1,3,4-Oxadiazoletriazole compounds as histone deacetylase 6 inhibitors and pharmaceutical compositions containing the same

Abstract

The present invention relates to a compound with a novel structure having histone deacetylase 6 (HDAC6) inhibitory activity, its stereoisomers, its pharmaceutically acceptable salts, their uses for manufacturing drugs, pharmaceutical compositions containing them, a method for prevention or treatment, and a method for manufacturing a novel 1,3,4-oxadiazole triazole. The compound with a novel structure having selective HDAC6 inhibitory activity is represented by the following chemical formula I. [Chemical formula I] JPEG2025523127000047.jpg3086

Description

Technical Field

[0001] The present invention relates to a compound having a novel structure with histone deacetylase 6 (HDAC6) inhibitory activity, its stereoisomers, its pharmaceutically acceptable salts, its use in the manufacture of prophylactic or therapeutic agents, a pharmaceutical composition containing the same, a prophylactic or therapeutic method thereof, and a method for producing the same.

Background Art

[0002] Post-translational modifications such as acetylation in cells are very important regulatory modules in biological processes and are strictly controlled by a number of enzymes. Histones are the main proteins that make up chromatin, which serve as the axis around which DNA is wound and assist in the condensation of DNA. Also, the balance between histone acetylation and deacetylation plays a very important role in gene expression. Histone deacetylases (HDACs) are enzymes that remove the acetyl group from the lysine residue of histone proteins that make up chromatin, are associated with gene silencing, and are known to induce cell cycle arrest, suppression of angiogenesis, immune regulation, cell death, etc. (Hassig et al., Curr. Opin. Chem. Biol. 1997, 1, 300-308). In addition, it has been reported that inhibition of the enzymatic function of HDAC induces the death of cancer cells by reducing the activity of factors related to the survival of cancer cells in vivo and activating factors related to the death of cancer cells (Warrell et al., J. Natl. Cancer Inst. 1998, 90, 1621-1625). In humans, 18 HDACs are known and are classified into four classes based on their homology to yeast HDACs. Of these, 11 HDACs that use zinc as a cofactor are divided into three groups: Class I (HDAC1,2,3,8), Class II (IIa:HDAC4,5,7,9, IIb:HDAC6,10), and Class IV (HDAC11). Furthermore, seven HDACs in Class III (SIRT1-7) use NAD+ as a cofactor instead of zinc (Bolden et al., Nat. Rev. Drug Discov. 2006, 5(9), 769-784).

[0003] While various HDAC inhibitors are in preclinical or clinical development stages, only non-selective HDAC inhibitors have been known as anticancer drugs to date. Vorinostat (SAHA) and romidepsin (FK228) are approved for the treatment of cutaneous T-cell lymphoma, and panobinostat (LBH-589) is approved for the treatment of multiple myeloma. However, non-selective HDAC inhibitors are generally known to cause side effects such as fatigue and nausea at high doses (Piekarz et al., Pharmaceuticals 2010, 3, 2751-2767). These side effects have been reported to be caused by the suppression of Class I HDACs, and due to these side effects, the development of non-selective HDAC inhibitors in other areas of anticancer drugs has been limited (Witt et al., Cancer Letters 277, (2009), 8-21). On the other hand, there are reports that selective Class II HDAC inhibition does not exhibit the toxicity seen with Class I HDAC inhibition. Therefore, developing selective HDAC inhibitors can resolve the side effects such as toxicity caused by non-selective HDAC inhibition, and selective HDAC inhibitors have the potential to be developed as effective treatments for various diseases (Matthias et al., Mol.Cell.Biol.2008,28,1688-1701). HDAC6, a Class IIb HDAC, is primarily found in the cytoplasm and is known to be involved in the deacetylation of numerous non-histone substrates (such as HSP90 and cortactin), including tubulin proteins (Yao et al., Mol. Cell 2005, 18, 601-607). HDAC6 has two catalytic domains, and its C-terminal zinc finger domain can bind to ubiquitinated proteins. HDAC6 has numerous non-histone proteins as substrates and is known to play an important role in various diseases, including cancer, inflammatory diseases, autoimmune diseases, neurological diseases, and neurodegenerative disorders (Santo et al., Blood 2012 119, 2579-2589; Vishwakarma et al., International Immunopharmacology 2013, 16, 72-78; Hu et al., J.Neurol.Sci.2011, 304, 1-8).

[0004] A common structural feature of various HDAC inhibitors is that they consist of a cap group, a linker group, and a zinc binding group (ZBG), as shown in the structure of vorinostat below. Many researchers have studied the inhibitory activity and selectivity of enzymes through structural modifications of the cap group and linker group. Among these, the zinc binding group is known to play a more important role in enzyme inhibitory activity and selectivity (Wiest et al., J.Org.Chem.2013 78:5051-5055; Methot et al., Bioorg.Med.Chem.Lett.2008,18,973-978). [ka] Most of the zinc-binding group consists of hydroxamic acid or benzamide. Of these, hydroxamic acid derivatives exhibit potent HDAC inhibitory effects but suffer from low bioavailability and serious off-target activity. In the case of benzamide, because it contains aniline, there is a risk of producing toxic metabolites in the body (Woster et al., Med. Chem. Commun. 2015, online publication). Therefore, there is a need for the development of selective HDAC6 inhibitors with zinc-binding groups that have no side effects and improved bioavailability, unlike non-selective inhibitors that have side effects, for the treatment of cancer, inflammatory diseases, autoimmune diseases, neurological diseases, and neurodegenerative disorders. [Prior art documents] [Patent Documents]

[0005] International Patent Publication No. WO2011 / 091213 (Published July 28, 2011): ACY-1215 International Published Patent Publication WO2011 / 011186 (Published January 27, 2011): Tubastatin International Published Patent Publication WO2013 / 052110 (Published April 11, 2013): Sloan-K International Patent Publication No. WO2013 / 041407 (Published March 28, 2013): Cellzome International Published Patent Publication WO2013 / 134467 (Published September 12, 2013): Kozi International Patent Publication No. WO2013 / 008162 (Published January 17, 2013): Novartis International Patent Publication No. WO2013 / 080120 (Published June 6, 2013): Novartis International Published Patent Publication WO2013 / 066835 (Published May 10, 2013): Tempero International Published Patent Publication WO2013 / 066838 (Published May 10, 2013): Tempero International Patent Publication No. WO2013 / 066833 (Published May 10, 2013): Tempero International Published Patent Publication WO2013 / 066839 (Published May 10, 2013): Tempero [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide compounds having selective HDAC6 inhibitory activity, stereoisomers thereof, or pharmaceutically acceptable salts thereof. Another object of the present invention is to provide a pharmaceutical composition comprising a compound having selective HDAC6 inhibitory activity, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. Another object of the present invention is to provide a method for manufacturing the same. Another object of the present invention is to provide pharmaceutical compositions for the prevention or treatment of HDAC6-mediated diseases. Another object of the present invention is to provide its use for the manufacture of agents for the prevention or treatment of HDAC6-mediated diseases. Another object of the present invention is to provide a method for preventing or treating HDAC6-mediated diseases, comprising administering a therapeutically effective amount of the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof. Another object of the present invention is to provide its use for prevention or treatment of HDAC6-mediated diseases. [Means for solving the problem]

[0007] The present inventors have discovered an oxadiazole compound having histone deacetylase 6 (HDAC6) inhibitory activity, and have completed the present invention by using this compound for the prevention or treatment of HDAC6-mediated diseases. This will be explained in detail below. All combinations of the various elements disclosed in this invention fall within the scope of the invention. Furthermore, the scope of the invention is not limited by the details of the following description.

[0008] Compound represented by compound I (1) The present invention provides a 1,3,4-oxadiazoletriazole compound represented by the following chemical formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof. [Chemical formula I] [ka] In the aforementioned chemical formula I, X1, X2, X3, and X4 are each independently either CH or N, and at least one of X1 to X4 is N. R1 is CF2H, L is a C1-C2 alkylene, R2 is H or C1-C5 alkyl. A is a C6-C12 aryl or a 5-6 member heteroaryl, where at least one of the H atoms in the C6-C12 aryl is substituted with a halogen. R3 is -NR4R5 or [Chemical formula] and R4 and R5 are each independently H or C1-C6 alkyl, R6 and R7 are each independently H, halogen, C1-C6 alkyl, or C1-C6 haloalkyl, n and m are each independently 1 or 2.

[0009] In the present invention, the halogen may be F, Cl, Br, or I. In the present invention, Cx-Cy (where x and y are each an integer of 1 or more) may indicate the carbon number range contained in the substituent. In the present invention, alkylene means a divalent functional group derived from a straight-chain or branched-chain saturated hydrocarbon. For example, C1 alkylene may be methylene. In the present invention, aryl means a monocyclic aromatic or polycyclic aromatic functional group consisting only of carbon and hydrogen. For example, aryl may include phenyl, naphthyl, and the like. In the present invention, heteroaryl means a monocyclic or polycyclic heterocyclic ring in which at least one or more carbons are substituted with heteroatoms. Examples of heteroatoms include nitrogen (N), oxygen (O), sulfur (S), and the like. When heteroaryl contains two or more heteroatoms, the two or more heteroatoms may be the same or different. For example, heteroaryl may include thiophenyl, pyridinyl, or thiazolyl. In the present invention, haloalkyl means a functional group in which at least one of the Hs of alkyl, which is a monovalent functional group derived from a straight-chain or branched-chain saturated hydrocarbon, is substituted with a halogen. For example, haloalkyl may include -CF3, -CH2-CF3, -CHF-CH3, -CF2H, -CFH2, and the like. In the present invention [Chemical formula] This indicates the connecting parts.

[0010] (2) In (1) above, X1, X3, and X4 of chemical formula I may each be CH and X2 may be N. (3) In (1) or (2) above, A in chemical formula I may be a phenyl in which one hydrogen is substituted with a halogen, or a 5-6 member heteroaryl containing at least one heteroatom selected from N and S. (4) In any of (1) to (3) above, the 5-6 member heteroaryl may include thiophenyl, pyridinyl, or thiazolyl. (5) In any of the above (1) to (4), the 1,3,4-oxadiazoletriazole compound according to the present invention can be provided, in this case, In the aforementioned chemical formula I, X1, X3, and X4 are each CH, and X2 is N. L is a C1 alkylene, R1, R2, A, and R3 are defined in the same way as in the definition of chemical formula I above.

[0011] (6) In the above (1), the 1,3,4-oxadiazoletriazole compound according to the present invention can be provided, at this time, The X1-X4, R1, L, and R2 in the aforementioned chemical formula I are the same as in the definition of the aforementioned chemical formula I. A is a C6 aryl atom, where at least one of the H atoms in the C6 aryl atom is replaced by a halogen. R3 is -NR4R5 or [ka] And, R4 and R5 are each independently C1-C6 alkyl groups. R6 and R7 are independently H or C1-C6 alkyl. n and m are independently either 1 or 2. (7) In the above (1) or (2), a 1,3,4-oxadiazoletriazole compound according to the present invention can be provided, in this case, X1 to X of the aforementioned chemical formula I 4, R1, L, and R2 are defined similarly to those in the definition of chemical formula I, A is a 6-membered heteroaryl compound. R3 is [ka] And, R6 and R7 are independently H or C1-C6 alkyl. n and m are independently either 1 or 2. (8) In (1) or (2) above, the 1,3,4-oxadiazoletriazole compound according to the present invention can be provided, in this case, The X1-X4, R1, L, and R2 in the aforementioned chemical formula I are the same as in the definition of the aforementioned chemical formula I. A is a 5-membered heteroaryl compound. R3 is -NR4R5 or [ka] And, R4 and R5 are each independently C1-C6 alkyl groups. R6 and R7 are independently H, halogen, C1-C6 alkyl, or C1-C6 haloalkyl. n and m are independently either 1 or 2.

[0012] In this invention, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, such as inorganic ion salts made from calcium, potassium, sodium, and magnesium; inorganic salts made from hydrochloric acid, nitric acid, phosphoric acid, bromate, iodate, perchloric acid, or sulfuric acid; acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, and asparagus. Examples include organic salts produced from nic acid, ascorbic acid, carboxylic acid, vanillic acid, hydroiodic acid, etc., sulfonates produced from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or naphthalenesulfonic acid, amino acid salts produced from glycine, arginine, lysine, etc., and amine salts produced from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts referred to in this invention are not limited by these salts. In the present invention, preferred salts include hydrochloric acid, trifluoroacetic acid, citric acid, bromate, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid. As an example, the pharmaceutically acceptable salt of the present invention may be a salt of compound 1 of this specification. The 1,3,4-oxadiazoletriazole compounds of the present invention may contain one or more chiral carbons, thereby allowing them to exist as racemates, racemic mixtures, single enantiomers, mixtures of partial stereoisomers, and their respective partial stereoisomers. These isomers of the compound represented by chemical formula I can be separated by conventional techniques, such as column chromatography or HPLC. Alternatively, each stereoisomer of the compound represented by chemical formula I can be synthesized stereospecifically using optically pure starting materials and / or reagents of known sequences. In the present invention, "stereoisomer" includes diastereomers and optical isomers, and optical isomers include not only enantiomers but also mixtures of enantiomers and racemates.

[0013] (9) The 1,3,4-oxadiazoletriazole compound according to the present invention may be any one selected from the compounds shown in Table 1 below. [Table 1-1] [Table 1-2]

[0014] Method for producing compounds of chemical formula I A preferred method for producing a 1,3,4-oxadiazoletriazole compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to the present invention is provided for Reaction Schemes 1 and 2, and also includes methods of production that are obvious to those skilled in the art. In the following reaction equations 1 and 2, those symbols used in chemical formula I, and not specifically explained, may be equivalent to the definition of chemical formula I, and therefore redundant explanations have been omitted. [Reaction Equation 1] [ka] According to the above reaction equation 1, compound 1-2 is synthesized by a reaction in which the halide portion of compound 1-1 is replaced with an azide. In the above reaction equation 1, X represents a halide. Compounds 1-2 can be used in the synthesis of all compounds having a triazole skeleton. [Reaction Equation 2] [ka] In the above reaction formula 2, each compound of reaction formula 2 [ka] Ring A, represented by , is a C6-C12 aryl (where at least one H is substituted with a halogen) or a 5- to 6-membered heteroaryl, in which case R3 is -NR4R5 (where R4 and R5 are independently H or C1-C5 alkyl) or [ka] (R6 and R7 may each be independently H, halogen, C1-C6 alkyl, or C1-C6 haloalkyl, and n and m may each independently represent 1 or 2.)

[0015] According to the above reaction formula 2, compound 2-3 having a trimethylsilane protecting group can be produced through CC coupling (Sonogashira coupling) between halide compound 2-1 and compound 2-2 having a triple bond, and then compound 2-4 having an aldehyde structure can be produced by removing the trimethylsilane protecting group. Compound 2-5, which has a triazole structure, can be produced by a click reaction between compound 2-4 and compound 1-2, and compound 2-6 can be produced by a reductive amination reaction. The 1,3,4-oxadiazoletriazole compound according to the present invention can be produced by the reaction formulas 1 and 2 described above.

[0016] Histone deacetylase 6-mediated diseases include cancer, inflammatory diseases, autoimmune diseases, neurological or degenerative neurological diseases, specifically lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, brain cancer, ovarian cancer, stomach cancer, skin cancer, pancreatic cancer, glioblastoma, glioblastoma carcinoma, leukemia, lymphoma, multiple myeloma, solid tumors, Wilson's disease, spinocerebellar ataxia, prion diseases, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, amyloidosis, Alzheimer's disease, alcoholic liver disease, spinal muscular atrophy, rheumatoid arthritis, or osteoarthritis, and other conditions or diseases associated with abnormal histone deacetylase function. Examples of histone deacetylase-mediated diseases include infectious diseases, neoplasms, endocrine, nutritional and metabolic disorders, mental and behavioral disorders, neurological disorders, ocular and adnexal disorders, cardiovascular diseases, respiratory diseases, digestive diseases, skin and subcutaneous tissue disorders, musculoskeletal and connective tissue disorders, or congenital malformations, deformities and chromosomal abnormalities. The endocrine, nutritional, and metabolic disorders are Wilson's disease, amyloidosis, or diabetes mellitus; the mental and behavioral disorders are depression or Rett syndrome; the neurological disorders are central nervous system atrophy, neurodegenerative diseases, motor disorders, neuropathy, motor neuron disorders, or demyelinating diseases of the central nervous system; the eye and adnexal disorders are uveitis; the skin and subcutaneous tissue disorders are psoriasis; the musculoskeletal and connective tissue disorders are rheumatoid arthritis, osteoarthritis, or systemic lupus erythematosus; the congenital malformations, deformities, and chromosomal abnormalities are autosomal dominant polycystic kidney disease; the infectious diseases are prion diseases; the neoplasms are benign or malignant tumors; the cardiovascular diseases are atrial fibrillation or stroke; the respiratory diseases are asthma; and the digestive diseases may be alcoholic liver disease, inflammatory bowel disease, Crohn's disease, or ulcerative bowel disease. The pharmaceutically acceptable salts are as described above in relation to the pharmaceutically acceptable salts of the 1,3,4-oxadiazoletriazole compounds according to the present invention.

[0017] The pharmaceutical composition of the present invention may further contain, in addition to a 1,3,4-oxadiazoletriazole compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers for administration. In this case, the pharmaceutically acceptable carrier can be a mixture of physiological saline, sterile water, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added, and the composition can be formulated into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Therefore, the composition of the present invention may be a patch, a liquid, a pill, a capsule, a granule, a tablet, or the like. These formulations can be manufactured by conventional methods used in the industry for formulation, or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and may be formulated as various formulations depending on the disease or component. The compositions of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage ranges depending on the patient's weight, age, sex, health condition, diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the compound represented by chemical formula I of the present invention is about 1 to 1000 mg / kg, preferably about 5 to 100 mg / kg, and can be administered in one to several divided doses per day. The pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar pharmacological effects as the compound represented by the aforementioned chemical formula I, or a 1,3,4-oxadiazoletriazole compound containing the compounds listed in Table 1, its stereoisomer, or a pharmaceutically acceptable salt thereof. The present invention provides a method for preventing or treating histone deacetylase 6-mediated diseases, comprising administering a therapeutically effective amount of a 1,3,4-oxadiazoletriazole compound containing the compound represented by the aforementioned chemical formula I, or the compounds listed in Table 1, its stereoisomer, or a pharmaceutically acceptable salt thereof. As used in this invention, the term "therapeutably effective amount" can refer to the amount of the compound represented by chemical formula I described above, or a 1,3,4-oxadiazoletriazole compound including the compounds listed in Table 1, that is effective in preventing or treating histone deacetylase 6-mediated diseases.

[0018] The present invention provides a method for selectively inhibiting HDAC6 by administering a 1,3,4-oxadiazoletriazole compound containing the compound represented by the aforementioned chemical formula I, or the compounds listed in Table 1, its stereoisomer, or a pharmaceutically acceptable salt thereof, to a mammal, including a human. The present invention provides a method for the prevention or treatment of histone deacetylase 6-mediated diseases, which may include not only treating the disease itself before the onset of symptoms, but also inhibiting or avoiding its symptoms, by administering 1,3,4-oxadiazoletriazole compounds, including the compound represented by the aforementioned chemical formula I, or the compounds listed in Table 1. In the management of the disease, the prophylactic or therapeutic dose of a particular active ingredient varies depending on the nature and severity of the disease or condition, and the route through which the active ingredient is administered. The dose and frequency of administration vary depending on the age, weight, and response of the individual patient. A suitable dose and administration method can be easily selected by a person with ordinary knowledge of the art, taking such factors into consideration. Furthermore, the method for preventing or treating histone deacetylase 6-mediated diseases of the present invention may further include administering a therapeutically effective amount of a further active agent useful for treating the disease, along with the compound represented by chemical formula I, or a 1,3,4-oxadiazole triazole compound containing the compounds listed in Table 1, and the further active agent may exert a synergistic or adjunctive effect together with the compound of chemical formula I.

[0019] The present invention also aims to provide uses for 1,3,4-oxadiazoletriazole compounds, stereoisomers thereof, or pharmaceutically acceptable salts thereof, including the compound represented by the aforementioned chemical formula I, or the compounds listed in Table 1, in the manufacture of agents for the prevention or treatment of histone deacetylase 6-mediated diseases. 1,3,4-oxadiazoletriazole compounds, including the compound represented by the aforementioned chemical formula I, or the compounds listed in Table 1, for the manufacture of agents can be mixed with acceptable adjuvants, diluents, carriers, etc., and manufactured as complex formulations with other active agents, which can have synergistic effects of the active ingredients. The uses, compositions, and therapeutic methods mentioned in this invention can be applied similarly, provided they do not contradict each other. [Effects of the Invention]

[0020] The 1,3,4-oxadiazoletriazole compounds of the present invention, their stereoisomers, or pharmaceutically acceptable salts thereof can selectively inhibit HDAC6 and are highly effective in preventing or treating diseases associated with histone deacetylase 6 activity. [Modes for carrying out the invention]

[0021] Example 1: Synthesis of Compound 1, 2-(difluoromethyl)-5-(6-((4-(5-((4-methylpiperidine-1-yl)methyl)thiophen-2-yl)-1H-1,2,3-triazole-1-yl)methyl)pyridine-3-yl)-1,3,4-oxadiazole [Step 1] Synthesis of 2-(6-(azidomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole [ka] 2-(6-(bromomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (1,000 g, 3,447 mmol) was dissolved in N,N-dimethylformamide (10 mL) at room temperature. Sodium azide (0.224 g, 3,447 mmol) was added to the solution, and the mixture was stirred at 40°C for 2 hours. The temperature was then lowered to room temperature to terminate the reaction. Water was added to the reaction mixture, and it was extracted with dichloromethane. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, and the mixture was filtered and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 24 g cartridge; ethyl acetate / hexane = 0%~50%) to obtain 2-(6-(azidomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.800 g, 92.0%) as a yellow solid.

[0022] [Step 2] Synthesis of 5-((trimethylsilyl)ethinyl)thiophene-2-carbaldehyde [ka] 5-bromothiophene-2-carbaldehyde (0.622 mL, 5.210 mmol), bis(triphenylphosphine)palladium dichloride (0.073 g, 0.104 mmol), copper iodide (I / II, 0.010 g, 0.052 mmol), and diethylamine (10.778 mL, 104.199 mmol) were dissolved in tetrahydrofuran. Trimethylsilylacetylene (0.810 mL, 5.731 mmol) was added at 0°C and the mixture was stirred at the same temperature for 0.5 hours, followed by further stirring at room temperature for 18 hours. The reaction mixture was subjected to reduced pressure to remove the solvent, and the resulting concentrate was added with water and extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, water was removed with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12g cartridge; dichloromethane / hexane = 0%~50%) to obtain 5-((trimethylsilyl)ethynyl)thiophene-2-carbaldehyde (0.600g, 55.3%) as a brown solid.

[0023] [Step 3] Synthesis of 5-ethinylthiophene-2-carbaldehyde [ka] The solution of 5-((trimethylsilyl)ethynyl)thiophene-2-carbaldehyde (0.550 g, 2.640 mmol) and potassium carbonate (1.094 g, 7.919 mmol) prepared in Step 2 was dissolved in methanol (5 mL) at room temperature and stirred at the same temperature for 18 hours. Water was added to the reaction mixture and extracted with dichloromethane. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, and the mixture was filtered and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane = 0%~20%) to obtain 5-ethynylthiophene-2-carbaldehyde (0.300 g, 83.5%) as a pale yellow solid.

[0024] [Step 4] Synthesis of 5-(1-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-1H-1,2,3-triazole-4-yl)thiophene-2-carbaldehyde [ka] 5-ethynylthiophene-2-carbaldehyde (0.250 g, 1.836 mmol) prepared in Step 3 and 2-(6-(azidomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.463 g, 1.836 mmol) prepared in Step 1 were dissolved in tert-butanol (5 mL) / water (5 mL) at room temperature. Sodium ascorbate (1.00 M solution, 0.184 mL, 0.184 mmol) and copper sulfate (I / II, 0.50 M solution, 0.184 mL, 0.092 mmol) were added to the solution, and the mixture was stirred at the same temperature for 18 hours. Saturated ammonium chloride aqueous solution was poured over the reaction mixture and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12g cartridge; ethyl acetate / hexane = 0%~70%) to obtain 5-(1-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-1H-1,2,3-triazole-4-yl)thiophene-2-carbaldehyde (0.300g, 42.1%) as a pale yellow solid.

[0025] [Step 5] Synthesis of Compound 1 [ka] 5-(1-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-1H-1,2,3-triazole-4-yl)thiophene-2-carbaldehyde (0.040 g, 0.103 mmol) and 4-methylpiperidine (0.020 g, 0.206 mmol), prepared in Step 4, were dissolved in dichloromethane (1 mL) at room temperature. Sodium triacetoxyborohydride (0.109 g, 0.515 mmol) was added to the solution and the mixture was stirred at the same temperature for 18 hours. Saturated sodium bicarbonate aqueous solution was poured over the reaction mixture and extracted with dichloromethane. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12g cartridge; dichloromethane / methanol = 100%~20%) to obtain 2-(difluoromethyl)-5-(6-((4-(5-((4-methylpiperidine-1-yl)methyl)thiophen-2-yl)-1H-1,2,3-triazole-1-yl)methyl)pyridine-3-yl)-1,3,4-oxadiazole (0.032g, 65.9%) as a white solid.

[0026] 1 H NMR(400MHz,CD3OD)δ9.27(d,J=1.6Hz,1H),8.53(dd,J=8.2,2.2Hz,1H),8.46(s,1H),7.62(d,J=8.4Hz,1H),7.40(d,J=3.6Hz,1H),7.26(t,J=51.4Hz) ,1H),7.20(d,J=3.2Hz,1H),6.71(s,2H),5.91(s,2H),4.27(s,2H),2.70(t ,J=12.6Hz,2H),1.86(d,J=12.8Hz,2H),1.62(s,1H);LRMS(ES)m / z472.3(M + +1)

[0027] Examples 7-16 Compounds 7 to 16 were synthesized using substantially the same steps as the method for producing compound 1 according to Example 1, except that in step 5, the reaction product shown in Table 2 below was used instead of 4-methylpiperidine. [Table 2]

[0028] Example 2: Synthesis of Compound 2, 2-(6-((4-(5-(azetidine-1-ylmethyl)pyridine-2-yl)-1H-1,2,3-triazole-1-yl)methyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole [Step 1] Synthesis of 6-((trimethylsilyl)ethynyl)nicotinaldehyde [ka] 6-bromonicotinaldehyde (1,000 g, 5.376 mmol), bis(triphenylphosphine)palladium dichloride (0.151 g, 0.215 mmol), copper iodide (I / II, 0.102 g, 0.538 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (Xantphos, 0.124 g, 0.215 mmol) were dissolved in triethylamine (15 mL), and trimethylsilylacetylene (0.836 mL, 5.914 mmol) was added at room temperature. The mixture was stirred at the same temperature for 18 hours. The reaction mixture was filtered through a Celite pad to remove the solid, and the filtrate was removed under reduced pressure to remove the solvent. The concentrate was then subjected to column chromatography (SiO₂). 2, The solution was purified and concentrated in a 24g cartridge with ethyl acetate / hexane (0%-50%) to obtain 6-((trimethylsilyl)ethynyl)nicotinaldehyde (0.400g, 36.6%) as a light brown solid.

[0029] [Step 2] Synthesis of 6-ethynylnicotinaldehyde [ka] The 6-(trimethylsilyl)ethynyl)nicotinaldehyde (0.370 g, 1.820 mmol) prepared in Step 1 and potassium carbonate (0.755 g, 5.459 mmol) were dissolved in methanol (5 mL) at room temperature and the solution was stirred at the same temperature for 18 hours. Water was added to the reaction mixture and extracted with dichloromethane. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, and the mixture was filtered and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane = 0%~40%) to obtain 6-ethynylnicotinaldehyde (0.200 g, 83.8%) as a beige solid.

[0030] [Step 3] Synthesis of 6-(1-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-1H-1,2,3-triazole-4-yl)nicotinaldehyde [ka] 6-ethynylnicotinaldehyde (0.100 g, 0.763 mmol) prepared in Step 2 and 2-(6-(azidomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.192 g, 0.763 mmol) prepared in Step 1 of Example 1 were dissolved in tert-butanol (2 mL) / water (2 mL) at room temperature. Sodium ascorbate (1.00 M solution, 0.076 mL, 0.076 mmol) and copper sulfate (I / II, 1.00 M solution, 0.038 mL, 0.038 mmol) were added to the solution, and the mixture was stirred at the same temperature for 18 hours. Saturated ammonium chloride aqueous solution was poured over the reaction mixture and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12g cartridge; ethyl acetate / hexane = 0%~50%) to obtain 6-(1-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-1H-1,2,3-triazole-4-yl)nicotinaldehyde (0.180g, 61.6%) as a pale yellow solid.

[0031] [Step 4] Synthesis of Compound 2 [ka] In Step 3, 6-(1-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-1H-1,2,3-triazole-4-yl)nicotinaldehyde (0.040 g, 0.104 mmol) and azetidine hydrochloride (0.020 g, 0.209 mmol) prepared in Step 3 were dissolved in dichloromethane (1 mL) at room temperature. Sodium triacetoxyborohydride (0.111 g, 0.522 mmol) was added to this solution and the mixture was stirred at the same temperature for 18 hours. Saturated sodium bicarbonate aqueous solution was poured over the reaction mixture and extracted with dichloromethane. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was analyzed by column chromatography (SiO₂).2, The solution was purified and concentrated using a 4g cartridge of dichloromethane / methanol (100%-80%) to obtain 2-(6-((4-(5-(azetidine-1-ylmethyl)pyridine-2-yl)-1H-1,2,3-triazole-1-yl)methyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.021g, 47.4%) as a white solid.

[0032] Examples 3-6 and 17 Compounds 3-6 and 17 were synthesized according to Examples 3-6 and 17, respectively, by following substantially the same steps as the method for producing compound 2 according to Example 2, except that in step 4, the reaction product shown in Table 3 below was used instead of azetidine. [Table 3]

[0033] Examples 18-39, 41 and 42 In the method for producing compound 2 according to Example 2, in step 3, instead of 6-ethynylnicotinaldehyde, reactant 1 from Table 4 and 2-(6-(azidomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole were reacted with the product obtained from this reaction and reactant 2 from Table 4 below, following substantially the same procedure as in step 4 of Example 2, to produce compounds 18-39, 41, and 42 according to Examples 18-39, 41, and 42, respectively. [Table 4-1] [Table 4-2]

[0034] Example 40: Synthesis of compound 40, 2-(difluoromethyl)-5-(6((4-(2-(piperidine-1-ylmethyl)thiazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)pyridine-3-yl)-1,3,4-oxadiazole In the method for producing compound 2 according to Example 2, in step 3, 4-ethynylthiazole-2-carbaldehyde was reacted with 2-(6-(azidomethyl)pyridine-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole instead of 6-ethynylnicotinaldehyde to produce the product obtained by reacting this product with piperidine in substantially the same manner as in step 4 of Example 2 to produce compound 40 of Example 40 (yield 61%).

[0035] The compounds 2 to 42 obtained as final products in Examples 2 to 42 and their analytical data are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10]

[0036] Activity measurement and analysis protocol for the compound of the present invention Experimental Example 1. Search for HDAC enzyme activity inhibition (in vitro) Experiments were conducted to confirm the selectivity of the 1,3,4-oxadiazoletriazole compound of the present invention for HDAC6 by inhibiting HDAC1 and HDAC6 enzyme activity. HDAC enzyme activity was measured using the HDAC Fluorimetric Drug Discovery Kit (BML-AK511, 516) from Enzo Life Science. For the HDAC1 enzyme activity test, human recombinant HDAC1 (BML-SE456) was used as the enzyme source, and Fluor de Lys®-"SIRT1" (BNL-KI177) was used as the substrate. After dispensing the compound diluted 5-fold into a 96-well plate, 0.3 μg of enzyme and 10 μM of substrate were added to each well, and the reaction was incubated at 30°C for 60 minutes. Then, Fluor de Lys® Developer II (BML-KI176) was added, and the reaction was incubated for 30 minutes. After the reaction was completed, fluorescence values ​​(Ex360, Em460) were measured using a multi-plate reader (Flexstation 3, Molecular Device). For the HDAC6 enzyme, human recombinant HDAC6 (382180) from Calbiochem was used, and the experiment was conducted using the same protocol as the HDAC1 enzyme activity test. The final results were recorded using the GraphPad Prism 4.0 program for each IC 50 The value was calculated. [Table 6]

[0037] As shown in Table 6 above, the results of activity inhibition tests against HDAC1 and HDAC6 confirmed that the 1,3,4-oxadiazoletriazole compound of the present invention, its stereoisomer, or its pharmaceutically acceptable salt exhibits excellent selective HDAC6 inhibitory activity of approximately 1048 to 3731 times.

[0038] Experimental Example 2. Analysis of the effects of HDAC6-specific inhibitors on mitochondrial axonal transport (in vitro) Experiments were conducted to investigate whether the 1,3,4-oxadiazoletriazole compound of the present invention selectively inhibits HDAC6 activity and increases the acetylation of tubulin, a major substrate of HDAC6, thereby improving the mitochondrial migration rate in neuronal axons, which is reduced by amyloid-beta treatment. Hippocampal neurons from Sprague-Dawley (SD) rat fetuses at 17-18 days (E17-18) were cultured for 7 days in imaging vessels coated with extracellular matrix, and treated with amyloid-beta protein sections at a concentration of 1M. After 24 hours, on day 8 of in-vessel culture, the cells were treated with the compound, and mitochondrial staining was performed by treating with MitoTracker Red CMXRos (Life Technologies, NY, USA) for a final 5 minutes after 3 hours. Axonal transport of stained neuronal mitochondria was measured using a confocal microscope (Leica SP8; Leicamicrosystems, UK), with images taken at 1-second intervals for 1 minute, and the mitochondrial migration speed was measured using the IMARIS analysis program (BITPLANE, Zurich, Switzerland). As a result, the improvement in mitochondrial axonal transport rate exhibited by the 1,3,4-oxadiazoletriazole compound of the present invention, its stereoisomer, or a pharmaceutically acceptable salt thereof was confirmed by setting a range in which mitochondrial transport rate was significantly reduced in the amyloid beta-treated group compared to the vehicle, then normalizing with 100% vehicle and 0% amyloid beta-treated group, and finally indicating the rate distribution of the compound as *, 0%~50%;**, 50%~100%;***, >100%.

[0039] [Table 7]

Claims

1. A 1,3,4-oxadiazoletriazole compound represented by the following chemical formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof: [Chemical formula 1] 【Chemistry 1】 In the aforementioned chemical formula I, X 1 , X 2 , X 3 and X 4 Each is independently CH or N, and X 1 ~X 4 At least one of them is N, R 1 CF 2 It is H, L is a C1-C2 alkylene, R 2 is H or C1-C5 alkyl, A is a five-membered heteroaryl compound. R 3 Ha-NR 4 R 5 or 【Chemistry 2】 And, R 4 and R 5 Each of them is independently a C1-C6 alkyl group, R 6 and R 7 Each of these is independently H, halogen, C1-C6 alkyl, or C1-C6 haloalkyl. n and m are independently either 1 or 2.

2. X of the aforementioned chemical formula I 1 , X 3 and X 4 Each of them is CH, and X 2 is N, L is C1 alkylene, R 1 , R 2 , A and R 3 Each of these is the same as the definition in claim 1. A 1,3,4-oxadiazoletriazole compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

3. A 1,3,4-oxadiazoletriazole compound selected from the group consisting of the compounds shown in the table below, its stereoisomer, or a pharmaceutically acceptable salt thereof: Table 1

4. A 1,3,4-oxadiazoletriazole compound according to any one of claims 1 to 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is provided as an active ingredient. Pharmaceutical compositions for the prevention or treatment of histone deacetylase-mediated diseases.

5. The aforementioned histone deacetylase-mediated diseases are Infectious diseases; neoplasms; endocrine, nutritional and metabolic disorders; mental and behavioral disorders; neurological disorders; eye and adnexal disorders; respiratory diseases; digestive disorders; skin and subcutaneous tissue disorders; musculoskeletal and connective tissue disorders; or congenital malformations, deformities and chromosomal abnormalities. The pharmaceutical composition according to claim 4.

6. The aforementioned endocrine, nutritional, and metabolic disorders include Wilson's disease, amyloidosis, or diabetes mellitus. The aforementioned mental and behavioral disorders are depression or Rett syndrome. The aforementioned neurological disorders include central nervous system atrophy, neurodegenerative diseases, motor disorders, neuropathy, motor neuron disorders, or demyelinating diseases of the central nervous system. The aforementioned eye and ocular adnexal disease is uveitis. The aforementioned skin and subcutaneous tissue disease is psoriasis. The aforementioned musculoskeletal and connective tissue diseases are rheumatoid arthritis, osteoarthritis, or systemic lupus erythematosus. The aforementioned congenital malformations, deformities, and chromosomal abnormalities are autosomal dominant polycystic kidney disease. The aforementioned infectious disease is a prion disease. The aforementioned neoplasm may be a benign or malignant tumor. The aforementioned respiratory disease is asthma. The aforementioned digestive disorders are alcoholic liver disease, inflammatory bowel disease, Crohn's disease, or ulcerative bowel disease. The pharmaceutical composition according to claim 5.

7. Use of a 1,3,4-oxadiazoletriazole compound according to any one of claims 1 to 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of an agent for the prevention or treatment of histone deacetylase-mediated diseases.

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