1,3,4-oxadiazole derivative compounds as histone deacetylase 6 inhibitor, and uses thereof

Abstract

This invention relates to a novel compound having histone deacetylase 6 (HDAC6) inhibitory activity, its preparation method, and its uses. The novel compound of this invention, its stereoisomers, or pharmaceutically acceptable salts thereof possess HDAC6 inhibitory activity and are effective in preventing or treating HDAC6-mediated diseases (including cancer, inflammatory diseases, autoimmune diseases, and neurodegenerative diseases).

Description

[Technical Field]

[0001] This invention relates to a 1,3,4-diazole derivative compound with histone deacetylase 6 (HDAC6) inhibitory activity, its preparation method and its uses. [Previous Technology]

[0002] Post-translational modifications (such as acetylation) in cells are crucial regulatory modules in core biological processes and are strictly controlled by many enzymes. Histones are core proteins that make up chromatin and help DNA condense by acting as the axis around which DNA is surrounded. Furthermore, the balance between histone acetylation and deacetylation plays a key role in gene expression.

[0003] Histone deacetylase (HDAC) is an enzyme that removes the acetyl group from the lysine residues of histone proteins that make up chromatin, and is known to be associated with gene silencing and induction of cell cycle arrest, angiogenesis inhibition, immune regulation, and cell death (Hassig et al., Curr. Opin. Chem. Biol. 1997, 1, 300-308). Furthermore, it has been reported that inhibiting HDAC enzyme function reduces the activity of cancer cell survival-related factors and activates cancer cell death-related factors, thereby inducing cancer cell apoptosis (Warrell et al., J. Natl. Cancer Inst. 1998, 90, 1621-1625).

[0004] In humans, 18 HDAC enzymes are known and are divided into four classes based on their homology with yeast HDAC. Among them, 11 HDACs that use zinc as a cofactor can be divided into three groups: Class I (HDAC1, 2, 3 and 8), Class II (IIa: HDAC4, 5, 7 and 9; IIb: HDAC6 and 10) and Class IV (HDAC11). In addition, the seven Class III HDACs (SIRT 1-7) use NAD+ instead of zinc as a cofactor (Bolden et al., Nat. Rev. Drug. Discov. 2006, 5(9), 769-784).

[0005] Although many HDAC inhibitors are in preclinical or clinical development stages, only non-selective HDAC inhibitors are known to be anticancer agents to date. Vorinostat (SAHA) and romidepsin (FK228) have been approved for the treatment of cutaneous T-cell lymphoma, and panobinostat (LBH-589) has been approved for the treatment of multiple myeloma. However, non-selective HDAC inhibitors are known to produce side effects at high doses, such as fatigue, nausea, and the like (Piekarz et al., Pharmaceuticals 2010, 3, 2751-2767). These side effects have been reported to be caused by inhibition of class I HDACs, and due to these side effects, the development of non-selective HDAC inhibitors in areas other than anticancer drugs is limited (Witt et al., Cancer Letters 277 (2009) 8.21).

[0006] Meanwhile, it has been reported that selective class II HDAC inhibition does not exhibit the toxicity seen in class I HDAC inhibition, and the development of selective HDAC inhibitors can resolve side effects (such as toxicity) caused by non-selective HDAC inhibition. Therefore, selective HDAC inhibitors have the potential to be developed as effective treatments for a variety of diseases (Matthias et al., Mol. Cell. Biol. 2008, 28, 1688-1701).

[0007] HDAC6 (a type of class IIb HDAC) is mainly found in the cytoplasm and is known to contain tubulin proteins, thus participating in the deacetylation of various non-histone receptors (HSP90, cortical actin, and the like) (Yao et al., Mol. Cell 2005, 18, 601-607). HDAC6 may have two catalytic domains, and the C-terminus of the zinc finger domain can bind to ubiquitinated proteins. Because HDAC6 has multiple non-histone proteins as receptors, it is known to play important roles in various diseases, such as cancer, inflammatory diseases, autoimmune diseases, neurological diseases, and neurodegenerative diseases, and the like (Santo et al., Blood 2012 119: 2579-258; Vishwakarma et al., International Immunopharmacology 2013, 16, 72-78; Hu et al., J. Neurol. Sci. 2011, 304, 1-8).

[0008] The common structural features of various HDAC inhibitors consist of a capping group, a linker group, and a zinc-binding group (ZBG), as shown in the structure of vorinostat below. Many researchers have conducted studies on the inhibitory activity and selectivity of enzymes through structural modifications of the capping and linker groups. 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: 5051-5065; Methot et al., Bioorg. Med. Chem. Lett. 2008, 18, 973-978).

[0009] Most of these zinc-binding groups are hydroxamic acids or benzoylamines, among which hydroxamic acid derivatives exhibit strong HDAC inhibition, but have problems such as low bioavailability and severe off-target activity. Benzoylamines contain aniline, and therefore also have the problem of producing toxic metabolites in vivo (Woster et al., Med. Chem. Commun. 2015, online publication).

[0010] Therefore, for the treatment of cancer, inflammatory diseases, autoimmune diseases, neurological diseases and neurodegenerative diseases and the like, there is a need to develop a selective inhibitor HDAC6 that has no side effects and improved bioavailability, while not having the side effects of non-selective inhibitors. [Summary of the Invention]

[0011] [Technical Problem] One objective of the present invention is to provide a 1,3,4-diazole derivative compound having selective histone deacetylase 6 (HDAC6) inhibitory activity, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising a 1,3,4-diazole derivative having selective HDAC6 inhibitory activity, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0013] Another objective of the present invention is to provide a method for its preparation.

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising such compounds for the prevention or treatment of HDAC6-related diseases including: infectious diseases; tumors; endocrine, nutritional and metabolic diseases; mental and behavioral disorders; neurological diseases; eye and ocular adnexa diseases; circulatory system diseases; respiratory diseases; digestive system diseases; skin and subcutaneous tissue diseases; musculoskeletal and connective tissue diseases; or congenital malformations, alterations and chromosomal abnormalities.

[0015] Another objective of the present invention is to provide the use of these compounds in the preparation of medicaments for the prevention or treatment of HDAC6 activity-related diseases.

[0016] Another objective of the present invention is to provide a method for treating HDAC6 activity-related diseases, the method comprising: administering a therapeutically effective amount of a pharmaceutical composition containing the above-mentioned compound.

[0017] [Technical Solution] The inventors have discovered a 1,3,4-diazole-derived compound with histone deacetylase 6 (HDAC6) inhibitory activity and used this 1,3,4-diazole-derived compound to inhibit or treat HDAC6 activity-related diseases, thereby completing this invention. 1,3,4-diazole-derived compound

[0018] In a general embodiment, the present invention provides 1,3,4-diazole derivatives, stereoisomers thereof, or pharmaceutically acceptable salts thereof represented by the following chemical formula I: [Chemical Formula I] In the above chemical formula I, R1 and R2 are each independently -(C1-C4 alkyl), or R1 and R2 are connected together with N atoms to form a heterocyclic alkyl group, wherein at least one H in the heterocyclic alkyl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo or heterocyclic alkyl; X is -H or -F; Y1 to Y5 are each independently N or CR3, wherein Y1 to Y5 cannot simultaneously be 3 or more Ns; R3 is -H, -(C1-C4 alkyl), ... -4-aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, aryl, or heteroaryl, wherein at least one -H in the aryl or heteroaryl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl), or -halo; Z1 to Z4 are each independently N or CR4, wherein Z1 to Z4 cannot simultaneously be 3 or more N; and R4 is -H, -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 ... 4-H-alkyl), -haloyl, aryl or heteroaryl, wherein at least one -H in the aryl or heteroaryl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl) or halogen.

[0019] According to one embodiment of the present invention, the compound represented by Formula I, its stereoisomers, or pharmaceutically acceptable salts thereof may be within the following ranges: R1 and R2 are connected together with N atoms to form a heterocyclic alkyl group, wherein at least one H in the heterocyclic alkyl ring may be independently substituted by -(C1-C4 alkyl) or heterocyclic alkyl; X is -H or -F; Y1 to Y5 are each independently CR3; R3 is -H, -halo or heteroaryl, wherein at least one -H in the heteroaryl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl) or -halo; Z1 to Z4 are each independently N or CR4, wherein Z1 to Z4 cannot simultaneously be 3 or more N atoms; and R 4-H or -halogen group.

[0020] According to one embodiment of the present invention, the compound represented by chemical formula I, its stereoisomers or pharmaceutically acceptable salts thereof may be in the following range: R1 and R2 are connected together with N atoms to form a 4 to 12-membered heterocyclic alkyl group, wherein at least one H in the heterocyclic alkyl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, or 3 to 6-membered heterocyclic alkyl groups.

[0021] According to one embodiment of the present invention, the compound represented by chemical formula I, its stereoisomers or pharmaceutically acceptable salts thereof may be within the following ranges: R1 and R2 are connected together with N atoms to form, or, wherein, or at least one H in the ring may be independently substituted with -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo; W is NR5, O, S or S(=O)2; R5 is -(C1-C4 alkyl) or 3 to 6-membered heterocyclic alkyl; and n1, n2, m1 and m2 are each independently 0, 1 or 2.

[0022] According to one embodiment of the present invention, the compound represented by chemical formula I, its stereoisomers or pharmaceutically acceptable salts thereof may be in the following range: R1 and R2 are connected together with N atoms to form , , , or , wherein at least one H in the , , , or rings may be independently substituted with -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo; and R5 is -(C1-C4 alkyl) or .

[0023] Furthermore, according to one embodiment of the present invention, specific compounds represented by chemical formula I of the present invention are shown in Table 1 below: [Table 1] Compound numbering structure Compound numbering structure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four .

[0024] In this invention, unless otherwise stated, the term "alkyl" as used herein may refer to a straight-chain or branched acyclic, cyclic, or saturated hydrocarbon with carbon atoms linked together. For example, "C1-4 alkyl" may mean an alkyl group containing 1 to 4 carbon atoms. The acyclic alkyl group may include, for example, methyl, ethyl, n-propyl, n-butyl, isopropyl, dibutyl, isobutyl, tributyl, and the like, but is not limited thereto. The term "cyclic alkyl" is used interchangeably with "cycloalkyl" in this specification and may include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, but is not limited thereto.

[0025] In this invention, "alkoxy" may refer to -(O-alkyl) as an alkyl ether group, wherein the alkyl group is the same as defined above. For example, "C1-4 alkoxy" may mean an alkoxy containing a C1-4 alkyl group, i.e., -(OC1-4 alkyl); and examples of alkoxy may include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, dibutoxy, terbutoxy, and the like.

[0026] In this invention, the "halogen group" may be F, Cl, Br or I.

[0027] In this invention, the term "haloalkyl" may mean a straight-chain or branched-chain alkyl (hydrocarbon) having a carbon atom substituted with at least one halogen group as defined herein. Examples of haloalkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, or n-butyl, which are independently substituted with at least one halogen such as F, Cl, Br, or I.

[0028] In this invention, the term "hydroxyalkyl" may refer to a straight-chain or branched-chain alkyl (hydrocarbon) having a carbon atom substituted with -OH. Examples of hydroxyalkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, or n-butyl, which are independently substituted with at least one hydroxyl group.

[0029] In this invention, the term "aminoalkyl" may refer to a straight-chain or branched-chain alkyl (hydrocarbon) having a carbon atom substituted with an amino group (NR'R"). In this document, R' and R" may each be independently selected from the group consisting of hydrogen and C1-4 alkyl groups, and the selected R' and R" may each be independently substituted or unsubstituted.

[0030] In this invention, the term "heterocyclic alkyl" may mean a ring containing 1 to 5 heteroatoms selected from N, O, and S as ring-forming atoms, and may be saturated or partially unsaturated. Hereinafter, when unsaturated, a heterocyclic alkyl may be referred to as a heterocyclic alkene. Unless otherwise stated, a heterocyclic alkyl may be monocyclic or polycyclic, such as a spirocyclic, bridged, or fused ring. Furthermore, "3 to 12-membered heterocyclic alkyl" may mean a heterocyclic alkyl containing 3 to 12 ring-forming atoms. Examples of heterocyclic alkyl groups may include, but are not limited to, pyrrolidine, piperidine, imidazoidine, pyrazolidine, butyrolactam, valproic acid, imidazolidinone, hydantoin, dioxacyclopentane, phthalimide, piperidine, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, piperan, Pyridones, 3-pyrrolidones, thiarans, pyranones, tetrahydrofurans, tetrahydrothiophenes, quinine rings, scopolanes, 2-azaspiro[3.3]heptane, (1R,5S)-3-azabicyclo[3.2.1]octane, (1s,4s)-2-azabicyclo[2.2.2]octane or (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane and the like.

[0031] In this invention, "aromatic hydrocarbon" may refer to an aromatic hydrocarbon ring. Aromatic hydrocarbons may be monocyclic or polycyclic aromatic hydrocarbons. The number of ring carbon atoms in an aromatic hydrocarbon may be 5 or more and 30 or less, 5 or more and 20 or less, or 5 or more and 15 or less. Examples of aromatic hydrocarbons include, but are not limited to, benzene, naphthalene, anthracene, phenanthrene, biphenyl, terphenyl, tetraphenyl, pentphenyl, hexaphenyl, biphenylene oxide, pyrene, benzo[a]fluoranthene, benzo[b]pyrene, and the like. In this specification, the portion obtained by removing one hydrogen atom from the aforementioned "aromatic hydrocarbon" is called an "aryl group".

[0032] In this invention, "heteroaromatic hydrocarbon" may be a ring containing one or more of O, N, P, Si, and S as heterogeneous elements. The number of carbon atoms in the ring of the heteroaromatic hydrocarbon may be 2 or more and 30 or less, or 2 or more and 20 or less. The heteroaromatic hydrocarbon may be a monocyclic heteroaromatic hydrocarbon or a polycyclic heteroaromatic hydrocarbon. Polycyclic heteroaromatic hydrocarbons may have, for example, a bicyclic or tricyclic structure. Examples of heteroaromatic hydrocarbons include, but are not limited to, thiophene, purine, pyrrole, pyrazole, imidazole, thiazole, acetazole, isothiazole, acetidine, bipyridine, triazine, acryloxy, pyridazine, pyrazine, quinoline, quinazolinine, quinazolinine, phenoxazine, pyrazine, pyrimidine, pyridopyrimidine, pyridopyrazine, pyrazinopyrazine, isoquinoline, indole, carbazole, imidazopyridazine, imidazole Azopyridine, imidazopyrimidine, pyrazolopyrimidine, imidazopyrazine or pyrazolopyrimidine, N-arylcarbazole, N-heteroarylcarbazole, N-alkylcarbazole, benzo[a]azole, benzimidazole, benzothiazole, benzo[a]carbazole, benzothiophene, dibenzothiophene, thiophene-thiophene, benzofuran, phenoroline, iso[a]azole, acediazole, thiadiazole, benzothiazole, tetrazolium, phenothiazine, dibenzothiophene, dibenzofuran, and the like. In one embodiment of the invention, the heteroaromatic hydrocarbon may also comprise bicyclic heterocyclic aromatic hydrocarbons, comprising an aromatic ring fused with a heterocyclic alkyl ring or a heteroaromatic hydrocarbon fused with a cycloalkyl ring. In this specification, the portion obtained by removing a hydrogen atom from the above-mentioned "heteroaromatic hydrocarbon" is referred to as "heteroaryl".

[0033] The compounds represented by Formula I of the present invention may contain at least one asymmetric carbon, and therefore may exist as racemic compounds, racemic mixtures, single mirror-image isomers, mixtures of non-mirror-image isomers, and various non-mirror-image isomers. These stereoisomers can be separated using conventional techniques, and for example, the compounds represented by Formula I can be separated by column chromatography, HPLC, or similar methods. Furthermore, the stereoisomers of the compounds represented by Formula I can be stereospecifically synthesized using optically pure starting materials and / or reagents with known configurations.

[0034] In this invention, the term "mirror image isomer" as used herein refers to a compound or its salt having the same chemical formula or molecular formula but different spatial arrangements. Each of these mirror image isomers and mixtures thereof are also included within the scope of this invention. Unless otherwise stated, the solid bonds (-) connected to the asymmetric carbon atom may comprise wedge-shaped solid bonds or wedge-shaped virtual bonds representing the absolute arrangement of the stereocenter.

[0035] The compounds represented by Formula I of the present invention may exist in the form of "medically acceptable salts". As salts, acid addition salts formed from pharmaceutically acceptable free acids are useful. As used herein, the term "medically acceptable salt" refers to a concentration that has a relatively non-toxic and harmless effect on the patient, comprising any organic or inorganic acid addition salt of the compound represented by Formula I, wherein the side effects caused by such salts do not diminish the beneficial efficacy of the compound.

[0036] The preparation of acid addition salts is carried out by conventional methods, for example, by dissolving the compound in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent (such as methanol, ethanol, acetone, or acetonitrile). A moderate molar amount of the compound and the acid or alcohol can be heated in water, and the mixture can then be evaporated to dryness, or the salt can be precipitated by suction filtration.

[0037] In this case, for the free acid, both organic and inorganic acids can be used, wherein the inorganic acids may include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and the like, and the organic acids may include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutamate, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like. However, organic and inorganic acids are not limited to these.

[0038] In addition, pharmaceutically acceptable metal salts can be prepared using alkalis. Alkali metal salts or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the insoluble complex salt, and then evaporating and drying the filtrate. Sodium, potassium, or calcium salts are particularly suitable for pharmaceutical applications, but the metal salts are not limited to these. Furthermore, corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0039] Unless otherwise indicated, pharmaceutically acceptable salts of the present invention comprise salts of acidic or basic groups that may be present in compounds represented by the above-described chemical formula I. For example, pharmaceutically acceptable salts may comprise sodium, calcium, and potassium salts of hydroxyl groups, while other pharmaceutically acceptable salts of amino groups may comprise hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, amygdalinate, methanesulfonate, and p-toluenesulfonate salts, and the like, which can be prepared by salt preparation methods known in this art. Methods for preparing 1,3,4-diazole derivative compounds

[0040] The present invention provides a method for preparing 1,3,4-diazole derivatives, stereoisomers thereof or pharmaceutically acceptable salts thereof represented by the following chemical formula I: [Chemical Formula I] The chemical formula I is as defined above.

[0041] In this invention, a preferred method for preparing a diazole derivative compound represented by chemical formula I, its stereoisomers or pharmaceutically acceptable salts thereof is shown in [Reaction Flow 1] to [Reaction Flow 4], which may also include a preparation method modified to a level obvious to those skilled in the art.

[0042] [Reaction Procedure 1] The above-described [Reaction Procedure 1] is a method for synthesizing 1,3,4-diazole derivatives having a cyclobutenedione structure. First, a compound containing an amino group, represented by Chemical Formula 1-1, is reacted with a compound represented by Chemical Formula 1-2 to prepare a compound containing an amino substituent, represented by Chemical Formula 1-3. Then, a compound represented by Chemical Formula 1-3 is reacted with a compound represented by Chemical Formula 1-4 to prepare a compound represented by Chemical Formula 1-5 having a cyclobutenedione structure containing a diamine substituent. A substitution reaction is carried out between the compound represented by Chemical Formula 1-5 and the compound represented by Chemical Formula 1-6 to prepare a compound represented by Chemical Formula 1-7. Simultaneously, the compound represented by Chemical Formula 1-5 can be prepared by reacting the compound represented by Chemical Formula 1-4 with the compound represented by Chemical Formula 1-2 to prepare a compound represented by Chemical Formula 1-8, which is then reacted with the compound represented by Chemical Formula 1-1. In this invention, compounds 2, 3, 4, 5, 6, 7, 10 and 24 can be prepared by the above reaction process 1.

[0043] [Reaction Procedure 2] The above-described [Reaction Procedure 2] is another method for synthesizing 1,3,4-diazole derivative compounds having a cyclobutenedione structure. First, the compound represented by chemical formula 2-1 is reacted with the compound represented by chemical formula 1-2 to prepare the compound represented by chemical formula 2-2 containing an amine substituent. Then, the compound represented by chemical formula 2-2 is reacted with the compound represented by chemical formula 1-4 to prepare the compound represented by chemical formula 1-7 having a cyclobutenedione structure containing a diamine substituent. In this invention, compound 1 and similar compounds can be prepared through the above-described reaction procedure 2.

[0044] [Reaction Procedure 3] The above-described [Reaction Procedure 3] is another method for synthesizing 1,3,4-diazole derivatives having a cyclobutenedione structure. The compounds represented by chemical formulas 1-7 prepared in [Reaction Procedure 1] and the compound represented by chemical formula 3-1 are subjected to CC coupling (Suzuki reaction) to prepare the compound represented by chemical formula 3-2. In this invention, compounds 8 and 9, and similar compounds, can be prepared through the above-described Reaction Procedure 3.

[0045] [Reaction Procedure 4] The above-described [Reaction Procedure 4] is another synthetic method for synthesizing 1,3,4-diazole derivatives having a cyclobutenedione structure. The compound represented by chemical formula 4-2 is prepared by removing the protecting group from the compound represented by chemical formula 4-1 prepared in [Reaction Procedure 1]. Then, the compound represented by chemical formula 4-3 is prepared by alkylation or reduction reaction. The compound represented by chemical formula 4-3 and the compound represented by chemical formula 3-1 are subjected to CC coupling (Suzuki reaction) to prepare the compound represented by chemical formula 4-4. In this invention, the above-described reaction procedure 4 can be used to prepare the compounds represented by chemical formula 4-3, i.e., compounds 10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 22 and 23 and the like, and to prepare the compounds represented by chemical formula 4-4, i.e., compounds 18 and 19 and the like. Uses of 1,3,4-diazole derivatives

[0046] The present invention provides the use of compounds represented by the following chemical formula I, their stereoisomers or pharmaceutically acceptable salts thereof: [Chemical Formula I] The chemical formula I is as defined above.

[0047] According to one embodiment of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases related to histone deacetylasin 6 activity, comprising, as described above, a compound represented by chemical formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition of the present invention selectively inhibits histone deacetylasin 6, thereby exhibiting significant effects in the prevention or treatment of diseases related to histone deacetylasin 6 activity.

[0048] Diseases associated with histone deacetylase 6 activity include infectious diseases such as prion disease; tumors such as benign tumors (e.g., myelomecosis syndrome) or malignant tumors (e.g., multiple myeloma, lymphoma, leukemia, lung cancer, colorectal cancer, colon cancer, prostate cancer, urothelial carcinoma, breast cancer, melanoma, skin cancer, liver cancer, brain cancer, stomach cancer, ovarian cancer, pancreatic cancer, head and neck cancer, oral cancer, or glioma); endocrine, nutritional, and metabolic diseases such as Wilson's disease, amyloidosis, or diabetes; mental and behavioral disorders such as depression or Rett syndrome; neurological diseases such as central nervous system atrophy (e.g., Huntington's disease, spinal muscular atrophy (SMA), spinocerebellar ataxia (SCA)); and neurodegenerative diseases (e.g., Alzheimer's disease). Diseases including: movement disorders (e.g., Parkinson's disease); neuropathy (e.g., hereditary neuropathy (Charcot-Marie-Tooth disease), sporadic neuropathy, inflammatory neuropathy, drug-induced neuropathy), motor neuropathy (e.g., amyotrophic lateral sclerosis (ALS)); and central nervous system demyelination (e.g., multiple sclerosis (MS)); eye and ocular adnexa diseases, such as uveitis; circulatory system diseases, such as atrial fibrillation or stroke; respiratory system diseases, such as asthma; and digestive system diseases, such as alcoholic liver disease, inflammatory bowel disease, and Crohn's disease. Diseases such as ulcerative colitis and the like; skin and subcutaneous tissue diseases such as psoriasis; musculoskeletal and connective tissue diseases such as rheumatoid arthritis, osteoarthritis or systemic lupus erythematosus (SLE); or congenital malformations, alterations and chromosomal abnormalities such as autosomal dominant polycystic kidney disease, and in addition, symptoms or diseases related to histone deacetase dysfunction.

[0049] Stereoisomers and pharmaceutically acceptable salts as described above in the section on stereoisomers and pharmaceutically acceptable salts of compounds represented by chemical formula I of the present invention.

[0050] For administration, in addition to compounds represented by Formula I, their stereoisomers, or pharmaceutically acceptable salts, the pharmaceutical compositions of the present invention may further comprise at least one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, or mixtures of one or more of these components, and (if desired) may contain other known additives, such as antioxidants, buffers, antibacterial agents, and the like. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added, and they may be formulated into injectable formulations, such as aqueous solutions, suspensions, emulsions, and the like, pills, capsules, granules, or tablets. Therefore, the pharmaceutical compositions of the present invention may be patches, liquids, pills, capsules, granules, tablets, suppositories, and the like. These formulations can be prepared by conventional methods for formulations in this field or by methods disclosed in the documents [see Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA], and can be formulated into various formulations according to different diseases or components.

[0051] The compositions of the present invention can be administered orally or non-enterically (e.g., intravenously, subcutaneously, intraperitoneally, or topically) according to the desired method, and the dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose of the compound represented by chemical formula I of the present invention is about 1 to 1000 mg / kg, preferably 5 to 100 mg / kg, and can be divided into doses and administered once or multiple times daily.

[0052] In addition to compounds represented by chemical formula I, their stereoisomers or pharmaceutically acceptable salts, the pharmaceutical compositions of the present invention may further include at least one active ingredient exhibiting the same or similar efficacy.

[0053] According to one embodiment of the present invention, the present invention provides a method for preventing or treating diseases related to histone deacetylase 6 activity, the method comprising: administering to an individual in need a therapeutically effective amount of a compound represented by chemical formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof. The individual may be a mammal (including humans).

[0054] Methods for preventing or treating diseases related to histone deacetylase 6 activity include not only treating the disease itself before symptom onset, but also inhibiting or preventing its symptoms by administering a compound represented by Formula I, its stereoisomers, or pharmaceutically acceptable salts. Furthermore, the methods of the present invention for preventing or treating diseases related to histone deacetylase 6 activity may further administer a therapeutically effective amount of an additional active agent that helps to treat the disease together with the compound represented by Formula I, wherein the additional active agent may exhibit a synergistic or adjuvant effect with the aforementioned compound represented by Formula I.

[0055] As used herein, the term "therapeutic effective amount" refers to an amount of a compound represented by Formula I that is effective in treating or preventing diseases related to histone deacetylase 6 activity. Specifically, "therapeutic effective amount" means an amount sufficient to treat the disease at a reasonable benefit / risk ratio suitable for drug therapy, and the effective dose can be determined based on factors including individual type and severity, age, sex, disease type, drug activity, drug sensitivity, time of administration, route of administration, excretion rate, duration of treatment, factors of concurrently used drugs, and other factors well known in the medical field. The pharmaceutical compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. Furthermore, the pharmaceutical compositions of the present invention can be administered in single doses or in multiple doses. Considering all the above factors, it is important to administer an amount that achieves maximum effect with minimal quantity and without side effects, which can be easily determined by those skilled in the art. The dosage of the pharmaceutical compositions of the present invention can be determined by experts based on various factors (such as the patient's condition, age, sex, complications, and the like). Because the active ingredient in the pharmaceutical composition of the present invention has excellent safety, the active ingredient can even be used at a higher than predetermined dose.

[0056] According to one embodiment of the present invention, the present invention provides a method for selectively inhibiting histone deacetase 6 (HDAC6) by administering a compound represented by chemical formula I, its stereoisomer or a pharmaceutically acceptable salt thereof to mammals (including humans).

[0057] According to one embodiment of the present invention, the present invention provides the use of a compound represented by chemical formula I, its stereoisomers, or pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating diseases related to histone deacetylase 6 activity. The compound represented by chemical formula I for the manufacture of the medicament can be mixed with acceptable adjuvants, diluents, carriers, and the like, and can be prepared with other active agents into a combination formulation to have a synergistic effect with the active ingredient.

[0058] The matters mentioned in the uses, compositions and methods of the present invention shall apply equally as long as they do not contradict each other.

[0059] Exemplary embodiments of the present invention may be modified in many other ways, and the scope of the present invention is not limited to the exemplary embodiments described below. Furthermore, exemplary embodiments of the present invention are provided to explain the invention more completely to those skilled in the art. Moreover, unless otherwise stated, the term "comprising" a component throughout this specification does not exclude other components, but rather implies that other components may be further included.

[0060] [Advantageous Effects] The compound represented by chemical formula I, its stereoisomer or its pharmaceutically acceptable salt according to the present invention can selectively inhibit HDAC6, thereby having a significant and excellent preventive or therapeutic effect on diseases related to histone deacetase 6 activity.

Implementation Method

[0061] The invention will be described in more detail below by way of examples and experimental examples. However, such examples and the like are presented only as examples of the invention, and the scope of the invention is not limited to such examples.

[0062] Preparation of 1,3,4-diazole derivatives A specific method for preparing compounds represented by chemical formula I is described below.

[0063] Example 1: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 1)

[0064] [Step 1] Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione: N-(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)aniline (0.200 g, 0.626 mmol) and 3,4-dimethoxycyclobut-3-ene-1,2-dione (0.098 g, 0.689 mmol) were dissolved in methanol (5 mL) at room temperature, and the reaction solution was stirred at 60 °C for 18 hours. The reaction was then carried out by lowering the reaction temperature to room temperature. The reaction mixture was filtered through a glass filter to remove the solids, and the solvent was removed from the filtrate under reduced pressure. The concentrate was then purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; ethyl acetate / hexane = 10% to 50%) to obtain the title compound (0.069 g, 25.7%), which was a yellow oil.

[0065] [Step 2] Synthesis of Compound 1 The 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (0.100 g, 0.233 mmol) and 1-methylpiperazine (0.052 mL, 0.466 mmol) prepared in Step 1 were dissolved in methanol (3 mL) at room temperature, and the reaction solution was stirred at 60 °C for 18 hours. Then, the reaction was completed by lowering the reaction temperature to room temperature. After the solvent was removed from the reaction mixture under reduced pressure, the concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 5%) to give the title compound (0.016 g, 13.8%) as a pale yellow solid. 1H NMR (400MHz, CDCl 3) δ 7.88 (d, J= 8.0Hz, 1H), 7.77 (d, J= 8.7Hz, 1H), 7.63 (t, J= 7.5Hz, 1H), 7.36 (t, J= 7.9Hz, 2H), 7.19 (t, J= 7.4Hz, 1H), 7.04-6.79 (m, 3H), 5.58 (s, 2H), 3.28 (brs, 4H), 2.25 (brs, 4H), 2.22 (s, 3H); LRMS (ES) m / z 498.5 (M ++1).

[0066] Example 2: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-morpholinylcyclobut-3-ene-1,2-dione (Compound 2)

[0067] [Step 1] Synthesis of 3-morpholino-4-(phenylamino)cyclobut-3-ene-1,2-dione 3-methoxy-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 2.461 mmol), morpholine (0.213 mL, 2.461 mmol), and N,N-diisopropylethylamine (0.429 mL, 2.461 mmol) were dissolved in methanol (10 mL) at 50 °C, and the reaction solution was stirred at room temperature for 2 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Diethyl ether was added to the concentrate and stirred to precipitate a solid. The precipitate was filtered, washed with diethyl ether, and dried to give the title compound (0.445 g, 70.0%) as a white solid.

[0068] [Step 2] Synthesis of Compound 2: 3-morpholino-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.387 mmol) prepared in Step 1 and sodium hydride (60.00%, 0.017 g, 0.426 mmol) were dissolved in N,N-dimethylformamide (3 mL) at 0 °C. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (0.131 g, 0.426 mmol) was added to the reaction solution and stirred at room temperature for 1 hour. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium bicarbonate was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; ethyl acetate / hexane = 20% to 70%) to give the title compound (0.019 g, 10.1%) as a white solid. 1H NMR (400MHz, CDCl 3) δ 7.88 (d, J= 8.1Hz, 1H), 7.77 (d, J= 10.1Hz, 1H), 7.64 (t, J= 7.6Hz, 1H), 7.38 (t, J= 7.8Hz, 2H), 7.22 (t, J= 7.4Hz, 1H), 7.05-6.79 (m, 3H), 5.59 (s, 2H), 3.52-3.50 (m, 4H), 3.26 (brs, 4H); LRMS (ES) m / z 485.4 (M ++1).

[0069] Example 3: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(1,1-dioxothiomorpholino)cyclobut-3-ene-1,2-dione (Compound 3)

[0070] [Step 1] Synthesis of 3-(1,1-dioxothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione 3-methoxy-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 2.461 mmol), thiomorpholino 1,1-dioxide (0.333 g, 2.461 mmol), and N,N-diisopropylethylamine (0.429 mL, 2.461 mmol) were dissolved in methanol (10 mL) at 50 °C, and the reaction solution was stirred at room temperature for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The precipitated solid was filtered, washed with methanol, and dried to give the title compound (0.554 g, 73.5%) as a pale yellow solid.

[0071] [Step 2] Synthesis of Compound 3: 3-(1,1-dioxothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.326 mmol) prepared in Step 1 and sodium hydride (60.00%, 0.014 g, 0.359 mmol) were dissolved in N,N-dimethylformamide (3 mL) at 0 °C. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (0.110 g, 0.359 mmol) was added to the reaction solution and the mixture was stirred at room temperature for 1 hour. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium chloride was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous calcium chloride (II), filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; ethyl acetate / hexane = 10% to 40%) to give the title compound (0.023 g, 13.2%) as a pale yellow solid. 1H NMR (400MHz, CDCl 3) δ 7.89 (dd, J= 8.0, 1.4Hz, 1H), 7.77 (dd, J= 10.0, 1.4Hz, 1H), 7.61 (t, J= 7.6Hz, 1H), 7.43 (t, J= 7.8Hz, 2H), 7.31 (d, J = 7.4Hz, 1H), 7.09 (d, J = 7.8Hz, 2H), 7.05-6.79 (m, 1H), 5.60 (s, 2H), 3.68 (brs, 4H), 2.89-2.87 (m, 4H); 533.5 (M++1).

[0072] Example 4: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 4)

[0073] [Step 1] Synthesis of 4-(2-methoxy-3,4-dimethoxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid tributyl ester: 3,4-dimethoxycyclobut-3-en-1,2-dione (1.520 g, 10.696 mmol) and piperazine-1-carboxylic acid tributyl ester (1.992 g, 10.696 mmol) were dissolved in ethanol (30 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. The precipitated solid was filtered, washed with hexane, and dried to obtain the desired title compound (1.860 g, 58.7%) as a white solid.

[0074] [Step 2] Synthesis of 4-(3,4-di-side-oxy-2-(phenylamino)cyclobut-1-en-1-yl)piperazin-1-carboxylic acid tributyl ester: The 4-(2-methoxy-3,4-di-side-oxycyclobut-1-en-1-yl)piperazin-1-carboxylic acid tributyl ester (1.860 g, 6.277 mmol), aniline (0.573 mL, 6.277 mmol), and triethylamine (1.750 mL, 12.554 mmol) prepared in Step 1 were dissolved in ethanol (20 mL) at 78 °C, and the reaction solution was stirred at the same temperature for 18 hours. Then, the reaction was completed by lowering the reaction temperature to room temperature. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. Water was added to the concentrate, and then it was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (0.300 g, 13.4%) as a yellow solid.

[0075] [Step 3] Synthesis of 4-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester: The 4-(3,4-dioxy-2-(phenylamino)cyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester (0.176 g, 0.492 mmol) prepared in Step 2 was dissolved in N,N-dimethylformamide (10 mL) at 0 °C. Sodium hydride (60.00%, 0.030 g, 0.739 mmol) was added to the reaction solution and stirred at the same temperature for 30 minutes. 2-(4-(bromomethyl)phenyl)-5-(difluoromethyl)-1,3,4-diazole (0.17 g, 0.59 mmol) was added to the reaction mixture and stirred for another 3 hours at room temperature. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. Water was added to the concentrate, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (0.135 g, 48.5%) as a white solid.

[0076] [Step 4] Synthesis of 2,2,2-trifluoroacetate of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(piperazin-1-yl)cyclobut-3-en-1,2-dione: The 4-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazin-1-carboxylic acid tributyl ester (0.135 g, 0.239 mmol) and trifluoroacetic acid (0.183 mL, 2.387 mmol) prepared in step 3 were dissolved in dichloromethane (10 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. After the solvent was removed from the reaction mixture under reduced pressure, the resulting product was ready for use without further purification (0.150 g, 108.4%, yellow oil).

[0077] [Step 5] Synthesis of Compound 4: 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione 2,2,2-trifluoroacetate (0.150 g, 0.259 mmol), N,N-diisopropylethylamine (0.045 mL, 0.259 mmol), formaldehyde (0.016 g, 0.518 mmol), and sodium triacetoxyborohydride (0.110 g, 0.518 mmol) prepared in Step 4 were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was added to the reaction mixture, and the mixture was then extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; methanol / dichloromethane = 0% to 10%) to obtain the title compound (0.066 g, 53.2%) as a white solid. 1H NMR (400MHz, CDCl 3) δ 8.05 (d, J= 7.5Hz, 2H), 7.47 (d, J= 8.2Hz, 2H), 7.35 (t, J= 7.9Hz, 2H), 7.20 (t, J= 7.4Hz, 1H), 7.04 (s, LRMS (ES) m / z 480.5 (M++1).

[0078] Example 5: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(1,1-dioxothiomorpholino)cyclobut-3-ene-1,2-dione (compound 5) The 3-(1,1-dioxothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.326 mmol) prepared in step 1 of Example 3 and sodium hydride (60.00%, 0.014 g, 0.359 mmol) were dissolved in N,N-dimethylformamide (3 mL) at room temperature. 2-(4-(bromomethyl)phenyl)-5-(difluoromethyl)-1,3,4-diazole (0.104 g, 0.359 mmol) was added to the reaction solution and stirred at the same temperature for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The extract was then filtered through a plastic filter to remove the solid residue and aqueous layer and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; ethyl acetate / hexane = 10% to 40%). Ethyl acetate and diethyl ether were added to the resulting product and stirred. The precipitated solid was filtered, washed with diethyl ether, and dried to give the title compound (0.016 g, 9.5%) as a white solid. 1H NMR (400MHz, DMSO- d 6) δ 8.00 (d, J= 8.2Hz, 2H), 7.60-7.41 (m, 3H), 7.39 (t, J= 6.4Hz, 1H), 7.23-7.19 (m, 3H), 5.52 (s, 2H), 3.53 (brs, 4H), 3.14 (brs, 4H); LRMS (ES) m / z 515.4 (M ++1).

[0079] Example 6: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-4-(1,1-dioxothiomorpholino)cyclobut-3-ene-1,2-dione (compound 6) The 3-(1,1-dioxothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.326 mmol) prepared in step 1 of Example 3 and sodium hydride (60.00%, 0.014 g, 0.359 mmol) were dissolved in N,N-dimethylformamide (3 mL) at room temperature. 2-(6-(bromomethyl)-5-(fluoropyridin-3-yl)-5-(difluoromethyl)-1,3,4-diazole (0.111 g, 0.359 mmol) was added to the reaction solution and stirred at the same temperature for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; ethyl acetate / hexane = 10% to 40%). Ethyl acetate and diethyl ether were added to the resulting product and stirred to precipitate the solid. The precipitate was filtered, washed with diethyl ether, and dried to give the title compound (0.017 g, 9.8%) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.41 (dd, J = 9.9, 1.5 Hz, 1H), 7.71-7.45 (m, 1H), 7.41 (t, J = 7.8Hz, 2H), 7.21-7.17 (m, 3H), 5.70 (brs, 2H), 3.62 (brs, 4H), 3.17 (s, 4H); (M++1).

[0080] Example 7: Synthesis of 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 7)

[0081] [Step 1] Synthesis of 3-((3-bromo-5-fluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione: 3,4-dimethoxycyclobut-3-ene-1,2-dione (2.000 g, 14.074 mmol) and 3-bromo-5-fluoroaniline (2.674 g, 14.074 mmol) were dissolved in methanol (50 mL) at room temperature and stirred at the same temperature for 18 hours. The precipitated solid was filtered, washed with hexane, and dried to obtain the desired title compound (3.500 g, 82.9%) as a white solid.

[0082] [Step 2] Synthesis of 4-(2-((3-bromo-5-fluorophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid tributyl ester: The 3-((3-bromo-5-fluorophenyl)amino)-4-methoxycyclobut-3-en-1,2-dione (2.000 g, 6.665 mmol), piperazine-1-carboxylic acid tributyl ester (1.862 g, 9.997 mmol), and N,N-diisopropylethylamine (2.322 mL, 13.330 mmol) prepared in Step 1 were dissolved in methanol (30 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. The solvent was removed from the reaction mixture under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 40 g filter cartridge; ethyl acetate / hexane = 0% to 30%) to obtain the title compound (2.900 g, 95.8%) as a black foamy solid.

[0083] [Step 3] Synthesis of 4-(2-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester: The 4-(2-((3-bromo-5-fluorophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester (2.900 g, 6.383 mmol) prepared in step 2 was dissolved in N,N-dimethylformamide (30 mL) at 0 °C. Sodium hydride (60.00%, 0.383 g, 9.575 mmol) was added to the reaction solution and stirred at the same temperature for 30 minutes. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (2.548 g, 8.298 mmol) was added to the reaction mixture and stirred for another 3 hours at room temperature. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 40 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (2.000 g, 46.0%) as a yellow, foamy solid.

[0084] [Step 4] Synthesis of 2,2,2-trifluoroacetate of 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-4-(piperazin-1-yl)cyclobut-3-en-1,2-dione: The tert-butyl 4-(2-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazin-1-carboxylic acid prepared in Step 3 (2.000 g, 2.939 mmol) and trifluoroacetic acid (2.251 mL, 29.392 mmol) were dissolved in dichloromethane (20 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours (mL). Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting product was ready for use without further purification (2,000 g, 98.0%, brown oil).

[0085] [Step 5] Synthesis of Compound 7: 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)amino)-4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione 2,2,2-trifluoroacetate (2.000 g, 2.880 mmol), N,N-diisopropylethylamine (0.502 mL, 2.880 mmol), formaldehyde (0.173 g, 5.761 mmol), and sodium triacetoxyborohydride (1.221 g, 5.761 mmol) prepared in Step 4 were dissolved in dichloromethane (20 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 40 g filter cartridge; methanol / dichloromethane = 0% to 10%) to give the title compound (1.500 g, 87.6%) as a yellow solid. 1H NMR (400MHz, CDCl 3) δ 7.92 (dd, J= 8.0, 1.5Hz, 1H), 7.85 (dd, J= 10.2, 1.5Hz, 1H), 7.59 (t, J= 7.7Hz, 1H), 7.06-7.02 (m, 1H), 7.04 (s, 0.25H), 6.93 (s, 0.5H), 6.85 (s, 1H), 6.80 (s, 0.25H), 6.71-6.67 (m, 1H), 5.49 (s, 2H), 3.45 (br s, 4H), 2.39 (br s, 4H), 2.30 (s, 3H); LRMS (ES) m / z 595.4 (M ++1).

[0086] Example 8: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)(3-fluoro-5-(pyridin-3-yl)phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 8) prepared in Example 7: 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (0.200 g, 0.336 mmol), pyridin-3-ylboronic acid (0.054 g, 0.437 mmol) [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (Pd(dtbpf)Cl₂, 0.022 g, 0.034 mmol) and cesium carbonate (0.219 g, 0.673 mmol) were mixed with 1,4-dimethylamine (9 mL) / water (3 mL) and heated at 100 °C for 20 min by microwave irradiation. The reaction was then brought to a complete temperature by lowering the reaction temperature to room temperature. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; methanol / dichloromethane = 0% to 20%) to give the title compound (0.080 g, 40.1%) as a brown oil. 1H NMR (400MHz, CDCl 3) δ 8.78 (d, J= 1.9Hz, 1H), 8.67 (dd, J= 4.7, 1.1Hz, 1H), 7.91 (dd, J= 8.0, 1.4Hz, 1H), 7.85-7.80 (m, 2H), 7.65 (t, J= 7.7Hz, 1H), 7.43 (dd, J= 7.9, 4.9Hz, 1H), 7.11-7.08 (m, 1H), 7.05 (s, 0.25H), 6.96 (s, 1H), 6.92 (s, 0.5H), 6.79 (s, 0.25H), 6.78-6.74 (m, 1H), 5.58 (s, 2H), 3.49 (br s, 4H), 2.28 (br s, 4H), 2.19 (s, 3H); LRMS (ES) m / z 593.5 (M ++1).

[0087] Example 9: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(6-(oxacyclobutane-3-yl)-2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 9) prepared in Example 7: 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (0.200 g, 0.336 mmol), pyridin-4-ylboronic acid (0.054 g, 0.437 mmol) [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (Pd(dtbpf)Cl₂, 0.022 g, 0.034 mmol) and cesium carbonate (0.219 g, 0.673 mmol) were mixed with 1,4-dimethylamine (9 mL) / water (3 mL) and heated at 100 °C for 20 min by microwave irradiation. The reaction was then brought to a complete temperature by lowering the reaction temperature to room temperature. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; methanol / dichloromethane = 0% to 20%) to give the title compound (0.070 g, 35.1%) as a brown oil. 1H NMR (400MHz, CDCl 3) δ 8.73 (d, J= 5.6Hz, 1H), 7.92-7.90 (m, 1H), 7.86-7.83 (m, 1H), 7.66 (t, J= 7.7Hz, 1H), 7.43 (d, J= 5.9Hz, 2H), 7.15-7.13 (m, 1H), 7.05 (s, 0.25H), 7.00 (s, 1H), 6.92 (s, 0.5H), 6.81 (s, 0.25H), 6.80-6.78 (m, 1H), 5.58 (s, 2H), 3.30 (br s, 4H), 2.27 (br s, 4H), 2.19 (s, 3H); LRMS (ES) m / z 593.4 (M ++1).

[0088] Example 10: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(6-(oxacyclobutane-3-yl)-2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 10)

[0089] [Step 1] Synthesis of 6-(2-methoxy-3,4-di-side-oxycyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tributyl ester: 3,4-dimethoxycyclobut-3-en-1,2-dione (1.710 g, 12.033 mmol) and 2,6-diazaspiro[3.3]heptane-2-carboxylic acid tributyl ester (2.386 g, 12.033 mmol) were dissolved in ethanol (30 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. The precipitated solid was filtered, washed with hexane, and dried to obtain the desired title compound (0.700 g, 18.9%) as a white solid.

[0090] [Step 2] Synthesis of 6-(3,4-di-side-oxy-2-(phenylamino)cyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tributyl ester: The 6-(2-methoxy-3,4-di-side-oxy-cyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tributyl ester (0.700 g, 2.270 mmol), aniline (0.207 mL, 2.270 mmol), and triethylamine (0.633 mL, 4.541 mmol) prepared in Step 1 were dissolved in ethanol (20 mL) at 78 °C, and the reaction solution was stirred at the same temperature for 18 hours. Then, the reaction was completed by lowering the reaction temperature to room temperature. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. Water was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (0.600 g, 71.5%) as a white solid.

[0091] [Step 3] Synthesis of 6-(2-((4-(5-(difluoromethyl)-1,3,4-diazolazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid third butyl ester The 6-(3,4-dioxy-2-(phenylamino)cyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid third butyl ester (0.687 g, 1.860 mmol) prepared in step 2 was dissolved in N,N-dimethylformamide (5 mL) at 0 °C. Sodium hydride (60.00%, 0.112 g, 2.790 mmol) was added to the reaction solution and stirred at the same temperature for 30 minutes. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (0.685 g, 2.232 mmol) was added to the reaction mixture and stirred again at room temperature for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (0.700 g, 63.2%) as a white solid.

[0092] [Step 4] Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione The 6-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-3,4-di-side-oxycyclobut-1-ene-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tributyl ester (0.700 g, 1.175 mmol) and trifluoroacetic acid (0.900 mL, 11.753 mmol) prepared in step 3 were dissolved in dichloromethane (10 The reaction solution was stirred at the same temperature for 18 hours (mL). A saturated aqueous sodium bicarbonate solution was poured into the reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting product was ready for use without further purification (0.400 g, 68.7%, white solid).

[0093] [Step 5] Synthesis of Compound 10: 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (0.100 g, 0.202 mmol), 3-oxacyclobutanone (0.029 g, 0.404 mmol), and sodium triethoxyborohydride (0.086 g, 0.404 mmol) prepared in Step 4 were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; methanol / dichloromethane = 0% to 20%) to obtain the title compound (0.080 g, 71.9%) as a colorless oil. 1H NMR (400MHz, CDCl 3) δ 7.84 (dd, J= 8.0, 1.4Hz, 1H), 7.69 (dd, J= 9.8, 1.4Hz, 1H), 7.61 (t, J= 7.6Hz, 1H), 7.39-7.33 (m, 3H), 7.06-7.04 (m, 2H), 7.06 (s, 0.25H), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.44 (s, 2H), 4.62-4.58 (m, 2H), 4.34-4.31 (m, 2H), 3.80 (br s, 4H), 3.60-3.58 (m, 1H), 3.23 (s, 4H); LRMS (ES) m / z 552.5 (M ++1).

[0094] Example 11: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(6-methyl-2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 11)

[0095] [Step 1] Synthesis of 6-(2-((4-(5-(difluoromethyl)-1,3,4-diazolazol-2-yl)benzyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid third butyl ester The 6-(3,4-dioxy-2-(phenylamino)cyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid third butyl ester (0.177 g, 0.479 mmol) prepared in step 2 of Example 10 was dissolved in N,N-dimethylformamide (10 mL) at 0 °C. Sodium hydride (60.00%, 0.029 g, 0.719 mmol) was added to the reaction solution and stirred at the same temperature for 30 minutes. 2-(4-(bromomethyl)phenyl)-5-(difluoromethyl)-1,3,4-diazole (0.166 g, 0.575 mmol) was added to the reaction mixture and stirred again at room temperature for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (0.150 g, 54.2%) as a white solid.

[0096] [Step 2] Synthesis of 2,2,2-trifluoroacetate of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione: The 6-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-3,4-dioxycyclobut-1-ene-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tributyl ester (0.180 g, 0.312 mmol) and trifluoroacetic acid (0.239 mL, 3.116 mmol) prepared in Step 1 were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours (mL). After removing the solvent from the reaction mixture under reduced pressure, the resulting product (0.180 g, 97.6%, yellow oil) was ready for use without further purification.

[0097] [Step 3] Synthesis of Compound 11: The 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)benzyl)(phenyl)amino)-4-(2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione 2,2,2-trifluoroacetate (0.180 g, 0.304 mmol), N,N-diisopropylethylamine (0.053 mL, 0.304 mmol), formaldehyde (0.018 g, 0.609 mmol), and sodium triacetoxyborohydride (0.129 g, 0.609 mmol) prepared in Step 2 were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; methanol / dichloromethane = 0% to 20%) to give the title compound (0.056 g, 37.4%) as a white solid. 1H NMR (400MHz, CDCl 3) δ 8.02 (d, J= 8.2Hz, 2H), 7.40-7.35 (m, 5H), 7.04 (s, 0.25H), 7.03-7.01 (m, 2H), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.33 (s, 2H), 3.68 (s, 4H), 2.45 (s, 3H), 1.45 (s, 4H); LRMS (ES) m / z 492.4 (M ++1).

[0098] Example 12: Synthesis of 3-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 12)

[0099] [Step 1] Synthesis of 3-((3-bromophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione: 3,4-dimethoxycyclobut-3-ene-1,2-dione (2.000 g, 14.074 mmol) and 3-bromoaniline (2.421 g, 14.074 mmol) were dissolved in methanol (50 mL) at room temperature and stirred at the same temperature for 18 hours. The precipitated solid was filtered, washed with hexane, and dried to obtain the desired title compound (3.500 g, 88.2%) as a white solid.

[0100] [Step 2] Synthesis of 4-(2-((3-bromophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid tributyl ester: The 3-((3-bromophenyl)amino)-4-methoxycyclobut-3-en-1,2-dione (1.250 g, 4.431 mmol), piperazine-1-carboxylic acid tributyl ester (1.238 g, 6.647 mmol), and N,N-diisopropylethylamine (1.544 mL, 8.862 mmol) prepared in Step 1 were dissolved in ethanol (20 mL) at 78 °C, and the reaction solution was stirred at the same temperature for 18 hours. Then, the reaction was completed by lowering the reaction temperature to room temperature. The precipitated solid was filtered, washed with hexane, and dried to obtain the title compound (1.200 g, 62.1%) as a white solid.

[0101] [Step 3] Synthesis of 4-(2-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester: The 4-(2-((3-bromophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester (1.200 g, 2.750 mmol) prepared in step 2 was dissolved in N,N-dimethylformamide (30 mL) at 0 °C. Sodium hydride (60.00%, 0.143 g, 3.575 mmol) was added to the reaction solution and stirred at the same temperature for 30 minutes. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (1.013 g, 3.300 mmol) was added to the reaction mixture and stirred for another 3 hours at room temperature. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 40 g filter cartridge; ethyl acetate / hexane = 0% to 50%) to give the title compound (1.400 g, 76.8%) as a colorless oil.

[0102] [Step 4] Synthesis of 2,2,2-trifluoroacetic acid salt of 3-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-4-(piperazin-1-yl)cyclobut-3-en-1,2-dione: The tert-butyl 4-(2-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-2-fluorobenzyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazin-1-carboxylic acid prepared in Step 3 (1.400 g, 2.113 mmol) and trifluoroacetic acid (1.618 mL, 21.13 mmol) were dissolved in dichloromethane (20 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours (mL). After the solvent was removed from the reaction mixture under reduced pressure, the resulting product (1.400 g, 97.9%, brown oil) was ready for use without further purification.

[0103] [Step 5] Synthesis of Compound 12: 3-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)amino)-4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione 2,2,2-trifluoroacetate (1.400 g, 2.070 mmol), formaldehyde (0.124 g, 4.140 mmol), N,N-diisopropylethylamine (0.361 mL, 2.070 mmol), and sodium triacetoxyborohydride (0.877 g, 4.140 mmol) prepared in Step 4 were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; methanol / dichloromethane = 0% to 10%) to give the title compound as a white solid (0.900 g, 75.4%). 1H NMR (400MHz, CDCl 3) δ 7.89 (dd, J= 8.0, 1.5Hz, 1H), 7.80 (dd, J= 10.1, 1.5Hz, 1H), 7.60 (t, J= 7.6Hz, 1H), 7.30-7.28 (m, 1H), 7.23 (t, J= 8.0Hz, 1H), 7.08 (t, J= 1.9Hz, 1H), 7.05 (s, 0.25H), 6.98 (dd, J= 8.0, 1.5 Hz, 1H), 6.92 (s, 0.5H), 6.79 (s, 0.25H), 5.53 (s, 2H), 3.36 (br s, 4H), 2.45 (br s, 4H), 2.25 (s, 3H); LRMS (ES) m / z 577.4 (M ++1).

[0104] Example 13: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-((1S,4S)-5-(oxacyclobutane-3-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 13)

[0105] [Step 1] Synthesis of (1S,4S)-5-(3,4-dioxy-2-(phenylamino)cyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1] heptane-2-carboxylic acid tributyl ester: 3-methoxy-4-(phenylamino)cyclobut-3-en-1,2-dione (0.500 g, 2.461 mmol), (1S,4S)-2,5-diazabicyclo[2.2.1] heptane-2-carboxylic acid tributyl ester (0.537 g, 2.707 mmol) and N,N-diisopropylethylamine (0.85 mL, 4.921 mmol) were dissolved in methanol (10 mL) at room temperature, and the reaction solution was stirred at room temperature for 18 hours. After the solvent was removed from the reaction mixture under reduced pressure, the concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 20% to 40%) to give the title compound (0.871 g, 95.8%) as a pale yellow solid.

[0106] [Step 2] Synthesis of (1S,4S)-5-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1] heptane-2-carboxylic acid third butyl ester prepared in step 1 (0.300 g, 0.812 g) 1,3,4-dimethylformamide (0.274 g, 0.893 mmol), 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (0.274 g, 0.893 mmol), potassium carbonate (0.224 g, 1.624 mmol), and potassium iodide (0.013 g, 0.081 mmol) were dissolved in N,N-dimethylformamide (4 mL) at room temperature, and the reaction solution was stirred at 60 °C for 18 hours. The reaction was then brought to a complete state by lowering the reaction temperature to room temperature. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium bicarbonate was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 30% to 60%) to obtain the title compound (0.315 g, 65.1%) as a yellow solid.

[0107] [Step 3] Synthesis of 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)cyclobut-3-en-1,2-dione The (1S,4S)-5-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tributyl ester (0.315 g, 0.529 mmol) and trifluoroacetic acid (0.284 g) prepared in step 2 were used. Sodium bicarbonate (3.702 mmol) was dissolved in dichloromethane (4 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The resulting product was ready for use without further purification (0.221 g, 84.3%, pale yellow solid).

[0108] [Step 4] Synthesis of Compound 13 The 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)cyclobut-3-ene-1,2-dione (0.100 g, 0.202 mmol) and formaldehyde (0.018 mL, 0.303 mmol) prepared in Step 3 were dissolved in dichloromethane (4 mL) at room temperature. Triacetoxyborohydride sodium (0.086 g, 0.404 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 2.5%) to obtain the title compound (0.075 g, 67.4%) as a yellow solid. 1H NMR (400MHz, CDCl 3) δ 7.87 (dd, J= 8.0, 1.4Hz, 1H), 7.72-7.66 (m, 2H), 7.35 (t, J= 7.6Hz, 2H), 7.29-7.25 (m, 1H), 7.07 (d, J= 7.6Hz, 2H), 7.05-6.79 (m, 1H), 5.56 (d, J= 15.4Hz, 1H), 5.47 (d, J= 15.4Hz, 1H), 4.63-4.57 (m, 2H), 4.40 (t, J= 5.9Hz, 2H), 3.74-3.71 (m, 1H), 3.13 (brs, 1H), 2.78 (brs, 1H), 2.67 (brs, 1H), 1.95 (brs, 1H), 1.76 (d, J= 9.8Hz, 1H), 1.54 (d, J= 10.1Hz, 1H), 1.30-1.28 (m, 1H); LRMS (ES) m / z 552.8 (M++1).

[0109] Example 14: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 14) prepared in step 3 of Example 14. 0.022 mL of 38.00% solution (0.303 mmol) and formaldehyde (38.00% solution, 0.303 mmol) were dissolved in dichloromethane (4 mL) at room temperature. Sodium triethoxyborohydride (0.086 g, 0.404 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, followed by extraction with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and an aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 4 g filter cartridge; methanol / dichloromethane = 0% to 5%) to give the title compound (0.049 g, 47.7%) as a yellow solid. 1H NMR (400MHz, CDCl 3) δ 7.88 (dd, J= 8.0, 1.5Hz, 1H), 7.74-7.67 (m, 2H), 7.35 (t, J= 7.6Hz, 2H), 7.28-7.25 (m, 1H), 7.07-6.79 (m, 3H), 5.58-5.47 (m, 2H), 3.13 (brs, 1H), 2.71 (brs, 1H), 2.63 (brs, 1H), 2.28 (s, 3H), 1.99-1.83 (m, 3H), 1.52 (d, J= 10.1Hz, 1H), 1.31-1.29 (m, 1H); LRMS (ES) m / z 510.8 (M++1).

[0110] Example 15: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 15)

[0111] [Step 1] Synthesis of (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1] heptane-2-carboxylic acid third butyl ester prepared in Step 1 of Example 13 (0.500 g, 1.353 g) 1,4,4-dimethylformamide (0.432 g, 1.489 mmol), 2-(6-(bromomethyl)pyridin-3-yl)-5-(difluoromethyl)-1,3,4-diazole (0.281 g, 2.030 mmol), potassium carbonate (0.022 g, 0.135 mmol), and potassium iodide (0.022 g, 0.135 mmol) were dissolved in N,N-dimethylformamide (4 mL) at room temperature, and the reaction solution was stirred at 60 °C for 18 hours. The reaction was then brought to a complete state by lowering the reaction temperature to room temperature. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium bicarbonate was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 30% to 65%) to obtain the title compound (0.440 g, 56.2%) as a light brown solid.

[0112] [Step 2] Synthesis of 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobutane-3-en-1,2-dione The (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-3,4-dioxycyclobutane-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tertiary butyl ester (0.440 g, 0.760 g) prepared in step 1 0.291 mL of sodium bicarbonate and 3.802 mmol of trifluoroacetic acid were dissolved in dichloromethane (4 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The resulting product was ready for use without further purification (0.277 g, 76.1%, brown solid).

[0113] [Step 3] Synthesis of Compound 15: 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-en-1,2-dione (0.090 g, 0.188 mmol) prepared in Step 2 and formaldehyde (38.00% aqueous solution, 0.021 mL, 0.282 mmol) were dissolved in dichloromethane (4 mL) at room temperature. Triacetoxyborohydride sodium (0.080 g, 0.376 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and an aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 5%) to obtain the title compound (0.054 g, 58.3%) as an orange solid. 1H NMR (400MHz, CDCl 3) δ 9.25 (d, J= 2.0Hz, 1H), 8.37 (dd, J= 8.2, 2.2Hz, 1H), 7.64 (d, J= 8.2Hz, 1H), 7.36 (t, J= 7.8Hz, 2H), 7.24 (t, J= 7.4Hz, 1H), 7.19 (d, J= 7.6Hz, 2H), 7.08-6.82 (m, 1H), 5.63-5.50 (m, 2H), 3.18 (brs, 1H), 2.76 (brs, 1H), 2.63 (brs, 1H), 2.31 (s, 3H), 2.16-2.14 (m, 2H), 1.87 (d, J= 9.9Hz, 1H), 1.57-1.53​​ (m, 1H), 1.32-1.28 (m, 1H); LRMS (ES) m / z 493.8 (M ++1).

[0114] Example 16: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 16) prepared in step 2 of Example 15. Sodium triacetoxyborohydride (0.080 g, 0.376 mmol) and acetone (0.021 mL, 0.282 mmol) were dissolved in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.080 g, 0.376 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, followed by extraction with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 4 g filter cartridge; methanol / dichloromethane = 0% to 5%) to give the title compound (0.056 g, 57.2%) as an orange solid. 1H NMR (400MHz, CDCl 3) δ 9.25 (d, J= 2.0Hz, 1H), 8.38 (dd, J= 8.2, 2.1Hz, 1H), 7.65 (d, J= 8.1Hz, 1H), 7.37 (t, J= 7.8Hz, 2H), 7.24 (t, J= 7.4Hz, 1H), 7.19 (d, J= 7.7Hz, 2H), 7.08-6.82 (m, 1H), 5.62-5.52 (m, 2H), 3.49 (brs, 1H), 3.03 (s, 1H), 2.46 (s, 2H), 1.88-1.57 (m, 4H), 1.04-1.03 (m, 3H), 0.99-0.95 (m, 4H); LRMS (ES) m / z 521.8 (M ++1).

[0115] Example 17: 3-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-(oxacyclobutane-3-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 17) The synthesis involved dissolving 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-en-1,2-dione (0.100 g, 0.209 mmol) and 3-oxacyclobutanone (0.018 mL, 0.314 mmol) in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.089 g, 0.418 mmol) was added to the reaction solution, and the mixture was stirred for 18 hours at the same temperature. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, followed by extraction with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 2.5%) to obtain the title compound (0.036 g, 32.2%) as an orange solid. 1H NMR (400MHz, CDCl 3) δ 9.27 (s, 1H), 8.39 (dd, J= 8.1, 1.8Hz, 1H), 7.64 (d, J= 8.2Hz, 1H), 7.38 (t, J= 7.7Hz, 2H), 7.26-7.24 (m, 1H), 7.21 (d, J= 8.0Hz, 2H), 7.08-6.82 (m, 1H), 5.62-5.51 (m, 2H), 4.65-4.59 (m, 2H), 4.44 (brs, 2H), 3.77 (brs, 2H), 3.20 (brs, 2H), 2.80-2.72 (m, 2H), 1.82-1.52 (m, 4H); LRMS (ES) m / z 535.8 (M ++1).

[0116] Example 18: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)pyridin-2-yl)methyl)(3-(pyridin-3-yl)phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 18) 3-((3-bromophenyl)((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)pyridin-2-yl)methyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (0.250 g, 0.447 mmol), pyridin-3-ylboronic acid (0.071 g, 0.581 mmol) [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (Pd(dtbpf)Cl₂, 0.029 g, 0.045 mmol) and cesium carbonate (0.364 g, 1.117 mmol) were mixed with 1,4-dimethylamine (9 mL) / water (3 mL) and heated at 100 °C for 20 hours by microwave irradiation. The reaction was then brought to a complete temperature by lowering the reaction temperature to room temperature. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; methanol / dichloromethane = 0% to 10%) to give the title compound (0.100 g, 40.1%) as a brown oil. 1H NMR (400MHz, CDCl 3) δ 8.78 (d, J= 1.6Hz, 1H), 8.64 (d, J= 3.8Hz, 1H), 7.88-7.76 (m, 3H), 7.66 (t, J= 7.6Hz, 1H), 7.47 (t, J= 7.8Hz, 1H), 7.42-7.39 (m, 2H), 7.18 (s, 1H), 7.07 (dd, J= 7.9, 1.3Hz, 1H), 7.04 (s, 0.25H), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.61 (s, 2H), 3.32 (br s, 4H), 2.23 (br s, 4H), 2.16 (s, 3H); LRMS (ES) m / z 558.4 (M ++1).

[0117] Example 19: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)pyridin-2-yl)methyl)(3-(pyridin-4-yl)phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-en-1,2-dione (Compound 19) 3-((3-bromophenyl)((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)pyridin-2-yl)methyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-en-1,2-dione (0.200 g, 0.358 mmol), pyridin-4-ylboronic acid (0.057 g, 0.465 mmol) [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (Pd(dtbpf)Cl₂, 0.023 g, 0.036 mmol) and cesium carbonate (0.291 g, 0.894 mmol) were mixed with 1,4-dimethylamine (9 mL) / water (3 mL) and heated at 100 °C for 15 minutes by microwave irradiation. The reaction was then brought to a complete temperature by lowering the reaction temperature to room temperature. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 12 g filter cartridge; methanol / dichloromethane = 0% to 10%) to give the title compound (0.060 g, 30.1%) as a black oil. 1H NMR (400MHz, CDCl 3) δ 8.71 (d, J= 4.4Hz, 2H), 7.89 (d, J= 8.1Hz, 1H), 7.80 (dd, J= 10.1, 1.1Hz, 1H), 7.67 (t, J= 7.6Hz, 1H), 7.50-7.43 (m, 4H), 7.23 (s, 1H), 7.11-7.09 (m, 1H), 7.04 (s, 0.25), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.62 (s, 2H), 3.31 (br s, 4H), 2.22 (br s, 4H), 3.16 (s, 3H); LRMS (ES) m / z 558.4 (M++1).

[0118] Example 20: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 20)

[0119] [Step 1] Synthesis of (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1] heptane-2-carboxylic acid third butyl ester prepared in Step 1 of Example 13 (0.500 g, 1.353 g) 1.459 g (1.489 mmol), 2-(bromomethyl)-3-fluoro-5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)pyridine (0.281 g, 2.030 mmol), potassium carbonate (0.022 g, 0.135 mmol), and potassium iodide (0.022 g, 0.135 mmol) were dissolved in N,N-dimethylformamide (4 mL) at room temperature, and the reaction solution was stirred at 60 °C for 18 hours. The reaction was then brought to a complete state by lowering the reaction temperature to room temperature. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium bicarbonate was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 30% to 70%) to obtain the title compound (0.285 g, 35.3%) as a brown solid.

[0120] [Step 2] Synthesis of 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)cyclobutane-3-en-1,2-dione The (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-adiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-3,4-dioxycyclobutane-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tertiary butyl ester (0.285) prepared in step 1 The reaction mixture (0.256 mL, 3.344 mmol) and trifluoroacetic acid (0.478 mmol, 0.478 g) were dissolved in dichloromethane (4 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 4 g filter cartridge; ethyl acetate / hexane = 0% to 30%) to give the title compound (0.206 g, 86.9%) as a pale brown solid.

[0121] [Step 3] Synthesis of Compound 20: 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-1,2-dione (0.100 g, 0.201 mmol) and formaldehyde (0.009 g, 0.302 mmol) prepared in Step 2 were dissolved in dichloromethane (4 mL) at room temperature. Triacetoxyborohydride sodium (0.085 g, 0.403 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and an aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 5%) to obtain the title compound (0.067 g, 65.2%) as a yellow solid. 1H NMR (400MHz, CDCl 3) δ 9.05 (s, 1H), 8.06 (d, J= 9.3Hz, 1H), 7.34 (t, J= 7.1Hz, 2H), 7.22 (t, J= 7.4Hz, 1H), 7.14 (d, J= 8.0Hz, 2H), 7.08-6.82 (m, 1H), 5.71-5.66 (m, 2H), 3.18 (brs, 1H), 2.77 (brs, 3H), 2.61-2.59 (m, 1H), 2.30 (brs, 3H), 1.86 (d, J= 9.8Hz, 1H), 1.57 (d, J= 10.1Hz, 1H); LRMS (ES) m / z 511.8 (M ++1).

[0122] Example 21: 3-(((5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-(oxacyclobutane-3-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 21) The synthesis involved dissolving 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-en-1,2-dione (0.100 g, 0.201 mmol) and 3-oxacyclobutanone (0.019 mL, 0.302 mmol) in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.085 g, 0.403 mmol) was added to the reaction solution, and the mixture was stirred for 18 hours at the same temperature. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, followed by extraction with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 2.5%) to obtain the title compound (0.019 g, 17.1%) as a yellow solid. 1H NMR (400MHz, CDCl 3) δ 9.09 (s, 1H), 8.10 (dd, J= 29.3, 20.0Hz, 1H), 7.37 (t, J= 7.7Hz, 2H), 7.26-7.24 (m, 1H), 7.18 (d, J= 7.6Hz, 2H), 7.08-6.82 (m, 1H), 5.68 (s, 2H), 4.68-4.63 (m, 2H), 4.50-4.45 (m, 2H), 3.81-3.80 (m, 1H), 3.22 (brs, 1H), 2.82 (brs, 1H), 2.75 (brs, 1H), 1.83-1.59 (m, 4H), 1.31-1.30 (m, 1H); LRMS (ES) m / z 553.7 (M ++1).

[0123] Example 22: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 22)

[0124] [Step 1] Synthesis of 3-((3,4-difluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione: 3,4-difluoroaniline (1.000 g, 7.745 mmol) and 3,4-dimethoxycyclobut-3-ene-1,2-dione (1.101 g, 7.745 mmol) were dissolved in methanol (30 mL) at room temperature and stirred at the same temperature for 18 hours. The precipitated solid was filtered, washed with methanol, and dried to obtain the title compound (1.610 g, 86.9%) as a pale yellow solid.

[0125] [Step 2] Synthesis of 4-(2-((3,4-difluorophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid tributyl ester: The 3-((3,4-difluorophenyl)amino)-4-methoxycyclobut-3-en-1,2-dione (1.500 g, 6.271 mmol), piperazine-1-carboxylic acid tributyl ester (1.168 g, 6.271 mmol), and N,N-diisopropylethylamine (1.092 mL, 6.271 mmol) prepared in Step 2 were dissolved in methanol (30 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Methanol was added to the reaction mixture and stirred to precipitate a solid. The precipitate was filtered, washed with methanol, and dried to obtain the title compound (1.770 g, 71.7%) as a pale green solid.

[0126] [Step 3] Synthesis of 4-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester: The 4-(2-((3,4-difluorophenyl)amino)-3,4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylic acid third butyl ester (0.500 g, 1.271 mmol) prepared in step 2 and sodium hydride (60.00%, 0.056 g, 1.398 mmol) were dissolved in N,N-dimethylformamide (20 mL) at 0 °C. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-diazole (0.429 g, 1.398 mmol) was added to the reaction solution and the mixture was stirred at room temperature for 18 hours. After removing the solvent from the reaction mixture under reduced pressure, the concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 5% to 40%) to give the title compound (0.516 g, 65.5%) as a pale yellow solid.

[0127] [Step 4] Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(piperazin-1-yl)cyclobut-3-en-1,2-dione The 4-(2-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-3,4-di-side-oxycyclobut-1-en-1-yl)piperazin-1-carboxylic acid tributyl ester (0.516 g, 0.833 mmol) and trifluoroacetic acid (0.510 mL, 6.663 mmol) prepared in step 3 were dissolved in dichloromethane (5 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate and dichloromethane were added to the reaction mixture and stirred to precipitate a solid. The precipitated solid was then filtered. The filtrate was washed with dichloromethane and dried to give the title compound (0.361 g, 83.4%) as a yellow solid.

[0128] [Step 5] Synthesis of Compound 22: 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione (0.100 g, 0.193 mmol) prepared in Step 4 and formaldehyde (38.00% solution, 0.021 mL, 0.289 mmol) were dissolved in dichloromethane (4 mL) at room temperature. Triacetoxyborohydride sodium (0.082 g, 0.385 mmol) was added to the reaction solution and the mixture was stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, and then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove solid residues and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g filter cartridge; methanol / dichloromethane = 0% to 5%) to obtain the title compound (0.044 g, 42.8%) as a white solid. 1H NMR (400MHz, CDCl 3) δ 7.90 (d, J= 8.0Hz, 1H), 7.81 (d, J= 10.2Hz, 1H), 7.63 (t, J= 7.7Hz, 1H), 7.17 (q, J= 9.0Hz, 1H), 7.06-6.85 (m, 2H), 6.77-6.75 (m, 1H), 5.52 (s, 1H), 3.37 (s, 4H), 2.35 (s, 4H), 2.28 (s, 3H); LRMS (ES) m / z 534.7 (M ++1).

[0129] Example 23: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(4-(oxacyclobutane-3-yl)piperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 23) prepared in step 4 of Example 23: 3-((4-(5-(difluoromethyl)-1,3,4-diazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione (0.065 g, 0.125 mmol) and oxacyclobut-3-one (0.012 mL, 0.188 mmol) were prepared in step 4 of Example 23. The 0.250 mmol sodium triethoxyborohydride was dissolved in dichloromethane (4 mL) at room temperature. Sodium triethoxyborohydride (0.053 g, 0.250 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. A saturated aqueous solution of sodium bicarbonate was poured into the reaction mixture, followed by extraction with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO₂, 4 g filter cartridge; methanol / dichloromethane = 0% to 2.5%) to give the title compound (0.016 g, 22.2%) as a white solid. 1H NMR (400MHz, CDCl 3) δ 7.90 (dd, J= 8.0, 1.2Hz, 1H), 7.80 (dd, J= 10.1, 1.2Hz, 1H), 7.62 (t, J= 7.7Hz, 1H), 7.17 (q, J= 9.0Hz, 1H), 7.06-6.85 (m, 2H), 6.77-6.75 (m, 1H), 5.52 (s, 2H), 4.64 (t, J= 6.5Hz, 2H), 4.52 (t, J= 6.1Hz, 2H), 3.50-3.39 (m, 5H), 2.25 (brs, 4H); LRMS (ES) m / z 576.3 (M ++1).

[0130] Example 24: Synthesis of 3-morpholino-4-[N-[[4-[5-(trifluoromethyl)-1,3,4-diazol-2-yl]phenyl]methyl]aniline]cyclobut-3-ene-1,2-dione (compound 24) The 3-morpholino-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 1.936 mmol) prepared in step 1 of Example 2 and sodium hydride (60.00%, 0.085 g, 2.125 mmol) were dissolved in N,N-dimethylformamide (10 mL) at 0 °C. 2-[4-(bromomethyl)phenyl)-5-(trifluoromethyl)-1,3,4-diazole (0.624 g, 2.03 mmol) was added to the reaction solution and stirred at room temperature for 18 hours. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium bicarbonate was added to the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and then concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g filter cartridge; ethyl acetate / hexane = 10% to 60%). Dichloromethane and diethyl ether were added to the resulting product and stirred to precipitate the solid, which was then filtered off. The filtrate was washed with diethyl ether and dried to give the title compound (0.196 g, 20.90%, white solid). 1H NMR (400MHz, DMSO- d 6) δ 8.06 (d, J= 8.4Hz, 2H), 7.60 (d, J= 8.4Hz, 2H), 7.41 (t, J= 7.8Hz, 2H), 7.26-7.21 (m, 3H), 5.57 (s, 2H), 3.51 (t, J= 4.8Hz, 4H), 3.27 (brs, 4H); LRMS (ES) m / z 485.7 (M ++1).

[0131] Activity determination and analysis scheme of compounds according to the present invention

[0132] <Experimental Example 1> Identification of HDAC Enzyme Activity Inhibition (In Vitro) Selectivity of HDAC6 inhibitors for HDAC1 inhibition is crucial, as this selectivity is the cause of side effects. To identify this importance, HDAC1 / 6 enzyme selectivity and cell selectivity (HDAC1: histone acetylation / HDAC6: tubulin acetylation) were identified.

[0133] 1. Experimental Methods The HDAC enzyme inhibitory activity of test substances was measured using the HDAC1 fluorescence drug discovery assay kit (Enzo Life Sciences: BML-AK511) and the HDAC6 human recombinant (Calbiochem: 382180). In the HDAC1 assay, test substances were treated at concentrations of 100, 1000, and 10000 nM, and in the HDAC6 assay, test substances were treated at concentrations of 0.1, 1, 10, 100, and 1000 nM. After sample treatment, the samples were reacted at 37°C for 60 minutes, then treated with a developer and reacted at 37°C for 30 minutes. Subsequently, the fluorescence intensity (Ex 390, Em 460) was measured using a FlexStatin3 (molecular device).

[0134] 2. Experimental Results Table 2 shows the results of the HDAC enzyme activity inhibition analysis obtained according to the above experimental method. [Table 2] compound HDAC6 IC 50 (uM) HDAC1 IC 50 (uM) 1 0.022 >10 2 0.026 >10 3 0.027 >10 4 0.031 >10 5 0.029 >10 6 0.032 >10 7 0.036 >10 8 0.035 >10 9 0.054 >10 10 0.056 >10 11 0.060 >10 12 0.030 ＞10 13 0.024 ＞10 14 0.024 ＞10 15 0.022 ＞10 16 0.028 ＞10 17 0.043 ＞10 18 0.051 ＞10 19 0.102 ＞10 20 0.039 ＞10 21 0.054 ＞10 22 0.064 ＞10 23 0.050 ＞10 24 0.161 ＞10

Claims

1. A 1,3,4-diazole derivative compound, its stereoisomer, or a pharmaceutically acceptable salt thereof represented by the following chemical formula I, [Chemical Formula I] In the above chemical formula I, R1 and R2 are connected together with an N atom to form a 3 to 12-membered heterocyclic alkyl group containing 1 to 5 heteroatoms selected from N, O, and S, wherein at least one H in the 3 to 12-membered heterocyclic alkyl ring containing 1 to 5 heteroatoms selected from N, O, and S may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, or a 3 to 12-membered heterocyclic alkyl group containing 1 to 5 heteroatoms selected from N, O, and S; X is -H or -F; Y1 to Y5 are each independently N or CR3, wherein Y1 to Y5 cannot simultaneously be 3 or more N atoms; When Y1, Y3 to Y5 are each independently CR3, R3 is -H, -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo group; When Y2 is CR3, R3 is a heteroaryl group consisting of -H, -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, 6-membered aryl, or a heteroaryl group containing one of O, N, P, Si, and S and having 4 or 5 ring carbon atoms. At least one -H in the 6-membered aryl group or the heteroaryl group containing one of O, N, P, Si, and S and having 4 or 5 ring carbon atoms can be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl), or a halo. Z1 to Z4 are each independently N or CR4, wherein Z1 to Z4 cannot simultaneously be 3 or more N; and R4 is -H, -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo.

2. The 1,3,4-diazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof as claimed in claim 1, wherein in the above chemical formula I, R1 and R2 are connected together with an N atom to form a 3 to 12-membered heterocyclic alkyl group containing 1 to 5 heteroatoms selected from N, O, and S, wherein at least one H in the 3 to 12-membered heterocyclic alkyl ring containing 1 to 5 heteroatoms selected from N, O, and S may be independently substituted by a -(C1-C4 alkyl) or a 3 to 12-membered heterocyclic alkyl group containing 1 to 5 heteroatoms selected from N, O, and S; X is -H or -F; Y1 to Y5 are each independently CR3; when Y1, Y3 to Y5 are each independently CR3, R3 is -H or -halogen; When Y2 is CR3, R3 is a heteroaryl group containing one of O, N, P, Si, and S and having 4 or 5 ring carbon atoms, wherein at least one -H in the heteroaryl ring containing one of O, N, P, Si, and S and having 4 or 5 ring carbon atoms can be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl), or a halogroup; Z1 to Z4 are each independently N or CR4, wherein Z1 to Z4 cannot simultaneously be 3 or more N; and R4 is a -H or -halo group.

3. The 1,3,4-diazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein in the above chemical formula I, R1 and R2 are connected together with an N atom to form a 4 to 12-membered heterocyclic alkyl group containing 1 to 5 heteroatoms selected from N, O, and S, wherein at least one H in the 4 to 12-membered heterocyclic alkyl ring containing 1 to 5 heteroatoms selected from N, O, and S may be independently substituted with -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, or a 3 to 6-membered heterocyclic alkyl group containing 1 to 5 heteroatoms selected from N, O, and S.

4. The 1,3,4-diazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof as claimed in claim 3, wherein in the above chemical formula I, R1 and R2 are connected together with an N atom to form, or, wherein at least one H in the ring may be independently substituted with -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), or -halo; W is NR5, O, S, or S(=O)2; R5 is -(C1-C4 alkyl) or a 3 to 6-membered heterocyclic alkyl containing 1 to 5 heteroatoms selected from N, O, and S; and n1, n2, m1, and m2 are each independently 0, 1, or 2.

5. The 1,3,4-diazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof as claimed in claim 4, wherein in the above chemical formula I, R1 and R2 are connected together with N atoms to form , , , or , wherein at least one H in , , , or rings may be independently substituted with -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo; and R5 is -(C1-C4 alkyl) or .

6. The 1,3,4-diazole derivative of claim 1, its stereoisomers or pharmaceutically acceptable salts thereof, wherein it is any one of the compounds listed in the table below: or its stereoisomers or pharmaceutically acceptable salts thereof.

7. A pharmaceutical composition for treating histone deacetase 6-mediated diseases by inhibiting histone deacetase 6, comprising, as claimed in any one of claims 1 to 6, a compound represented by chemical formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof as an active ingredient.

8. Use of a compound represented by chemical formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, in the preparation of a medicament for treating histone deacetase 6-mediated diseases by inhibiting histone deacetase 6.

9. For the purposes described in claim 8, wherein the diseases mediated by histone deacetase 6 are infectious diseases; tumors; endocrine, nutritional and metabolic diseases; mental and behavioral disorders; neurological diseases; eye and ocular adnexa diseases; respiratory diseases; digestive system diseases; skin and subcutaneous tissue diseases; musculoskeletal and connective tissue diseases; or congenital malformations, alterations and chromosomal abnormalities.