diseases mediated by TDP-43 proteinopathies, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia. . In particular, in pathologies with post-translational modifications of TDP-43. These compounds are CDC7 inhibitors in TDP-43 phosphorylation.
    [0021]
    [0022] Therefore, in a first aspect, the present invention relates to the use of a compound of formula (I)
    [0023]
    [0024]
    [0025]
    [0026] where:
    [0027] X is selected from CH2, CO;
    [0028] n has a value of 0 or 1;
    [0029] Y is selected from N, CH;
    [0030] Ar is selected from the following groups:
    [0031]
    [0032]
    [0033]
    [0034]
    [0035] where R1 to R7 are independently selected from H, NH2, -O (C1-C4 alkyl), NH (C1-C4 alkyl) or halogen and ------ represents the point of attachment to the rest of the molecule;
    [0036]
    [0037] or any of its pharmaceutically acceptable salts, solvates or isomers for use in the treatment and / or prevention of pathologies related to the TDP-43 protein;
    [0038]
    [0039] with the proviso that the compound of formula (I) is not the following compound:
    [0040]
    [0041]
    [0042]
    [0043]
    [0044] The term "alkyl" refers, in the present invention, to saturated, linear or branched hydrocarbon chains having 1 to 10 carbon atoms, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl , tere-butyl, sec-butyl, n-pentyl, nhexyl, etc. Preferably the alkyl group has from 1 to 4 carbon atoms The alkyl groups can optionally be substituted by one or more substituents 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, halogen, CN and NO2.
    [0045]
    [0046] By "halogen" in the present invention is meant a bromine, chlorine, iodine or fluorine atom.
    [0047]
    [0048] In a preferred embodiment of this first aspect, in the compound for use as described, Ar is the following group:
    [0049]
    [0050]
    [0051]
    [0052] In a more preferred embodiment, in the compound for use as described, X is CO and Y is N.
    [0053]
    [0054] In another more preferred embodiment, in the compound for use as described, X is CH2 and Y is N.
    [0055]
    [0056] In another more preferred embodiment, in the compound for use as described, n is 0 and Y is CH.
    [0057]
    [0058] In another more preferred embodiment, in the compound for use as described, at least one of R1 to R5 is -O (C1-C4 alkyl), preferably O-methyl.
    [0059]
    [0060] In another more preferred embodiment, in the compound for use as described, at least one of R1 to R5 is halogen.
    [0061]
    [0062] In another preferred embodiment, in the compound for use as described, Ar is the following group:
    [0063]
    [0064] In one embodiment
    [0065] is CO and Y is N.
    [0066]
    [0067] In another more preferred embodiment, in the compound for use as described, at least one of R1 to R7 is -O (C1-C4 alkyl), preferably is -O-methyl.
    [0068]
    [0069] In another preferred embodiment, in the compound for use as described above, at least one of R1 to R7 is halogen.
    [0070]
    [0071] In another preferred embodiment, the compound for use as described is selected from the following list:
    [0072] • 1- (4-methoxyphenyl) -2 - ((9H-purin-6-yl) thio) - ethan-1-one (2)
    [0073] • 1- (naphthalen-2-yl) -2 - ((9H-purin-6-yl) thio) - ethan-1-one (3)
    [0074] • 1- (3-methoxyphenyl) -2 - ((9H-purin-6-yl) thio) -ethan-1-one (4)
    [0075] • 1- (4-iodophenyl) -2 - ((9H-purin-6-yl) thio) -ethan-1-one (5)
    [0076] • 6 - ((4-chlorophenethyl) thio) -9H-purine (6)
    [0077] • 6 - ((3-chlorophenethyl) thio) -9H-purine (7)
    [0078] • 6 - ((2-chlorophenethyl) thio) -9H-purine (8)
    [0079] • 6 - ((4-fluorophenethyl) thio) -9H-purine (9)
    [0080] • 4- (benzylthio) -7H-pyrrolo [2,3-d] pyrimidine (10)
    [0081]
    [0082] In another preferred embodiment, the TDP-43 protein related disease is a neurological disease. In a more preferred embodiment, the TDP-43 protein related disease is a neurological disease that is selected from amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, age-associated cognitive decline, and chronic traumatic encephalopathy. In an even more preferred embodiment, the TDP-43 protein-related disease is selected from amyotrophic lateral sclerosis, frontotemporal dementia, and Alzheimer's disease.
    [0083] In a second aspect, the invention relates to a compound that is selected from:
    [0084] • 6 - ((4-Chlorophenethyl) thio) -9H-purine (6)
    [0085] • 6 - ((3-Chlorophenethyl) thio) -9H-purine (7)
    [0086] • 6 - ((2-Chlorophenethyl) thio) -9H-purine (8)
    [0087] • 6 - ((4-Fluorophenethyl) thio) -9H-purine (9)
    [0088]
    [0089] In a third aspect, the invention relates to a pharmaceutical composition comprising a compound of the aforementioned second aspect of the invention, together with a pharmaceutically acceptable carrier. Optionally, this composition may contain another active ingredient.
    [0090]
    [0091] The compounds of the present invention represented by formula (I) or by the compounds of the second aspect of the invention, can include isomers, depending on the presence of multiple bonds (for example, Z, E), including optical isomers or enantiomers, depending on the presence of chiral centers. The individual isomers, enantiomers, or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to any mixture of isomers, such as diastereomers, racemics, etc., including their optically isomers. active ingredients or mixtures in different proportions thereof. The individual enantiomers or diastereoisomers, as well as their mixtures, can be separated by conventional techniques.
    [0092]
    [0093] All the compounds described in the invention can be in crystalline form 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 of formula (I) or of the compounds of the second aspect that can be used in the manufacture of a medicament , as pharmaceutically unacceptable solvates, which may be useful in the preparation of pharmaceutically acceptable solvates or salts. 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 known to those skilled in the art.
    [0094] For application in therapy, the compounds of formula (I) or the compounds of the second aspect of the present invention, their salts, solvates or isomers, 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 even more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), (II) or the compounds of the second aspect of the present invention, or of their salts, solvates or isomers.
    [0095]
    [0096] In another aspect, the present invention relates to pharmaceutical compositions comprising at least one compound of the invention, or an isomer, a pharmaceutically acceptable salt or a derivative thereof, together with a pharmaceutically acceptable carrier or carrier, an excipient or a vehicle, for administration to a patient.
    [0097]
    [0098] The pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of therapeutic compositions.
    [0099]
    [0100] Another aspect of the invention is a method of treating a neurological or neurodegenerative disease, comprising administering to a patient a therapeutically effective amount of a compound of formula (I), a compound of the second aspect of the invention, or a pharmaceutical composition comprising them, where the neurological or neurodegenerative disease is a disease related to the TDP-43 protein that can be fundamentally selected from amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, cognitive impairment associated with age and chronic traumatic encephalopathy. .
    [0101]
    [0102] In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the agent or compound capable of carrying out the therapeutic action determined by its pharmacological properties, calculated for produce the desired effect and, in general, will be determined, among other causes, by the characteristics of the compounds, including the age, condition of the patient, the severity of the alteration or disorder, and the route and frequency of administration.
    [0103]
    [0104] The compounds described in the present invention, their salts or solvates, as well as the pharmaceutical compositions containing them can be used together with other drugs, or additional active ingredients, to provide a combination therapy. Said additional drugs may form 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 formula (I), or a salt or solvate thereof.
    [0105]
    [0106] In another particular embodiment, said therapeutic composition is prepared in the form of a solid form or aqueous suspension, in a pharmaceutically acceptable diluent. The therapeutic composition provided by this invention can be administered by any appropriate route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. In a particular embodiment, the administration of the therapeutic composition provided by this invention is carried out orally, topically, rectally or parenterally (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, etc.).
    [0107]
    [0108] In a preferred embodiment of the present invention, the pharmaceutical compositions are suitable for oral administration, in solid or liquid form. Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, such as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone), fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinyl pyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate.
    [0109] Compositions for oral administration can be prepared by conventional Pharmaceutical Galenic methods, such as mixing and dispersing. The tablets can be coated following methods known in the pharmaceutical industry.
    [0110]
    [0111] The pharmaceutical compositions can be adapted for parenteral administration, as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dose. Suitable excipients can be used, such as pH buffering agents or surfactants.
    [0112]
    [0113] The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
    [0114]
    [0115] The administration of the compounds or compositions of the present invention can be carried out by any suitable method, such as intravenous infusion and oral, intraperitoneal or intravenous routes. Oral administration is preferred for the convenience of the patients and for the chronic nature of the diseases to be treated.
    [0116]
    [0117] The amount of a compound of the present invention administered will depend on the relative efficacy of the chosen compound, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times a day, with a total dose between 0.1 and 1000 mg / Kg / day. It is important to bear in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.
    [0118]
    [0119] The compounds and compositions of the present invention can be used together with other drugs in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.
    [0120]
    [0121] The use of the compounds of the invention is compatible with their use in protocols in which the compounds of formula (I), or their mixtures are used by themselves or in combinations with other treatments or any medical procedure.
    [0122] 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.
    [0123]
    [0124] BRIEF DESCRIPTION OF THE FIGURES
    [0125]
    [0126] FIG. 1. Shows the linear correlation between the described and experimental permeability of 10 commercial compounds using the PAMPA methodology.
    [0127]
    [0128] FIG. 2. Shows the neuroprotective effect of the CDC7 inhibitor (compound 3) on SH-SY5Y human neuroblastoma cells pretreated with ethacrinic acid (AE, 20 pM) for 12 hours in the presence or absence of the 10 pM inhibitor and two compounds of reference (GSK-3 inhibitors). Data represent the mean of four different experiments ± SEM (* p <0.05).
    [0129]
    [0130] EXAMPLES
    [0131]
    [0132] Next, the invention will be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.
    [0133]
    [0134] Example 1: synthesis of the new compounds of the invention.
    [0135]
    [0136] 6 - ((4-Chlorophenethyl) thio) -9 H -purine (6):
    [0137] 6-Mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K2CO3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 mL). The reaction mixture is kept under stirring for 1 h at room temperature. 4-Chlorophenethyl bromide (387.0 mg, 1.76 mmol) is added and it is kept under stirring for 2 hr and 30 min at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 mL) is added. The organic phase is washed with distilled water (3 x 100 mL) adding a little NaCl. Dry over anhydrous Mg2SO4, filter, and concentrate to dryness. The crude is purified by chromatographic column using CH2Cl2 / MeOH (10: 1) as eluent. In this way, product 6 is obtained as a white solid (315.4 mg, 62%). 1 H-NMR (300 MHz, DMSO- c fe): 513.51 (s, 1H), 8.71 (s, 1H), 8.44 (s, 1H), 7.40 - 7.29 (m, 4H), 3.59 (t, J = 7.5 Hz, 2H), 3.02 (t, J = 7.5 Hz, 2H). 13 C-NMR (75 MHz, DMSO- d6 ): 5158.2 (1C), 151.5 (1C), 149.6 (1C), 143.3 (1C), 139.1 (1C), 131.0 (1C), 130.5 (2C), 130.0 ( 1C), 128.3 (2C), 34.4 (1C), 29.0 (1C). Mp 190-192 ° C.
    [0138] Elemental Analysis (C ^ HuCl ^ S) Calculated: C 53.70%, H 3.81%, N 19.27%, S 11.03%. Found: C 53.27%, H 3.79%, N 19.03%, S 10.85%.
    [0139]
    [0140] 6 - ((3-Chlorophenethyl) thio) -9 H -purine (7):
    [0141] 6-Mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K2CO3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 mL). The reaction mixture is kept under stirring for 1 h at room temperature. 3-Chlorophenethyl bromide (387.0 mg, 1.76 mmol) is added and it is kept under stirring for 3 hr and 30 min at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 mL) is added. The organic phase is washed with distilled water (3 x 100 mL) adding a little NaCl. Dry over anhydrous Mg2SO4, filter, and concentrate to dryness. The crude is purified by chromatographic column using CH2Cl2 / MeOH (20: 1) as eluent. In this way, product 7 is obtained in the form of a white solid (402.2 mg, 78%). 1 H-NMR (300 MHz, DMSO- cfe ): 513.51 (s, 1H), 8.71 (s, 1H), 8.42 (s, 1H), 7.39 (t, J = 1.6 Hz, 1H), 7.37 - 7.24 ( m, 3H), 3.62 (t, J = 7.5 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H) .13 C-NMR (75 MHz, DMSO- cfe ): 5 158.5 (1C), 151.5 ( 1C), 149.2 (1C), 142.9 (1C), 142.6 (1C), 133.0 (1C), 130.4 (1C), 130.2 (1C), 128.5 (1C), 127.4 (1C), 126.4 (1C), 34.7 ( 1C), 28.8 (1C). Mp 143-145 ° C. Elemental Analysis (C ^ HuCl ^ S) Calculated: C 53.70%, H 3.81%, N 19.27%, S 11.03%. Found: C 53.34%, H 3.80%, N 19.09%, S 11.00%.
    [0142]
    [0143] 6 - ((2-Chlorophenethyl) thio) -9 H -purine (8):
    [0144] 6-Mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K2CO3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 mL). The reaction mixture is kept under stirring for 1 h at room temperature. 2-Chlorophenethyl bromide (387.0 mg, 1.76 mmol) is added and it is kept under stirring for 5 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 mL) is added. The organic phase is washed with distilled water (3 x 100 mL) adding a little NaCl. Dry over anhydrous Mg2SO4, filter, and concentrate to dryness. The crude is purified by chromatographic column using CH2Cl2 / MeOH (10: 1) as eluent. In this way, product 8 is obtained as a white solid (250.4 mg, 49%). 1 H-NMR (300 MHz, DMSO- cfe ): 513.49 (s, 1H), 8.70 (s, 1H), 8.43 (s, 1H), 7.44 (dd, J = 6.9, 2.5 Hz, 1H), 7.43 ( dd, J = 6.9, 2.4 Hz, 1H), 7.33 - 7.22 (m, 2H), 3.62 (dd, J = 8.3, 6.6 Hz, 2H), 3.17 (dd, J = 8.2, 6.7 Hz, 2H). 13C-NMR (75 MHz, DMSO- de ):
    [0145] 5 157.6 (1C), 151.4 (1C), 150.3 (1C), 143.3 (1C), 137.3 (1C), 133.1 (1C), 131.1 (1C), 129.2 (2C), 128.4 (1C), 127.2 (1C) , 32.7 (1C), 27.6 (1C). Mp 154-156 ° C. Elemental Analysis (C ^ HuCl ^ S) Calculated: C 53.70%, H 3.81%, N 19.27%, S 11.03%. Found: C 53.60%, H 3.86%, N 19.14%, S 10.85%.
    [0146]
    [0147] 6 - ((4-Fluorophenethyl) thio) -9 H -purine (9):
    [0148] 6-Mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K2CO3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 mL). The reaction mixture is kept under stirring for 1 h at room temperature. 4-Fluorophenethyl bromide (358.0 mg, 1.76 mmol) is added and it is kept under stirring for 5 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 mL) is added. The organic phase is washed with distilled water (3 x 100 mL) adding a little NaCl. Dry over anhydrous Mg2SO4, filter, and concentrate to dryness. The crude is purified by chromatographic column using CH2Cl2 / MeOH (10: 1) as eluent. In this way product 9 is obtained as a white solid (285.0 mg, 59%). 1H-NMR (300 MHz, DMSO- cfe ): 513.51 (s, 1H), 8.70 (s, 1H), 8.42 (s, 1H), 7.45 - 7.26 (m, 2H), 7.22 - 6.94 (m, 2H) , 3.59 (t, J = 7.6 Hz, 2H), 3.02 (t, J = 7.5 Hz, 2H). 13C-NMR (75 MHz, DMSO- cfe ): 5 160.9 (d, J = 241.7 Hz, 1C), 158.6 (1C), 151.5 (1C), 149.2 (1C), 142.9 (1C), 136.3 (d, J = 2.7 Hz, 1C), 130.4 (d, J = 7.9 Hz, 2C), 130.4 (1C), 115.0 (d, J = 21.0 Hz, 2C), 34.3 (1C), 29.2 (1C). HPLC: Purity> 99%, tr = 6.99 min. MS (ES): m / z 275 [M + 1]. Mp 184-186 ° C. Elemental Analysis (C13H11FN4S) Calculated: C 56.82%, H 4.04%, N 20.42%, S 11.69%. Found: C 56.38%, H 4.01%, N 20.04%, S 11.43%.
    [0149]
    [0150] Example 2: Measurement of CDC7 inhibition
    [0151] The LanthaScreen Eu kinase inhibition assay uses an Alexa Fluor ™ marker that binds to a kinase and is detected by adding an Eu-labeled antibody. Binding of the marker and the antibody to the kinase results in a high degree of FRET, whereas displacement of the marker by an inhibitor results in a loss of FRET. Unlike many other kinase activity assays, this is a simple mix-read assay, with no development steps. This assay method has been developed by "Life Technologies" and they identify competitive ATP kinase inhibitors, making them suitable for the detection of any compound that binds to the ATP site.
    [0152] Compounds are evaluated in 1% DMSO (final) in the well. A mixture of recombinant human CDC7 / DBF4 (0.5 nM), Eu-antiGST antibody (2 nM) and AlexaFluor marker (1nM) was used in a 50 mM HEPES buffer pH 7.5, 0.01% BRIJ- 35, 10 mM MgCl2, 1 mM EGTA. In coded low volume, 348-well white plates (Greiner cat. # 784207), add 160 nL (100 x compound in 100% DMSO), 3.84 pL (buffer with CDC7 / DBF4), 8.0 pL (antibody), 4.0 pL (marker). Shake 30 s and incubate at room temperature for 60 min. The fluorescence is then measured in the plate reader and the data analyzed (Table 1).
    [0153]
    [0154] Table 1 . CDC7 inhibition values of the compounds of formula (I):
    [0155]
    [0156]
    [0157]
    [0158]
    [0159] * Comp. (1): 1- (4-Bromophenyl) -2 - ((9H-purin-6-yl) thio) -ethan-1-one, included for comparison.
    [0160]
    [0161] Example 3: Prediction of the blood-brain barrier passage
    [0162]
    [0163] An essential requirement that must be met by drugs intended for the treatment of neurodegenerative diseases is the ability to cross the blood-brain barrier (EHB) since, otherwise, they could not act on the target of interest. Therefore, for compounds that are not permeable or located in the area of uncertainty, it may be necessary that they be adequately transported in a pharmaceutical formulation by methods known to a person skilled in the art, such as by encapsulation. This ability can be predicted in vitro using a method known by the acronym PAMPA ( Parallel Artificial Membrane Permeability Assay) described by Di et al. (Di, L .; Kerns, EH; Fan, K .; McConnell, OJ; Carter, GT Eur. J. Med. Chem., 2003, 38 (3), 223-232) and which has subsequently been developed in our research group. This method allows predicting the effective permeability through artificial membranes through a passive diffusion process.
    [0164]
    [0165] Firstly, it is necessary to validate the method, for which 10 commercial compounds whose penetration capacity in the central nervous system (CNS) is known are used, which will be specified below, obtaining in this case a good linear correlation between the values of experimental permeability ( Pe ) and those described (FIG. 1). This correlation line obtained following the pattern described in the bibliography allows establishing the limits to predict whether or not a compound can cross the blood-brain barrier. Thus, a compound is considered to be permeable to EHB (CNS +) if it has a permeability of> 4.48 x 10 "6 cm" 1.
    [0166]
    [0167] For the procedure, 3-5 mg of caffeine, desipramine, enoxacin, hydrocortisone, ofloxacin, piroxicam and testosterone, 12 mg of promazine and 25 mg of atenolol and verapamil were taken and dissolved in EtOH (1000 pL). 100 pL of these solutions were taken and EtOH (1400 pL) and phosphate buffer (PBS) pH = 7.4 (3500 pL) were added in order to reach a final EtOH concentration of 30% v / v in solution. Finally, the solutions were filtered.
    [0168]
    [0169] On the other hand, a PBS / EtOH solution (70:30) was added to each well of the acceptor plate (180 pL). The donor plate was impregnated with a porcine brain lipid solution (4 pL) dissolved in dodecane (20 mg-mL "1). After 5 min, solution of each compound was added on this plate (180 pL).
    [0170]
    [0171] Of the compounds evaluated, between 1-2 mg were taken and dissolved in EtOH (1500 pL) and phosphate buffer (PBS) pH = 7.4 (3500 pL), filtered and added to the donor plate. With these solutions, the wavelengths at which the compounds absorb are determined and the initial absorbance levels at these wavelengths are measured using a UV absorbance reader. Each sample was analyzed from two to five wavelengths, in three wells and in two independent experiments.
    [0172]
    [0173] Next, the donor plate was deposited on the acceptor forming a kind of "sandwich" and they were allowed to incubate for 2 h and 30 min at 25 ° C. In this way, the compounds will pass from the donor plate to the acceptor plate through of porcine brain lipid by passive diffusion After this time, the donor plate is carefully removed and the final concentration and absorbance of both the commercial and synthesized compounds are determined.The results obtained are expressed as the mean of the measurements [deviation standard (SD)] of the different experiments performed and are collected in Table 2.
    [0174]
    [0175] Table 2 . Permeability values ( Pe 10-6 cm s-1) in the PAMPA-BHE experiment and prediction of penetration into the central nervous system (CNS) of the compounds of formula (II) as also described in Table 1:
    [0176]
    [0177]
    [0178] Example 4: Neuroprotective effect of CDC7 inhibitors against ethacrynic acid
    [0179] The SH-SY5Y human neuroblastoma cell line was grown at 37 ° C with 5% CO2 in DMEN (Dulbecco's Modified Eagle Medium) medium enriched with L-glutamine (2mM), 1% non-essential amino acids, 1% of Penicillin / Streptomycin and 10% bovine fetal serum. In the semiconfluence state, the cells were treated with the CDC7 inhibitor (compound 3) at different concentrations for 1.30 hours post-addition of the agent causing phosphorylation of TPD-43; etacrinic acid (20 pM) (Sigma). At 24 hours, cell viability was assessed with MTT ([3 (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) following a described procedure (Denizot F, Lang R. J Immunol Mefhods. 1987; 89: 271-7). Figure 2 shows how the cells have a 40% mortality due to excess phosphorylation of TDP-43 (ethacrynic acid) and how this death is reversed with the addition of the reference standards, as well as with the CDC7 inhibitor.
    权利要求:
    Claims (20)
    [1]
    1. Compound of formula (I)

    [2]
    2. Compound for use according to claim 1 where Ar is the following group:
    [3]

    [4]
    4. Compound for use according to claim 2 where X is CH2 and Y is N.
    [5]
    5. Compound for use according to claim 2 where n is 0 and Y is CH.
    [6]
    6. Compound for use according to any one of claims 2 to 4 wherein at least one of R1 to R5 is -O (C1-C4 alkyl).
    [7]
    7. Compound for use according to claim 6 wherein at least one of R1 to R5 is -O-methyl.
    [8]
    8. Compound for use according to any of claims 2 to 5 wherein at least one of R1 to R5 is halogen.
    [9]
    9. Compound for use according to claim 1 where Ar is the following group:

    [10]
    10. Compound
    [11]
    11. Compound for use according to any of claims 9 or 10 wherein at least one of R1 to R7 is -O (C1-C4-alkyl).
    [12]
    12. Compound for use according to claim 11 wherein at least one of R1 to R7 is -O-methyl.
    [13]
    13. Compound for use according to any of claims 9 or 10 wherein at least one of R1 to R7 is halogen.
    [14]
    14. Compound for use according to claim 1 which is selected from the following list:
    • 1- (4-methoxyphenyl) -2 - ((9H-purin-6-yl) thio) - ethan-1-one
    • 1- (naphthalen-2-yl) -2 - ((9H-purin-6-yl) thio) - ethan-1-one
    • 1- (3-methoxyphenyl) -2 - ((9H-purin-6-yl) thio) -ethan-1-one
    • 1- (4-iodophenyl) -2 - ((9H-purin-6-yl) thio) -ethan-1-one
    • 6 - ((4-chlorophenethyl) thio) -9H-purine
    • 6 - ((3-chlorophenethyl) thio) -9H-purine
    • 6 - ((2-chlorophenethyl) thio) -9H-purine
    • 6 - ((4-fluorophenethyl) thio) -9H-purine
    • 4- (benzylthio) -7H-pyrrolo [2,3-d] pyrimidine
    [15]
    15. Compound for use according to any of claims 1 to 14, wherein the TDP-43 protein related disease is a neurological disease.
    [16]
    16. Compound for use according to claim 15, wherein the TDP-43 protein related disease is a neurological disease selected from amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer, age-related cognitive decline and chronic traumatic encephalopathy.
    [17]
    17.
    [18]
    18. Compound selected from:
    • 6 - ((4-Chlorophenethyl) thio) -9H-purine
    • 6 - ((3-Chlorophenethyl) thio) -9H-purine
    • 6 - ((2-Chlorophenethyl) thio) -9H-purine
    • 6 - ((4-Fluorophenethyl) thio) -9H-purine
    [19]
    19. Pharmaceutical composition comprising a compound according to claim 18 together with a pharmaceutically acceptable carrier.
    [20]
    20. Compound according to claim 18 for use as a medicine.
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    同族专利:
    公开号 | 公开日
    US20210346388A1|2021-11-11|
    ES2749743B2|2020-12-14|
    EP3854401A1|2021-07-28|
    AU2019344041A1|2021-04-22|
    WO2020058558A1|2020-03-26|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP0350742A1|1988-07-09|1990-01-17|Hoechst Aktiengesellschaft|6-Merkaptopurines derivatives, their preparation and their use against retrovirus infections|
    EP1444982A1|2003-02-06|2004-08-11|Merckle Gmbh|The use of purine derivatives as selective kinase inhibitors|
    WO2014123332A1|2013-02-07|2014-08-14|한국과학기술연구원|7h-pyrrolo[2,3-d]pyrimidine-4-thiol derivative useful as jak-3 inhibitor|
    CN103319485A|2013-06-26|2013-09-25|吉林省标达科技有限责任公司|6-mercaptopurine compound, and synthesis method and application thereof|
    ES2686909A1|2017-03-22|2018-10-22|Consejo Superior De Investigaciones Científicas |CDC-7 INHIBITORS COMPOUNDS AND THEIR USE FOR THE TREATMENT OF NEUROLOGICAL PATHOLOGIES |
    WO2007124288A1|2006-04-19|2007-11-01|Novartis Ag|Indazole compounds and methods for inhibition of cdc7|
    ES2483594T3|2009-07-15|2014-08-06|Abbott Laboratories|Kinase inhibitor pyrrolopyrazines|
    US20130072506A1|2011-09-16|2013-03-21|Lenka ZAHAJSKA|6,8-disubstituted purine compositions|
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    申请号 | 申请日 | 专利标题
    ES201830914A|ES2749743B2|2018-09-21|2018-09-21|PURINE DERIVATIVES INHIBITORS OF CDC7 AND THEIR USE FOR THE TREATMENT OF NEUROLOGICAL PATHOLOGIES|ES201830914A| ES2749743B2|2018-09-21|2018-09-21|PURINE DERIVATIVES INHIBITORS OF CDC7 AND THEIR USE FOR THE TREATMENT OF NEUROLOGICAL PATHOLOGIES|
    PCT/ES2019/070632| WO2020058558A1|2018-09-21|2019-09-23|Cdc7-inhibiting purine derivatives and their use for the treatment of neurological conditions|
    US17/277,921| US20210346388A1|2018-09-21|2019-09-23|CDC7-Inhibiting Purine Derivatives and their use for the Treatment of Neurological Conditions|
    AU2019344041A| AU2019344041A1|2018-09-21|2019-09-23|Cdc7-inhibiting purine derivatives and their use for the treatment of neurological conditions|
    EP19862056.9A| EP3854401A1|2018-09-21|2019-09-23|Cdc7-inhibiting purine derivatives and their use for the treatment of neurological conditions|
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