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

Abstract

The present invention relates to a novel compound having histone deacetylase 6 (HDAC6) inhibitory activity, and a preparation method and use thereof. The novel compound, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof according to the present invention has HDAC6 inhibitory activity and is effective in the prevention or treatment of HDAC6-mediated diseases (including cancers, inflammatory diseases, autoimmune diseases, neurological or neurodegenerative diseases).

Description

Technical Field

[0001] The present invention relates to a 1,3,4-oxadiazole derivative compound having histone deacetylase 6 (HDAC6) inhibitory activity, a preparation method thereof and use thereof. Background Art

[0002] Post-translational modifications in cells, such as acetylation, are very important 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 wrapped. In addition, the balance between acetylation and deacetylation of histones plays a key role in gene expression.

[0003] Histone deacetylase (HDAC) is an enzyme that removes acetyl groups from lysine residues of histone proteins that constitute chromatin, and is known to be associated with gene silencing and induction of cell cycle arrest, angiogenesis inhibition, immunomodulation, cell death, etc. (Hassig et al., Curr. Opin. Chem. Biol. 1997, 1, 300-308). In addition, it has been reported that inhibition of HDAC enzyme function reduces the activity of cancer cell survival-related factors in vivo and activates cancer cell death-related factors, thereby inducing the self-death of cancer cells (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 to yeast HDACs. Among them, the 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 HDACs of Class III (SIRT 1-7) use NAD + Replaces zinc as a cofactor (Bolden et al., Nat. Rev. Drug. Discov. 2006, 5(9), 769-784).

[0005] Although a variety of HDAC inhibitors are in preclinical or clinical development stages, so far, only non-selective HDAC inhibitors are known to be anticancer agents, of which 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, it is known that non-selective HDAC inhibitors generally produce side effects at high doses, such as fatigue, nausea and the like (Piekarz et al., Pharmaceuticals 2010, 3, 2751-2767). This side effect has been reported to be caused by inhibiting class I HDAC, and due to this side effect, non-selective HDAC inhibitors are limited in drug development in the field other than anticancer drugs (Witt et al., Cancer Letters 277 (2009) 8.21).

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

[0007] HDAC6 (which is one of class IIb HDACs) is mainly present in the cytoplasm and is known to contain tubulin proteins, and is therefore involved in the deacetylation of a variety of non-histone substrates (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 may bind to ubiquitinated proteins. Since HDAC6 has a variety of non-histone proteins as substrates, it is known that it plays an important role 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 are composed of a capping group, a linking 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 capping groups and linking groups. It is known that the zinc binding group plays 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] Capping group Linking group Zinc binding group

[0010]

[0011] Most of these zinc-binding groups are hydroxamic acid or benzamide, of which hydroxamic acid derivatives show strong HDAC inhibition, but have problems such as low bioavailability and severe off-target activity. Benzamide contains aniline and therefore also has the problem of producing toxic metabolites in vivo (Woster et al., Med. Chem. Commun. 2015, online publication).

[0012] 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 of HDAC6 with a zinc binding moiety that has no side effects and improved bioavailability, while not having the side effects of non-selective inhibitors. Summary of the invention

[0013] [Technical issues]

[0014] An object of the present invention is to provide a 1,3,4-oxadiazole derivative compound having selective histone deacetylase 6 (HDAC6) inhibitory activity, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0015] Another object of the present invention is to provide a pharmaceutical composition comprising a 1,3,4-oxadiazole derivative compound having selective HDAC6 inhibitory activity, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0016] Another object of the present invention is to provide a preparation method thereof.

[0017] Another object of the present invention is to provide a pharmaceutical composition comprising such a compound for preventing or treating HDAC6 activity-related diseases including: infectious diseases; neoplasms; endocrine, nutritional and metabolic diseases; mental and behavioral disorders; neurological diseases; eye and eye adnexal diseases; circulatory system diseases; respiratory diseases; digestive system diseases; skin and subcutaneous tissue diseases; musculoskeletal and connective tissue diseases; or congenital malformations, changes and chromosomal abnormalities.

[0018] Another object of the present invention is to provide use of the compound in preparing a medicament for preventing or treating diseases related to HDAC6 activity.

[0019] Another object of the present invention is to provide a method for treating diseases related to HDAC6 activity, which comprises: administering a therapeutically effective amount of a pharmaceutical composition containing the above-mentioned compound.

[0020] [Technical solution]

[0021] The present inventors have discovered a 1,3,4-oxadiazole derivative compound having histone deacetylase 6 (HDAC6) inhibitory activity and used the 1,3,4-oxadiazole derivative compound to inhibit or treat HDAC6 activity-related diseases, thereby completing the present invention.

[0022] 1,3,4-Oxadiazole derivatives

[0023] In a general embodiment, the present invention provides a 1,3,4-oxadiazole derivative compound represented by the following Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0024] [Chemical Formula I]

[0025]

[0026] In the above chemical formula I,

[0027] R1 and R2 are each independently -(C1-C4 alkyl), or R1 and R2 are joined together with the N atom to form a heterocycloalkyl, wherein at least one H in the heterocycloalkyl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo or heterocycloalkyl;

[0028] X is -H or -F;

[0029] Y1 to Y5 are each independently N or CR3, wherein Y1 to Y5 cannot be 3 or more N at the same time;

[0030] R3 is -H, -(C1-C4 alkyl), -(C1-C4 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;

[0031] Z1 to Z4 are each independently N or CR4, wherein Z1 to Z4 cannot be 3 or more N at the same time; and

[0032] R4 is -H, -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, aryl or heteroaryl, wherein at least one -H in the aryl or heteroaryl ring can be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl) or halo.

[0033] According to one embodiment of the present invention, the compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt may be within the following range:

[0034] R1 and R2 are linked together with the N atom to form a heterocycloalkyl group, wherein at least one H in the heterocycloalkyl ring may be independently substituted by -(C1-C4 alkyl) or a heterocycloalkyl group;

[0035] X is -H or -F;

[0036] Y1 to Y5 are each independently CR3;

[0037] 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;

[0038] Z1 to Z4 are each independently N or CR4, wherein Z1 to Z4 cannot be 3 or more N at the same time; and

[0039] R4 is -H or -halo.

[0040] According to one embodiment of the present invention, the compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt may be within the following range:

[0041] R1 and R2 are linked together with the N atom to form a 4- to 12-membered heterocycloalkyl group, wherein at least one H in the heterocycloalkyl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo or 3- to 6-membered heterocycloalkyl.

[0042] According to one embodiment of the present invention, the compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt may be within the following range:

[0043] R1 and R2 are connected together with the N atom to form in At least one H in the ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo;

[0044] W is NR5, O, S or S(=O)2;

[0045] R5 is -(C1-C4 alkyl) or 3- to 6-membered heterocycloalkyl; and

[0046] n1, n2, m1 and m2 are each independently 0, 1 or 2.

[0047] According to one embodiment of the present invention, the compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt may be within the following range:

[0048] R1 and R2 are connected together with the N atom to form in At least one H in the ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl) or -halo; and

[0050] R5 is -(C1-C4 alkyl) or

[0051] In addition, according to one embodiment of the present invention, the specific compound represented by the chemical formula I of the present invention is shown in Table 1 below: [Table 1]

[0052]

[0053]

[0054]

[0055] In the present invention, unless otherwise specified, the term "alkyl" used herein may refer to a linear or branched non-cyclic, cyclic or saturated hydrocarbon connected by carbon atoms. 1-4 "Alkyl" may mean an alkyl group containing 1 to 4 carbon atoms. The non-cyclic alkyl group may include, for example, methyl, ethyl, n-propyl, n-butyl, isopropyl, sec-butyl, isobutyl, tert-butyl and the like, but is not limited thereto. Cyclic alkyl groups may be used interchangeably with "cycloalkyl" in the present specification, and may include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like, but are not limited thereto.

[0056] In the present invention, "alkoxy" may refer to -(O-alkyl) as an alkyl ether group, wherein the alkyl group has the same meaning as above. 1-4 "Alkoxy" may refer to 1-4 Alkoxy of alkyl, i.e. -(OC 1-4 and examples of alkoxy groups may include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

[0057] In the present invention, "halo" may be F, Cl, Br or I.

[0058] In the present invention, the term "haloalkyl" may refer to a straight or branched alkyl (hydrocarbon) having a carbon atom substituted with at least one halogen as defined herein. Examples of haloalkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl or n-butyl independently substituted with at least one halogen such as F, Cl, Br or I.

[0059] In the present invention, the term "hydroxyalkyl" may refer to a straight or branched 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 independently substituted with at least one hydroxyl group.

[0060] In the present invention, the term "aminoalkyl" may refer to a straight or branched alkyl (hydrocarbon) group having a carbon atom substituted by an amino group-(NR'R"). Herein, R' and R" may each be independently selected from hydrogen and C 1-4 The selected R' and R" may be independently substituted or unsubstituted.

[0061] In the present invention, the term "heterocycloalkyl" may mean a ring containing 1 to 5 heteroatoms selected from N, O and S as atoms forming the ring, and may be saturated or partially unsaturated. Herein, when unsaturated, the heterocycloalkyl may be referred to as heterocycloalkene. Unless otherwise specified, the heterocycloalkyl may be a monocyclic or polycyclic ring, such as a spirocyclic ring, a bridged ring or a condensed ring. In addition, "3 to 12 membered heterocycloalkyl" may mean a heterocycloalkyl containing 3 to 12 atoms forming the ring. Examples of heterocycloalkyl may include, but are not limited to, pyrrolidine, piperidine, imidazolidinyl, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4 (1H, 3H) -dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, piperan, Pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, tropane, 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.

[0062] In the present invention, "aromatic hydrocarbon" may mean an aromatic hydrocarbon ring. The aromatic hydrocarbon may be a monocyclic aromatic hydrocarbon or a polycyclic aromatic hydrocarbon. The number of ring carbon atoms in the 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 may include, but are not limited to, benzene, naphthalene, fluorene, anthracene, phenanthrene, biphenyl, terphenyl, quaterphenyl, pentaphenyl, sexphenyl, diphenyl, pyrene, benzofluoranthene, In the present specification, a moiety obtained by removing one hydrogen atom from the above-mentioned "arene" is referred to as an "aryl group".

[0063] In the present invention, "heteroaromatic hydrocarbon" may be a ring containing one or more of O, N, P, Si and S as heteroelements. The number of ring carbon atoms of heteroaromatic hydrocarbons may be 2 or more and 30 or less or 2 or more and 20 or less. Heteroaromatic hydrocarbons may be monocyclic heteroaromatic hydrocarbons or polycyclic heteroaromatic hydrocarbons. 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, oxazole, isothiazole, oxadiazole, triazole, pyridine, bipyridine, triazine, acridinyl, pyridazine, pyrazine, quinoline, quinazoline, quinoxaline, phenoxazine, pyrazine, pyrimidine, pyridopyrimidine, pyridopyrazine, pyrazinopyrazine, isoquinoline, indole, carbazole, imidazopyridazine, imidazopyridine, imidazopyridine, pyrazolopyrimidine, pyrazolopyrazine, isoquinoline, indole, carbazole, imidazopyridazine, imidazopyridine, imidazopyridine, pyrazolopyrimidine , imidazopyrazine or pyrazolopyridine, N-arylcarbazole, N-heteroarylcarbazole, N-alkylcarbazole, benzoxazole, benzimidazole, benzothiazole, benzocarbazole, benzothiophene, dibenzothiophene, thienothiophene, benzofuran, phenanthroline, isoxazole, oxadiazole, thiadiazole, benzothiazole, tetrazole, phenothiazine, dibenzosilole, dibenzofuran and the like. In one embodiment of the present invention, the heteroaromatic hydrocarbon may also include a bicyclic heteroaromatic hydrocarbon, including an aromatic hydrocarbon ring fused to a heterocycloalkyl ring or a heteroaromatic hydrocarbon fused to a cycloalkyl ring. In the present specification, the portion obtained by removing a hydrogen atom from the above-mentioned "heteroaromatic hydrocarbon" is referred to as a "heteroaryl group".

[0064] The compound represented by the chemical formula I of the present invention may contain at least one asymmetric carbon, and thus may exist in the form of a racemate, a racemic mixture, a single mirror image isomer, a mixture of diastereomers, and each diastereomer. Such stereoisomers can be separated by conventional techniques, and for example, the compound represented by the chemical formula I can be separated by column chromatography, HPLC, or the like. In addition, each stereoisomer of the compound represented by the chemical formula I can be stereospecifically synthesized using optically pure starting materials and / or reagents having known configurations.

[0065] In the present invention, the term "mirror image isomer" as used herein means a compound or a salt thereof having the same chemical formula or molecular formula but different in spatial arrangement. Each of such mirror image isomers and mixtures thereof are also included within the scope of the present invention. Unless otherwise specified, a real bond (-) connected to an asymmetric carbon atom may include a wedge-shaped real bond indicating the absolute arrangement of a stereocenter. or wedge-shaped virtual key

[0066] The compound represented by the chemical formula I of the present invention may exist in the form of a "pharmaceutically acceptable salt". As the salt, an acid addition salt formed from a pharmaceutically acceptable free acid is useful. The term "pharmaceutically acceptable salt" as used herein refers to a concentration having a relatively non-toxic and harmless effective effect on patients, which includes any organic acid or inorganic acid addition salt of the compound represented by the chemical formula I, wherein the side effects caused by such salt do not reduce the beneficial effects of the compound.

[0067] Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and the acid or alcohol may be heated in water and the mixture evaporated to dryness, or the precipitated salt may be filtered off with suction.

[0068] In this case, for the free acid, an organic acid and an inorganic acid may be used, wherein the inorganic acid may include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid and the like, and the organic acid 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, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like. However, the organic acid and the inorganic acid are not limited thereto.

[0069] In addition, pharmaceutically acceptable metal salts can be prepared using a base. Alkali metal salts or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble composite salt, and then evaporating and drying the filtrate. Here, for metal salts, sodium, potassium or calcium salts are particularly suitable for pharmaceutical purposes, but metal salts are not limited thereto. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0070] Unless otherwise indicated, the pharmaceutically acceptable salts of the present invention include salts of acidic or basic groups that may be present in the compounds represented by the above Chemical Formula I. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxyl groups, while other pharmaceutically acceptable salts of amino groups may include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (toluenesulfonate) salts and the like, which can be prepared by salt preparation methods known in the art.

[0071] Method for preparing 1,3,4-oxadiazole derivative compounds

[0072] The present invention provides a method for preparing a 1,3,4-oxadiazole derivative compound represented by the following chemical formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0073] [Chemical Formula I]

[0074]

[0075] The chemical formula I is as defined above.

[0076] In the present invention, a preferred method for preparing an oxadiazole derivative compound represented by Chemical Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof is shown in [Reaction Scheme 1] to [Reaction Scheme 4], which may also include a preparation method modified to a level obvious to those skilled in the art.

[0077]

[0078] The above-mentioned [reaction flow 1] is a method for synthesizing a 1,3,4-oxadiazole derivative compound having a cyclobutenedione structure. First, the amino-containing compound represented by Chemical Formula 1-1 is reacted with the compound represented by Chemical Formula 1-2 to prepare the compound represented by Chemical Formula 1-3 containing an amino substituent. Then, the compound represented by Chemical Formula 1-3 is reacted with the compound represented by Chemical Formula 1-4 to prepare the compound represented by Chemical Formula 1-5 having a cyclobutenedione structure containing a diamino substituent. The compound represented by Chemical Formula 1-5 and the compound represented by Chemical Formula 1-6 are subjected to a substitution reaction to prepare the compound represented by Chemical Formula 1-7. At the same time, 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 the compound represented by Chemical Formula 1-8, and then reacting with the compound represented by Chemical Formula 1-1. In the present invention, compounds 2, 3, 4, 5, 6, 7, 10 and 24 can be prepared by the above reaction scheme 1.

[0079] [Reaction Scheme 2]

[0080]

[0081] The above [reaction flow 2] is another method for synthesizing a 1,3,4-oxadiazole derivative compound having a cyclobutenedione structure. First, a compound represented by chemical formula 2-1 is reacted with a compound represented by chemical formula 1-2 to prepare a compound represented by chemical formula 2-2 containing an amino substituent. Then, a compound represented by chemical formula 2-2 is reacted with a compound represented by chemical formula 1-4 to prepare a compound represented by chemical formula 1-7 having a cyclobutenedione structure containing a diamino substituent. In the present invention, compound 1 and the like can be prepared by the above reaction flow 2.

[0082] [Reaction Scheme 3]

[0083]

[0084] The above [Reaction Scheme 3] is another method for synthesizing a 1,3,4-oxadiazole derivative compound having a cyclobutenedione structure. The compound represented by Chemical Formula 1-7 prepared in [Reaction Scheme 1] and the compound represented by Chemical Formula 3-1 are subjected to CC coupling (Suzuki reaction) to prepare a compound represented by Chemical Formula 3-2. In the present invention, compounds 8 and 9 and the like can be prepared by the above Reaction Scheme 3.

[0085] [Reaction Scheme 4]

[0086]

[0087] The above [reaction flow 4] is another method for synthesizing a 1,3,4-oxadiazole derivative compound having a cyclobutenedione structure. The compound represented by chemical formula 4-2 is prepared by removing the protective group from the compound represented by chemical formula 4-1 prepared in [reaction flow 1]. Then, an alkylation reaction or a reduction reaction is used to prepare a compound represented by chemical formula 4-3. 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 a compound represented by chemical formula 4-4. In the present invention, the above reaction flow 4 can be used to prepare the compound 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 compound represented by chemical formula 4-4, i.e., compounds 18 and 19 and the like.

[0088] Uses of 1,3,4-oxadiazole derivatives

[0089] The present invention provides the use of a compound represented by the following chemical formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0090] [Chemical Formula I]

[0091]

[0092] The chemical formula I is as defined above.

[0093] According to one embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diseases related to histone deacetylase 6 activity, which comprises a compound represented by chemical formula I as described above, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition of the present invention selectively inhibits histone deacetylase 6, thereby exhibiting a significant effect in preventing or treating diseases related to histone deacetylase 6 activity.

[0094] The histone deacetylase 6 activity-related diseases include infectious diseases, such as prion disease; neoplasms, such as benign tumors (e.g., myelodysplastic syndrome) or malignant tumors (e.g., multiple myeloma, lymphoma, leukemia, lung cancer, colorectal cancer, colon cancer, prostate cancer, urothelial cell 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, amyloid degeneration 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)), neurodegenerative diseases (e.g., Alzheimer's disease disease), 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 eye adnexal diseases, such as uveitis; circulatory system diseases, such as atrial fibrillation or stroke; respiratory tract diseases, such as asthma; digestive system diseases, such as alcoholic liver disease, inflammatory bowel disease, Crohn's disease, disease), ulcerative bowel disease 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, changes and chromosomal abnormalities, such as autosomal dominant polycystic kidney disease, and in addition include symptoms or diseases related to abnormal function of histone deacetylase.

[0095] The stereoisomers and pharmaceutically acceptable salts are as described above in the stereoisomers and pharmaceutically acceptable salts of the compound represented by Chemical Formula I of the present invention.

[0096] For administration, in addition to the compound represented by Formula I, its stereoisomer or pharmaceutically acceptable salt, the pharmaceutical composition of the present invention may further include at least one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol or a mixture of one or more of these ingredients, and (if necessary) may contain other well-known additives such as antioxidants, buffers, antibacterial agents and the like. In addition, diluents, dispersants, surfactants, adhesives 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 composition of the present invention may be a patch, a liquid, a pill, a capsule, a granule, a tablet, a suppository and the like. Such formulations can be prepared by well-known methods used in the art for formulations or by methods disclosed in documents [see, Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA], and can be formulated into a variety of formulations according to each disease or component.

[0097] The composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is changed according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the disease. The daily dosage of the compound represented by the 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 batches and administered once or more daily.

[0098] In addition to the compound represented by Chemical Formula I, its stereoisomer or pharmaceutically acceptable salt, the pharmaceutical composition of the present invention may further include at least one active ingredient exhibiting the same or similar efficacy.

[0099] According to one embodiment of the present invention, the present invention provides a method for preventing or treating a disease related to histone deacetylase 6 activity, the method comprising: administering to an individual in need thereof a therapeutically effective amount of a compound represented by Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The individual may be a mammal (including a human).

[0100] The method for preventing or treating a disease associated with histone deacetylase 6 activity includes not only treating the disease itself before the onset of symptoms, but also inhibiting or avoiding its symptoms by administering a compound represented by Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. In addition, the method for preventing or treating a disease associated with histone deacetylase 6 activity of the present invention may further administer a therapeutically effective amount of an additional active agent that helps treat the disease together with the compound represented by Chemical Formula I, wherein the additional active agent may exhibit a synergistic or auxiliary effect with the above-mentioned compound represented by Chemical Formula I.

[0101] The term "therapeutically effective amount" used herein refers to the amount of the compound represented by chemical formula I that is effective for treating or preventing histone deacetylase 6 activity-related diseases. Specifically, "medicine effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for drug therapy, and the effective dose can be determined according to factors including individual type and severity, age, sex, disease type, drug activity, sensitivity to drugs, administration time, administration route, excretion rate, treatment duration, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered in individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. In addition, the pharmaceutical composition of the present invention can be administered in a single dose or in multiple doses. Taking all the above factors into account, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention can be determined by experts according to a variety of factors (such as the patient's illness, age, sex, complications, and the like). Because the active ingredient of the pharmaceutical composition of the present invention has excellent safety, it can even be used at a higher dose than the predetermined dose.

[0102] According to one embodiment of the present invention, the present invention provides a method for selectively inhibiting histone deacetylase 6 (HDAC6) by administering a compound represented by Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a mammal (including a human).

[0103] According to one embodiment of the present invention, the present invention provides the use of a compound represented by Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease associated with the activity of histone deacetylase 6. The compound represented by Chemical Formula I for the manufacture of a medicament can be mixed with an acceptable adjuvant, diluent, carrier, and the like, and can be prepared as a combined preparation with other active agents to have a synergistic effect of the active ingredients.

[0104] Matters mentioned in the uses, compositions and methods of treatment of the present invention are equally applicable unless they conflict with each other.

[0105] The exemplary embodiments of the present invention can be modified in a variety of other forms, and the scope of the present invention is not limited to the exemplary embodiments described below. In addition, exemplary embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. In addition, unless otherwise stated, "comprising" a component throughout this specification does not mean excluding other components, but means that other components may be further included.

[0106] [Beneficial Effects]

[0107] The compound represented by Chemical Formula I, its stereoisomer or a pharmaceutically acceptable salt thereof according to the present invention can selectively inhibit HDAC6, thereby having a significant and excellent preventive or therapeutic effect on diseases related to histone deacetylase 6 activity. DETAILED DESCRIPTION

[0108] The present invention will be described in more detail below by examples and experimental examples. However, these examples and the like are only presented as examples of the present invention, and the scope of the present invention is not limited to these examples.

[0109] Preparation of 1,3,4-oxadiazole derivatives

[0110] A specific method for preparing the compound represented by Chemical Formula I is described below.

[0111] Example 1: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 1)

[0112] [Step 1] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4- Synthesis of Methoxycyclobut-3-ene-1,2-dione

[0113]

[0114] N-(4-(5-(difluoromethyl)-1,3,4-oxadiazole-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. Then, the reaction was completed by lowering the reaction temperature to room temperature. The reaction mixture was filtered through a glass filter, the solid was removed therefrom, a filtrate was obtained, and the solvent was removed from the filtrate under reduced pressure. Then, the concentrate was purified and concentrated by column chromatography (SiO2, 4 g cartridge; ethyl acetate / hexane=10% to 50%) to obtain the title compound (0.069 g, 25.7%) as a yellow oil.

[0115] [Step 2] Synthesis of Compound 1

[0116]

[0117] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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 by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane=0% to 5%) and concentrated to obtain the title compound (0.016 g, 13.8%) as a pale yellow solid.

[0118] 1 H NMR (400MHz, CDCl3) δ7.88(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).

[0119] Example 2: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-morpholinylcyclobut-3-ene-1,2-dione (Compound 2)

[0120] [Step 1] Synthesis of 3-morpholino-4-(phenylamino)cyclobut-3-ene-1,2-dione

[0121]

[0122] 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 poured into 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 and the precipitated solid was filtered, washed with diethyl ether and dried to obtain the title compound (0.445 g, 70.0%) as a white solid.

[0123] [Step 2] Synthesis of Compound 2

[0124]

[0125] 3-Morpholinyl-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-oxadiazole (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. Saturated aqueous sodium bicarbonate solution was poured into the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; ethyl acetate / hexanes = 20% to 70%) and concentrated to afford the title compound (0.019 g, 10.1%) as a white solid.

[0126] 1 H NMR (400MHz, CDCl3) δ7.88(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).

[0127] Example 3: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(1,1-dioxothiomorpholinyl)cyclobut-3-ene-1,2-dione (Compound 3)

[0128] [Step 1] Synthesis of 3-(1,1-dioxothiomorpholinyl)-4-(phenylamino)cyclobut-3-ene-1,2-dione

[0129]

[0130] 3-Methoxy-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 2.461 mmol), thiomorpholine 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 poured into the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with 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 obtain the title compound (0.554 g, 73.5%) as a light yellow solid.

[0131] [Step 2] Synthesis of compound 3

[0132]

[0133] 3-(1,1-dioxothiomorpholinyl)-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-oxadiazole (0.110 g, 0.359 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 sodium chloride solution was poured into 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 (II) chloride, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; ethyl acetate / hexane = 10% to 40%) and concentrated to afford the title compound (0.023 g, 13.2%) as a light yellow solid.

[0134] 1H NMR (400MHz, CDCl3) δ7.89(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),7.3 1(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); LRMS(ES)m / z533.5(M + +1).

[0135] Example 4: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 4)

[0136] [Step 1] Synthesis of tert-butyl 4-(2-methoxy-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate

[0137]

[0138] 3,4-Dimethoxycyclobut-3-ene-1,2-dione (1.520 g, 10.696 mmol) and tert-butyl piperazine-1-carboxylate (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.

[0139] [Step 2] Tert-butyl 4-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)piperazine-1-carboxylate synthesis

[0140]

[0141] The tert-butyl 4-(2-methoxy-3,4-dioxocyclobut-1-ene-1-yl)piperazine-1-carboxylate (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 poured into the concentrate, and then extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane=0% to 50%) to obtain the title compound (0.300 g, 13.4%) as a yellow solid.

[0142] [Step 3] 4-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-3, Synthesis of tert-butyl 4-(4-dioxycyclobut-1-en-1-yl)piperazine-1-carboxylate

[0143]

[0144] Tert-butyl 4-(3,4-dioxo-2-(phenylamino)cyclobut-1-ene-1-yl)piperazine-1-carboxylate (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-oxadiazole (0.17 g, 0.59 mmol) was added to the reaction mixture and stirred at room temperature for another 3 hours. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. Water was poured into the concentrate, and then extracted with ethyl acetate. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexanes = 0% to 50%) and concentrated to afford the title compound (0.135 g, 48.5%) as a white solid.

[0145] [Step 4] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-4-(piperidin Synthesis of 2,2,2-trifluoroacetate of (1-oxazine-1-yl)cyclobut-3-ene-1,2-dione

[0146]

[0147] Tert-butyl 4-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (0.135 g, 0.239 mmol) prepared in step 3 and trifluoroacetic acid (0.183 mL, 2.387 mmol) 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 obtained product was used without further purification (0.150 g, 108.4%, yellow oil).

[0148] [Step 5] Synthesis of Compound 4

[0149]

[0150] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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) prepared in step 4, 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) were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was poured into the reaction mixture, which was then extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 10%) and concentrated to afford the title compound (0.066 g, 53.2%) as a white solid.

[0151] 1 H NMR (400MHz, CDCl3) δ8.05(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,0.25H),6.98(d,J=7.7Hz,2H),6.92(s,0.5H),6.79(s,0.25H),5.50(s,2H),3.28(br s,4H),2.26(s,4H),2.22(s,3H); LRMS(ES)m / z 480.5(M + +1).

[0152] Example 5: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-4-(1,1-dioxothiomorpholinyl)cyclobut-3-ene-1,2-dione (Compound 5)

[0153]

[0154] 3-(1,1-dioxothiomorpholinyl)-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-oxadiazole (0.104 g, 0.359 mmol) was added to the reaction solution and stirred at the same temperature for 3 hours. Water was poured into the reaction mixture, and then extracted with dichloromethane. The extract was then filtered through a plastic filter to remove the solid residue and the aqueous layer and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; ethyl acetate / hexane=10% to 40%) and concentrated. 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.

[0155] 1 H NMR(400MHz,DMSO-d6)δ8.00(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 / z515.4(M + +1).

[0156] Example 6: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-4-(1,1-dioxothiomorpholinyl)cyclobut-3-ene-1,2-dione (Compound 6)

[0157]

[0158] 3-(1,1-Dioxothiomorpholinyl)-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. To the reaction solution was added 2-(6-(bromomethyl)-5-(fluoropyridin-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.111 g, 0.359 mmol) and stirred at the same temperature for 3 hours. Water was poured into the reaction mixture, and the mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; ethyl acetate / hexane=10% to 40%) and concentrated. Ethyl acetate and diethyl ether were added to the resulting product and stirred to precipitate a solid and the precipitated solid was filtered, washed with diethyl ether and dried to obtain the title compound (0.017 g, 9.8%) as a pale yellow solid.

[0159] 1 H NMR(400MHz, DMSO-d6)δ8.99(s,1H),8.41(dd,J=9.9,1.5Hz,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); LRMS(ES)m / z 534.4(M + +1).

[0160] Example 7: Synthesis of 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 7)

[0161] [Step 1] Synthesis of 3-((3-bromo-5-fluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione

[0162]

[0163] 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.

[0164] [Step 2] 4-(2-((3-bromo-5-fluorophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-yl Synthesis of tert-Butyl Formate

[0165]

[0166] 3-((3-bromo-5-fluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (2.000 g, 6.665 mmol) prepared in step 1, tert-butyl piperazine-1-carboxylate (1.862 g, 9.997 mmol) and N,N-diisopropylethylamine (2.322 mL, 13.330 mmol) 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 by column chromatography (SiO2, 40 g cartridge; ethyl acetate / hexane=0% to 30%) and concentrated to obtain the title compound (2.900 g, 95.8%) as a black foamy solid.

[0167] [Step 3] 4-(2-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluoro Synthesis of tert-butyl (( ...

[0168]

[0169] Tert-butyl 4-(2-((3-bromo-5-fluorophenyl)amino)-3,4-dioxocyclobut-1-ene-1-yl)piperazine-1-carboxylate (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-oxadiazole (2.548 g, 8.298 mmol) was added to the reaction mixture and stirred at room temperature for another 3 hours. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 40 g cartridge; ethyl acetate / hexane = 0% to 50%) and concentrated to afford the title compound (2.000 g, 46.0%) as a yellow foamy solid.

[0170] [Step 4] 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl Synthesis of 2,2,2-trifluoroacetate of 4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione

[0171]

[0172] Tert-butyl 4-(2-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (2.000 g, 2.939 mmol) prepared in step 3 and trifluoroacetic acid (2.251 mL, 29.392 mmol) were dissolved in dichloromethane (20 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Water was poured into the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained product was used without further purification (2.000 g, 98.0%, brown oil).

[0173] [Step 5] Synthesis of Compound 7

[0174]

[0175] 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-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) prepared in step 4, 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) were dissolved in dichloromethane (20 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was poured into the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 40 g cartridge; methanol / dichloromethane = 0% to 10%) and concentrated to afford the title compound (1.500 g, 87.6%) as a yellow solid.

[0176] 1H NMR(400MHz, CDCl3) δ7.92(dd,J=8.0,1.5Hz,1H),7.85(dd,J=10.2,1.5Hz,1H),7.59(t,J=7.7Hz,1H),7.06-7.0 2(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).

[0177] Example 8: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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)

[0178]

[0179] 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-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]dichloropalladium(II) (Pd(dtbpf)Cl2, 0.022 g, 0.034 mmol) and cesium carbonate (0.219 g, 0.673 mmol) prepared in Example 7 were mixed with 1,4-dioxane (9 mL) / water (3 mL) and heated at 100°C for 20 minutes by microwave irradiation. Then, the reaction was completed by lowering the reaction temperature to room temperature. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 20%) and concentrated to obtain the title compound (0.080 g, 40.1%) as a brown oil.

[0180] 1H NMR (400MHz, CDCl3) δ8.78(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),7.4 3(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).

[0181] Example 9: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(6-(oxetane-3-yl)-2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 9)

[0182]

[0183] 3-((3-bromo-5-fluorophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-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]dichloropalladium(II) (Pd(dtbpf)Cl2, 0.022 g, 0.034 mmol) and cesium carbonate (0.219 g, 0.673 mmol) prepared in Example 7 were mixed with 1,4-dioxane (9 mL) / water (3 mL) and heated at 100°C for 20 minutes by microwave irradiation. Then, the reaction was completed by lowering the reaction temperature to room temperature. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 20%) and concentrated to obtain the title compound (0.070 g, 35.1%) as a brown oil.

[0184] 1H NMR (400MHz, CDCl3) δ8.73(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,2 H),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).

[0185] Example 10: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(6-(oxetane-3-yl)-2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 10)

[0186] [Step 1] 6-(2-methoxy-3,4-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane- Synthesis of tert-butyl 2-formate

[0187]

[0188] 3,4-Dimethoxycyclobut-3-ene-1,2-dione (1.710 g, 12.033 mmol) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (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.

[0189] [Step 2] 6-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,6-diazaspiro[3.3] Synthesis of tert-butyl heptane-2-carboxylate

[0190]

[0191] 6-(2-methoxy-3,4-dioxocyclobut-1-ene-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl 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 poured into the concentrate, and the reaction mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexanes = 0% to 50%) and concentrated to afford the title compound (0.600 g, 71.5%) as a white solid.

[0192] [Step 3] 6-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino -3,4- Synthesis of tert-butyl (2-(2-oxo)cyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate

[0193]

[0194] Tert-butyl 6-(3,4-dioxo-2-(phenylamino)cyclobut-1-ene-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (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-oxadiazole (0.685 g, 2.232 mmol) was added to the reaction mixture and stirred at room temperature for another 3 hours. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexanes = 0% to 50%) and concentrated to afford the title compound (0.700 g, 63.2%) as a white solid.

[0195] [Step 4] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4- Synthesis of (2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione

[0196]

[0197] Tert-butyl 6-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (0.700 g, 1.175 mmol) prepared in step 3 and trifluoroacetic acid (0.900 mL, 11.753 mmol) were dissolved in dichloromethane (10 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution was poured into the reaction mixture, which was then extracted with dichloromethane. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained product was used without further purification (0.400 g, 68.7%, white solid).

[0198] [Step 5] Synthesis of compound 10

[0199]

[0200] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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-oxetanone (0.029 g, 0.404 mmol) and sodium triacetoxyborohydride (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 poured into the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 20%) and concentrated to obtain the title compound (0.080 g, 71.9%) as a colorless oil.

[0201] 1H NMR (400MHz, CDCl3) δ7.84(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),7.0 6-7.04(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).

[0202] Example 11: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-4-(6-methyl-2,6-diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 11)

[0203] [Step 1] 6-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-3,4- Synthesis of tert-butyl (1-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate

[0204]

[0205] Tert-butyl 6-(3,4-dioxo-2-(phenylamino)cyclobut-1-ene-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (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-oxadiazole (0.166 g, 0.575 mmol) was added to the reaction mixture and stirred at room temperature for another 3 hours. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexanes = 0% to 50%) and concentrated to afford the title compound (0.150 g, 54.2%) as a white solid.

[0206] [Step 2] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-4-(2,6- Synthesis of diazaspiro[3.3]hept-2-yl)cyclobut-3-ene-1,2-dione 2,2,2-trifluoroacetate

[0207]

[0208] Tert-butyl 6-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (0.180 g, 0.312 mmol) prepared in step 1 and trifluoroacetic acid (0.239 mL, 3.116 mmol) 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 obtained product was used without further purification (0.180 g, 97.6%, yellow oil).

[0209] [Step 3] Synthesis of compound 11

[0210]

[0211] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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) prepared in step 2, 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) were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was poured into the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 20%) and concentrated to afford the title compound (0.056 g, 37.4%) as a white solid.

[0212] 1 H NMR (400MHz, CDCl3) δ8.02 (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).

[0213] Example 12: Synthesis of 3-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 12)

[0214] [Step 1] Synthesis of 3-((3-bromophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione

[0215]

[0216] 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.

[0217] [Step 2] tert-4-(2-((3-bromophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylic acid Synthesis of Butyl Ester

[0218]

[0219] 3-((3-bromophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (1.250 g, 4.431 mmol) prepared in step 1, tert-butyl piperazine-1-carboxylate (1.238 g, 6.647 mmol) and N,N-diisopropylethylamine (1.544 mL, 8.862 mmol) 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.

[0220] [Step 3] 4-(2-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl) Synthesis of tert-butyl (amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate

[0221]

[0222] Tert-butyl 4-(2-((3-bromophenyl)amino)-3,4-dioxocyclobut-1-ene-1-yl)piperazine-1-carboxylate (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-oxadiazole (1.013 g, 3.300 mmol) was added to the reaction mixture and stirred at room temperature for another 3 hours. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 40 g cartridge; ethyl acetate / hexanes = 0% to 50%) and concentrated to obtain the title compound (1.400 g, 76.8%) as a colorless oil.

[0223] [Step 4] 3-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)amino Synthesis of 4-(piperazine-1-yl)cyclobut-3-ene-1,2-dione 2,2,2-trifluoroacetate

[0224]

[0225] Tert-butyl 4-(2-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (1.400 g, 2.113 mmol) prepared in step 3 and trifluoroacetic acid (1.618 mL, 21.13 mmol) were dissolved in dichloromethane (20 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 obtained product was used without further purification (1.400 g, 97.9%, brown oil).

[0226] [Step 5] Synthesis of compound 12

[0227]

[0228] 3-((3-bromophenyl)(4-(5-(difluoromethyl)-1,3,4-oxadiazol-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) prepared in step 4, 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) were dissolved in dichloromethane (10 mL) at room temperature. The reaction solution was stirred at the same temperature for 18 hours. Water was poured into the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 10%) and concentrated to afford the title compound (0.900 g, 75.4%) as a white solid.

[0229] 1 H NMR (400MHz, CDCl3) δ7.89(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=8.0,1.5Hz,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 / z577.4(M + +1).

[0230] Example 13: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-((1S,4S)-5-(oxetane-3-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 13)

[0231] [Step 1] (1S,4S)-5-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,5-diazepine Synthesis of tert-butyl bicyclo[2.2.1]heptane-2-carboxylate

[0232]

[0233] 3-Methoxy-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 2.461 mmol), (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl 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 by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane=20% to 40%) and concentrated to obtain the title compound (0.871 g, 95.8%) as a pale yellow solid.

[0234] [Step 2] (1S,4S)-5-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(benzene tert-butyl)-3,4-dioxocyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate synthesis

[0235]

[0236] Tert-butyl (1S,4S)-5-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.300 g, 0.812 mmol), 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.274 g, 0.893 mmol), potassium carbonate (0.224 g, 1.624 mmol) and potassium iodide (0.013 g, 0.081 mmol) prepared in step 1 were dissolved in N,N-dimethylformamide (4 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. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous sodium bicarbonate solution was poured into the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane = 30% to 60%) and concentrated to obtain the title compound (0.315 g, 65.1%) as a yellow solid.

[0237] [Step 3] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-((4-(5-(difluoromethyl)- Synthesis of (1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)cyclobut-3-ene-1,2-dione

[0238]

[0239] Tert-butyl (1S,4S)-5-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.315 g, 0.529 mmol) and trifluoroacetic acid (0.284 mL, 3.702 mmol) prepared in step 2 were dissolved in dichloromethane (4 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The obtained product was used without further purification (0.221 g, 84.3%, pale yellow solid).

[0240] [Step 4] Synthesis of compound 13

[0241]

[0242] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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. Sodium triacetoxyborohydride (0.086 g, 0.404 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 2.5%) and concentrated to afford the title compound (0.075 g, 67.4%) as a yellow solid.

[0243] 1H NMR (400MHz, CDCl3) δ7.87 (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 / z552.8(M + +1).

[0244] Example 14: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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)

[0245]

[0246] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)cyclobut-3-ene-1,2-dione (0.100 g, 0.202 mmol) and formaldehyde (38.00% solution, 0.022 mL, 0.303 mmol) prepared in Step 3 of Example 14 were dissolved in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.086 g, 0.404 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 5%) and concentrated to afford the title compound (0.049 g, 47.7%) as a yellow solid.

[0247] 1H NMR (400MHz, CDCl3) δ7.88(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).

[0248] Example 15: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-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)

[0249] [Step 1] (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl tert-(2-(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid Synthesis of Butyl Ester

[0250]

[0251] Tert-butyl (1S,4S)-5-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.500 g, 1.353 mmol), 2-(6-(bromomethyl)pyridin-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.432 g, 1.489 mmol), potassium carbonate (0.281 g, 2.030 mmol) and potassium iodide (0.022 g, 0.135 mmol) prepared in step 1 of Example 13 were dissolved in N,N-dimethylformamide (4 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. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. To the concentrate was poured a saturated aqueous sodium bicarbonate solution, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane=30% to 65%) and concentrated to obtain the title compound (0.440 g, 56.2%) as a light brown solid.

[0252] [Step 2] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)- Synthesis of (1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-1,2-dione

[0253]

[0254] Tert-butyl (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.440 g, 0.760 mmol) and trifluoroacetic acid (0.291 mL, 3.802 mmol) prepared in step 1 were dissolved in dichloromethane (4 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The obtained product was used without further purification (0.277 g, 76.1%, brown solid).

[0255] [Step 3] Synthesis of compound 15

[0256]

[0257] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-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. Sodium triacetoxyborohydride (0.080 g, 0.376 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 5%) and concentrated to afford the title compound (0.054 g, 58.3%) as an orange solid.

[0258] 1H NMR(400MHz, CDCl3) δ9.25(d,J=2.0Hz,1H),8.37(dd,J=8.2,2.2Hz,1H),7.64(d,J=8.2H z,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),2.1 6-2.14(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).

[0259] Example 16: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-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)

[0260]

[0261] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-1,2-dione (0.090 g, 0.188 mmol) prepared in step 2 of Example 15 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. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 5%) and concentrated to afford the title compound (0.056 g, 57.2%) as an orange solid.

[0262] 1H NMR (400MHz, CDCl3) δ9.25 (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=7.7H z,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).

[0263] Example 17: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-(oxetane-3-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 17)

[0264]

[0265] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-1,2-dione (0.100 g, 0.209 mmol) and 3-oxetanone (0.018 mL, 0.314 mmol) prepared in Step 2 of Example 15 were dissolved in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.089 g, 0.418 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 2.5%) and concentrated to afford the title compound (0.036 g, 32.2%) as an orange solid.

[0266] 1H NMR (400MHz, CDCl3) δ9.27 (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).

[0267] Example 18: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-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)

[0268]

[0269] 3-((3-bromophenyl)((5-(5-(difluoromethyl)-1,3,4-oxadiazol-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]dichloropalladium(II) (Pd(dtbpf)Cl2, 0.029 g, 0.045 mmol) and cesium carbonate (0.364 g, 1.117 mmol) were mixed with 1,4-dioxane (9 mL) / water (3 mL) and heated at 100°C for 20 hours by microwave irradiation. Then, the reaction was completed by lowering the reaction temperature to room temperature. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 10%) and concentrated to obtain the title compound (0.100 g, 40.1%) as a brown oil.

[0270] 1H NMR (400MHz, CDCl3) δ8.78(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 / z558.4(M + +1).

[0271] Example 19: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)(3-(pyridin-4-yl)phenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 19)

[0272]

[0273] 3-((3-bromophenyl)((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)methyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-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]dichloropalladium(II) (Pd(dtbpf)Cl2, 0.023 g, 0.036 mmol) and cesium carbonate (0.291 g, 0.894 mmol) were mixed with 1,4-dioxane (9 mL) / water (3 mL) and heated at 100°C for 15 minutes by microwave irradiation. Then, the reaction was completed by lowering the reaction temperature to room temperature. Water was poured into the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% to 10%) and concentrated to obtain the title compound (0.060 g, 30.1%) as a black oil.

[0274] 1H NMR (400MHz, CDCl3) δ8.71(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(brs,4H),3.16(s,3H); LRMS(ES)m / z 558.4(M + +1).

[0275] Example 20: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-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)

[0276] [Step 1] (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-3-fluoropyridine-2-yl) (phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-yl Synthesis of tert-Butyl Formate

[0277]

[0278] Tert-butyl (1S,4S)-5-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.500 g, 1.353 mmol), 2-(bromomethyl)-3-fluoro-5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridine (0.459 g, 1.489 mmol), potassium carbonate (0.281 g, 2.030 mmol) and potassium iodide (0.022 g, 0.135 mmol) prepared in step 1 of Example 13 were dissolved in N,N-dimethylformamide (4 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. The solvent was removed from the reaction mixture under reduced pressure to obtain a concentrate. To the concentrate was poured a saturated aqueous sodium bicarbonate solution, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane=30% to 70%) and concentrated to obtain the title compound (0.285 g, 35.3%) as a brown solid.

[0279] [Step 2] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)- Synthesis of (1,3,4-oxadiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-1,2-dione

[0280]

[0281] Tert-butyl (1S,4S)-5-(2-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.285 g, 0.478 mmol) prepared in step 1 and trifluoroacetic acid (0.256 mL, 3.344 mmol) were dissolved in dichloromethane (4 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; ethyl acetate / hexanes = 0% to 30%) and concentrated to afford the title compound (0.206 g, 86.9%) as a light brown solid.

[0282] [Step 3] Synthesis of Compound 20

[0283]

[0284] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-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. Sodium triacetoxyborohydride (0.085 g, 0.403 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 5%) and concentrated to afford the title compound (0.067 g, 65.2%) as a yellow solid.

[0285] 1H NMR (400MHz, CDCl3) δ9.05 (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).

[0286] Example 21: Synthesis of 3-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)-4-((1S,4S)-5-(oxetane-3-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl)cyclobut-3-ene-1,2-dione (Compound 21)

[0287]

[0288] 3-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-(((5-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-3-fluoropyridin-2-yl)methyl)(phenyl)amino)cyclobut-3-ene-1,2-dione (0.100 g, 0.201 mmol) and 3-oxetanone (0.019 mL, 0.302 mmol) prepared in step 2 of Example 20 were dissolved in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.085 g, 0.403 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution was poured into the reaction mixture, which was then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 2.5%) and concentrated to afford the title compound (0.019 g, 17.1%) as a yellow solid.

[0289] 1H NMR (400MHz, CDCl3) δ9.09 (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,2 H),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).

[0290] Example 22: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(4-methylpiperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 22)

[0291] [Step 1] Synthesis of 3-((3,4-difluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione

[0292]

[0293] 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.

[0294] [Step 2] 4-(2-((3,4-difluorophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate Synthesis of tert-butyl ester

[0295]

[0296] 3-((3,4-difluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (1.500 g, 6.271 mmol) prepared in step 2, tert-butyl piperazine-1-carboxylate (1.168 g, 6.271 mmol) and N,N-diisopropylethylamine (1.092 mL, 6.271 mmol) 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, and the precipitated solid was filtered, washed with methanol and dried to obtain the title compound (1.770 g, 71.7%) as a light green solid.

[0297] [Step 3] 4-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(3,4-difluorobenzene Synthesis of tert-butyl (3,4-dioxy)cyclobut-1-en-1-yl)piperazine-1-carboxylate

[0298]

[0299] Tert-butyl 4-(2-((3,4-difluorophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (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-oxadiazole (0.429 g, 1.398 mmol) was added to the reaction solution and stirred at room temperature for 18 hours. After the solvent was removed from the reaction mixture under reduced pressure, the concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane=5% to 40%) and concentrated to obtain the title compound (0.516 g, 65.5%) as a pale yellow solid.

[0300] [Step 4] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl) Synthesis of 4-(amino)-4-(piperazin-1-yl)cyclobut-3-ene-1,2-dione

[0301]

[0302] Tert-butyl 4-(2-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (0.516 g, 0.833 mmol) prepared in step 3 and trifluoroacetic acid (0.510 mL, 6.663 mmol) were dissolved in dichloromethane (5 mL) at room temperature, and the reaction solution was stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution and dichloromethane were added to the reaction mixture and stirred to precipitate a solid, and the precipitated solid was filtered. The filtrate was washed with dichloromethane and dried to obtain the title compound (0.361 g, 83.4%) as a yellow solid.

[0303] [Step 5] Synthesis of Compound 22

[0304]

[0305] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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. Sodium triacetoxyborohydride (0.082 g, 0.385 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution 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 the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 5%) and concentrated to afford the title compound (0.044 g, 42.8%) as a white solid.

[0306] 1 H NMR (400MHz, CDCl3) δ7.90(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).

[0307] Example 23: Synthesis of 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(3,4-difluorophenyl)amino)-4-(4-(oxetane-3-yl)piperazin-1-yl)cyclobut-3-ene-1,2-dione (Compound 23)

[0308]

[0309] 3-((4-(5-(difluoromethyl)-1,3,4-oxadiazol-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 oxetan-3-one (0.012 mL, 0.188 mmol) prepared in step 4 of Example 23 were dissolved in dichloromethane (4 mL) at room temperature. Sodium triacetoxyborohydride (0.053 g, 0.250 mmol) was added to the reaction solution and stirred at the same temperature for 18 hours. Saturated aqueous sodium bicarbonate solution was poured into the reaction mixture, which was then extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% to 2.5%) and concentrated to afford the title compound (0.016 g, 22.2%) as a white solid.

[0310] 1 H NMR (400MHz, CDCl3) δ7.90(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),7.06-6.8 5(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).

[0311] Example 24: Synthesis of 3-morpholinyl-4-[N-[[4-[5-(trifluoromethyl)-1,3,4-oxadiazol-2-yl]phenyl]methyl]phenylamino]cyclobut-3-ene-1,2-dione (Compound 24)

[0312]

[0313] 3-Morpholinyl-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-oxadiazole (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. Saturated aqueous sodium bicarbonate solution was poured into the concentrate, and the reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and the aqueous layer, and then concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, 12 g cartridge; ethyl acetate / hexane=10% to 60%) and concentrated. Dichloromethane and diethyl ether were added to the obtained product and stirred to precipitate a solid, and the precipitated solid was filtered. The filtrate was washed with diethyl ether and dried to obtain the title compound (0.196 g, 20.90%, white solid).

[0314] 1 H NMR (400MHz, DMSO-d6) δ8.06(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).

[0315] Activity assays and analytical protocols for compounds according to the invention

[0316] <Experimental Example 1> Identification of inhibition of HDAC enzyme activity (in vitro)

[0317] Selective HDAC6 inhibitors are crucial for the selectivity of HDAC1 inhibition, which is responsible for the side effects. To identify this importance, HDAC1 / 6 enzyme selectivity and cellular selectivity (HDAC1: histone acetylation / HDAC6: tubulin acetylation) were identified.

[0318] 1. Experimental Methods

[0319] The HDAC enzyme inhibition ability of the test substance was measured using the HDAC1 fluorescent drug discovery assay kit (Enzo life sciences: BML-AK511) and HDAC6 human recombinant (Calbiochem: 382180). In the HDAC1 assay, the test substance was treated at a concentration of 100, 1000 and 10000 nM, and in the HDAC6 assay, the test substance was treated at a concentration of 0.1, 1, 10, 100 and 1000 nM. After sample treatment, the sample was reacted at 37°C for 60 minutes, then treated with a developer, reacted at 37°C for 30 minutes, and then its fluorescence intensity (Ex 390, Em 460) was measured using FlexStatin3 (Molecular Devices).

[0320] 2. Experimental results

[0321] Table 2 shows the results of HDAC enzyme activity inhibition analysis obtained according to the above experimental method.

[0322] [Table 2]

[0323]

[0324]

Claims

1. A 1,3,4-oxadiazole derivative compound represented by the following chemical formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above chemical formula I, R1 and R2 are each independently -(C1-C4 alkyl), or R1 and R2 are joined together with the N atom to form a heterocycloalkyl group, wherein at least one H in the heterocycloalkyl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo or heterocycloalkyl; X is -H or -F; Y1 to Y5 are each independently N or CR3, wherein Y1 to Y5 cannot be 3 or more N at the same time; R3 is -H, -(C1-C4 alkyl), -(C1-C4 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 be 3 or more N at the same time; and R4 is -H, -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo, aryl or heteroaryl, wherein at least one -H in the aryl or heteroaryl ring can be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -OH, -O(C1-C4 alkyl) or -halo.

2. The 1,3,4-oxadiazole derivative compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, wherein in the above chemical formula I, R1 and R2 are linked together with the N atom to form a heterocycloalkyl group, wherein at least one H in the heterocycloalkyl ring may be independently substituted by -(C1-C4 alkyl) or a heterocycloalkyl group; 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 be 3 or more N at the same time; and R4 is -H or -halo.

3. The 1,3,4-oxadiazole derivative compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, wherein in the above chemical formula I, R1 and R2 are linked together with the N atom to form a 4- to 12-membered heterocycloalkyl group, wherein at least one H in the heterocycloalkyl ring may be independently substituted by -(C1-C4 alkyl), -(C1-C4 aminoalkyl), -(C1-C4 hydroxyalkyl), -(C1-C4 haloalkyl), -halo or 3- to 6-membered heterocycloalkyl.

4. The 1,3,4-oxadiazole derivative compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 3, wherein in the above chemical formula I, R1 and R2 are connected together with the N atom to form in At least one H in the ring may be independently substituted by -(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 heterocycloalkyl; and n1, n2, m1 and m2 are each independently 0, 1 or 2.

5. The 1,3,4-oxadiazole derivative compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 4, wherein in the above chemical formula I, R1 and R2 are connected together with the N atom to form in At least one H in the ring may be independently substituted by -(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-oxadiazole derivative compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, wherein it is any one of the compounds listed in the following table:

7. A pharmaceutical composition for preventing or treating a disease mediated by histone deacetylase 6, comprising the compound represented by Chemical Formula I according to any one of claims 1 to 6, its stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient.

8. The pharmaceutical composition for preventing or treating a disease mediated by histone deacetylase 6 according to claim 7, wherein the disease mediated by histone deacetylase 6 is an infectious disease; a neoplasm; an endocrine, nutritional and metabolic disease; a mental and behavioral disorder; a neurological disease; an eye and eye appendage disease; a circulatory system disease; a respiratory tract disease; a digestive system disease; a skin and subcutaneous tissue disease; a musculoskeletal and connective tissue disease; or a congenital malformation, change and chromosomal abnormality.

9. A method for preventing or treating a disease mediated by histone deacetylase 6, comprising administering a therapeutically effective amount of a compound represented by Chemical Formula I according to any one of claims 1 to 6, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

10. Use of the compound represented by Chemical Formula I, its stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a medicament for preventing or treating a disease mediated by histone deacetylase 6.

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