RU2858220C2 - 1,3,4-oxadiazole compound derivatives as histone deacetylase 6 inhibitor and uses thereof - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: group of inventions includes a compound of Chemical Formula I, a stereoisomer, pharmaceutically acceptable salts thereof, a pharmaceutical composition based thereon, a method for inhibiting histone deacetylase 6 and use thereof. In Chemical Formula I, R₁ and R₂ are linked to each other to form, together with the N atom, a 3-12-membered heterocycloalkyl containing 1-5 heteroatoms selected from N, O and S, in which at least one H of the 3-12-membered heterocycloalkyl ring containing 1-5 heteroatoms selected from N, O and S may each be independently substituted by C₁-C₄ alkyl, C₁-C₄ aminoalkyl, C₁-C₄ hydroxyalkyl, C₁-C₄ haloalkyl, halogen or 3-6-membered heterocycloalkyl containing one O atom or one S atom; X represents H or F; Y₁-Y₅ each independently represents CR₃; R₃ represents H, halogen, 6-membered aryl or heteroaryl containing one N atom or one P atom, and having 4-5 ring carbon atoms, wherein at least one H of the 6-membered aryl or heteroaryl ring containing one N atom or one P atom, and having 4-5 ring carbon atoms, may each be independently substituted by C₁-C₄ alkyl, C₁-C₄ aminoalkyl, C₁-C₄ hydroxyalkyl, C1-C4 haloalkyl, OH, O(C₁-C₄ alkyl) or halogen; Z₁-Z₄ each independently represents N or CR₄, wherein three or more of Z₁-Z₄ cannot simultaneously represent N, and R₄ represents H, C₁-C₄ alkyl, C₁-C₄ aminoalkyl, C₁-C₄ hydroxyalkyl, C₁-C₄ haloalkyl or halogen.

EFFECT: compound of Chemical Formula I having inhibitory activity against histone deacetylase 6 (HDAC6).

10 cl, 2 tbl, 25 ex

Description

[Field of technology]

The present invention relates to 1,3,4-oxadiazole derivatives having histone deacetylase 6 (HDAC6) inhibitory activity, methods for their preparation and uses thereof.

[State of the art]

Post-translational modifications, such as acetylation in cells, are crucial regulatory modules at the heart of biological processes and are tightly controlled by a number of enzymes. Histones are nuclear proteins that make up chromatin and facilitate DNA condensation by acting as a pivot around which DNA is wound. Furthermore, the balance between histone acetylation and deacetylation plays a critical role in gene expression.

Histodeacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues in histone proteins that make up chromatin and are known to be associated with gene silencing and inducing cell cycle arrest, inhibition of angiogenesis, immunoregulation, cell death (apoptosis), etc. (Hassig et al., Curr. Opin. Chem. Biol. 1997, 1, 300-308). Furthermore, it has been reported that inhibition of the enzymatic function of HDACs reduces the activity of factors associated with cancer cell survival in vivo and activates factors associated with cancer cell death, thereby inducing spontaneous death (apoptosis) of cancer cells (Warrell et al, J. Natl. Cancer Inst. 1998, 90, 1621-1625).

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

Even though a wide variety of HDAC inhibitors are in preclinical or clinical development, only non-selective HDAC inhibitors are currently known as antitumor agents, with vorinostat (SAHA) and romidepsin (FK228) approved for the treatment of cutaneous T-cell lymphoma, and panobinostat (LBH-589) approved for the treatment of multiple myeloma. However, non-selective HDAC inhibitors in general are known to cause side effects such as fatigue, nausea, and the like at high doses (Piekarz et al., Pharmaceuticals 2010, 3, 2751-2767). These side effects have been reported to be caused by Class I HDAC inhibition, and due to these side effects, non-selective HDAC inhibitors have been limited in their use in drug development in areas other than anticancer drug production (Witt et al., Cancer Letters 277 (2009) 8.21).

Meanwhile, it has been reported that selective inhibition of Class II HDACs may not exhibit the toxicity observed with Class I HDAC inhibition, and the development of selective HDAC inhibitors could address the problem of side effects, such as toxicity, caused by non-selective HDAC inhibition. Therefore, selective HDAC inhibitors have good potential for further development to produce effective agents for the treatment of a wide variety of diseases (Matthias et al., Mol. Cell. Biol. 2008, 28, 1688–1701).

HDAC6, which is a Class IIb HDAC, is primarily present in the cytoplasm and is known to recruit the protein tubulin to participate in the deacetylation of a number of non-histone substrates (HSP90, cortactin, etc.) (Yao et al., Mol. Cell 2005, 18, 601-607). HDAC6 may have two catalytic domains, and the C-terminal zinc-finger domain can bind to ubiquitinated proteins. HDAC6 is known to play an important role in various diseases such as cancer, inflammatory diseases, autoimmune diseases, neurological diseases, neurodegenerative disorders, etc., since it contains a number of non-histone proteins as substrates (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).

Common structural features of various HDAC inhibitors include a cap group, a linker group, and a zinc binding group (ZBG), as shown in the structure of vorinostat below. Several researchers have studied the inhibitory activity and enzyme selectivity through structural modifications in the cap group and linker group. It is known that the zinc binding group plays a more important role in the inhibitory activity and enzyme selectivity (Wiest et al., J. Org. Chem. 2013 78: 5051–5065; Methot et al., Bioorg. Med. Chem. Lett. 2008, 18, 973–978).

Most zinc-binding groups are hydroxamic acid or benzamide, with the hydroxamic acid derivative exhibiting potent HDAC inhibitory effects but having problems such as low bioavailability and significant off-target activity. Benzamide contains aniline and thus also presents problems associated with the production of toxic metabolites in vivo (Woster et al., Med. Chem. Commun. 2015, online edition).

Accordingly, for the treatment of cancer, inflammatory diseases, autoimmune diseases, neurological diseases, neurodegenerative disorders, etc., there is a need to develop a selective HDAC6 inhibitor containing a zinc-binding group, without side effects and with improved bioavailability, in contrast to non-selective inhibitors that have significant side effects.

[Brief description of the invention]

[Technical problem]

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

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.

Another object of the present invention is to provide a method for producing such a derivative.

Another objective of the present invention is also to provide a pharmaceutical composition containing said compounds for the prevention or treatment of diseases associated with HDAC6 activity, including infectious diseases, neoplasms, endocrine, nutritional and metabolic diseases, mental and behavioral disorders, neurological diseases, diseases of the eye and adnexa of the eye, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system; diseases of the skin and subcutaneous tissues, diseases of the musculoskeletal system and connective tissues or congenital developmental defects, alterations and chromosomal abnormalities.

Another object of the present invention is also to provide options for using said compounds in the production of medicaments for the prevention or treatment of diseases associated with HDAC6 activity.

Another object of the present invention is also to provide a method for treating diseases associated with HDAC6 activity, comprising administering a therapeutically effective amount of a pharmaceutical composition containing the compounds described above.

[Technical solution]

The present invention discloses a 1,3,4-oxadiazole derivative having an inhibitory activity against histone deacetylase 6 (HDAC6), and shows the use of said 1,3,4-oxadiazole derivative for suppressing the development or treatment of diseases associated with HDAC6 activity, thereby ensuring the completeness of the present invention.

1,3,4-oxadiazole derivative

According to one general aspect of the present invention, there is provided a 1,3,4-oxadiazole derivative, namely a compound represented by the following Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

where in the above Chemical Formula I:

R ₁ and R ₂ each independently represent C ₁ -C ₄ alkyl, or R ₁ and R ₂ are linked together to form, together with the N atom, a heterocycloalkyl in which at least one H of the heterocycloalkyl ring may each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, halogen or heterocycloalkyl;

X represents H or F;

Y ₁ - Y ₅ each independently represent N or CR ₃ , and three or more of Y ₁ - Y ₅ cannot simultaneously represent N;

R ₃ is H, C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, halogen, aryl or heteroaryl, wherein at least one H of the aryl or heteroaryl ring may each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, OH, O(C ₁ -C ₄ alkyl) or halogen;

Z ₁ - Z ₄ each independently represents N or CR4, and three or more of Z ₁ - Z ₄ cannot simultaneously represent N and

R4 is H, _C1 - _C4 alkyl, _C1 - _C4 aminoalkyl, _C1 - _C4 hydroxyalkyl, _C1 - _C4 haloalkyl, halogen, aryl or heteroaryl, wherein at least one H of the aryl or heteroaryl ring may each independently be substituted with _C1 - _C4 alkyl, _C1 - _C4 aminoalkyl, _C1 - _C4 hydroxyalkyl, _C1 - _C4 haloalkyl, OH, O( _C1 - _C4 alkyl) or halogen.

According to an embodiment of the present invention, a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof may meet the following conditions:

R ₁ and R ₂ are linked together to form, together with the N atom, a heterocycloalkyl, in which at least one H of the heterocycloalkyl ring can each be independently substituted by C ₁ -C ₄ alkyl or heterocycloalkyl;

X represents H or F;

Y ₁ - Y ₅ each independently represents CR ₃ ;

R ₃ is H, halogen or heteroaryl, wherein at least one H of the heteroaryl ring may each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, OH, O(C ₁ -C ₄ alkyl) or halogen;

Z ₁ - Z ₄ each independently represents N or CR ₄ , and three or more of Z ₁ - Z ₄ cannot simultaneously represent N and

R ₄ is H or halogen.

According to an embodiment of the present invention, a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof may meet the following conditions:

R ₁ and R ₂ are linked together to form, together with the N atom, a 4-12-membered heterocycloalkyl, in which at least one H of the heterocycloalkyl ring can each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, halogen, or 3-6-membered heterocycloalkyl.

According to an embodiment of the present invention, a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof may meet the following conditions:

R ₁ and R ₂ are linked together to form, together with the N atom,

may each be independently substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl or halogen;

W is NR ₅ , O, S or S(=O) ₂ ;

R ₅ is C ₁ -C ₄ alkyl or 3-6-membered heterocycloalkyl and

n1, n2, m1 and m2 are each independently equal to 0, 1 or 2.

According to an embodiment of the present invention, a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof may meet the following conditions:

R ₁ and R ₂ are linked together to form, together with the N atom,

may each be independently substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl or halogen;

R ₅ is C ₁ -C ₄ alkyl or

In addition, in accordance with embodiments of the present invention, Table 1 below shows specific compounds corresponding to Chemical Formula I according to the present invention:

In the context of the present invention, the term "alkyl" as used herein may refer to an acyclic, cyclic, or saturated hydrocarbon with a straight or branched chain in which all carbon atoms are connected to each other, unless otherwise specified. For example, the term "C ₁ -C ₄ alkyl" may mean an alkyl containing from 1 to 4 carbon atoms. Acyclic alkyl, for example, may mean methyl, ethyl, n-propyl, n-butyl, isopropyl, sec-butyl, isobutyl, tert-butyl, and the like, but is not limited to them. The term "cyclic alkyl" in the context of the present description may be used interchangeably with the term "cycloalkyl" and may mean, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, but is not limited to them.

In the context of the present description, the term "alkoxy group" may refer to an -O-alkyl group as a simple alkyl ether group in which alkyl is as defined above. For example, the term "C ₁ -C ₄ alkoxy group" may mean an alkoxy group containing C ₁ -C ₄ alkyl, i.e., an -O-(O-C 4 alkyl) group, and examples of alkoxy groups may include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy groups, and the like.

In the context of the present description, the term "halogen" may mean F, Cl, Br or I.

As used herein, the term "haloalkyl" may mean a straight-chain or branched-chain alkyl (hydrocarbon) containing carbon atoms substituted with at least one halogen atom 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 atom, such as F, Cl, Br, or I.

As used herein, the term "hydroxyalkyl" may refer to a straight-chain or branched-chain alkyl (hydrocarbon) having carbon atoms substituted with a hydroxyl group (-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.

As used herein, the term "aminoalkyl" may refer to a straight-chain or branched-chain alkyl (hydrocarbon) having carbon atoms substituted with an amino group (-NR'R"). Each of R' and R" may be independently selected from the group consisting of hydrogen and _C1 - _C4 alkyl, and each of the selected R' and R" may independently be substituted or unsubstituted.

As used herein, the term "heterocycloalkyl" may refer to a ring containing from one to five heteroatoms selected from N, O, and S as constituent atoms of the ring structure, which may be saturated or partially unsaturated. In this specification, if a heterocycloalkyl is unsaturated, it may be referred to as heterocycloalkenyl. Unless otherwise specified, a heterocycloalkyl may be a single ring or multiple rings joined as spiro-linked rings, bridged rings, or fused rings. Furthermore, the term "3- to 12-membered heterocycloalkyl" may mean a heterocycloalkyl containing from 3 to 12 ring-forming atoms. Examples of heterocycloalkyl may include, but are not limited to, pyrrolidine, piperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-8,8-oxide, piperazine, pyran, 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, etc.

As used herein, the term "arene" may refer to an aromatic hydrocarbon ring. The arene may be a monocyclic arene or a polycyclic arene. The number of carbon atoms that make up the ring in an arene 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 arenes may include, but are not limited to, benzene, naphthalene, fluorene, anthracene, phenanthrene, bibenzene, terbenzene, quaterbenzene, quickwebenzene, sexybenzene, triphenylene, pyrene, benzofluoranthene, chrysene, and the like. As used herein, the moiety obtained by removing one hydrogen atom from the molecule of the above "arene" is referred to as "aryl".

In the context of the present description, the term "heteroarene" may mean a ring containing one or more of O, N, P, Si, and S as heteroatoms. The number of carbon atoms in the ring of a heteroarene may be 2 or more and 30 or less, or 2 or more and 20 or less. The heteroarene may be a monocyclic heteroarene or a polycyclic heteroarene. The polycyclic heteroarene may have, for example, a bicyclic or tricyclic structure. Examples of heteroarenes include, but are not limited to, thiophene, purine, pyrrole, pyrazole, imidazole, thiazole, oxazole, isothiazole, oxadiazole, triazole, pyridine, bipyridyl, triazine, acridyl, pyridazine, pyrazine, quinoline, quinazoline, quinoxaline, phenoxazine, phthalazine, pyrimidine, pyridopyrimidine, pyridopyrazine, pyrazinopyrazine, isoquinoline, indole, carbazole, imidazopyridazine, imidazopyridine, imidazopyrimidine, pyrazolopyrimidine, imidazopyrazine or pyrazolopyridine, N-arylcarbazole, N-heteroarylcarbazole, N-alkyl carbazole, benzoxazole, benzoimidazole, benzothiazole, benzocarbazole, benzothiophene, dibenzothiophene, thienothiophene, benzofuran, phenanthroline, isoxazole, oxadiazole, thiadiazole, benzothiazole, tetrazole, phenothiazine, dibenzosilene, dibenzofuran, etc. In an embodiment of the present invention, the heteroarene may also include a bicyclic heterocycloarene comprising an arene ring fused to a heterocycloalkyl ring or a heteroarene ring fused to a cycloalkyl ring. In the context of the present description, a moiety obtained by removing one hydrogen atom from the molecule of the above "heteroarene" is called "heteroaryl".

The compound represented by Chemical Formula I according to the present invention may contain at least one asymmetric carbon atom and, thus, may be present as a racemate, a racemic mixture, a single enantiomer, a diastereomeric mixture, and each diastereomer individually. These stereoisomers can be separated by conventional methods, and, for example, the compound represented by Chemical Formula I can be separated using column chromatography, HPLC, or other similar techniques. Alternatively, each stereoisomer of the compound represented by Chemical Formula I can be synthesized stereospecifically using optically pure starting materials and/or reagents with known configurations.

In the context of the present description, the term "enantiomer" as used herein means a compound having the same chemical formula or molecular formula but differing in the spatial arrangement of the atoms, or a salt thereof. Each of these enantiomers and mixtures thereof are also included within the scope of the present invention. Unless otherwise specified, a solid bond (-) attached to an asymmetric carbon atom may include a wedge-shaped solid bond 1 ^ 1 or a wedge-shaped dotted bond, which represent the absolute arrangement of stereocenters in space.

The compound represented by Chemical Formula I according to the present invention may be present in the form of a "pharmaceutically acceptable salt." A suitable salt is an acid addition salt formed from a pharmaceutically acceptable free acid. The term "pharmaceutically acceptable salt," as used herein, refers to a concentration that produces a relatively non-toxic and harmless effect on patients and includes any salt formed by the addition of an organic acid or inorganic acid to a compound represented by Chemical Formula I, wherein the side effects caused by these salts do not outweigh the beneficial efficacy of the compound.

Acid addition salts are prepared by conventional methods, such as dissolving the compound in an excess aqueous acid solution and precipitating the resulting salt with a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Equimolar amounts of the compound and the acid or alcohol can be heated in water, after which the mixture can be evaporated to dryness, or the precipitated salt can be filtered off using a Buchner funnel.

In this case, organic acids and inorganic acids can be used as the free acid, wherein the inorganic acid can be hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, etc., and the organic acid can be 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, etc. However, organic acids and inorganic acids are not limited to them.

Furthermore, a pharmaceutically acceptable metal salt can be prepared using an appropriate base. An alkali metal salt or alkaline earth metal salt is prepared, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, separating the precipitated insoluble salt by filtration, and then evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are particularly suitable as pharmaceutically acceptable metal salts, but the choice of metal salt is not limited to these. Additionally, the corresponding silver salt can be prepared by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

Pharmaceutically acceptable salts according to the present invention, unless otherwise indicated, include salts of acidic or basic groups that may be present in the compounds represented by Chemical Formula I above. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts formed by the hydroxyl group, other pharmaceutically acceptable salts formed by the amino group, which may include hydrobromides, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates, acetates, succinates, citrates, tartrates, lactates, mandelates, methanesulfonates (mesylates) and p-toluenesulfonates (tosylates), and the like, which can be prepared by methods for preparing salts known in the art.

Method for producing 1,3,4-oxadiazole derivative

The present invention provides a method for producing a 1,3,4-oxadiazole derivative represented by the following Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof:

The definition of Chemical Formula I is given in the description above.

In the present description, the schemes [Reaction Scheme 1] to [Reaction Scheme 4] show a preferred method for producing an oxadiazole derivative compound represented by Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which may also include production methods modified to a level understandable to those skilled in the art.

The above Reaction Scheme 1 is a method for synthesizing a 1,3,4-oxadiazole derivative having a cyclobutenedione structure. First, a compound represented by Chemical Formula 1-1 containing an amino group is reacted with a compound represented by Chemical Formula 1-2 to obtain a compound represented by Chemical Formula 1-3 containing an amine substituent. Then, the compound represented by Chemical Formula 1-3 is reacted with a compound represented by Chemical Formula 1-4 to obtain a compound represented by Chemical Formula 1-5 having a cyclobutenedione structure containing a diamine substituent. The compound represented by Chemical Formula 1-5 and the compound represented by Chemical Formula 1-6 undergo a substitution reaction to thereby obtain a compound represented by Chemical Formula 1-7. Meanwhile, the compound represented by Chemical Formula 1-5 can be obtained by reacting the compound represented by Chemical Formula 1-4 with the compound represented by Chemical Formula 1-2 to obtain 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 obtained by the above Reaction Scheme 1.

The above Reaction Scheme 2 is another synthetic method for producing a 1,3,4-oxadiazole derivative 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 obtain a compound represented by Chemical Formula 2-2 containing an amine substituent. Then, the compound represented by Chemical Formula 2-2 is reacted with a compound represented by Chemical Formula 1-4 to obtain a compound represented by Chemical Formula 1-7 having a cyclobutenedione structure containing a diamine substituent. In the present invention, Compound 1 and compounds similar to it can be produced through the above Reaction Scheme 2.

The above Reaction Scheme 3 is another method for synthesizing a 1,3,4-oxadiazole derivative having a cyclobutenedione structure. The compound represented by Chemical Formula 1-7 obtained according to Reaction Scheme 1 and the compound represented by Chemical Formula 3-1 are subjected to a C-C coupling reaction (Suzuki reaction) to obtain a compound represented by Chemical Formula 3-2. In the present invention, Compounds 8, 9 and similar compounds can be prepared through the above Reaction Scheme 3.

The above Reaction Scheme 4 is another method for synthesizing a 1,3,4-oxadiazole derivative having a cyclobutenedione structure. The compound represented by Chemical Formula 4-2 is obtained by deprotecting the compound represented by Chemical Formula 4-1 having the protecting group obtained according to Reaction Scheme 1. Then, a compound represented by Chemical Formula 4-3 is obtained through an alkylation reaction or a reduction reaction. The compound represented by Chemical Formula 4-3 and the compound represented by Chemical Formula 3-1 are subjected to a C-C coupling reaction (Suzuki reaction) to obtain a compound represented by Chemical Formula 4-4. In the present invention, the above Reaction Scheme 4 can be used to prepare compounds represented by Chemical Formula 4-3, i.e., Compounds 10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 22 and 23, etc., and obtaining compounds represented by Chemical Formula 4-4, i.e. Compounds 18, 19, etc.

Applications of 1,3,4-oxadiazole derivative

The present invention provides uses of a compound represented by the following Chemical Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

The definition of Chemical Formula I is given in the description above.

According to an embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of diseases associated with histone deacetylase 6 activity is provided, comprising a compound represented by the above-described Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to the present invention selectively inhibits histone deacetylase 6, thereby demonstrating significant effects in the prevention or treatment of diseases associated with histone deacetylase 6 activity.

Diseases associated with histone deacetylase 6 activity include infectious diseases such as prion disease, neoplasms such as benign tumors (e.g., myelodysplastic syndrome) or malignancies (e.g., multiple myeloma, lymphoma, leukemia, lung cancer, colorectal cancer, colon cancer, prostate cancer, urinary tract epithelial 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, amyloidosis, or diabetes, mental and behavioral disorders such as depression or Rett syndrome, neurological diseases such as central nervous system atrophy (e.g., Huntington's disease, spinal muscular atrophy (SMA), spinal cerebellar ataxia (SCA)), neurodegenerative diseases (e.g. Alzheimer's 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 demyelination of the central nervous system (e.g. multiple sclerosis (MS)), diseases of the eye and adnexa of the eye such as uveitis, diseases of the circulatory system such as atrial fibrillation or stroke, respiratory diseases such as asthma, diseases of the digestive system such as alcoholic liver disease, inflammatory bowel disease, Crohn's disease, peptic ulcer disease, etc., diseases of the skin and subcutaneous tissue such as psoriasis, diseases of the musculoskeletal system and connective tissue such as rheumatoid arthritis, osteoarthritis or systemic systemic lupus erythematosus (SLE), or congenital malformations, alterations, and chromosomal abnormalities such as autosomal dominant polycystic kidney disease, and also include symptoms or diseases associated with abnormal function of histone deacetylases.

The stereoisomers and pharmaceutically acceptable salts are as described above for the stereoisomers and pharmaceutically acceptable salts of the compound represented by Chemical Formula I according to the present invention.

For the purposes of administration to a patient, the pharmaceutical composition according to the present invention, in addition to the said compound represented by Chemical Formula I, its stereoisomer or pharmaceutically acceptable salt, may further comprise at least one or more pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier may be a physiological solution, sterile water, Ringer's solution, buffered physiological solution, dextrose solution, maltodextrin solution, glycerol, ethanol or a mixture of one or more of the said ingredients, and, if necessary, may contain other traditional additives such as antioxidants, buffers, bacteriostatic agents, etc. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added, which can also be included in the composition of injection preparations such as aqueous solutions, suspensions, emulsions and the like, pills, capsules, granules or tablets. Accordingly, 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. These formulations may be prepared by conventional methods used for preparing drug formulations in the art or by the method disclosed herein [see Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA], and may be formulated in various ways depending on each disease or component.

The composition according to the present invention can be administered to a patient orally or parenterally (e.g., by intravenous, subcutaneous, intraperitoneal, or local administration) according to the desired route, and the dosage varies depending on the patient's body weight, age, sex, health condition, diet, time of administration, route of administration, excretion rate, and severity of the disease. The daily dose of the compound represented by Chemical Formula I according to the present invention is from about 1 to 1000 mg/kg, preferably from 5 to 100 mg/kg, and can be divided and administered at one time or in several separate doses per day.

The pharmaceutical composition according to the present invention, in addition to the compound represented by Chemical Formula I, its stereoisomer or pharmaceutically acceptable salt, may further contain at least one active ingredient having the same or similar effectiveness.

According to an embodiment of the present invention, a method for the prevention or treatment of diseases associated with histone deacetylase 6 activity is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof. The subject may be a mammal, including a human.

A method for the prevention or treatment of diseases associated with histone deacetylase 6 activity includes not only combating the disease itself before its symptoms appear, but also suppressing or eliminating existing symptoms by administering a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt. Furthermore, the method for the prevention or treatment of diseases associated with histone deacetylase 6 activity according to the present invention may, in addition to the compound represented by Chemical Formula I, also include administering a therapeutically effective amount of an additional active agent that is useful in the treatment of said diseases, wherein the additional active agent may exhibit synergistic or adjuvant effects in combination with the compound represented by the aforementioned Chemical Formula I.

The term "therapeutically effective amount" as used herein refers to an amount of a compound represented by Chemical Formula I effective for treating or preventing diseases associated with histone deacetylase 6 activity. In particular, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined depending on factors including the type of subject and the severity of his condition, age, gender, type of existing diseases, drug activity, drug susceptibility, time of administration, route of administration, excretion rate, duration of treatment, concomitantly administered drugs and other factors well known in the medical art. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with a commercially available therapeutic agent. Furthermore, the pharmaceutical composition according to the present invention can be administered simultaneously or in multiple divided doses. Taking into account all of the above factors, it is important to administer an amount that provides the maximum effect with minimal or no side effects and that can be easily determined by those skilled in the art. The administered dose of the pharmaceutical composition according to the present invention can be determined by specialists taking into account various factors, such as the patient's condition, age, gender, the presence of complications, etc. Since the active ingredient of the pharmaceutical composition according to the present invention exhibits exceptional safety, the active ingredient can be used even in excess of the prescribed dose.

According to an embodiment of the present invention, a method for selectively inhibiting histone deacetylase 6 (HDAC6) is provided by administering to mammals, including humans, a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof.

According to an embodiment of the present invention, the use of a compound represented by Chemical Formula I, its stereoisomer or a pharmaceutically acceptable salt thereof for the production of medicinal products for the treatment of diseases associated with the activity of histone deacetylase 6 is proposed. To produce said medicinal products, the compound represented by Chemical Formula I can be mixed with acceptable adjuvants, diluents, carriers, etc., and can be prepared as a combination drug with other active agents to obtain a synergistic effect from the combination of various active ingredients.

The provisions mentioned in the uses, compositions and methods of treatment according to the present invention are applicable to the extent that they do not contradict each other.

The illustrative embodiments of the present invention may be modified and presented in various other forms, and the scope of the present invention is not limited only to the illustrative embodiments described below. Furthermore, these illustrative embodiments of the present invention are provided for the purpose of more fully explaining the present invention to one of ordinary skill in the art. Furthermore, the "inclusion" of a particular component in the description does not mean the exclusion of other components, but rather means that other components may be additionally included, unless otherwise specified.

[Advantages of the invention]

The compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt according to the present invention can selectively inhibit HDAC6, thereby exerting an exceptionally remarkable preventive or therapeutic effect on diseases associated with histone deacetylase 6 activity.

[Best implementation option]

The present invention will now be illustrated in more detail using examples and experimental procedures. However, these examples and the like are presented only as particular instances of the present invention, and the scope of the present invention is not limited to these examples.

Obtaining a derivative of 1,3,4-oxadiazole

A specific method for preparing the compound represented by Chemical Formula I is described as follows.

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)

[Step 1] Synthesis of 3-((4-(5-(difluoromethyl)-1,3.4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione

N-(4-(5-(difluoromethyl)-1,3,4-oxadiazol-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 resulting reaction solution was stirred at 60 °C for 18 hours. Then, the reaction was terminated by lowering the reaction temperature to room temperature. The reaction mixture was filtered through a glass funnel to obtain a filtrate free of solids, and the solvent in the filtrate was distilled off under reduced pressure. The resulting concentrate was then purified by column chromatography ( _SiO2 , 4 g cartridge, ethyl acetate/hexane = 10% to 50%) and concentrated to give the title compound (0.069 g, yield 25.7%) as a yellow oily liquid.

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) obtained in Step 1 and 1-methylpiperazine (0.052 ml, 0.466 mmol) were dissolved in methanol (3 ml) at room temperature, and the resulting reaction solution was stirred at 60°C for 18 hours. Then, the reaction was terminated by lowering the reaction temperature to room temperature. After distilling off the solvent 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 give the title compound (0.016 g, yield 13.8%) as a pale yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88 (d, J=8.0 Hz, 1H), 7.77 (d, J=8.7 Hz, 1H), 7.63 (t, J=7.5 Hz, 1H), 7.36 (t, J=7.9 Hz, 2H), 7.19 (t, J=7.4 Hz, 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 ⁺ +l).

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

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 resulting reaction solution was stirred for 2 hours at room temperature. Then, water was poured into the reaction mixture, and the resulting 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. Then, diethyl ether was added to the concentrate and stirred until a solid precipitated, and the precipitated solid was collected by filtration, washed with diethyl ether and dried to give the title compound (0.445 g, yield 70.0%) as a white solid.

3-Morpholino-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.387 mmol) obtained 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 resulting reaction solution, which was stirred for 1 hour at room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium hydrogen carbonate was poured into the concentrate, and the resulting reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 4 g cartridge, ethyl acetate/hexane mixture = 20% to 70%) and concentrated to give the title compound (0.019 g, yield 10.1%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88 (d, J=8.1 Hz, 1H), 7.77 (d, J=10.1 Hz, 1H), 7.64 (t, J=7.6 Hz, 1H), 7.38 (t, J=7.8 Hz, 2H), 7.22 (t, J=7.4 Hz, 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).

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

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

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 5 (0.429 ml, 2.461 mmol) were dissolved in methanol (10 ml) at 50 °C, and the resulting reaction solution was stirred at room temperature for 4 hours. Then, water was poured into the reaction mixture, and the resulting 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 collected by filtration, washed with methanol and dried to give the title compound (0.554 g, yield 73.5%) as a pale yellow solid.

[Step 2] Synthesis of Compound 3

3-(1,1-Dioxidothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.326 mmol) obtained 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 resulting reaction solution, which was stirred at room temperature for 1 hour. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous sodium chloride solution was poured into the concentrate, and the resulting 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 resulting 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, yield 13.2%) as a pale yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.89 (dd, J=8.0, 1.4 Hz, 1H), 7.77 (dd, J=10.0, 1.4 Hz, 1H), 7.61 (t, J=7.6 Hz, 1H), 7.43 (t, J=7.8 Hz, 2H), 7.31 (d, J=7.4 Hz, 1H), 7.09 (d, J=7.8 Hz, 2H), 7.05 - 6.79 (m, 1H), 5.60 (s, 2H), 3.68 (brs, 4H), 2.89 - 2.87 (m, 4H); LRMS (ES)m/z 533.5 (M ⁺ +1).

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

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

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 resulting reaction solution was stirred at this temperature for 18 hours. The precipitated solid was collected by filtration, washed with hexane, and dried to give the title target compound (1.860 g, yield 58.7%) as a white solid.

[Step 2] Synthesis of tert-butyl 4-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)piperazine-1-carboxylate

Tert-butyl 4-(2-methoxy-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (1.860 g, 6.277 mmol) obtained in Step 1, aniline (0.573 mL, 6.277 mmol) and triethylamine (1.750 mL, 12.554 mmol) were dissolved in ethanol (20 mL) at 78 °C, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, the reaction was completed by lowering the reaction temperature to room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. Water was poured into the concentrate, which was then extracted with dichloromethane. The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge, ethyl acetate/hexane = 0% to 50%) and concentrated to give the title compound (0.300 g, yield 13.4%) as a yellow solid.

[Step 3] Synthesis of 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

Tert-butyl 4-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)piperazine-1-carboxylate (0.176 g, 0.492 mmol) obtained 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 resulting reaction solution, which was stirred for 30 minutes at the same temperature. 2-(4-(bromomethyl)phenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.17 g, 0.59 mmol) was added to the resulting reaction mixture, which was further stirred at room temperature for 3 hours. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. Water was added to the concentrate, and the solution was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge, ethyl acetate/hexane = 0% to 50%) and concentrated to afford the title compound (0.135 g, yield 48.5%) as a white solid.

[Step 4] Synthesis of 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

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) obtained in Step 3 and trifluoroacetic acid (0.183 ml, 2.387 mmol) were dissolved in dichloromethane (10 ml) at room temperature, and the resulting reaction solution was stirred for 18 h at the same temperature. After distilling off the solvent from the reaction mixture under reduced pressure, the resulting product was used without further purification (0.150 g, yield 108.4%, yellow oily liquid).

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) obtained 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 resulting reaction solution was stirred at the same temperature for 18 hours. Then, water was poured into the reaction mixture, which 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 concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; methanol/dichloromethane = 0% to 10%) and concentrated to give the title compound (0.066 g, yield 53.2%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (d, J=7.5 Hz, 2H), 7.47 (d, J=8.2 Hz, 2H), 7.35 (t, J=7.9 Hz, 2H), 7.20 (t, J=7.4 Hz, 1H), 7.04 (s, 0.25H), 6.98 (d, J=7.7 Hz, 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).

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

3-(1,1-Dioxidothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.326 mmol) obtained 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 resulting reaction solution was added 2-(4-(bromomethyl)phenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.104 g, 0.359 mmol), and the resulting mixture was stirred for 3 hours at the same temperature. Then, water was poured into the reaction mixture, after which it was extracted with dichloromethane. Then, the extract was 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 collected by filtration, washed with diethyl ether and dried to give the title compound (0.016 g, yield 9.5%) as a white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.00 (d, J = 8.2 Hz, 2H), 7.60 - 7.41 (m, 3H), 7.39 (t, J = 6.4 Hz, 1H), 7.23 - 7.19 (m, 3H), 5.52 (s, 2H), 3.53 (brs, 4H), 3.14 (brs, 4H); LRMS (ES) m/z 515.4 (M ⁺ +1).

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

3-(1,1-Dioxidothiomorpholino)-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.100 g, 0.326 mmol) obtained in Step 1 of Example 3 and sodium hydride (60.00%, 0.014 g, 0.359 mmol) were dissolved in N,N-dimethylformamide (3 ml) at room temperature. 2-(6-(bromomethyl)-5-(fluoropyridin-3-yl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.111 g, 0.359 mmol) was added to the resulting reaction solution, and stirred for 3 hours at the same temperature. Then, water was poured into the reaction mixture, and the resulting 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 (SiO ₂ , 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 collected by filtration, washed with diethyl ether, and dried to give the subject compound the title compound (0.017 g, yield 9.8%) as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.99 (s, 1H), 8.41 (dd, J=9.9, 1.5 Hz, 1H), 7.71 - 7.45 (m, 1H), 7.41 (t, J=7.8 Hz, 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).

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)

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

3,4-Dimethoxycyclobut-3-ene-1,2-dione (2000 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 the resulting solution was stirred for 18 h at the same temperature. The precipitated solid was collected by filtration, washed with hexane, and dried to give the title compound (3.500 g, yield 82.9%) as a white solid.

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

3-((3-Bromo-5-fluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (2000 g, 6.665 mmol) obtained in Step 1, tert-butylpiperazine-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 resulting reaction solution was stirred for 18 hours at the same temperature. The solvent was distilled off from the reaction mixture under reduced pressure. The resulting concentrate was purified by column chromatography ( _SiO2 , 40 g cartridge; ethyl acetate/hexane = 0% to 30%) and concentrated to give the title compound (2900 g, 95.8% yield) as a black porous solid.

[Step 3] Synthesis of 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

Tert-butyl 4-(2-((3-bromo-5-fluorophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (2.900 g, 6.383 mmol) obtained 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 resulting reaction solution, and the resulting mixture was stirred for 30 minutes at the same temperature. Then, 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (2.548 g, 8.298 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for another 3 hours. Then, water was poured into the reaction mixture and extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 40 g cartridge, ethyl acetate/hexane = 0% to 50%) and concentrated to afford the title compound (2.000 g, yield 46.0%) as a porous yellow solid.

[Step 4] Synthesis of 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

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 (2000 g, 2.939 mmol) obtained in Step 3 and trifluoroacetic acid (2.251 ml, 29.392 mmol) were dissolved in dichloromethane (20 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, water was poured into the reaction mixture, and the resulting mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting product was used without further purification (2000 g, yield 98.0%, brown oily liquid).

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 (2000 g, 2.880 mmol) obtained 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 resulting reaction solution was stirred at the same temperature for 18 hours. Then water was poured into the reaction mixture, and the resulting mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 40 g cartridge; methanol/dichloromethane mixture = 0% to 10%) and concentrated to give the title compound (1.500 g, yield 87.6%) as a yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.92 (dd, J=8.0, 1.5 Hz, 1H), 7.85 (dd, J=10.2, 1.5 Hz, 1H), 7.59 (t, J=7.7 Hz, 1H), 7.06 - 7.02 (m, 1H), 7.04 (s, 0.25H), 6.93 (s, 0.5H), 6.85 (s, 1H), 6.80 (s, 0.25H), 6.71 - 6.67 (m, 1H), 5.49 (s, 2H), 3.45 (br s, 4H), 2.39 (br s, 4H), 2.30 (s, 3H); LRMS (ES) m/z 595.4 (M ⁺⁺ 1).

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)

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) obtained in Example 7, pyridin-3-ylboronic acid (0.054 g, 0.437 mmol), [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (Pd(dtbpf)Cl ₂ , 0.022 g, 0.034 mmol) and cesium carbonate (0.219 g, 0.673 mmol) were mixed with a mixture of 1,4-dioxane (9 ml) and water (3 ml) and heated at 100°C for 20 minutes under microwave irradiation. Then, the reaction was terminated by lowering the reaction temperature to room temperature. Then, water was poured into the reaction mixture and 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 give the title compound (0.080 g, yield 40.1%) as a brown oily liquid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.78 (d, J=1.9 Hz, 1H), 8.67 (dd, J=4.7, 1.1 Hz, 1H), 7.91 (dd, J=8.0, 1.4 Hz, 1H), 7.85 - 7.80 (m, 2H), 7.65 (t, J=7.7 Hz, 1H), 7.43 (dd, J=7.9, 4.9 Hz, 1H), 7.11 - 7.08 (m, 1H), 7.05 (s, 0.25H), 6.96 (s, 1H), 6.92 (s, 0.5 H), 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).

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

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) obtained in Example 7, pyridin-4-ylboronic acid (0.054 g, 0.437 mmol), [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (Pd(dtbpf)Cl ₂ , 0.022 g, 0.034 mmol) and cesium carbonate (0.219 g, 0.673 mmol) were mixed with a mixture of 1,4-dioxane (9 ml) and water (3 ml) and heated at 100°C for 20 min under microwave irradiation. Then, the reaction was terminated by lowering the reaction temperature to room temperature. Then, water was poured into the reaction mixture and 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 give the title compound (0.070 g, yield 35.1%) as a brown oily liquid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.73 (d, J=5.6 Hz, 1H), 7.92 - 7.90 (m, 1H), 7.86 - 7.83 (m, 1H), 7.66 (t, J=7.7 Hz, 1H), 7.43 (d, J=5.9 Hz, 2H), 7.15-7.13 (m, 1H), 7.05 (s, 0.25H), 7.00 (s, 1H), 6.92 (s, 0.5H), 6.81 (s, 0.25H), 6.80 - 6.78 (m, 1H), 5.58 (s, 2H), 3.30 (br s, 4H), 2.27 (br s, 4H), 2.19 (s, 3H); LRMS (ES) m/z 593.4 (M ⁺ +1).

Example 10: Synthesis of 3-((4-(5-(difluoromethyl)-1,314-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(6-(oxetan-3-yl)-2,6-diazaspiro[3,3]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 10)

[Step 1] Synthesis of tert-butyl 6-(2-methoxy-3.4-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3,3]heptane-2-carboxylate

3,4-Dimethoxycyclobut-3-ene-1,2-dione (1.710 g, 12.033 mmol) tert-butyl 2,6-diazaspiro[3,3]heptane-2-carboxylate (2.386 g, 12.033 mmol) was dissolved in ethanol (30 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. The precipitated solid was collected by filtration, washed with hexane and dried to give the title compound (0.700 g, yield 18.9%) as a white solid.

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

Tert-butyl 6-(2-methoxy-3,4-dioxocyclobut-1-en-1-yl)-2,6-diazaspiro[3,3]heptane-2-carboxylate (0.700 g, 2.270 mmol) obtained in Step 1, aniline (0.207 mL, 2.270 mmol) and triethylamine (0.633 mL, 4.541 mmol) were dissolved in ethanol (20 mL) at 78 °C, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, the reaction was completed by lowering the reaction temperature to room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. Water was poured into the concentrate, and the resulting reaction 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 concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge, ethyl acetate/hexane = 0% to 50%) and concentrated to afford the title compound (0.600 g, yield 71.5%) as a white solid.

[Step 3] Synthesis of 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

Tert-butyl 6-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,6-diazaspiro[3,3]heptane-2-carboxylate (0.687 g, 1.860 mmol) obtained 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 resulting reaction solution, which was stirred for 30 minutes at the same temperature. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.685 g, 2.232 mmol) was added to the reaction mixture, which was stirred at room temperature for another 3 hours. Then, water was poured into the reaction mixture and extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge, ethyl acetate/hexane = 0% to 50%) and concentrated to afford the title compound (0.700 g, yield 63.2%) as a white solid.

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

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) obtained in Step 3 and trifluoroacetic acid (0.900 ml, 11.753 mmol) were dissolved in dichloromethane (10 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, a saturated aqueous sodium hydrogen carbonate 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 resulting product was used without further purification (0.400 g, yield 68.7%, white solid).

3-((4-(5-(Difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-(2,6-diazaspiro[3,3]heptan-2-yl)cyclobut-3-ene-1,2-dione (0.100 g, 0.202 mmol) obtained in Step 4, 3-oxetanone (0.029 g, 0.404 mmol) and sodium triacetoxyborohydride (0.086 g, 0.404 mmol) were dissolved in dichloromethane (10 ml) at room temperature. The resulting reaction solution was stirred at the same temperature for 18 hours. Then, water was poured into the reaction mixture, and the resulting 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 concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; methanol/dichloromethane = 0% to 20%) and concentrated to give the title compound (0.080 g, yield 71.9%) as a colorless oily liquid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.84 (dd, J=8.0, 1.4 Hz, 1H), 7.69 (dd, J=9.8, 1.4 Hz, 1H), 7.61 (t, J=7.6 Hz, 1H), 7.39 - 7.33 (m, 3H), 7.06 - 7.04 (m, 2H), 7.06 (s, 0.25H), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.44 (s, 2H), 4.62 - 4.58 (m, 2H), 4.34 - 4.31 (m, 2H), 3.80 (br s, 4H), 3.60 - 3.58 (m, 1H), 3.23 (s, 4H); LRMS (ES) m/z 552.5 (M ⁺ +1).

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]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 11)

[Step 1] Synthesis of 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

Tert-butyl 6-(3,4-dioxo-2-(phenylamino)cyclobut-1-en-1-yl)-2,6-diazaspiro[3,3]heptane-2-carboxylate (0.177 g, 0.479 mmol) obtained 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 resulting reaction solution, which was stirred for 30 minutes at the same temperature. 2-(4-(bromomethyl)phenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.166 g, 0.575 mmol) was added to the reaction mixture, which was stirred at room temperature for another 3 hours. Then, water was poured into the reaction mixture and extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge, ethyl acetate/hexane = 0% to 50%) and concentrated to afford the title compound (0.150 g, yield 54.2%) as a white solid.

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

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) obtained in Step 1 and trifluoroacetic acid (0.239 mL, 3.116 mmol) were dissolved in dichloromethane (10 mL) at room temperature, and the resulting reaction solution was stirred for 18 h at the same temperature. After distilling off the solvent from the reaction mixture under reduced pressure, the resulting product was used without further purification (0.180 g, yield 97.6%, yellow oily liquid).

3-((4-(5-(Difluoromethyl)-1,3,4-oxadiazol-2-yl)benzyl)(phenyl)amino)-4-(2,6-diazaspiro[3.3]heptan-2-yl)cyclobut-3-ene-1,2-dione-2,2,2-trifluoroacetate (0.180 g, 0.304 mmol) obtained 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 resulting reaction solution was stirred at the same temperature for 18 hours. Then water was poured into the reaction mixture, and the resulting mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; methanol/dichloromethane mixture = 0% to 20%) and concentrated to give the title compound (0.056 g, yield 37.4%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (d, J=8.2 Hz, 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).

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)

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

3,4-Dimethoxycyclobut-3-ene-1,2-dione (2000 g, 14.074 mmol) and 3-bromoaniline (2.421 g, 14.074 mmol) were dissolved in methanol (50 mL) at room temperature and stirred for 18 h at the same temperature. The precipitated solid was collected by filtration, washed with hexane and dried to give the title compound (3.500 g, yield 88.2%) as a white solid.

[Step 2] Synthesis of tert-butyl 4-(2-((3-bromophenyl)amino)-3.4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate

3-((3-Bromophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (1.250 g, 4.431 mmol) obtained in Step 1, tert-butylpiperazine-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 resulting reaction solution was stirred at the same temperature for 18 hours. Then, the reaction was terminated by lowering the reaction temperature to room temperature. The precipitated solid was collected by filtration, washed with hexane and dried to give the title compound (1.200 g, yield 62.1%) as a white solid.

[Step 3] Synthesis of 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

Tert-butyl 4-(2-((3-bromophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (1.200 g, 2.750 mmol) obtained 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 resulting reaction solution, which was stirred for 30 minutes at the same temperature. 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (1.013 g, 3.300 mmol) was added to the reaction mixture, which was stirred at room temperature for another 3 hours. Then, water was poured into the reaction mixture and extracted with ethyl acetate. The organic layer was washed with a saturated aqueous solution of sodium chloride, 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 obtain the title compound (1.400 g, yield 76.8%) as a colorless oily liquid.

[Step 4] Synthesis of 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

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) obtained in Step 3 and trifluoroacetic acid (1.618 mL, 21.13 mmol) were dissolved in dichloromethane (20 mL) at room temperature, and the resulting reaction solution was stirred for 18 h at the same temperature. After distilling off the solvent from the reaction mixture under reduced pressure, the resulting product was used without further purification (1.400 g, yield 97.9%, brown oily liquid).

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) obtained 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 resulting reaction solution was stirred at the same temperature for 18 hours. Then, water was poured into the reaction mixture, and the resulting mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; methanol/dichloromethane mixture = 0% to 10%) and concentrated to give the title compound (0.900 g, yield 75.4%) as a white solid.

¹ NMR (400 MHz, CDCl ₃ ) δ 7.89 (dd, J=8.0, 1.5 Hz, 1H), 7.80 (dd, J=10.1, 1.5 Hz, 1H), 7.60 (t, J=7.6 Hz, 1H), 7.30 - 7.28 (m, 1H), 7.23 (t, J=8.0 Hz, 1H), 7.08 (t, J=1.9 Hz, 1H), 7.05 (s, 0.25H), 6.98 (dd, J=8.0, 1.5 Hz, 1H), 6.92 (s, 0.5H), 6.79 (s, 0.25H), 5.53 (s, 2H), 3.36 (br s, 4H), 2.45 (br s, 4H), 2.25 (s, 3H); LRMS (ES) m/z 577.4 (M ⁺ +1).

Example 13: Synthesis of 3-((4-(5-(difluoromethyl)-1,314-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-((1S,4S)-5-(oxetan-3-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 13)

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

3-Methoxy-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 2.461 mmol), tert-butyl (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (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 resulting reaction solution was stirred at room temperature for 18 hours. After removing the solvent from the reaction mixture under reduced pressure, the resulting concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; ethyl acetate/hexane = 20% to 40%) and concentrated to give the title compound (0.871 g, yield 95.8%) as a pale yellow solid.

[Step 2] Synthesis of 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

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) obtained in Step 1, 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) were dissolved in N,N-dimethylformamide (4 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at 60°C. The reaction was then terminated by lowering the reaction temperature to room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium hydrogen carbonate was poured into the concentrate, and the resulting reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The resulting concentrate was purified by column chromatography ( _SiO2 cartridge 12 g, ethyl acetate/hexane mixture = 30% to 60%) and concentrated to obtain the title compound (0.315 g, yield 65.1%) as a yellow solid.

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

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) obtained in Step 2 and trifluoroacetic acid (0.284 ml, 3.702 mmol) were dissolved in dichloromethane (4 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, a saturated aqueous sodium hydrogen carbonate 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 aqueous layer and then concentrated under reduced pressure. The resulting product was used without further purification (0.221 g, yield 84.3%, pale yellow solid).

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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) obtained in Step 3 and formaldehyde (0.018 ml, 0.303 mmol) were dissolved in dichloromethane (4 ml) at room temperature. Sodium triacetoxyborohydride (0.086 g, 0.404 mmol) was added to the resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, saturated aqueous sodium hydrogen carbonate solution was poured into the reaction mixture, which was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 4 g cartridge; methanol/dichloromethane mixture = 0% to 2.5%) and concentrated to give the title compound (0.075 g, yield 67.4%) as a yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (dd, J=8.0, 1.4 Hz, 1H), 7.72 - 7.66 (m, 2H), 7.35 (t, J=7.6 Hz, 2H), 7.29 - 7.25 (m, 1H), 7.07 (d, J=7.6 Hz, 2H), 7.05 - 6.79 (m, 1H), 5.56 (d, J=15.4 Hz, 1H), 5.47 (d, J=15.4 Hz, 1H), 4.63 - 4.57 (m, 2H), 4.40 (t, J=5.9 Hz, 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=9.8 Hz, 1H), 1.54 (d, J=10.1 Hz, 1H), 1.30 - 1.28 (m, 1H); LRMS (ES) m/z 552.8 (M ⁺¹ ).

Example 14: Synthesis of 3-((4-(5-(difluoromethyl)-1,314-oxadiazol-2-yl)-2-fluorobenzyl)(phenyl)amino)-4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 14)

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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) obtained in Step 3 of Example 14 and formaldehyde (38.00% solution, 0.022 ml, 0.303 mmol) were dissolved in dichloromethane (4 ml) at room temperature. Sodium triacetoxyborohydride (0.086 g, 0.404 mmol) was added to the resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate was poured into the reaction mixture, after which it 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; methanol/dichloromethane mixture = 0% to 5%) and concentrated to give the title compound (0.049 g, yield 47.7%) as a yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88 (dd, J=8.0, 1.5 Hz, 1H), 7.74 - 7.67 (m, 2H), 7.35 (t, J=7.6 Hz, 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=10.1 Hz, 1H), 1.31 - 1.29 (m, 1H); LRMS (ES) m/z 510.8 (M ⁺⁺ 1).

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]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 15)

[Step 1] Synthesis of 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

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) obtained in Step 1 of Example 13, 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) were dissolved in N,N-dimethylformamide (4 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at 60°C. The reaction was then terminated by lowering the reaction temperature to room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium hydrogen carbonate was poured into the concentrate, and the resulting reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The resulting 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, yield 56.2%) as a pale brown solid.

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

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) obtained in Step 1 and trifluoroacetic acid (0.291 ml, 3.802 mmol) were dissolved in dichloromethane (4 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, a saturated aqueous sodium hydrogen carbonate 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 aqueous layer and then concentrated under reduced pressure. The resulting product was used without further purification (0.277 g, 76.1% yield, brown solid).

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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) obtained in Step 2 and formaldehyde (38.00% solution in water, 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 resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate was poured into the reaction mixture, after which it 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; methanol/dichloromethane mixture = 0% to 5%) and concentrated to give the title compound (0.054 g, yield 58.3%) as an orange solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.25 (d, J=2.0 Hz, 1H), 8.37 (dd, J=8.2, 2.2 Hz, 1H), 7.64 (d, J=8.2 Hz, 1H), 7.36 (t, J=7.8 Hz, 2H), 7.24 (t, J=7.4 Hz, 1H), 7.19 (d, J=7.6 Hz, 2H), 7.08 - 6.82 (m, 1H), 5.63 - 5.50 (m, 2H), 3.18 (brs, 1H), 2.76 (brs, 1H), 2.63 (brs, 1H), 2.31 (s, 3H), 2.16 - 2.14 (m, 2H), 1.87 (d, J=9.9 Hz, 1H), 1.57 - 1.53 (m, 1H), 1.32 - 1.28 (m, 1H); LRMS (ES) m/z 493.8 (M ⁺ +1).

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]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 16)

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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) obtained 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 resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate was poured into the reaction mixture, after which it 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; methanol/dichloromethane mixture = 0% to 5%) and concentrated to give the title compound (0.056 g, yield 57.2%) as an orange solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.25 (d, J=2.0 Hz, 1H), 8.38 (dd, J=8.2, 2.1 Hz, 1H), 7.65 (d, J=8.1 Hz, 1H), 7.37 (t, J=7.8 Hz, 2H), 7.24 (t, J=7.4 Hz, 1H), 7.19 (d, J=7.7 Hz, 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).

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

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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) obtained in Step 2 of Example 15 and 3-oxetanone (0.018 ml, 0.314 mmol) were dissolved in dichloromethane (4 ml) at room temperature. Sodium triacetoxyborohydride (0.089 g, 0.418 mmol) was added to the resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate was poured into the reaction mixture, after which it 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; methanol/dichloromethane mixture = 0% to 2.5%) and concentrated to give the title compound (0.036 g, yield 32.2%) as an orange solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.27 (s, 1H), 8.39 (dd, J=8.1, 1.8 Hz, 1H), 7.64 (d, J=8.2 Hz, 1H), 7.38 (t, J=7.7 Hz, 2H), 7.26 - 7.24 (m, 1H), 7.21 (d, J=8.0 Hz, 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).

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)

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]palladium(II) dichloride (Pd(dtbpf)Cl ₂ , 0.029 g, 0.045 mmol) and cesium carbonate (0.364 g, 1.117 mmol) were mixed with a mixture of 1,4-dioxane (9 mL) and water (3 mL) and heated at 100°C for 20 hours under microwave irradiation. Then, the reaction was terminated by lowering the reaction temperature to room temperature. Then, water was poured into the reaction mixture and 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 give the title compound (0.100 g, yield 40.1%) as a brown oily liquid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.78 (d, J=1.6 Hz, 1H), 8.64 (d, J=3.8 Hz, 1H), 7.88 - 7.76 (m, 3H), 7.66 (t, J=7.6 Hz, 1H), 7.47 (t, J=7.8 Hz, 1H), 7.42 - 7.39 (m, 2H), 7.18 (s, 1H), 7.07 (dd, J=7.9, 1.3 Hz, 1H), 7.04 (s, 0.25H), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.61 (s, 2H), 3.32 (br s, 4H), 2.23 (br s, 4H), 2.16 (s, 3H); LRMS (ES) m/z 558.4 (M ⁺ +1).

Example 19: Synthesis of 3-((5-(5-(difluoromethyl)-1,314-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)

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]palladium(II) dichloride (Pd(dtbpf)Cl ₂ , 0.023 g, 0.036 mmol) and cesium carbonate (0.291 g, 0.894 mmol) were mixed with a mixture of 1,4-dioxane (9 mL) and water (3 mL) and heated at 100°C for 15 minutes under microwave irradiation. Then, the reaction was terminated by lowering the reaction temperature to room temperature. Then, water was poured into the reaction mixture and 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 give the title compound (0.060 g, yield 30.1%) as a black oily liquid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.71 (d, J=4.4 Hz, 2H), 7.89 (d, J=8.1 Hz, 1H), 7.80 (dd, J=10.1, 1.1 Hz, 1H), 7.67 (t, J=7.6 Hz, 1H), 7.50 - 7.43 (m, 4H), 7.23 (s, 1H), 7.11 - 7.09 (m, 1H), 7.04 (s, 0.25), 6.91 (s, 0.5H), 6.78 (s, 0.25H), 5.62 (s, 2H), 3.31 (br s, 4H), 2.22 (br s, 4H), 3.16 (s, 3H); LRMS (ES) m/z 558.4 (M ⁺ +1).

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]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 20)

[Step 1] Synthesis of 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

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) obtained in Step 1 of Example 13, 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) were dissolved in N,N-dimethylformamide (4 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at 60°C. Then the reaction was terminated by lowering the reaction temperature to room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium hydrogen carbonate was poured into the concentrate, and the resulting reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The resulting concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge, ethyl acetate/hexane = 30% to 70%) and concentrated to give the title compound (0.285 g, yield 35.3%) as a brown solid.

[Step 2] Synthesis of 3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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

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) obtained in Step 1 and trifluoroacetic acid (0.256 ml, 3.344 mmol) were dissolved in dichloromethane (4 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. Then, saturated aqueous sodium hydrogen carbonate solution was poured into the reaction mixture, which was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The resulting concentrate was purified by column chromatography ( _SiO2 , 4 g cartridge, ethyl acetate/hexane = 0% to 30%) and concentrated to give the title compound (0.206 g, yield 86.9%) as a brown solid.

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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) obtained in Step 2 and formaldehyde (0.009 g, 0.302 mmol) were dissolved in dichloromethane (4 ml) at room temperature. Sodium triacetoxyborohydride (0.085 g, 0.403 mmol) was added to the resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate was poured into the reaction mixture, after which it 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; methanol/dichloromethane mixture = 0% to 5%) and concentrated to give the title compound (0.067 g, yield 65.2%) as a yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.05 (s, 1H), 8.06 (d, J=9.3 Hz, 1H), 7.34 (t, J=7.1 Hz, 2H), 7.22 (t, J=7.4 Hz, 1H), 7.14 (d, J=8.0 Hz, 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=9.8 Hz, 1H), 1.57 (d,J=10.1 Hz, 1H); LRMS (ES) m/z 511.8 (M ⁺⁺ 1).

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-(oxetan-3-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)cyclobut-3-ene-1,2-dione (Compound 21)

3-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-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 (0.000 g, 0.201 mmol) obtained in Step 2 of Example 20 and 3-oxetanone (0.019 ml, 0.302 mmol) were dissolved in dichloromethane (4 ml) at room temperature. Sodium triacetoxyborohydride (0.085 g, 0.403 mmol) was added to the resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate was poured into the reaction mixture, after which it 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; methanol/dichloromethane mixture = 0% to 2.5%) and concentrated to give the title compound (0.019 g, yield 17.1%) as a yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.09 (s, 1H), 8.10 (dd, J=29.3, 20.0 Hz, 1H), 7.37 (t, J=7.7 Hz, 2H), 7.26 - 7.24 (m, 1H), 7.18 (d, J=7.6 Hz, 2H), 7.08 - 6.82 (m, 1H), 5.68 (s, 2H), 4.68 - 4.63 (m, 2H), 4.50 - 4.45 (m, 2H), 3.81 - 3.80 (m, 1H), 3.22 (brs, 1H), 2.82 (brs, 1H), 2.75 (brs, 1H), 1.83 - 1.59 (m, 4H), 1.31 - 1.30 (m, 1H); LRMS (ES) m/z 553.7 (M ⁺ +1).

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)

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

3,4-Difluoroaniline (1.000 g, 7.745 mmol) and 3,4-dimethoxycyclobut-3-ene-1,2-dione (1.101 g, 7.745 mmol) were dissolved in methanol (30 mL) at room temperature and stirred for 18 h at the same temperature. The precipitated solid was collected by filtration, washed with methanol and dried to give the title compound (1.610 g, yield 86.9%) as a pale yellow solid.

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

3-((3,4-Difluorophenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (1.500 g, 6.271 mmol) obtained in Step 2, tert-butylpiperazine-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 resulting reaction solution was stirred for 18 hours at the same temperature. Methanol was added to the resulting reaction mixture and stirred until a solid was precipitated, and the precipitated solid was collected by filtration, washed with methanol and dried to give the title compound (1.770 g, 71.7%) as a pale green solid.

[Step 3] Synthesis of 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

Tert-butyl 4-(2-((3,4-difluorophenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)piperazine-1-carboxylate (0.500 g, 1.271 mmol) obtained 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. To the resulting reaction solution, 2-(4-(bromomethyl)-3-fluorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (0.429 g, 1.398 mmol) was added, and stirred for 18 hours at room temperature. After removing the solvent from the reaction mixture under reduced pressure, the resulting concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; ethyl acetate/hexane = 5% to 40%) and concentrated to give the title compound (0.516 g, yield 65.5%) as a pale yellow solid.

[Step 4] Synthesis of 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

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) obtained in Step 3 and trifluoroacetic acid (0.510 ml, 6.663 mmol) were dissolved in dichloromethane (5 ml) at room temperature, and the resulting reaction solution was stirred for 18 hours at the same temperature. Saturated aqueous sodium hydrogen carbonate solution and dichloromethane were added to the resulting reaction mixture, and the mixture was stirred until a solid was precipitated, and the precipitated solid was collected by filtration. The filtrate was washed with dichloromethane and dried to give the title compound (0.361 g, yield 83.4%) as a yellow solid.

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) obtained 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 resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, saturated aqueous sodium hydrogen carbonate 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 aqueous layer and then concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 4 g cartridge; methanol/dichloromethane mixture = 0% to 5%) and concentrated to give the title compound (0.044 g, yield 42.8%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.90 (d, J=8.0 Hz, 1H), 7.81 (d, J=10.2 Hz, 1H), 7.63 (t, J=7.7 Hz, 1H), 7.17 (q, J=9.0 Hz, 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)

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

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) obtained in Step 4 of Example 22 and oxetan-3-one (0.012 ml, 0.188 mmol) were dissolved in dichloromethane (4 ml) at room temperature. Sodium triacetoxyborohydride (0.053 g, 0.250 mmol) was added to the resulting reaction solution, which was stirred for 18 hours at the same temperature. Then, a saturated aqueous solution of sodium hydrogen carbonate 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 aqueous layer and then concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 4 g cartridge; methanol/dichloromethane mixture = 0% to 2.5%) and concentrated to afford the title compound (0.016 g, yield 22.2%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.90 (dd, J=8.0, 1.2 Hz, 1H), 7.80 (dd, J=10.1, 1.2 Hz, 1H), 7.62 (t, J=7.7 Hz, 1H), 7.17 (q, J=9.0 Hz, 1H), 7.06 - 6.85 (m, 2H), 6.77 - 6.75 (m, 1H), 5.52 (s, 2H), 4.64 (t, J=6.5 Hz, 2H), 4.52 (t, J=6.1 Hz, 2H), 3.50 -3.39 (m, 5H), 2.25 (brs, 4H); LRMS (ES) m/z 576.3 (M ⁺⁺ 1).

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

3-Morpholino-4-(phenylamino)cyclobut-3-ene-1,2-dione (0.500 g, 1.936 mmol) obtained 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 resulting reaction solution, and stirred for 18 hours at room temperature. The solvent was distilled off from the reaction mixture under reduced pressure to obtain a concentrate. A saturated aqueous solution of sodium hydrogen carbonate was poured into the concentrate, and the resulting reaction mixture was extracted with dichloromethane. The mixture was filtered through a plastic filter to remove the solid residue and aqueous layer and then concentrated under reduced pressure. The concentrate was purified by column chromatography ( _SiO2 , 12 g cartridge; ethyl acetate/hexane = 10% to 60%) and concentrated. Dichloromethane and diethyl ether were added to the resulting product and stirred until a solid precipitated, and the precipitated solid was collected by filtration. The filtrate was washed with diethyl ether and dried to give the title compound (0.196 g, yield 20.90%, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.06 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 7.8 Hz, 2H), 7.26 - 7.21 (t, 3H), 5.57 (s, 2H), 3.51 (t, J = 4.8 Hz, 4H), 3.27 (brs, 4H); LRMS (ES) m/z 485.7 (M ⁺ +1).

Protocol for measuring the activity and analysis of compounds according to the present invention

Experimental Example 1 Confirmation of HDAC enzyme activity inhibition (in vitro)

A selective HDAC6 inhibitor is also important for the selectivity of HDAC1 inhibition as a cause of side effects. To confirm this importance, selectivity and cellular selectivity for HDAC 1/6 enzymes (HDAC1 - histone acetylation, HDAC6 - tubulin acetylation) were confirmed.

1. Experimental methodology

The ability of the test substance to inhibit the HDAC enzyme was measured using the HDAC1 Fluorimetric Drug Discovery Assay Kit (Enzo life sciences: BML-AK511) and recombinant human HDAC6 enzyme (Calbiochem: 382180). In the HDAC1 assay, the test substance was treated at concentrations of 100, 1000, and 10,000 nM, and in the HDAC6 assay, the test substance was treated at concentrations of 0.1, 1, 10, 100, and 1000 nM. After processing, the samples were left to react at 37°C for 60 minutes, then treated with developer and left to react at 37°C for another 30 minutes, and their fluorescence intensity values (Ex 390, Em 460) were measured using a FlexStation 3 multifunctional reader (Molecular Devices).

2. Result of the experiment

Table 2 shows the results of quantitative determinations of the degree of inhibition of HDAC enzyme activity obtained in accordance with the above experimental procedure.

Claims (33)

1. A 1,3,4-oxadiazole derivative compound represented by the following Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] , where in the above Chemical Formula I: R ₁ and R ₂ are linked together to form, together with the N atom, a 3- to 12-membered heterocycloalkyl ring containing 1-5 heteroatoms selected from N, O and S, in which at least one H of the 3- to 12-membered heterocycloalkyl ring containing 1-5 heteroatoms selected from N, O and S can each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, halogen or 3- to 6-membered heterocycloalkyl containing one O atom or one S atom; X represents H or F; Y ₁ - Y ₅ each independently represents CR ₃ ; R ₃ is H, halogen, 6-membered aryl or heteroaryl containing one N atom or one P atom and having 4-5 ring carbon atoms, wherein at least one H of the 6-membered aryl or heteroaryl ring containing one N atom or one P atom and having 4-5 ring carbon atoms may each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, OH, O(C ₁ -C ₄ alkyl) or halogen; Z ₁ - Z ₄ each independently represents N or CR ₄ , and three or more of Z ₁ - Z ₄ cannot simultaneously represent N and R ₄ is H, C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl or halogen. 2. A 1,3,4-oxadiazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, wherein in the above Chemical Formula I: R ₁ and R ₂ are linked together to form, together with the N atom, a 3-12-membered heterocycloalkyl containing 1-5 heteroatoms selected from N, O and S, in which at least one H of the 3-12-membered heterocycloalkyl ring containing 1-5 heteroatoms selected from N, O and S can each independently be substituted with C ₁ -C ₄ alkyl or 3-6-membered heterocycloalkyl containing one O atom or one S atom; X represents H or F; Y ₁ - Y ₅ each independently represents CR ₃ ; R ₃ is H, halogen or heteroaryl containing one N atom or one P atom and having 4-5 ring carbon atoms, in which at least one H of the heteroaryl ring containing one N atom or one P atom and having 4-5 ring carbon atoms may each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, OH, O(C ₁ -C ₄ alkyl) or halogen; Z ₁ - Z ₄ each independently represents N or CR ₄ , and three or more of Z ₁ - Z ₄ cannot simultaneously represent N and R ₄ is H or halogen. 3. A 1,3,4-oxadiazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, wherein in the above Chemical Formula I: R ₁ and R ₂ are linked together to form, together with the N atom, a 4-12-membered heterocycloalkyl containing 1-5 heteroatoms selected from N, O and S, in which at least one H of the 4-12-membered heterocycloalkyl ring containing 1-5 heteroatoms selected from N, O and S can each independently be substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, halogen or 3-6-membered heterocycloalkyl containing one O atom or one S atom. 4. A 1,3,4-oxadiazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 3, wherein in the above Chemical Formula I: R ₁ and R ₂ are linked together to form, together with the N atom, , or , in which there is at least one H ring , or may each be independently substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl or halogen; W is NR ₅ , O, S or S(=O) ₂ ; R ₅ is C ₁ -C ₄ alkyl or 3-6 membered heterocycloalkyl containing one O atom or one S atom; and n1, n2, m1 and m2 are each independently equal to 0 or 1. 5. A 1,3,4-oxadiazole derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 4, wherein in the above Chemical Formula I: R ₁ and R ₂ are linked together to form, together with the N atom, , , , or , in which there is at least one H ring , , , or may each be independently substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ aminoalkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl or halogen; and R ₅ is C ₁ -C ₄ alkyl or . 6. A 1,3,4-oxadiazole derivative, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1, wherein said derivative is any of the compounds listed in the following table: Connection No. Structural formula Connection No. Structural formula 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 7. A pharmaceutical composition having inhibitory activity against histone deacetylase 6, containing a therapeutically effective amount of a compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1-6 as an active ingredient; and a pharmaceutically acceptable carrier. 8. A pharmaceutical composition according to claim 7, wherein said composition is used for the prevention or treatment of diseases mediated by histone deacetylase 6, wherein the diseases mediated by histone deacetylase 6 are infectious diseases, neoplasms, endocrine, nutritional and metabolic diseases, mental and behavioral disorders, neurological diseases, diseases of the eye and adnexa of the eye, respiratory diseases, diseases of the digestive organs, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue or congenital malformations, alterations or chromosomal abnormalities. 9. A method for inhibiting histone deacetylase 6, comprising administering a therapeutically effective amount of a compound represented by Chemical Formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6 as an active ingredient. 10. Use of a compound represented by Chemical Formula I, its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 6 for the production of a medicinal product intended for inhibiting histone deacetylase 6.