Tau-tubulin kinase (TTBK) inhibitor compounds

Abstract

The present invention relates to TTBK enzyme inhibitors of formula (I) and their use for the treatment and / or prevention of tauopathies, such as Alzheimer's disease (AD) and / or TDP-43 pathologies, such as amyotrophic lateral sclerosis (ALS). [Formula 1] JPEG2024527587000037.jpg37169

Description

Detailed description of the invention

[0001] This invention relates to TTBK enzyme inhibitors and their use for the treatment and / or prevention of tauopathy and / or TDP-43 diseases. Therefore, this invention may fall under the fields of medical chemistry and pharmaceuticals.

[0002] [Background of the Invention] Tau-tubulin kinase (TTBK) is a recently discovered family of serine / threonine and tyrosine kinases involved in the phosphorylation of important substrates such as tau, tubulin, or TDP-43. Its two isoforms, TTBK1 and TTBK2, exhibit different expression patterns and are also involved in different critical physiological mechanisms, including mitosis, ciliation, and neurotransmission.

[0003] Recently, it has been shown that TTBK is also involved in the phosphorylation of TDP-43 (Liachko NF, et al. The tau tubulin kinases TTBK1 / 2 promote accumulation of pathological TDP-43. PLoS Genet. 2014 Dec 4;10(12):e1004803), and therefore, their phosphorylation activity is also associated with the development of neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. These neurodegenerative diseases are associated with the pathological aggregation of tau protein and TDP-43 protein in the human brain and are therefore called tauopathies and TDP-43 diseases, respectively.

[0004] In the following publication: Nozal, A. Martinez, Tau Tubulin Kinase 1 (TTBK1), a new player in the fight against neurodegenerative diseases, Eur J Med Chem 2019 Jan 1;161:39-47, the importance of the aforementioned TTBK kinase in neurodegenerative diseases such as Alzheimer's disease (AD), progressive supranuclear palsy, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) has already been clarified, and TTBK1 and TTBK2 have been proposed as new therapeutic targets for these diseases, and selective inhibitors of TTBK1 and TTBK2 have been proposed as effective drugs for their treatment. However, it has only recently been demonstrated that TTBK1 and TTBK2 are involved in the neurodegenerative process not only through tau hyperphosphorylation but also through TDP-43 hyperphosphorylation (Taylor LM, McMillan PJ, Liachko NF, Strovas TJ, Ghetti B, Bird). TD, Keene CD, Kraemer BC. Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration. Mol Neurodegener.2018 Feb 6;13(1):7).

[0005] Based on the above, TTBK inhibition is considered a promising approach for the treatment of neurodegenerative diseases involving tau proteins and TDP-43 proteins, namely tauopathies and TDP-43 diseases.

[0006] Given the current state of affairs in this field, there are virtually no known compounds that can act as inhibitors of the aforementioned enzyme. Therefore, the present invention proposes a novel compound that acts as an inhibitor of this enzyme, which is useful for the treatment and / or prevention of neurodegenerative diseases involving this enzyme. Accordingly, the present invention is a replacement or improvement on compounds already known for the treatment of these types of diseases.

[0007] [Description of the Invention] This invention presents a family of compounds that have the ability to inhibit the TTBK1 enzyme and / or the TTBK2 enzyme at a micromolar level or less, and that demonstrate usefulness in the treatment and / or prevention of tauopathy and / or TDP-43 disease.

[0008] In a first embodiment, the present invention relates to a compound of formula (I) or its isomer, or any pharmaceutically acceptable salt thereof, [ka] During the ceremony, R1 is selected from H and NH2. X is selected from O, S, CO, and CH2O. R2, R3, and R4 are each independently selected from H, halogen, CF3, CN, NO2, NH2, C1-C3 alkyl, and -O-C1-C3 alkyl. At least one of R2, R3, and R4 is H. This relates to any compound, its isomer, or any pharmaceutically acceptable salt thereof for use in the treatment and / or prevention of tauopathy and / or TDP-43 disease.

[0009] The compound of formula (I) acts as an inhibitor of the TTBK1 enzyme and / or the TTBK2 enzyme. These enzymes are involved in tauopathy and TDP-43 disease. These are types of neurodegenerative diseases in the nervous system that are associated with the pathological aggregation of the respective tau protein or TDP-43, as described in the Current Status section of the Art. Thus, these are neurodegenerative diseases involving tau-tubulin kinases (TTBKs), such as TTBK1 and TTBK2.

[0010] Preferably, the tauopathy is selected from the following list: Alzheimer's disease (AD), progressive supranuclear palsy, frontotemporal dementia (FTD), Pick's disease, and Down syndrome. More preferably, the tauopathy is selected from Alzheimer's disease (AD) and frontotemporal dementia (FTD), and even more preferably, the tauopathy is Alzheimer's disease.

[0011] Preferably, the TDP-43 disease is selected from the following list: amyotrophic lateral sclerosis (ALS), Alexander disease, limbic-dominant age-related TDP-43 encephalopathy (LATE), frontotemporal dementia (FTD), and Huntington's disease. More preferably, the TDP-43 disease is selected from frontotemporal dementia (FTD), limbic-dominant age-related TDP-43 encephalopathy (LATE), and amyotrophic lateral sclerosis (ALS), and even more preferably, the TDP-43 disease is amyotrophic lateral sclerosis.

[0012] In a preferred embodiment of the compound of the present invention for use, at least two of R2, R3, and R4 are H. In another preferred embodiment of the compound of the present invention for use, two of R2, R3, and R4 are H, and one of them is not H. In yet another preferred embodiment of the compound of the present invention for use, R2, R3, and R4 are H.

[0013] In preferred embodiments of the compound of formula (I) for use, R1 is H.

[0014] In another preferred embodiment of the compound of formula (I) for use, X is O.

[0015] In a more preferred embodiment, R1 is H and X is O. Even more preferably, R1 is H and X is O, and R2, R3 and R4 are independently selected from H, halogen, CN, NO2, NH2, and C1-C3 alkyl-(O)-C1-C3 alkyl.

[0016] In a preferred embodiment, R1 is H, X is O, R2 and R4 are H, and R3 is selected from H, halogen, CF3, CN, NO2, NH2, C1-C3 alkyl and -(O)-C1-C3 alkyl, more preferably R3 is selected from H, halogen, CN, NO2, NH2, C1-C3 alkyl and -(O)-C1-C3 alkyl.

[0017] In a preferred embodiment, R1 is H, X is O, R2 and R3 are H, and R4 is selected from halogens and -(O)-C1-C3 alkyl groups.

[0018] In a preferred embodiment, R1 is H, X is O, R3 and R4 are H, and R2 is selected from halogen, CF3, -(O)-C1-C3 alkyl and C1-C3 alkyl, more preferably R2 is selected from halogen, CF3, -(O)-C1-C3 alkyl and alkyl C1-C3.

[0019] In another preferred embodiment, X is S. More preferably, R1 is H and X is S. Even more preferably, R1 is H and X is S, and R2, R3 and R4 are independently selected from H, NO2 and NH2.

[0020] In another preferred embodiment, X is CO. More preferably, X is CO and R1 is H. Even more preferably, X is CO and R1 is H, and R2, R3 and R4 are independently selected from halogens and H.

[0021] In another embodiment of the present invention for use, R1 is NH2. More preferably, R1 is NH2 and X is O. Even more preferably, R1 is NH2, X is O, and R2, R3 and R4 are independently selected from H, halogen and CF3, and at least one of R2, R3 and R4 is different from H. Even more preferably, R1 is NH2, X is O, R4 is H, and R2 and R3 are independently selected from H, halogen and CF3, and at least one of R2 and R3 is different from H.

[0022] In a more preferred embodiment of this first aspect, the present invention relates to a compound of formula (I) for use, selected from the list including: N-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-fluorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-cyanophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-bromophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-nitrophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-aminophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2,4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N4-(4-(4-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-chlorophenoxy)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N-[4-((4-nitrophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-((4-aminophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(benzyloxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine (4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](phenyl)methanone (4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](4-fluorophenyl)methanone.

[0023] The structures of each compound listed above are shown in the examples (see Example 1).

[0024] A second aspect of the present invention is a compound of formula (I) or its isomer, or a pharmaceutically acceptable salt thereof, [ka] In the formula, R1, X, R2, R3, and R4 have the same meanings as described above in the first embodiment of the present invention, and at least one of R2, R3, and R4 is H, but the compound is not either of the following two (I') and (I''), and the invention relates to a compound, an isomer thereof, or a pharmaceutically acceptable salt thereof. [ka]

[0025] In preferred embodiments, the present invention relates to a compound of formula (I) selected from the following list: N-[4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-fluorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-cyanophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-bromophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-nitrophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-aminophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2,4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N4-(4-(4-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-chlorophenoxy)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N-[4-((4-nitrophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-((4-aminophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(benzyloxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N(4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](phenyl)methanone N(4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](4-fluorophenyl)methanone

[0026] A third aspect of the present invention relates to a compound of formula (I) as defined in the first aspect of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical, wherein the compound is not one of the formulas (I') and (I'') described above, or an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0027] The present invention also relates to a pharmaceutical composition comprising a compound of formula (I) as defined in a first aspect of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is not of formula (I') or (I'') as described above.

[0028] The term "pharmaceutical composition" refers to a composition comprising a compound of formula (I) other than (I') and (I''), its isomer, or a pharmaceutically acceptable salt thereof, and at least one excipient, a pharmaceutically acceptable adjuvant, and / or carrier.

[0029] The term “excipient, adjuvant, and / or carrier” refers to a molecular entity or substance through which an active ingredient is administered. Such pharmaceutically acceptable excipients, adjuvants, or carriers may be sterile liquids such as water and oils, and include oils of animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similar oils, as well as excipients, disintegrants, wetting agents, or diluents. Pharmaceutically acceptable excipients and carriers are listed by EW Martin in “Remington's Pharmaceutical Sciences.”

[0030] The compounds of formula (I) of the present invention for use have the ability to cross the blood-brain barrier, as shown in some of the following examples. This represents a further advantage of the compounds when used in the therapeutic treatment of central nervous system-related diseases such as tauopathy and TDP-43 disease.

[0031] The compound of the present invention represented by formula (I) may be in crystalline form as a free compound or a solvate, and both forms are intended to be within the scope of the present invention. The pharmaceutically acceptable properties of the solvate are not important as long as they are pharmaceutically acceptable. In certain embodiments, the solvate is a hydrate. The solvate can be obtained by conventional solvation methods known to those skilled in the art.

[0032] Compounds of formula (I) for therapeutic use are prepared in solid form or aqueous suspension in pharmaceutically acceptable diluents. These formulations can be administered by any suitable route of administration; for this purpose, the formulations are formulated in a pharmaceutically appropriate form for the selected route of administration. In certain embodiments, administration of compounds of formula (I) is carried out orally, topically, rectally, or parenterally (including subcutaneously, intraperitoneally, intradermally, intramuscularly, intravenously, etc.).

[0033] Another aspect of the present invention relates to a method for treating tauopathy and / or TDP-43 disease, comprising administering a therapeutically effective dose of the compound of formula (I) described in the first aspect of the present invention to a subject.

[0034] The final aspect of the present invention refers to the use of a compound of formula (I) described in the first aspect of the present invention for the preparation of a pharmaceutical for the treatment and / or prevention of tauopathy and / or TDP-43 disease.

[0035] The compounds described in the present invention, their pharmaceutically acceptable salts, and solvates, as well as pharmaceutical compositions containing them, can be used together with other additional agents to provide combination therapy. The additional agents may constitute part of the same pharmaceutical composition, or they may be provided in the form of separate compositions for administration, whether concurrently with or concurrently with the administration of the pharmaceutical composition containing the compound of formula (I), its isomer, or a pharmaceutically acceptable salt thereof.

[0036] Unless otherwise indicated, the compounds of the present invention also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, substitution of hydrogen with deuterium or tritium, or carbon 13 C or 14 C-rich carbon or 15 Except for substitution with nitrogen enriched with nitrogen, compounds having the above structure are within the scope of the present invention.

[0037] In the present invention, the term "C1-C3 alkyl" refers to a linear or branched hydrocarbon chain radical having 1 to 3 carbon atoms bonded to the rest of the molecule by a single bond, such as propyl, ethyl, methyl, isopropyl, etc. When this substituent is present in the compound of the present invention, the "C1-C3 alkyl" is preferably methyl.

[0038] The term "halogen" refers to F, Cl, Br, and I. When this substituent is present in the compound of the present invention, the halogen is preferably Cl.

[0039] As used herein, the expression “treatment and / or prevention” means, unless otherwise indicated, reversing, alleviating, slowing the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0040] Throughout this specification and the claims, the term "comprising" and its variations are not intended to exclude other technical features, additives, components or steps. For those skilled in the art, other objectives, advantages and features of the present invention can be partially inferred from both this specification and the embodiments of the present invention. The following examples are provided for illustrative purposes and are not intended to limit the present invention.

[0041] [Description of Drawings] Figure 1: Cell viability (MTT) 24 hours after exposure to 40 μM etacrynic acid (EA) in the presence or absence of the patented compound (5 μM) in the SH-SY5Y cell line. The commercially available GSK-3β inhibitor tideglusib (5 μM) was used as an internal control. The values represent the mean ± SEM of three different experiments ( * p < 0.05; ** p < 0.01, **** p < 0.0001 indicates a significant difference compared to cells treated with EA, ++++ p < 0.0001 is related to the control).

[0042] Figure 2: Representative immunoblot and quantification of p-TDP-43 levels 24 hours after exposure to 40 μM etacrynic acid (EA) in the presence or absence of the patented compound (5 μM) in the SH-SY5Y cell line. The values represent the mean ± SEM of three different experiments ( * p < 0.05, ** p < 0.01 indicates a significant difference compared to cells treated with EA, ++p < 0.01 is related to the control).

[0043] Figure 3: Cell viability (MTT) 24 hours after exposure to 30 nM okadaic acid (OA) in the presence or absence of the patented compound (1 μM) in the SH-SY5Y cell line. The values represent the mean ± SEM of three different experiments ( * p < 0.05; ** p < 0.01, **** p < 0.0001 indicates a significant difference compared to cells treated with AO, ++++ p < 0.0001 is related to the control).

[0044] [Examples] Next, the present invention will be described by an assay performed by the inventors to demonstrate the effectiveness of the product of the present invention.

[0045] [Example 1: Synthesis of the compound of the present invention] [ka] Summary of the method: Dissolve 1 equivalent (250 mg, 1.63 mmol) of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-2-amine or 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 1 equivalent (1.63 mmol) of the corresponding aniline (both reagents are commercially available from Sigma-Aldrich or Fluorochem) in 2 ml of tetrahydrofuran. Stir the crude mixture under microwave irradiation at 100°C until the reaction is complete. Next, extract the crude mixture with 20 ml of ethyl acetate, wash with saturated solutions of NaHCO3 and NaCl, combine the organic phases, dry over MgSO4, filter, and remove the solvent under reduced pressure. The resulting crude mixture is purified by column chromatography using a suitable mixture of DCM and MeOH as the eluent.

[0046] [(1)N-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-phenoxyaniline Melting point: 250℃~251℃. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.75(s,1H),9.34(s,1H),8.26(s,1H),7.89(d,J=9.0Hz,2H),7.37(dd,J=8.6,7.3 Hz,2H),7.23(dd,J=3.5,2.3Hz,1H),7.13-6.96(m,5H),6.77(dd,J=3.5,1.9Hz,1H).13 C-NMR(75MHz,DMSO-d6) δ(ppm) 157.6,153.5,150.8,150.75,150.7,136.3,129.9,122.7,122.1,122.0,119.4,117.6,103.5,98.7. Elemental analysis C 18 H 14 N4O Calculated values: %C 71.51, %H 7.67, %N 18.53. Detected values: %C 71.04, %H 4.70, %N 18.38.

[0047] [(2)N-[4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-chlorophenoxy)aniline Melting point: 250℃~251℃. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.77(s,1H),9.36(s,1H),8.26(s,1H),7.92(d,J=9.0Hz,2H),7.41(d,J=9.0Hz,2H),7.24(dd ,J=3.4,2.3Hz,1H),7.06(d,J=9.0Hz,2H),7.00(d,J=9.0Hz,2H),6.77(dd,J=3.5,1.9Hz,1H). 13 C-NMR (75MHz, DMSO-d6) δ 156.7,153.4,150.8,150.7,150.2,136.8,129.7,126.4,122.1,121.9,119.7,119.2,103.5,98.7. C 18 H 13 ClN4O theoretical values: %C 64.20, %H 3.89, %N 16.64. Experimental values: %C 65.15, %H 3.96, %N 16.69.

[0048] [(3)N-[4-(4-fluorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-fluorophenoxy)aniline Melting point: 227°C to 228°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.75(s,1H),9.33(s,1H),8.25(s,1H),7.88(d,J=9.0Hz,2H),7.26-7.15(m,3H),7.08-6.95(m,4H),6.76(dd,J=3.5,1.9Hz,1H). 13 C-NMR(75MHz,DMSO-d6):δ(ppm) 159.4(d,J=238.1Hz,153.6(d,J=2.3Hz),153.5,151.3,150.8,136.3,122 .1,122.0,119.6(d,J=8.4Hz),119.0,116.4(d,J=23.3Hz),103.5,98.7C. 18 H 13 FN4O theoretical values: %C 67.49, %H 4.09, %N 17.49. Experimental values: %C 67.09, %H 4.09, %N 17.38.

[0049] [(4)N-[4-(4-cyanophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-cyanophenoxy)aniline Melting point: 259°C to 260°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.77(s,1H),9.41(s,1H),8.28(s,1H),7.99(d,J=9.0Hz,2H),7.83(d,J=8.9Hz,2H),7.25(dd ,J=3.5,2.3Hz,1H),7.15(d,J=9.0Hz,2H),7.09(d,J=8.9Hz,2H),6.79(dd,J=3.5,1.8Hz,1H). 13C-NMR (75MHz, DMSO-d6): δ(ppm) 161.9,153.3,150.8,150.7,148.4,137.8,134.6,122.2,121.8,120.7,118.8,117.3,104.5,103.6,98.7. C 19 H 13 Theoretical values ​​for N5O: %C 69.71, %H 4.00, %N 21.39. Experimental values: %C 69.33, %H 4.15, %N 21.01.

[0050] [(5)N-[4-(4-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-methoxyphenoxy)aniline. Melting point: 202°C to 203°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.74(s,1H),9.30(s,1H),8.25(s,1H),7.84(d,J=9.0Hz,2H),7.23(dd,J=3 .5,2.3Hz,1H),7.04-6.94(m,6H),6.76(dd,J=3.1,1.9Hz,1H),3.76(s,3H). 13 C-NMR (75MHz, DMSO-d6): δ(ppm) 155.2,153.5,152.4,150.8,150.7,150.4,135.5,122.0,121.9,119.8,118.0,115.0,103.4,98.7,55.4. C 19 H 16 Theoretical values ​​for N4O2: %C 68.66, %H 4.85, %N 16.86. Experimental values: %C 67.97, %H 4.91, %N 16.64.

[0051] [(6)[N-[4-(4-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-trifluoromethylphenoxy)aniline Melting point: 233°C to 234°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.77(s,1H),9.40(s,1H),8.28(s,1H),7.98(d,J=9.0Hz,2H),7.72(d,J=8.7Hz,2 H),7.24(dd,J=3.5,2.3Hz,1H),7.14(t,J=9.1Hz,4H),6.79(dd,J=3.5,1.9Hz,1H). 13 C-MNR(75MHz,DMSO-d6):δ(ppm) 162.1,154.3,151.7,151.6,149.9,138.4,128.3(q,J=3.6Hz),125.2(q,J =271.7Hz),123.6(q,J=32.1Hz),123.1,122.7,121.4,118.0,104.5,99.6. C 19 H 13 F3N4O theoretical values: %C 61.62, %H 3.54, %N 15.13. Experimental values: %C 61.55, %H 3.51, %N 15.04.

[0052] [(7)[N-[4-(4-bromophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-bromophenoxy)aniline Melting point: 257°C to 258°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.75(s,1H),9.35(s,1H),8.27(s,1H),7.93(d,J=9.0Hz,2H),7.54(d,J=9.0Hz,2H),7.24(dd ,J=3.5,2.3Hz,1H),7.08(d,J=9.0Hz,2H),6.96(d,J=8.9Hz,2H),6.78(dd,J=3.5,1.9Hz,1H).13 C-NMR (75MHz, DMSO-d6) δ 157.7,153.9,151.3,151.2,150.6,137.3,133.1,122.6,122.4,120.2,120.1,114.7,104.0,99.2. C 18 H 13 Theoretical values ​​for BrN4O: %C 56.71, %H 3.44, %N 14.70. Experimental values: %C 56.21, %H 3.51, %N 14.43.

[0053] [(8)[N-[4-(4-nitrophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-nitrophenoxy)aniline Melting point: 270°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.86(s,1H),9.63(s,1H),8.35-8.16(m,3H),8.00(d,J=9.0Hz,2H),7.29-7.24( m,1H),7.20(d,J=9.0Hz,2H),7.14(d,J=9.3Hz,2H),6.84(dd,J=3.5,1.9Hz,1H). 13 C-NMR (75MHz,DMSO-d6) δ 163.6,153.1,150.5,150.1,148.7,142.0,137.7,126.2,122.5,122.3,120.9,116.9,103.6,99.1. C 18 H 13 N5O3[M+H] + The calculated ESI value is 348.1091, and the detected value is 348.1088.

[0054] [(9)[N-[4-(4-aminophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-aminophenoxy)aniline Melting point: 242°C to 243°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.68(s,1H),9.21(s,1H),8.21(s,1H),7.75(d,J=9.0Hz,2H),7.19(dd,J=3.5,2.3Hz,1H),6.87(d, J=9.0Hz,2H),6.76(d,J=8.8Hz,2H),6.71(dd,J=3.5,1.9Hz,1H),6.58(d,J=8.8Hz,2H),4.92(s,2H). 13 C-NMR (75MHz, DMSO-d6) δ 153.74,153.64,150.8,150.7,146.5,145.1,134.7,122.1,121.9,120.3,117.0,114.8,103.3,98.7. C 18 H 15 N5O[M+H] + The calculated ESI value is 318.1349, and the detected value is 318.1348.

[0055] [(10)[N-[4-(4-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(4-methylphenoxy)aniline Melting point: 229°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.72(s,1H),9.29(s,1H),8.24(s,1H),7.85(d,J=9.0Hz,2H),7.22(dd,J=3.5,2.4Hz,1H),7.17(dd,J =8.8,0.7Hz,2H),6.99(d,J=9Hz,2H),6.89(d,J=8.5Hz,2H),6.75(dd,J=3.5,1.9Hz,1H),2.28(s,3H). 13C-NMR (75MHz, DMSO-d6): δ(ppm) 155.2,153.5,151.5,150.8(C-2,4),136.0,131.9,130.3,122.03,121.98,118.9,117.9,103.5,98.7,20.2. C 19 H 16 Theoretical values ​​for N4O: %C 72.13, %H 5.10, %N 17.71. Experimental values: %C 71.84, %H 5.15, %N 17.96.

[0056] [(11)[N-[4-(3-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(3-methylphenoxy)aniline Melting point: 212°C to 213°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.73(s,1H),9.31(s,1H),8.25(s,1H),7.88(d,J=9.0Hz,2H),7.28-7.20(m,2H),7.0 2(d,J=9.0Hz,2H),6.91(ddt,J=7.5,1.7,0.8Hz,1H),6.82-6.78(m,3H),2.28(s,3H). 13 C-NMR(75MHz,DMSO-d6) δ(ppm) 157.7,153.5,150.9,150.79,150.76,139.6,136.3,129.6,123.5,122.1,121.9,119.4,118.1,114.7,103.5,98.7,21.0. C 19 H 16 N4O[M+H] + The calculated ESI value is 317.1397, and the detected value is 317.1393.

[0057] [(12)[N-[4-(3-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(3-chlorophenoxy)aniline Melting point: 224°C to 225°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.79(s,1H),9.39(s,1H),8.29(s,1H),7.96(d,J=9.0Hz,2H),7.41(t,J=8.1Hz,1H),7.26(dd,J=3.5,2.2Hz,1H),7.17(ddd,J=8 .0,2.0,0.9Hz,1H),7.12(d,J=9.0Hz,2H),7.03(t,J=2.2Hz,1H),6.97(ddd,J=8.3,2.3,0.8Hz,1H),6.80(dd,J=3.5,1.8Hz,1H). 13 C-NMR (75MHz, DMSO-d6): δ(ppm) 158.9,153.4,150.8,150.8,149.7,137.1,133.9,131.4,122.5,122.2,121.9,120.0,117.2,116.0,103.5,98.7. C 18 H 13 ClN4O theoretical values: %C 64.20, %H 3.89, %N 16.64. Experimental values: %C 64.06, %H 3.91, %N 16.75.

[0058] [(13)[N-[4-(3-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(3-trifluoromethylphenoxy)aniline Melting point: 176°C to 177°C. 1H-NMR(300MHz,DMSO-d6):δ(ppm) 11.75(s,1H),9.37(s,1H),8.27(s,1H),7.96(d,J=9.0Hz,2H),7.61(t,J=7.8Hz,1H),7.4 4(d,J=7.9Hz,1H),7.32-7.19(m,3H),7.13(d,J=8.9Hz,2H),6.78(dd,J=3.5,1.9Hz,1H). 13 C-NMR(75MHz,DMSO-d6):δ(ppm) 158.8,153.8,151.2,151.1,149.9,137.6,131.6,130.95(q,J=32Hz),127.7(d,J=272. 5Hz),122.5,122.3,121.5,120.5,119.5(d,J=3.9Hz),113.9(d,J=4.0Hz),103.9,99.1. C 19 H 13 F3N4O theoretical values: %C 61.62, %H 3.54, %N 15.13. Experimental values: %C 61.60, %H 3.47, %N 15.22.

[0059] [(14)[N-[4-(2-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(2-chlorophenoxy)aniline Melting point: 215℃. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.73(s,1H),9.33(s,1H),8.25(s,1H),7.89(d,J=9.0Hz,2H),7.58(dd,J=8.0,1.6Hz,1H),7.34(ddd,J=8.2,7.4,1.6 Hz,1H),7.22(dd,J=3.5,2.4Hz,1H),7.17(ddd,J=8.0,7.4,1.5Hz,1H),7.05-6.95(m,3H),6.76(dd,J=3.5,1.9Hz,1H). 13C-NMR(75MHz,DMSO-d6):δ(ppm) 153.5,152.6,150.80,150.74,150.71,136.5,130.6,128.7,124.6,123.8,122.1,122.0,119.8,118.4,103.5,98.7. C 18 H 13 ClN4O theoretical values: %C 64.20, %H 3.89, %N 16.64. Experimental values: %C 64.11, %H 3.96, %N 16.63.

[0060] [(15)[N-[4-(2-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(2-methoxyphenoxy)aniline Melting point: 214℃~215℃. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.69(s,1H),9.23(s,1H),8.21(s,1H),7.76(d,J=9.0Hz,2H),7.20(dd,J=3.5,2.3Hz,1H),7.18 -7.14(m,2H),7.02-6.92(m,2H),6.86(d,J=9.1Hz,2H),6.71(dd,J=3.5,1.9Hz,1H),3.77(s,3H). 13 C-NMR(75MHz,DMSO-d6) δ(ppm) 153.6,152.6,151.1,150.8,150.7,144.6,135.0,124.9,122.1,121.9,121.0,120.7,116.7,113.3,103.3,98.7,55.6. C 19 H 16 Theoretical values ​​for N4O2: %C 68.66, %H 4.85, %N 16.96. Experimental values: %C 68.006, %H 4.82, %N 16.97.

[0061] [(16)[N-[4-(2,4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(2,4-dichlorophenoxy)aniline Melting point: 252°C to 253°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.72(s,1H),9.37(s,1H),8.24(s,1H),7.89(d,J=9.1Hz,2H),7.72(d,J=2.5Hz,1H),7.39(dd,J=8. 8,2.6Hz,1H),7.22(dd,J=3.5,2.3Hz,1H),7.02(dd,J=8.9,2.1Hz,3H),6.75(dd,J=3.5,1.9Hz,1H). 13 C-NMR(75MHz,DMSO-d6)δ(ppm) 153.6,152.0,150.9,150.5,136.9,130.1,128.8,127.7,124.9,122.4,122.2,120.9,118.8,103.7,98.9. C 18 H 12 Cl2N4O theoretical values: %C 58.24, %H 3.26, %N 15.09. Experimental values: %C 58.13, %H 3.33, %N 15.17.

[0062] [(17)[N-[4-(3-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-(3-methoxyphenoxy)aniline Melting point: 210℃~211℃. 1H-NMR(300MHz,DMSO-d6):δ(ppm) 11.73(s,1H),9.32(s,1H),8.25(s,1H),7.89(d,J=9.0Hz,2H),7.31-7.20(m,2H),7.05(d,J=9.0Hz, 2H),6.76(dd,J=3.5,1.8Hz,1H),6.67(ddd,J=8.3,2.4,0.9Hz,1H),6.59-6.48(m,2H),3.73(s,3H), 13 C-NMR(75MHz,DMSO-d6):δ(ppm) 160.6,158.9,153.5,150.80,150.75,150.5,136.5,130.4,122.1,121.9,119.6,109.5,108.4,103.7,103.5,98.7,55.2,C 19 H 16 N4O2[M+H] + The calculated ESI value is 333.1346, and the detected value is 333.1342.

[0063] [(18)N4-(4-(4-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-2-amine and 4-(4-trifluoromethylphenoxy)aniline Melting point: 201℃~202℃. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 10.85(s,1H),8.99(s,1H),8.03(d,J=9.0Hz,2H),7.72(d,J=9.0Hz,2H),7.11(dd,J=9.0,0.8Hz, 2H),7.07(d,J=9.0Hz,2H),6.77(dd,J=3.5,2.2Hz,1H),6.55(dd,J=3.5,1.9Hz,1H),5.72(s,2H). 13C-NMR(75MHz, DMSO-d6) δ(ppm) 161.3, 159.4, 153.9, 153.5, 148.5, 138.2, 127.4(q, J = 3.7Hz), 124.4(d, J = 271.1Hz), 122.7(d, J = 32Hz), 121.2, 120.4, 117.9, 117.1, 108.5, 98.8, 96.9. C 19 H 14 F3N5O[M + H] + ESI calculated value 386.1223, detected value 386.1220.

[0064] [(19)N4-(4-(3-(Trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine]

Chemical formula

[0065] [(20)N4-(4-(4-Chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine [Chemical formula] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-2-amine and 4-(4-chlorophenoxy)aniline Melting point: 232 °C - 233 °C 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) 10.83 (s, 1H), 8.94 (s, 1H),​​​​​​​​​​​​​​​​​​​​​​​H-NMR(300MHz,DMSO-d6):δ(ppm) 10.84(s,1H),8.97(s,1H),8.00(d,J=9.0Hz,2H),7.39(t,J=8.1Hz,1H),7.14(ddd,J=8.0,2.0,0.9Hz,1H)7.03(d,J=9.0Hz,2H) 6.99(t,J=2.2Hz,1H),6.94(ddd,J=8.3,2.4,0.9Hz,1H),6.76(dd,J=3.5,2.2Hz,1H),6.54(dd,J=3.5,1.9Hz,1H),5.71(s,2H). 13 C-NMR(75MHz,DMSO-d6) δ(ppm) 159.4,159.1,153.9,153.5,149.1,137.8,133.9,131.3,122.5,121.3,119.9,117.8,117.1,116.0,98.8,96.9. C 18 H 14 ClN5O[M+H] + The calculated ESI value is 352.0960, and the detected value is 352.0955.

[0067] [(22)[N-[4-((4-nitrophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-((4-nitrophenyl)thio)aniline Melting point: 265℃~266℃ 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 12.46(s,1H),10.72(s,1H),8.43(s,1H),8.17(d,J=8.6Hz,2H),8.00(d,J=8.1Hz,2H),7 .67(d,J=8.2Hz,2H),7.44(t,J=2.6Hz,1H),7.33(d,J=8.6Hz,2H),7.03(t,J=2.4Hz,1H). 13C-MNR(75MHz,DMSO-d6) δ 151.7,148.3,147.6,146.6,144.9,139.9,135.7,126.4,124.3,124.1,123.7,123.5,103.7,100.7. C 18 H 13 Theoretical values ​​for N5S2O: %C 59.49, %H 3.61, %N 19.27, %S 8.82. Experimental values: %C 59.26, %H 3.70, %N 19.37, %S 8.95.

[0068] [(23)[N-[4-((4-aminophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4,4'-thiodianiline Melting point: 200℃~201℃. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.73(s,1H),9.30(s,1H),8.24(s,1H),7.80(d,J=8.8Hz,2H),7.23(dd,J=3.5,2.3Hz,1H),7.22(dd,J=3.5 ,2.3Hz,1H),7.14(dd,J=16.7,8.6Hz,4H),6.84(dd,J=3.5,1.9Hz,1H),6.61(d,J=8.5Hz,2H),5.48(s,1H). 13 C-NMR (75MHz, DMSO-d6) δ 153.3,150.8,150.6,149.3,138.5,135.0,131.3,128.2,122.1,120.9,117.1,114.7,103.6,98.7. C 18 H 15 N5S theoretical values: %C 59.49, %H 3.61, %N 19.27, %S 8.82. Experimental values: %C 59.26, %H 3.70, %N 19.37, %S 8.95.

[0069] [(24)[N-[4-(benzyloxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-benzyloxyaniline Melting point: 238°C to 239°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.66(s,1H),9.15(s,1H),8.20(s,1H),7.71(d,J=9.1Hz,2H),7.50-7.29(m,5H),7.18 (dd,J=3.5,2.4Hz,1H),7.00(d,J=9.1Hz,2H),6.67(dd,J=3.5,1.9Hz,1H),5.09(s,2H). 13 C-NMR (75MHz, DMSO-d6): δ(ppm) 153.80,153.76,150.9,150.7,137.3,133.5,128.4,127.74,127.66,122.3,122.0,114.7,103.2,98.7,69.4. C 19 H 16 N4O[M+H] + The calculated ESI value is 317.1397, and the detected value is 317.1395.

[0070] [(25)(4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](phenyl)methanone] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and (4-aminophenyl)phenylmethanone Melting point: 247°C to 248°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.91(s,1H),9.76(s,1H),8.38(s,1H),8.16(d,J=8.9Hz 2H),7.79(d,J=8.8Hz,2H),7.77-7.53(m,5H),7.32(dd,J=3.5,2.3Hz,1H),6.89(dd,J=3.5,1.9Hz,1H). 13C-NMR (75MHz, DMSO-d6): δ(ppm) 194.4,152.8,151.2,150.5,145.1,137.9,132.0,131.1,129.6,129.3,128.4,123.0,118.6,104.4,98.7. C 19 H 14 N4O[M+H] + The calculated ESI value is 315.1240, and the detected value is 315.1238.

[0071] [(26)(4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](4-fluorophenyl)methanone] [ka] Reagents: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and (4-aminophenyl)(4'-fluorophenyl)methanone Melting point: 275°C to 276°C. 1 H-NMR(300MHz,DMSO-d6):δ(ppm) 11.89(s,1H),9.75(s,1H),8.38(s,1H),8.16(d,J=8.9Hz,2H),7.93-7.72(m,4H ),7.39(t,J=8.9Hz,2H),7.32(dd,J=3.5,2.3Hz,1H),6.88(dd,J=3.5,2.3,1H). 13 C-NMR(75MHz,DMSO-d6):δ(ppm) 193.1,164.3(d,J=250.3Hz,152.9,151.2,150.5,145.2,134.5(d,J=3.2Hz),132 .2(d,J=9.2Hz),131.1,129.5,123.0,118.7,115.5(d,J=22.0Hz),104.4,98.8. 19 H 13 FN4O theoretical values: %C 68.67, %H 3.94, %N 16.86. Experimental values: %C 68.32, %H 3.90, %N 16.72.

[0072] [Example 2. Enzyme inhibition of TTBK1 and TTBK2] A buffer containing recombinant human enzyme TTBK1 (1-1321) or TTBK2 (1-450) (5 mU to 20 mU) and 50 mM Tris, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg / ml BSA, and 10 mM DTT at pH 7.5 was tested against the peptide RRKDLHDDEEDEAMSITA (SEQ ID NO: 1) in a final volume of 25.5 μl. The final reaction mixture contained the peptide at a concentration of 0.3 mM, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50 cpm / pmol to 1000 cpm / pmol). The reaction mixture was incubated at room temperature for 30 minutes, and the reaction was stopped after adding 5 μl of orthophosphate. This mixture was placed on a P81 unifilter plate, washed with 50 mM orthophosphate, and the data were read (see Table 1).

[0073] [Example 3. Permeability of the blood-brain barrier] Blood-brain barrier (BBB) ​​cross-section prediction was performed using the PAMPA (Parallel Artificial Membrane Permeability Assay) method (Kansy M, Senner F, Gubernator K. Physicochemical high throughput screening: parallel artificial membrane permeation assay in description of passive absorption processes. J Med Chem. 1998;41(7):1007-1010; Di L, Kerns EH, Fan K, McConnell OJ, Carter GT. High throughput artificial membrane permeability assay for blood-brain barrier. Eur J Med Chem. 2003;38(3):223-232). This technique consists of an artificial system of parallel plates separated by a membrane coated with porcine brain lipids that emulate the BBB. To validate this study, ten commercially available drugs approved by the FDA and known to permeate in humans (enoxacin, hydrocortisone, desipramine, caffeine, ofloxacin, piroxicam, testosterone, promazine, verapamil, and atenolol) were used (Di L, Kerns EH, Fan K, McConnell OJ, Carter GT. High throughput artificial membrane permeability assay for blood-brain barrier. Eur J Med Chem. 2003;38(3):223-232). The test compounds and controls (1 mg to 2 mg) were dissolved in 5 ml of assay buffer (phosphate-buffered saline (PBS, pH 7.4) and ethanol (EtOH) in a 70:30 ratio).Next, these were filtered, and then scanned from 220 nm to 400 nm wavelength using a Varioskan® ultraviolet (UV) reader (Thermo Fisher). As a result, the wavelengths absorbed by each compound were identified, and the spectra of each compound were obtained.

[0074] Once the initial concentration of each compound was determined by the absorbance obtained at a set wavelength (initial absorbance), 180 μl of each sample was added to a 96-well donor plate (the bottom of which was a semipermeable membrane pre-covered with 5 μl of porcine brain lipid dissolved in dodecane (20 mg / ml)). The 96-well acceptor plate was filled with 180 μl / well of assay buffer. Next, the donor plate was placed on top of the acceptor plate to form a "sandwich" at high humidity at room temperature for 2.5 hours. After the incubation period, the donor plate was removed, and the absorbance of the solution in the acceptor plate was read at a pre-set wavelength. The effective transmittance (Ep) of each compound was calculated based on the established correlation between the experimental Ep and the Ep of 10 control drugs as described in the literature, according to the protocol described in the aforementioned literature (Table 1). In this way, the probability of each test compound passing through the BBB is predicted to be high (CNS+), low (CNS-), or uncertain (CNS+ / CNS-) (Table 1). Each sample was analyzed using 3-5 wavelength, triple-chain, and two independent assays. The results are shown as the mean and standard deviation of the two tests.

[0075] [Table 1] JPEG0007893856000032.jpg120169

[0076] [Example 4. Neuroprotective effect of ethacrine] Human neuroblastoma cells (SH-SY5Y) were seeded in 96-well plates at a density of 60,000 cells / well. Once the cells reached confluence, they were pre-treated for 1 hour with a test compound and control dissolved in DMSO at a specified concentration (5 μM), and then exposed to 40 μM ethacrine (EA). Cell viability was evaluated after 24 hours of incubation following EA addition using the MTT assay (Morgan DM. Tetrazolium (MTT) assay for cellular viability and activity. Methods Mol Biol. 1998;79:179-183) (Figure 1).

[0077] [Example 5. Reduction of TDP-43 phosphorylation] The neuroprotective effect of protein expression levels was evaluated using Western blotting (Figure 2). Cells were placed in 6-well plates in a 2 × 10⁶ format. 6 Cells were seeded at a density of cells / well. The experiment was carried out according to the same protocol as detailed in the previous section. After incubation periods in and out of the presence of the compound, the medium was removed, the cells were washed with PBS 1×, and the cells were collected. 30 μl of lysis buffer (50 mM Tris pH 7.4, 1% Nonidet-40, 150 mM NaCl, 10 μl / ml protease and phosphatase inhibitors) was added. After a freeze-thaw cycle at -80°C, the lysis solution was centrifuged at 15,000 rpm for 20 minutes, and the protein fraction remained in the supernatant.

[0078] Protein quantification was performed using a commercially available Pierce® BCA protein quantification kit. Protein separation based on molecular weight was performed by electrophoresis on a 10% polyacrylamide gel, with loading between 50 μg per sample. The samples were transferred to a PVDF (polyvinylidene fluoride) membrane at 4°C. The membranes were blocked for 1 hour with 5% bovine serum albumin or BSA dissolved in TBS-T buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20). The membranes were incubated overnight at 4°C with primary antibodies (Proteintech's p-Ser409 / 410-TDP43 (1:800), TDP-43 (1:1000), and Santa Cruz Biotechnology's GAPDH (1:1000)). Secondary antibodies conjugated to horseradish peroxidase (HRP) were washed three times with TBS-T for 5 minutes each, and then incubated at room temperature for 1 hour. The secondary antibody signal was amplified using an ECL® (Thermo Scientific) chemiluminescence detection system. Bands were quantified using a ChemiDoc (Bio-Rad) kit. GAPDH protein was used as a loading control.

[0079] [Example 6. Neuroprotective effect on okadaic acid] Human neuroblastoma cells (SH-SY5Y) were seeded in 96-well plates at a density of 15,000 cells / well. Once the cells reached confluence, they were pre-treated for 1 hour with a test compound and control dissolved in DMSO at a specified concentration (1 μM), and then exposed to 30 μM okadaic acid (OA). Okadaic acid is a phosphatase inhibitor, and its cytotoxicity is associated with hyperphosphorylation of tau protein (Harris KA, Oyler GA, Doolittle GM, Vincent I, Lehman RA, Kincaid RL, Billingsley ML. Okadaic acid induces hyperphosphorylated forms of tau protein in human brain slices. Ann Neurol. 1993 Jan;33(1):77-87). Neuroprotection by the compound was evaluated using the MTT assay (Morgan DM. Tetrazolium (MTT) assay for cellular viability and activity. Methods Mol Biol. 1998;79:179-183) after 24 hours of incubation following OA addition (Figure 3).

[0080] [Conclusion] TTBK1 inhibitors can cross the blood-brain barrier and reduce tau and TDP-43 phosphorylation in cell models, making them potential candidates for therapeutic agents of central nervous system diseases that cause hyperphosphorylation of tau and TDP-43 proteins. Therefore, the inhibitors described in this patent can be used to treat and / or prevent neurodegenerative conditions, particularly Alzheimer's disease, amyotrophic lateral sclerosis, and frontotemporal dementia, in which half of patients show alterations in tau and the other half show alterations in TDP-43. [Brief explanation of the drawing]

[0081] [Figure 1]Cell viability (MTT) 24 hours after exposure to 40 μM ethacrine (EA) in the SH-SY5Y cell line, in or without the presence of the patented compound (5 μM). [Figure 2] Representative immunoblotting and quantification of p-TDP-43 levels 24 hours after exposure to 40 μM ethacrine (EA) in the SH-SY5Y cell line, in or without the presence of the patented compound (5 μM). [Figure 3] Cell viability (MTT) 24 hours after exposure to 30 nM okadaic acid (OA) in the SH-SY5Y cell line, in or without the presence of the patented compound (1 μM).

Claims

1. A pharmaceutical composition for the treatment and / or prevention of tauopathy and / or TDP-43 disease comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 During the ceremony, R １ H and NH ２ Selected from, X is O, S, CO, and CH ２ Selected from O, R ２ 、 R ３ and R ４ are each independently selected from H, halogen, CF ３ , CN, NO ２ , NH ２ , C １ -C ３ -alkyl, and -O-C １ -C ３ -alkyl, R ２ , R ３ and R ４ At least one of them is H.

2. R ２ , R ３ and R ４ The composition according to claim 1, wherein at least two of are H.

3. R １ The composition according to claim 1, wherein is H.

4. The composition according to claim 1, wherein X is O.

5. R １ H is, X is O, R ２ , R ３ and R ４ These are independently H, halogen, CN, NO ２ NH ２ , C １ -C ３ Alkyl and -O-C １ -C ３ The composition according to claim 1, selected from alkyl groups.

6. R ２ and R ４ H is R ３ However, H, halogen, CN, NO ２ NH ２ , C １ -C ３ Alkyl and -O-C １ -C ３ The composition according to claim 5, selected from alkyl groups.

7. R ２ and R ３ H is R ４ However, halogens and -O-C １ -C ３ The composition according to claim 5, selected from alkyl groups.

8. R ３ and R ４ H is R ２ However, halogen, -(O)-C １ -C ３ Alkyl and C １ -C ３ The composition according to claim 5, selected from alkyl groups.

9. The composition according to claim 1, wherein X is S.

10. R １ H is R ２ , R ３ and R ４ H, NO ２ and NH ２ A composition according to claim 9, selected from the following.

11. The composition according to claim 1, wherein X is CO.

12. R １ H is R ２ , R ３ and R ４ The composition according to claim 11, wherein is independently selected from halogen and H.

13. R １ NH ２ The composition according to claim 1.

14. The composition according to claim 13, wherein X is O.

15. R ２ , R ３ and R ４ H, halogen and CF ３ Selected from, R ２ , R ３ and R ４ The composition according to claim 14, wherein at least one of is different from H.

16. The composition according to claim 1, wherein the compound of formula (I) is selected from the list including the following: N-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-fluorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-cyanophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-bromophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-nitrophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-aminophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2,4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N4-(4-(4-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-chlorophenoxy)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N-[4-((4-nitrophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-((4-aminophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(benzyloxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine (4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](phenyl)methanone (4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](4-fluorophenyl)methanone.

17. The composition according to any one of claims 1 to 16, wherein the tauopathy is selected from the following list: Alzheimer's disease (AD), progressive supranuclear palsy, frontotemporal dementia (FTD), Pick's disease, Down syndrome.

18. The composition according to any one of claims 1 to 16, wherein the TDP-43 disease is selected from the following list: amyotrophic lateral sclerosis (ALS), Alexander disease, limbic-dominant age-related TDP-43 encephalopathy (LATE), frontotemporal dementia (FTD), and Huntington's disease.

19. Any compound of formula (I) or any pharmaceutically acceptable salt thereof, 【Chemistry 2】 In the formula, R １ X, R ２ , R ３ and R ４ This has the same meaning as described in claim 1, and R ２ , R ３ and R ４ A compound or a pharmaceutically acceptable salt thereof, wherein at least one of the elements is H, but the compound is neither of the following two: 【Transformation 3】

20. The compound according to claim 19, wherein the compound is selected from the following list: N-[4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[N-[4-(4-fluorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-cyanophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-bromophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-nitrophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-aminophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(4-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-trifluoromethylphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(2,4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(3-methoxyphenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N4-(4-(4-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-(trifluoromethyl)phenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(4-chlorophenoxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N4-(4-(3-chlorophenoxy)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine N-[4-((4-nitrophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-((4-aminophenyl)thio)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine N-[4-(benzyloxy)phenyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine (4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](phenyl)methanone (4-((7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)phenyl](4-fluorophenyl)methanone.

21. A compound of formula (I) according to claim 19 or 20 or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.

22. A pharmaceutical composition comprising a compound of formula (I) as defined in claim 19 or 20 or a pharmaceutically acceptable salt thereof, and at least one excipient, a pharmaceutically acceptable adjuvant, and / or carrier.

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