Imidazo [1,2-b] pyridazine-based novel compound as CDK inhibitor and use thereof

Abstract

Provided are a novel compound, a use thereof for inhibiting the activity of CDK12 and / or CDK13 and degrading cyclin K, a pharmaceutical composition comprising same, and a use for the treatment of diseases associated with CDK12 and / or CDK13 and / or cyclin K, such as cancer. According to the present invention, the compounds exhibit excellent inhibitory activity and selectivity against CDK12 and / or CDK13, and induce degradation of cyclin K, and thus can be useful for the treatment of cancers such as breast cancer or gastric cancer.

Description

Novel compounds of the imidazo[1,2-b]pyridazine series as CDK inhibitors and their uses

[0001] The present invention relates to novel compounds that inhibit the activity of cyclin-dependent kinases (CDKs), such as CDK12 and / or CDK13, and degrade cyclin K. The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and their use in the treatment of diseases mediated by CDKs.

[0002] One of the key features of cancer is uncontrolled cell growth, making targeted therapies that inhibit cell division effective in treating cancer. Cyclins and cyclin-dependent kinases are a group of proteins that primarily control cell division (Mengna et al. CDK inhibitors in cancer therapy, an overview of recent development. 2021, Am J Cancer Res;11(5):1913-1935). Cyclin-dependent protein kinases (CDKs) are the catalytic subunits of the serine / threonine protein kinase family. CDKs form complexes with cyclin proteins and phosphorylate serine or threonine residues on their substrates, activating them. Based on their cellular functions, CDKs can be classified into two categories: those that control the cell cycle and those that regulate cellular transcription.

[0003] The activity of CDKs is regulated by specific associations with cyclin regulatory units, such as cyclin A, cyclin B, cyclin C, cyclin D, and cyclin E. For example, in mammalian cells, CDK1, which forms a complex with cyclin A / B, regulates progression from G2 to M phase, CDK4 and CDK6, which form a complex with cyclin D, control progression from G1 to S phase and initiate DNA synthesis, and CDK2, which forms a complex with cyclin E / A, completes DNA synthesis in S phase. CDK7-9 form complexes with cyclins H, C, and T, respectively, to regulate gene transcription [Shigeaki et al. CDK1 inhibitor controls G2 / M phase transition and reverses DNA damage sensitivity. 2021, Biochem Biophys Res Commun. Apr 23;550:56-61], [Manuel et al. A Cdk4 / 6-dependent phosphorylation gradient regulates the early to late G1 phase transition. 2021, Sci Rep. Jul 19;11(1):14736], [Stefan et al. Structural basis for CDK7 activation by MAT1 and Cyclin H. 2020, Proc Natl Acad Sci US A. Oct 27;117(43):26739-26748].

[0004] CDK12 and its ortholog, CDK13, belong to the CDK family that regulate transcriptional and post-transcriptional processes. Among the CDK family, genetic mutations in CDK12 are reported to be relatively high in various cancers. Furthermore, CDK12 and CDK13 play a crucial role in regulating kinase activity by forming a complex with their cyclin partner, cyclin K, and then phosphorylating the C-terminal domain of RNA polymerase II. In addition, CDK12 is known to regulate major signaling pathways related to cancer induction and DNA damage response, and inhibition of these pathways is expected to have anticancer effects. SR-4835 (International Publication No. WO 2019 / 217421) is a well-known representative CDK12 / CDK13 inhibitor, and numerous experiments have been reported to confirm its anticancer effects (Victor et al. Therapeutic Targeting of CDK12 / CDK13 in Triple-Negative Breast Cancer. 2019, Cancer Cell. Nov 11;36(5):545-558.e7). However, the development of CDK12 / 13 inhibitors with improved efficacy and selectivity compared to existing substances is required in the art.

[0005] An object of the present invention is to provide a compound of the following formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, which inhibits the activity of CDK, e.g., CDK12 and / or CDK13, and degrades cyclin K.

[0006] [Chemical Formula I]

[0007]

[0008] In the above chemical formula I,

[0009] R 1is halo, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl-, C3-C6 cycloalkyl-C2-C6 alkenyl-, or C3-C6 cycloalkyl-C2-C6 alkynyl-, and the R 1 Any C1-C6 alkoxy group, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group and C3-C6 cycloalkyl group among them may be optionally substituted with halo, hydroxy or cyano;

[0010] R 2 is C6-C 10 Aryl; 5- or 6-membered heteroaryl comprising 1 to 2 nitrogen atoms; or a monocyclic or bridged or spiro 4- to 12-membered heterocyclyl comprising 1 nitrogen atom and linked to an imidazopyridazine ring of formula I through said nitrogen atom, wherein said heterocyclyl may optionally comprise 1 additional nitrogen atom or 1 oxygen atom,

[0011] The above R 2 may be optionally substituted with one or more substituents selected from:

[0012] (i) H, halo, hydroxy, cyano, C1-C6 alkyl, C3-C6 cycloalkyl,

[0013] (ii) C1-C6 alkyl substituted with halo, hydroxy or cyano,

[0014] (iii) C3-C6 cycloalkyl substituted with halo, hydroxy or cyano,

[0015] (iv) amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 hydroxyalkyl)amino, (C1-C6 alkyl)carbonylamino, (C1-C6 hydroxyalkyl)carbonylamino or (C1-C6 alkoxy)(C1-C6 alkyl)carbonylamino, and

[0016] (v) (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl, di(C1-C6 alkyl)aminocarbonyl, carboxy or (C1-C6 alkoxy)carbonyl;

[0017] R 3 silver or and;

[0018] X 1a Silver NR 3a , O or S, and X 2a is N or CR 3a But, X 2a Go CR 3a Back side X 1a Silver NR 3a and;

[0019] X 1b and X 2b One of them is N and the other is CR 3b and;

[0020] X 2c is N or CR 3c and;

[0021] X 3a , X 4a , X 5a and X 6a At least one of them is N, the rest are CR 3d and;

[0022] X 3b , X 4b , X 5b and X 6b At least one of them is N, the rest are CR 3e and;

[0023] X 3c , X 4c , X 5c and X 6c 1 or less of these are NR 3f and the rest are CR 3f R 3g and;

[0024] R 3a , R 3b and R 3care each independently H or C1-C6 alkyl,

[0025] R 3d , R 3e , R 3f and R 3g are each independently H, halo, cyano, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, C1-C6 alkoxy or C1-C6 alkyl, and R 3d , R 3e , R 3f and R 3g Any C1-C6 alkoxy group and C1-C6 alkyl group may be optionally substituted with halo, cyano or hydroxy.

[0026] An object of the present invention is to provide a pharmaceutical composition comprising a novel compound that inhibits the activity of CDK12 and / or CDK13 and degrades cyclin K, for example, a pharmaceutical composition for treating cancer.

[0027] An object of the present invention is to provide a method for inhibiting CDK12 and / or CDK13 and degrading cyclin K using novel compounds that inhibit the activity of CDK12 and / or CDK13 and degrade cyclin K, and a use of these compounds for inhibiting CDK12 and / or CDK13 and degrading cyclin K.

[0028] An object of the present invention is to provide a method for treating diseases related thereto, such as cancer, using a novel compound that inhibits the activity of CDK12 and / or CDK13 and degrades cyclin K.

[0029] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0030] One aspect of the present invention provides a compound of the following formula I, a stereoisomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof:

[0031] [Chemical Formula I]

[0032]

[0033] In the chemical formula I of the present invention, R 1 is halo, hydroxy, cyano, C1-C6alkoxy, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, C3-C6cycloalkyl-C1-C6alkyl-, C3-C6cycloalkyl-C2-C6alkenyl-, or C3-C6cycloalkyl-C2-C6alkynyl-. In one specific embodiment, R 1 , or R 1 Any C1-C6 alkoxy group, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group and C3-C6 cycloalkyl group contained in may be optionally substituted independently with halo, hydroxy or cyano.

[0034] In one specific example, R 1 may be C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, C3-C6cycloalkyl-C1-C6alkyl-, C3-C6cycloalkyl-C2-C6alkenyl-, or C3-C6cycloalkyl-C2-C6alkynyl- optionally substituted with halo, for example, R 1 It can be ethynyl, methylethynyl, cyclopropylethynyl, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, trifluoroethyl, iodo or chloro.

[0035] In one specific example, R 1 may be C1-C6 haloalkyl, for example CF3.

[0036] In the chemical formula I of the present invention, R 2 is C6-C 10Aryl; 5- or 6-membered heteroaryl containing 1 to 2 nitrogen atoms; or monocyclic or bridged or spiro 4- to 12-membered heterocyclyl containing 1 nitrogen atom and connected to the imidazopyridazine ring of formula I through said nitrogen atom.

[0037] In one specific example, R of formula I 2 may be a monocyclic or bridged or spiro 4- to 12-membered heterocyclyl. The 4- to 12-membered heterocyclyl may comprise one nitrogen atom and be linked to the imidazopyridazine ring of formula I via the nitrogen atom. In one embodiment, the heterocyclyl may optionally comprise one additional nitrogen atom or one additional oxygen atom.

[0038] In one embodiment, the 4- to 12-membered heterocyclyl may be azetidinyl, pyrrolidinyl, morpholinyl, piperidinyl, or piperazinyl. In one embodiment, any two non-adjacent carbon atoms of the morpholinyl, piperidinyl, or piperazinyl may be optionally linked to each other by a C1-C3 alkylene to form a bridged ring. For example, the bridged heterocyclyl is 3-oxa-8-azabicyclo[3.2.1]octan-8-yl, 8-oxa-3-azabicyclo[3.2.1]octan-3-yl, 3,8-diazabicyclo[3.2.1]octan-3-yl, 3,8-diazabicyclo[3.2.1]octan-8-yl, 2-oxa-5-azabicyclo[2.2.2]octan-5-yl, 8-azabicyclo[3.2.1]octan-8-yl, 3-azabicyclo[3.1.1]heptan-3-yl, 6-oxa-3-azabicyclo[3.1.1]heptan-3-yl, 3,6-diazabicyclo[3.1.1]heptan-3-yl or It may be, but is not limited to, 3,6-diazabicyclo[3.1.1]heptan-6-yl.

[0039] Alternatively, the 4- to 12-membered heterocyclyl may be a 7- to 11-membered azaspiro ring comprising one nitrogen atom connected to the imidazopyridazine ring of formula I, and optionally comprising one additional nitrogen atom or one oxygen atom. For example, the 7 to 11 membered azaspiro ring is 2-azaspiro[3.3]heptan-2-yl, 2,6-diazaspiro[3.3]heptan-2-yl, 6-oxa-2-azaspiro[3.3]heptan-2-yl, 2-azaspiro[3.4]octane-2-yl, 2,6-diazaspiro[3.4]octane-2-yl, 6-oxa-2-azaspiro[3.4]octane-2-yl, 6-azaspiro[3.4]octane-6-yl, 2,6-diazaspiro[3.4]octane-6-yl, 2-oxa-6-azaspiro[3.4]octane-6-yl, 2-azaspiro[4.4]nonan-2-yl, 2,7-diazaspiro[4.4]nonan-2-yl or 2-oxa-7-azaspiro[4.4]nonan-7-yl. For example, the azaspiro ring includes, but is not limited to, 2-azaspiro[3.3]heptan-2-yl, 2,6-diazaspiro[3.3]heptan-2-yl, 6-oxa-2-azaspiro[3.3]heptan-2-yl.

[0040] Alternatively, R of the present chemical formula I 2 may be a 5- or 6-membered heteroaryl containing 1 or 2 nitrogen atoms. In one embodiment, the 5- or 6-membered heteroaryl may be pyrimidinyl, pyridinyl, pyrrolyl, or imidazolyl. For example, the heteroaryl may be pyrimidinyl.

[0041] Alternatively, R of the present chemical formula I 2 is C6-C 10 It can be aryl. For example, the above C6-C 10 Aryl can be phenyl or naphthyl. For example, the above C6-C 10 Aryl can be phenyl.

[0042] R of the above chemical formula I 2 may be optionally substituted with one or more substituents selected from the following:

[0043] (i) H, halo, hydroxy, cyano, C1-C6 alkyl, C3-C6 cycloalkyl,

[0044] (ii) C1-C6 alkyl substituted with halo, hydroxy or cyano,

[0045] (iii) C3-C6 cycloalkyl substituted with halo, hydroxy or cyano,

[0046] (iv) amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 hydroxyalkyl)amino, (C1-C6 alkyl)carbonylamino, (C1-C6 hydroxyalkyl)carbonylamino or (C1-C6 alkoxy)(C1-C6 alkyl)carbonylamino, and

[0047] (v) (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl, di(C1-C6 alkyl)aminocarbonyl, carboxy or (C1-C6 alkoxy)carbonyl.

[0048] In one specific example, R of formula I 2 may not be substituted with the substituents listed in (i) to (v) above. Alternatively, R of the formula I 2 may be substituted by one or two substituents independently selected from the substituents listed in (i) to (iv) above.

[0049] As a specific example, R of the above chemical formula I 2 is substituted with H, hydroxy, cyano, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy-(C1-C6 alkyl), hydroxy-(C3-C6)cycloalkyl, amino, (C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 hydroxyalkyl)carbonylamino, (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl or carboxy, but is not limited thereto. For example, R of the above formula I2 may be substituted with, but is not limited to, H, hydroxy, cyano, methyl, ethyl, cyclopropyl, cyclobutyl, hydroxymethyl, hydroxyethyl, 2-hydroxyisopropyl, 1-hydroxycyclopropyl, 1-hydroxycyclobutyl, amino, methylamino, ethylamino, cyclopropylamino, hydroxyacetamido, methylcarbonyl (i.e., acetyl), hydroxyacetyl, aminocarbonyl, carbamoyl, or carboxy.

[0050] In one specific example, R of the chemical formula I of the present invention 2 is C6-C 10 Aryl, wherein said aryl is R selected from the group consisting of H, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino and (C3-C6 cycloalkyl)amino 2d can be arbitrarily substituted. For example, R 2 is phenyl, and R 2d can be hydroxy or amino.

[0051] In one specific example, R of the chemical formula I of the present invention 2 is a 5- or 6-membered heteroaryl containing 1 to 2 nitrogen atoms, wherein said heteroaryl is R selected from the group consisting of H, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino and (C3-C6 cycloalkyl)amino 2d can be arbitrarily substituted. For example, R 2 is pyridinyl or pyrimidinyl, and R 2d can be hydroxy or amino.

[0052] In one specific example, R of the chemical formula I of the present invention 2 may be selected from the following chemical formula A or chemical formula B:

[0053]

[0054] In the above chemical formula A, n1 and n2 are each independently 1 or 2. In one specific embodiment, the carbon atoms constituting the ring of chemical formula A may be optionally substituted with C1-C6 alkyl.

[0055] In the above chemical formula A, when both n1 and n2 are 2, Y 1 Silver CR 2a R 2b , NR 2c , or O. In this case, any two non-adjacent carbon atoms in the ring of chemical formula A are optionally C 1- can be linked to each other by C3 alkylene to form a bridged ring. Alternatively, when one or both of n1 and n2 are 1, Y 1 Silver CR 2a R 2b am.

[0056] In the above chemical formula B, n3, n4, n5 and n6 are each independently 1 or 2. For example, n3, n4, n5 and n6 can all be 1. In one specific embodiment, the carbon atoms constituting the ring of chemical formula B can be optionally substituted with C1-C6 alkyl. In the above chemical formula B, Y 2 is CR 2a R 2b , NR 2c Or O.

[0057] In the above chemical formulas A and B, R 2a Wow R 2bare each independently selected from the group consisting of H, halo, hydroxy, cyano, C1-C6 alkyl, or C3-C6 cycloalkyl; C1-C6 alkyl substituted with halo, hydroxy, or cyano; C3-C6 cycloalkyl substituted with halo, hydroxy, or cyano; amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 alkyl)carbonylamino, (C1-C6 hydroxyalkyl)carbonylamino; and (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl, di(C1-C6 alkyl)aminocarbonyl, and carboxy.

[0058] In one specific example, in the chemical formula A and chemical formula B, R 2a and R 2b One of which may be selected from the group consisting of H, halo, hydroxy, cyano, C1-C6 alkyl, or C3-C6 cycloalkyl; C1-C6 alkyl substituted with halo, hydroxy, or cyano; C3-C6 cycloalkyl substituted with halo, hydroxy, or cyano; amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 alkyl)carbonylamino, (C1-C6 hydroxyalkyl)carbonylamino; and (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl, di(C1-C6 alkyl)aminocarbonyl, and carboxy. In this case, R 2a and R 2b The remaining one may be H, halo, hydroxy, cyano or C1-C6 alkyl.

[0059] For example, in the above chemical formulas A and B, R 2a and R 2bmay be, but is not limited to, H, hydroxy, amino, cyclopropylamino, hydroxyacetamido, methyl, hydroxymethyl, 2-hydroxy-isopropyl, hydroxycyclopropyl, carboxy, carbamoyl, hydroxyacetyl or cyano.

[0060] In the above chemical formulas A and B, R 2c may be H, C1-C6 alkyl, C3-C6 cycloalkyl, (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl or di(C1-C6 alkyl)aminocarbonyl. For example, R 2c may be, but is not limited to, H, methyl, cyclopropyl, acetyl, hydroxyacetyl or carbamoyl.

[0061] In one specific example, R of the chemical formula I of the present invention 2 may be selected from the following groups:

[0062]

[0063] The above R 2 In the structure of R 2a , R 2b , R 2c and R 2d is as described above with respect to chemical formula A and chemical formula B.

[0064] For example, R of the chemical formula I of the present invention 2 may be selected from the following groups:

[0065]

[0066] In the chemical formula I of the present invention, R 3 is selected from the following structures:

[0067] or

[0068] In the above (1), X 1a Silver NR 3a , O or S, and X 2a is N or CR 3aIt is. However, X 2a Go CR 3a Back X 1a Silver NR 3a is. Also, X 3a , X 4a , X 5a and X 6a At least one of them is N, the rest are CR 3d am.

[0069] In the above (2), X 1b and X 2b One of them is N and the other is CR 3b is. Also, X 3b , X 4b , X 5b and X 6b At least one of them is N, the rest are CR 3e am.

[0070] In the above (3), X 2c is N or CR 3c is. Also, X 3c , X 4c , X 5c and X 6c 1 or less of these are NR 3f and the rest are CR 3f R 3g am.

[0071] In the above (1) to (3), R 3a , R 3b and R 3c are each independently H or C1-C6 alkyl. In addition, R 3d , R 3e , R 3f and R 3g are each independently H, halo, cyano, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, C1-C6 alkoxy or C1-C6 alkyl. In this case, R 3d , R 3e , R 3f and R 3g Any C1-C6 alkoxy group and C1-C6 alkyl group may be optionally substituted with halo, cyano or hydroxy.

[0072] In one specific example, R of the chemical formula I of the present invention 3 can be selected from the following structures:

[0073]

[0074] ; and .

[0075] The above R 3 In the structure of X 3a , X 4a , X 5a and X 6a At least one of them is N, the rest are CR 3d It is. X 3b , X 4b , X 5b and X 6b At least one of them is N, the rest are CR 3e It is. X 3c , X 4c , X 5c and X 6c 1 or less of these are NR 3f and the rest are CR 3f R 3g am.

[0076] In this case, R 3a , R 3b and R 3c are each independently H or C1-C6 alkyl. R 3d , R 3e , R 3f and R 3g are each independently H, halo, cyano, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, C1-C6 alkoxy or C1-C6 alkyl. The R 3d , R 3e , R 3f and R 3g Any C1-C6 alkoxy group and C1-C6 alkyl group among them may be optionally substituted with halo, cyano or hydroxy. In one specific embodiment, R 3d , R 3e , R 3f and R3g may be H, halo, hydroxy, amino, C1-C6 alkoxy, C1-C6 alkyl or C1-C6 haloalkyl. For example, R 3d , R 3e , R 3f and R 3g can each independently be H, F, Cl, hydroxy, amino, methoxy, methyl and trifluoromethyl. For example, R 3d , R 3e , R 3f and R 3g can each independently be a halogen, for example, F or Cl.

[0077] In one specific example, R 3 can be selected from the following structures:

[0078]

[0079]

[0080] The above R 3 In the structure of R 3a , R 3b and R 3c are each independently H or C1-C6 alkyl.

[0081] The above R 3 In the structure of R 3d , R 3e , R 3f and R 3g are each independently H, halo, hydroxy, amino, C1-C6 alkoxy, C1-C6 alkyl or C1-C6 haloalkyl. For example, R 3d , R 3e , R 3f and R 3g can each independently be H, F, Cl, hydroxy, amino, methoxy, methyl and trifluoromethyl.

[0082] For example, R of the chemical formula I of the present invention 3 may be selected from the following groups:

[0083]

[0084]

[0085] In one specific example, the compound of the present formula I may be a compound of the following formula IA:

[0086] [Chemical Formula IA]

[0087]

[0088] In the above chemical formula IA, R 1 may be halo, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl-, C3-C6 cycloalkyl-C2-C6 alkenyl-, or C3-C6 cycloalkyl-C2-C6 alkynyl-. The R 1 , or R 1 Any C1-C6 alkoxy group, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group and C3-C6 cycloalkyl group contained in may be optionally substituted with halo, hydroxy or cyano. R described with respect to chemical formula I 1 The various examples can be equally applied to chemical formula IA.

[0089] In the above formula IA, ring A is a monocyclic or bridged or spiro 4- to 12-membered heterocyclyl which may optionally include one additional nitrogen atom or one additional oxygen atom. R of formula I 2 The various examples of monocyclic or bridged or spiro 4- to 12-membered heterocyclyls described above can equally be applied to formula IA.

[0090] In the above chemical formula IA, ring A may be optionally substituted with one or more substituents selected from the following.

[0091] (i) H, halo, hydroxy, cyano, C1-C6 alkyl, C3-C6 cycloalkyl,

[0092] (ii) C1-C6 alkyl substituted with halo, hydroxy or cyano,

[0093] (iii) C3-C6 cycloalkyl substituted with halo, hydroxy or cyano,

[0094] (iv) amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 hydroxyalkyl)amino, (C1-C6 alkyl)carbonylamino, (C1-C6 hydroxyalkyl)carbonylamino or (C1-C6 alkoxy)(C1-C6 alkyl)carbonylamino, and

[0095] (v) (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl, di(C1-C6 alkyl)aminocarbonyl, carboxy or (C1-C6 alkoxy)carbonyl.

[0096] R of chemical formula I 2 The various examples given above for the substituents of and the corresponding substituents in formulae A and B can be equally applied to formula IA.

[0097] In the above chemical formula IA, R 3d is H, halo, cyano, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, C1-C6 alkoxy or C1-C6 alkyl. In this case, R 3d Any C1-C6 alkoxy group and C1-C6 alkyl group among them may be optionally substituted with halo, cyano or hydroxy. R of formula I 3d The various examples described above can equally be applied to chemical formula IA.

[0098] In the above chemical formula IA, p can be 0, 1, 2 or 3. In one specific embodiment, p can be 0, 1 or 2.

[0099] In one specific embodiment, the compound of the present formula I may be a compound of the following formula IB:

[0100] [Chemical Formula IB]

[0101]

[0102] In the above chemical formula IB, R 1 may be halo, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl-, C3-C6 cycloalkyl-C2-C6 alkenyl-, or C3-C6 cycloalkyl-C2-C6 alkynyl-. The R 1 , or R 1 Any C1-C6 alkoxy group, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group and C3-C6 cycloalkyl group contained in may be optionally substituted with halo, hydroxy or cyano. R described with respect to chemical formula I 1 The various examples can be equally applied to chemical formula IB.

[0103] In the above chemical formula IB, ring B is selected from: . In one specific example, ring B is am.

[0104] In the above chemical formula IB, R 2d can be H, hydroxy, amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino or (C3-C6 cycloalkyl)amino. For example, R 2d Includes, but is not limited to, hydroxy or amino.

[0105] In the above chemical formula IB, R 3d is H, halo, cyano, hydroxy, C1-C6 alkoxy or C1-C6 alkyl. In this case, R 3d Any C1-C6 alkoxy group and C1-C6 alkyl group among them may be optionally substituted with halo, cyano or hydroxy. R of formula I 3d The various examples described above can equally be applied to chemical formula IB.

[0106] In the above chemical formula IB, p can be 0, 1, 2 or 3. In one specific embodiment, p can be 0, 1 or 2.

[0107] For example, in the above chemical formula IB, this It could be R 2d is as described above with respect to chemical formula IB. For example, R 2d can be amino, (C1-C6 alkyl)amino or di(C1-C6 alkyl)amino.

[0108] In one specific example, the compound of the present invention's formula I may be a compound of the following formula IC-1 or IC-2:

[0109]

[0110] In the above chemical formula IC-1, n1 and n2 are each independently 1 or 2. Y 1 The carbon atoms of the ring containing N may be optionally substituted with C1-C6 alkyl. In one specific embodiment, when n1 and n2 are both 2, Y 1 Silver CR 2a R 2b , NR 2c , or O. Alternatively, if one or both of n1 and n2 are 1, Y 1 Silver CR 2a R 2b am.

[0111] In the above chemical formula IC-2, Y 2 is CR 2a R 2b , NR 2c Or O.

[0112] In the above chemical formulas IC-1 and IC-2, R 1 is halo, hydroxy, cyano, C1-C6alkoxy, C1-C6alkyl or C1-C6haloalkyl.

[0113] In the above chemical formulas IC-1 and IC-2, R 3dare each independently H, halo, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkyl, or C1-C6 haloalkyl.

[0114] In the above chemical formulas IC-1 and IC-2, p is an integer from 0 to 2. For example, p is 2.

[0115] In the above chemical formulas IC-1 and IC-2, the R 2a and R 2b are each independently selected from the group consisting of H, halo, hydroxy, cyano, C1-C6 alkyl, or C3-C6 cycloalkyl; C1-C6 alkyl substituted with halo, hydroxy, or cyano; C3-C6 cycloalkyl substituted with halo, hydroxy, or cyano; amino, (C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, (C3-C6 cycloalkyl)amino, (C1-C6 alkyl)carbonylamino, (C1-C6 hydroxyalkyl)carbonylamino; and (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl, di(C1-C6 alkyl)aminocarbonyl, and carboxy.

[0116] In the above chemical formulas IC-1 and IC-2, the R 2c is H, C1-C6 alkyl, C3-C6 cycloalkyl, (C1-C6 alkyl)carbonyl, (C1-C6 hydroxyalkyl)carbonyl, aminocarbonyl, (C1-C6 alkyl)aminocarbonyl or di(C1-C6 alkyl)aminocarbonyl.

[0117] n1, n2, Y of chemical formula IC-1 or IC-2 1 , Y 2 , R 1 , R 2a , R 2b , R 2c , R 3d And with respect to p, the specific examples described above with respect to Formula 1, Formula A, Formula B, Formula IA and Formula IB can be equally applied to the corresponding variables and structures of Formula IC-1 or Formula IC-2.

[0118] The compound of the above formula I may be a compound selected from the group consisting of the following formulae:

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] definition

[0127] All technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art, and unless otherwise stated, conventional measuring methods, manufacturing methods, conventional ingredients or materials based on conventional techniques of pharmacology, pharmaceutical manufacturing, mass spectrometry, NMR, HPLC, biochemistry, etc. are used.

[0128] The individual features and components of each embodiment described and illustrated in this specification may be combined with the features and components of any other embodiment without departing from the scope or spirit of the present disclosure.

[0129] Unless otherwise specified, in this specification and any attached claims, "and" and "or" mean "and / or." The terms "comprises" and "comprised" are open-ended, meaning that the compound, composition, or method may include additional features or components in addition to the specific features or components listed.

[0130] In this specification, a numerical range indicated using the term “to” refers to a range that includes the numerical values ​​described before and after the term “to” as the lower and upper limits, respectively.

[0131] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the term "optionally substituted" means that the occurrences include instances where the occurrences are either substituted or unsubstituted with the specified substituent.

[0132] compound

[0133] The term "halogen" refers to an atom belonging to Group 17 of the periodic table. Halogen atoms include fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). The term "halogen" may be used interchangeably with the term "halo," which refers to a monovalent functional group composed of a halogen.

[0134] The term "hydroxy" refers to the -OH functional group (hydroxyl group).

[0135] The term "-CN" or "cyano" refers to a functional group consisting of a triple bond between a carbon atom and a nitrogen atom.

[0136] The term "amino" refers to a nitrogen atom with hydrogen attached to it, i.e. -NH2.

[0137] The term "alkylamino" as used herein refers to a functional group in which one hydrogen of amino is replaced by alkyl. For example, C 1-6 Alkylamino is -NH(C 1- C6 alkyl) may include, but is not limited to, methylamino, ethylamino, propylamino, butylamino, etc.

[0138] The term "dialkylamino" as used herein refers to a functional group in which two hydrogens of amino are each replaced by alkyl. In this case, the substituted alkyls may be the same or different. For example, di(C 1-6 Alkyl)amino is -N(C 1-C6 alkyl)2 may include, but is not limited to, dimethylamino, diethylamino, dipropylamino, dibutylamino, ethylmethylamino, methylpropylamino, ethylpropylamino, etc.

[0139] The term "oxo" refers to =O, and "oxo-substituted" means that the carbon atom has an =O substituent in the form -C(=O)-. A carbon atom substituted with an oxo group may serve as a carbonyl group.

[0140] The term “carbonyl” as used herein refers to a divalent functional group of -C(=O)-.

[0141] The term “carboxy” as used herein refers to -COOH.

[0142] The term "alkyl" refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon. The alkyl may be a substituted or unsubstituted alkyl group. The alkyl may be an alkyl group having C1 to C8, C1 to C7, C1 to C6, C1 to C5, C1 to C4, C1 to C3, or C1 to C2. Non-limiting examples of the alkyl may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, iso-amyl, or n-hexyl.

[0143] The term "haloalkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group having a specified number of carbon atoms, substituted with one or more halogen atoms. Haloalkyl groups include perhaloalkyl groups, wherein all hydrogens of the alkyl group are replaced with halogens (e.g., -CF3, -CF2CF3). The halogens may be the same (e.g., CHF2, -CF3) or different (e.g., CF2Cl). Where specified, a haloalkyl group may be optionally substituted with one or more substituents other than halogen. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, dichloroethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl groups.

[0144] As used herein, the term "alkylene" refers to a divalent, fully saturated, branched or unbranched (or straight-chain or linear) hydrocarbon having the formula -C n H 2n - refers to a functional group expressed as a. For example, C 1-6 Alkylenes may include ethylene, propylene, butylene, and hexylene.

[0145] The term "alkenyl" refers to a linear or branched hydrocarbyl group having 2 to 8 carbon atoms, and in some embodiments, 2 to 6 carbon atoms or 2 to 4 carbon atoms, and having at least one site of vinyl unsaturation (>C=C<). For example, (Cx-Cy)alkenyl refers to an alkenyl group having x to y carbon atoms, and may include, for example, ethenyl, propenyl, isopropylene, 1,3-butadienyl, and the like.

[0146] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" may also include hydrocarbyl groups having one triple bond and one double bond. For example, (C2-C6)alkynyl may include ethynyl, propynyl, and the like.

[0147] The term "alkoxy" refers to a substituent in which a substituted or unsubstituted straight or branched chain alkyl moiety is linked to another chemical structure by an oxygen atom. The alkoxy may include, without limitation, all possible isomers thereof, such as methoxy, ethoxy, propoxy, and butoxy, or isopropoxy, isobutoxy, and t-butoxy.

[0148] The term "cycloalkyl" refers to a saturated or partially unsaturated hydrocarbon ring having a specified number of carbon atoms as ring elements. That is, C3-C6 cycloalkyl refers to a cycloalkyl group having 3, 4, 5, or 6 carbon atoms as ring elements. The term "cycloalkyl" herein may refer to, for example, C3-C6 cycloalkyl, C3-C5 cycloalkyl, or C3-C4 cycloalkyl. For example, it may be cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, or cyclohexenyl.

[0149] The term "heterocyclyl" or "heterocyclic ring" refers to a saturated or partially unsaturated cyclic hydrocarbon group containing at least one heteroatom. A heterocyclyl group can be a monocyclic, bicyclic, or tricyclic group. The two ring groups can be a spiro-ring, a bridged-ring, or a fused-ring. A spiro-ring group refers to a structure in which two rings share a common atom. A bridged-ring group refers to a structure in which two non-adjacent ring elements are connected to each other by one or more bridging elements. A heterocyclyl group can contain 3 to 20, 3 to 12, 3 to 10, 3 to 7, 3 to 6, 4 to 6, or 5 to 6 ring elements. The above heterocyclyl group may contain one or more heteroatoms selected from the group consisting of N, O and S.

[0150] One or more of the N or S atoms in a heterocyclyl group may be oxidized, and the term "heterocyclyl" includes such oxidized forms (e.g., N → O-, S(O), SO2). Non-limiting examples of monocyclic heterocyclic ring groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, azepanyl, and the like. For example, spiro heterocyclyl groups include 2-azaspiro[3.3]heptan-2-yl, 2,6-diazaspiro[3.3]heptan-2-yl, 6-oxa-2-azaspiro[3.3]heptan-2-yl, 2-azaspiro[3.4]octane-2-yl, 2,6-diazaspiro[3.4]octane-2-yl, 6-oxa-2-azaspiro[3.4]octane-2-yl, 6-azaspiro[3.4]octane-6-yl, 2,6-diazaspiro[3.4]octane-6-yl, 2-oxa-6-azaspiro[3.4]octane-6-yl, 2-azaspiro[4.4]nonan-2-yl, Includes, but is not limited to, 2,7-diazaspiro[4.4]nonan-2-yl or 2-oxa-7-azaspiro[4.4]nonan-7-yl. For example, the bridged heterocyclyl group is selected from the group consisting of 3-oxa-8-azabicyclo[3.2.1]octan-8-yl, 8-oxa-3-azabicyclo[3.2.1]octan-3-yl, 3,8-diazabicyclo[3.2.1]octan-3-yl, 3,8-diazabicyclo[3.2.1]octan-8-yl, 2-oxa-5-azabicyclo[2,2,2]octan-5-yl, 8-azabicyclo[3.2.1]octan-8-yl, 3-azabicyclo[3.1.1]heptan-3-yl, or 6-oxa-3-azabicyclo[3.1.1]heptan-3-yl, 3,6-diazabicyclo[3.1.1]heptan-3-yl or Including, but not limited to, 3,6-diazabicyclo[3.1.1]heptan-6-yl.

[0151] The term "aryl" also includes groups in which an aromatic ring or an aromatic ring is fused to one or more carbon rings, such as C6-C 12 The aryl group of is, for example, C6 to C 10Or it may be an aryl group having C6 to C8. Non-limiting examples of aryl include phenyl or naphthyl.

[0152] The term "heteroaryl" refers to a monocyclic or bicyclic group containing one or more heteroatoms selected from the group consisting of N, O, and S, with the remaining ring atoms being carbon. The heteroaryl group may contain, for example, 1 to 3 heteroatoms and may contain 5 to 10 ring atoms. For example, the heteroaryl may be a 5-membered or 6-membered monocyclic heteroaryl. The heteroaryl may contain, for example, 1 or 2 nitrogen atoms. The S or N may be oxidized to have multiple oxidation states. Examples of monocyclic heteroaryls include, but are not limited to, pyrimidinyl, pyridinyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like.

[0153] The term "substitution" in the above "optionally substituted" refers to introducing a substituted hydrogen atom in the case where one or more hydrogen atoms in an organic compound are replaced with another atomic group to form a derivative, and "substituent" refers to the introduced atomic group. As used herein, a "substituted" group is one in which one or more hydrogen atoms are replaced with one or more non-hydrogen atomic groups, provided that the valence requirement is satisfied and a chemically stable compound is generated from the substitution. As used herein, unless explicitly described as "unsubstituted," all substituents should be interpreted as being either substituted or unsubstituted.

[0154] In this specification, when a combination of substituents is referred to as one group, e.g., haloalkyl, hydroxyalkyl, etc., it generally contains the atom from which the last mentioned group is attached to the remainder of the molecule.

[0155] In this specification " ", "*" or "-" are used to indicate the position at which the substituent is bonded to the residue of the compound. For example, when - is displayed at the end of a substituent, it means that the end is bonded to the remaining residue of the compound. Also, when two or more substituents are connected with "-", it means that the substituent immediately before "-" is bonded to the substitutable atom of the substituent immediately after "-".

[0156] The term "solvate" as used herein may refer to a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric solvent bound by non-covalent intermolecular forces. Preferred solvents include those that are volatile, non-toxic, and / or suitable for human administration. Those skilled in the art will readily be able to prepare solvates, such as hydrates, of the compounds disclosed herein using appropriate techniques known in the art.

[0157] The term "stereoisomer" as used herein may mean a compound of the present invention or a salt thereof having the same chemical or molecular formula but different optically or sterically, and specifically may be a diastereomer, an enantiomer, or a geometric isomer.

[0158] In some embodiments, the compounds of the present invention may contain one or more asymmetric centers, and may be in the form of racemates, single enantiomers, mixtures of enantiomers, single diastereomers, mixtures of diastereomers, etc. In one embodiment, due to the nature or restricted rotation of the asymmetric center, the compounds of the present invention may exist in the form of enantiomers or diastereomers.

[0159] When two or more asymmetric centers are present in the compounds of the present invention, multiple diastereoisomers and enantiomers of the chemical structures disclosed herein may exist, and all such pure isomers, isolated isomers, partially pure isomers, or racemic mixtures are intended to fall within the scope of the present invention.

[0160] Purification of the above isomers and separation of the isomer mixture can be achieved by standard techniques known in the art. For example, a diastereomeric mixture can be separated into individual diastereoisomers by chromatographic processes or crystallization, and racemates can be separated into individual enantiomers by chiral phase chromatographic processes or resolution.

[0161] The term "salt" refers to inorganic and organic acid addition salts of a compound. The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. The pharmaceutically acceptable salts may be salts that do not cause serious irritation to the organism to which the compound is administered and do not impair the biological activity and physical properties of the compound. The inorganic salts may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salt may be formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl, trichloroacetic acid, trifluoroacetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate. In addition, the metal salt may be calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

[0162] A pharmaceutically acceptable salt of a compound according to the present invention can be prepared by dissolving the compound of formula I in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, adding an excess of an organic acid or an aqueous solution of an inorganic acid, and then precipitating or crystallizing the mixture. Subsequently, the solvent or the excess of acid is evaporated from the mixture, followed by drying to obtain an addition salt, or the precipitated salt can be prepared by suction filtration.

[0163] The compounds of the present invention, including stereoisomers, hydrates, solvates and salts, can be prepared by known organic synthetic methods and can be synthesized through a number of synthetic routes.

[0164] The reaction for preparing the compound of the present invention can be performed in a suitable solvent that can be appropriately selected by those skilled in the art of organic synthesis. Suitable solvents are those that are substantially non-reactive with the starting materials (reactants), intermediates, or target products at the temperature at which the reaction occurs. Those skilled in the art will be able to appropriately select the appropriate solvent for each specific reaction step.

[0165] During the synthesis of the compound of the present invention, protection and deprotection of various functional groups can be achieved. Those skilled in the art will readily be able to determine the necessity of protection and deprotection and select appropriate protecting groups.

[0166] Each reaction can be monitored by any suitable method known in the art. For example, the synthesis of the target compound can be monitored by spectroscopic means, e.g., NMR (e.g., 1 H or 13 C), mass spectroscopy, or chromatography (HPLC or TLC).

[0167] The compound of the present invention can be synthesized according to the synthetic process described in the examples below, and based on this, the target compound can be manufactured by appropriately changing the reactants and reaction conditions according to the structure of the target compound.

[0168] Medicinal uses, pharmaceutical compositions and methods of administration

[0169] Another aspect provides a pharmaceutical composition comprising a compound according to one aspect, a stereoisomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof. The compound, stereoisomer, solvate, and salt are as described above.

[0170] Compounds according to one aspect of the present invention can inhibit overexpression, excessive activity, mutation, and activation of signaling pathways associated with cyclin K of CDK12 and / or CDK13. Accordingly, the compounds can inhibit cell proliferation, inhibit cell cycle progression, and / or promote cell death.

[0171] CDK12 and its ortholog CDK13 belong to the CDK family that regulate transcriptional and posttranscriptional processes, and are known to mediate gene transcription by forming a complex with cyclin K to phosphorylate the C-terminal domain of RNA polymerase II (e.g., Greifenberg et al. 2016, "Structural and Functional Analysis of the CDK13 / Cyclin K Complex," Cell Rep. Vol. 14, No. 2, pp. 320-331.). The CDK12 / cyclin K and CDK13 / cyclin K complexes phosphorylate Ser2 of the C-terminal domain of RNA polymerase II, a process considered a crucial step in transitioning from transcription initiation to elongation.

[0172] Recently, various compounds have been reported to directly degrade cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase (Zuzanna et al., 2023, "Design principles for cyclin K molecular glue degraders", Nat Chem Biol Sep 7). Thus, the cyclin K degrading ability of these compounds is expected to exhibit characteristics distinct from those of conventional kinase inhibitors, which regulate the phosphorylation of downstream signaling molecules.

[0173] The compounds according to the present invention may exhibit cyclin K degrading activity together with CDK12 and / or CDK13 inhibitory activity.

[0174] Cyclin K degradation can induce novel anticancer effects in addition to direct inhibition of CDK12 and / or CDK13 in cells. Cyclin K is an essential partner of both CDK12 and / or CDK13 and is required for their activity. Independently of CDK12 and / or CDK13, cyclin K expression is increased in several cancer cells compared to normal cells, suggesting that cyclin K itself is directly involved in the proliferation of various cancer cells. Furthermore, cyclin K has been shown to increase the expression of specific proteins associated with treatment resistance, suggesting that it may improve responsiveness to existing treatments (Yi Xiao and Jixin Dong, 2023, "Coming of Age: Targeting Cyclin K in Cancers", Cells. Aug 11;12(16):2044).

[0175] Therefore, the compound according to the present invention is expected to have superior anticancer efficacy compared to existing therapeutic agents because it can exhibit increased and sustained efficacy compared to CDK12 / CDK13 inhibitors that only inhibit kinase activity.

[0176] Accordingly, a pharmaceutical composition according to one aspect of the present invention may be used for the treatment of diseases caused by overexpression, excessive activity, mutation, and / or activation of signaling pathways associated with CDK12 and / or CDK13 and / or cyclin K. In addition, the pharmaceutical composition may be used for inhibiting cell proliferation, inhibiting cell cycle progression, and / or promoting cell death.

[0177] In one specific example, the disease may be cancer, a proliferative disease, a neurodegenerative disease, an autoimmune disease, or a disease caused by an abnormality in protein translation function within a cell.

[0178] A "proliferative disease" as referred to herein is a disease characterized by excessive proliferation of cells. A proliferative disease is associated with: (1) pathological proliferation of normally quiescent or normally proliferating cells; (2) pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); (3) pathological expression of proteolytic enzymes, such as matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases), which may cause unwanted transformation of the cellular matrix; and / or (4) pathological angiogenesis, which occurs in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancer, benign neoplasms, and angiogenesis that accompanies and facilitates the disease state (as defined above as pathological angiogenesis).

[0179] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for treating cancer. In one embodiment, the cancer may be a cancer caused by overexpression, excessive activity, mutation of CDK12 and / or CDK13, and / or activation of a signaling pathway associated with cyclin K. For example, the cancer may be a cancer caused by overexpression, excessive activity, mutation of CDK12 and / or CDK13, and / or activation of a signaling pathway associated with cyclin K, for example, a cancer characterized by amplification of CDK12, CDK13, or cyclin K genes.

[0180] In one specific example, the cancer may be selected from the following groups:

[0181] (1) Carcinomas including breast, liver, lung, colon, kidney, bladder, small cell lung cancer, non-small cell lung cancer, head and neck cancer, thyroid cancer, esophageal cancer, stomach cancer, pancreatic cancer, ovarian cancer, gallbladder cancer, cervical cancer, prostate cancer, and skin cancer, including squamous cell carcinoma

[0182] (2) Sarcomas, such as osteosarcoma and osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma and mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), hemangiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (fatty tissue), glioma and astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), or mesenchymal and mixed mesodermal tumors (mixed connective tissue type);

[0183] (3) Hematopoietic tumors of the lymphoid system, such as leukemia, acute lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, hairy cell lymphoma, myeloma, mantle cell lymphoma, and Burkett's lymphoma.

[0184] (4) Hematopoietic tumors of the bone marrow, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), and promyelocytic leukemia.

[0185] (5) Tumors of the central and peripheral nervous system, such as astrocytoma, neuroblastoma, glioma, and schwannoma.

[0186] In one specific example, the neurodegenerative disease may be Alzheimer's disease, Parkinson's disease, or Lou Gehrig's disease. For example, the autoimmune disease may be rheumatoid arthritis, systemic lupus erythematosus, psoriasis, or Sjogren's syndrome. For example, the disease caused by an abnormality in the intracellular protein translation function may be muscular dystrophy, myotonic dystrophy, amyotrophic lateral sclerosis, spinal muscular atrophy, or fragile X syndrome.

[0187] As used herein, the term "treating" or "treatment" means inhibiting a disease, condition or disorder, e.g., preventing further development of the pathology and / or symptoms in a subject experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder, or ameliorating a disease, condition or disorder, e.g., ameliorating a disease, condition or disorder, e.g., reversing the pathology and / or symptoms, e.g., reducing disease severity.

[0188] "Cancer," a disease to be prevented or treated by the pharmaceutical composition, is a general term for diseases caused by cells that have aggressive characteristics in which cells divide and grow while ignoring normal growth limits, invasive characteristics in which cells infiltrate surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body. Cancers in which the compound of the present invention has therapeutic activity may include, for example, any cancer that can be treated, alleviated, delayed, inhibited, or prevented due to overexpression, excessive activity, mutation, and / or activation of a signaling pathway associated with cyclin K of CDK12 and / or CDK13.

[0189] For example, the cancer includes, but is not limited to, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, head and neck cancer, thyroid cancer, skin cancer, biliary tract cancer, esophageal cancer, hematopoietic tumors of the lymphoid and myeloid systems, tumors of the central and peripheral nervous systems, sarcoma, etc. In one embodiment, the cancer may include breast cancer, lung cancer, stomach cancer, pancreatic cancer, or colon cancer. In one embodiment, the cancer may be breast cancer, such as triple negative breast cancer.

[0190] For example, the pharmaceutical compositions described herein can be used to treat “high-grade” cancers (e.g., high-grade serous ovarian cancer, metastatic non-small cell lung cancer, metastatic breast cancer (e.g., triple-negative breast cancer), metastatic gastric cancer, metastatic liver cancer), tumors exhibiting a particular phenotype (e.g., estrogen receptor-positive (ER+) breast cancer, human epidermal growth factor receptor 2 (HER2) positive breast or gastric cancer, tyrosine kinase-positive non-small cell lung cancer, or PD-L1 positive solid tumors), and / or cancers that have become resistant to treatment with previously administered therapeutic agents (e.g., chemotherapeutic agents (e.g., cisplatin or fluorouracil), CDK4 / 6 inhibitors (e.g., palbociclib), estrogen receptor-degrading agents (e.g., fulvestrant), or PARP inhibitors (e.g., olaparib)).

[0191] The pharmaceutical composition may include an additional anticancer agent. The pharmaceutical composition may be a single composition or individual compositions. For example, the pharmaceutical composition according to one aspect may be a composition in an oral dosage form, and the anticancer agent may be a composition in a parenteral dosage form.

[0192] When used in the treatment of cancer, the compounds of the present invention may be used alone or in combination with conventional surgery or radiotherapy, chemotherapy or immunotherapy.

[0193] For example, the compounds of the present invention may be used in combination with other anticancer therapies, such as radiation therapy, taxane derivatives (e.g., paclitaxel, docetaxel, cabazitaxel), platinum compounds (e.g., cisplatin, carboplatin), antimetabolites (e.g., 5-FU, gemcitabine, cytarabine), anti-CTLA4 therapies (e.g., ipilimumab, tremelimumab), anti-PD1 therapies (e.g., nivolumab, pembrolizumab), anti-PD-L1 therapies (e.g., atezolizumab, durvalumab), anti-VEGF therapies (e.g., bevacizumab, ramucirumab, aflivacept), anti-EGFR therapies (e.g., cetuximab), topoisomerase inhibitors (e.g., irinotecan), anti-HER2 therapies (e.g., trastuzumab, pertuzumab), anti-hormonal therapies (e.g., tamoxifen, exemestane, letrozole, anastrozole), estrogen receptor inhibitors (e.g., elacestrant, fulvestrant), ERK inhibitors (e.g., ulixertinib), PARP inhibitors (e.g., olaparib, niraparib, talazoparib), mTOR inhibitors (e.g., everolimus, temsirolimus), CDK4 / 6 inhibitors (e.g., abemaciclib, palbociclib), EGFR inhibitors (e.g., afatinib, erlotinib, osimertinib, gefitinib, dacomitinib), HER2 inhibitors (e.g., neratinib, lapatinib), ALK inhibitors (e.g., crizotinib, alectinib, brigatinib, ceritinib), tyrosine kinase inhibitors (e.g., avapritinib, ripretinib, sunitinib, sorafenib, pazopanib, Regorafenib, cabozantinib, lenvatinib), MEK inhibitors (e.g., trametinib), BCR-ABL inhibitors (e.g., imatinib, nilotinib, dasatinib), PI3K inhibitors (e.g., alpelisib), FGFR inhibitors (e.g., putivatinib, pemigatinib), ROS1 inhibitors (e.g., crizotinib, entrectinib), androgen biosynthesis inhibitors (e.g., abiraterone acetate), androgen receptor inhibitors (e.g., enzalutamide, darolutamide, apalutamide), Hedgehog inhibitors (e.g., sonidegib, vismodegib), MET inhibitors (e.g., capmatinib, tepotinib), AXL inhibitors,It can be administered in combination with NTRK1 inhibitors, RET inhibitors (e.g., pralsetinib, selpercatinib), KRAS inhibitors (e.g., adagrasib, sotorasib), or RAF inhibitors (e.g., encorafenib, vemurafenib).

[0194] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used to mean an excipient, diluent, or auxiliary. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, saline, a buffer such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0195] The pharmaceutical composition described above may be prepared in any dosage form according to conventional methods. For example, the composition may be formulated as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral dosage form (e.g., injection). Furthermore, the composition may be prepared as a systemic dosage form or a topical dosage form.

[0196] In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid preparation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the liquid preparation for oral administration may be a suspension, an oral solution, an emulsion, or a syrup. The liquid preparation may include water or liquid paraffin. The liquid preparation may include an excipient such as a wetting agent, a sweetener, a flavoring agent, or a preservative. In the above pharmaceutical composition, the preparation for parenteral administration may be a sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized product, or suppository. The non-aqueous solvent or suspension may contain a vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, Tween 61, cocoa butter, laurin butter, or glycerogelatin.

[0197] The pharmaceutical composition comprises a compound according to one aspect, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient of the pharmaceutical composition. The term "active ingredient" refers to a physiologically active substance used to achieve pharmacological activity (e.g., anticancer).

[0198] The pharmaceutical composition may comprise an effective amount of a compound according to one aspect, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof. The term "effective amount" refers to an amount sufficient to exhibit the effect of preventing or treating a disease when administered to a subject in need of prevention or treatment. The effective amount can be appropriately selected by those skilled in the art depending on the cell or subject selected. The preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the subject, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, the compound, its stereoisomer, solvate, or pharmaceutically acceptable salt may be administered in divided doses of, for example, about 0.0001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 100 mg / kg, once to 24 times a day, once to 7 times every 2 days to 1 week, or once to 24 times every 1 month to 12 months. In the pharmaceutical composition, the compound, its stereoisomer, solvate, or pharmaceutically acceptable salt may be included in an amount of about 0.0001 wt% to about 10 wt%, or about 0.001 wt% to about 1 wt%, based on the total weight of the entire composition.

[0199] Administration may be oral or parenteral. For example, the route of administration may be oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal. The composition may be administered systemically or locally, and may be administered alone or in combination with other pharmaceutically active compounds.

[0200] Another aspect provides a method for treating a disease caused by overexpression, excessive activity, mutation and / or activation of a signaling pathway associated with CDK12 and CDK13, comprising administering to a subject a compound according to one aspect, a stereoisomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof. The compound, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, and the disease treatable therefor are as described above.

[0201] For example, the pharmaceutical compositions described herein can be used to treat “high-grade” cancers (e.g., high-grade serous ovarian cancer, metastatic non-small cell lung cancer, metastatic breast cancer (e.g., triple-negative breast cancer), metastatic gastric cancer, metastatic liver cancer), tumors exhibiting a particular phenotype (e.g., estrogen receptor-positive (ER+) breast cancer, human epidermal growth factor receptor 2 (HER2) positive breast or gastric cancer, tyrosine kinase-positive non-small cell lung cancer, or PD-L1 positive solid tumors), and / or cancers that have become resistant to treatment with previously administered therapeutic agents (e.g., chemotherapeutic agents (e.g., cisplatin or fluorouracil), CDK4 / 6 inhibitors (e.g., palbociclib), or estrogen receptor-degrading agents (e.g., fulvestrant) or PARP inhibitors (e.g., olaparib)).

[0202] In one embodiment, the method may comprise the steps of determining whether the subject has advanced cancer or tumor cells of a particular phenotype, or has developed resistance to a previously administered therapeutic agent; and administering a compound of the invention if the subject is determined to have advanced cancer or tumor cells of a particular phenotype, or has developed resistance.

[0203] The subject may be a mammal, such as a human, mouse, rat, cow, horse, pig, dog, monkey, sheep, goat, ape, or cat. The subject may be suffering from, or likely to suffer from, symptoms associated with cancer.

[0204] The method may further comprise administering to the subject a known effective ingredient for treating cancer. The known effective ingredient may be administered to the subject simultaneously, separately, or sequentially with the compound, a stereoisomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof according to one aspect.

[0205] The route of administration may be oral or parenteral. The route of administration may be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal.

[0206] Another aspect provides a method for inhibiting CDK12 and / or CDK13 and degrading cyclin K, comprising adding to a cell a compound according to one aspect, a stereoisomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof. In one embodiment, the method can be performed in vitro. The compound, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, and treatable disease are as described above.

[0207] According to a compound according to one aspect, a use thereof for inhibiting CDK12 and / or CDK13, a pharmaceutical composition comprising the same, and a use thereof for treating a disease related to CDK12 and / or CDK13, for example, cancer, the compound has excellent inhibitory activity and selectivity for CDK12 and / or CDK13, and thus may be useful for treating cancer, such as breast cancer, stomach cancer, lung cancer, pancreatic cancer, colon cancer, etc.

[0208] Figure 1 shows the results of measuring the cyclin K degradation ability of the compound of Example 8 in HCC70 breast cancer cells.

[0209] Figure 2 shows the results of measuring the cyclin K degradation ability of the compound of Example 30 in HCC70 breast cancer cells.

[0210] Figure 3 shows the results of measuring the cyclin K degradation ability of the compound of Example 40 in HCC70 breast cancer cells.

[0211] Figure 4 shows the results of measuring the cyclin K degradation ability of the compound of Example 8 in MKN45 gastric cancer cells.

[0212] Figure 5 shows the results of measuring the cyclin K degradation ability of the compound of Example 40 in MKN45 gastric cancer cells.

[0213] Figure 6 illustrates the relative tumor size reduction by the compound of Example 30 in the HCC70 zebrafish xenograft breast cancer model.

[0214] Figures 7 and 8 illustrate the relative tumor size reduction and reduction in the number of metastatic cancer cells by the compound of Example 8 in an AGS zebrafish xenograft gastric cancer model.

[0215] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0216] The meanings of the abbreviations used in the examples below are as follows, and abbreviations not listed below have the meanings commonly used in the relevant fields.

[0217] NCS:N-chlorosuccinimide

[0218] NBS:N-bromosuccinimide

[0219] NIS:N-iodosuccinimide

[0220] MeCN: Acetonitrile

[0221] THF: Tetrahydrofuran

[0222] BnBr: Benzyl bromide

[0223] PMB:p-methoxybenzyl

[0224] Cbz: Benzyloxycarbonyl

[0225] NaBH(OAc)3: Sodium triacetoxyborohydride

[0226] CbzOSu:N-(benzyloxycarbonyloxy)succinimide

[0227] TBDMSCl: tert-butyldimethylsilyl chloride

[0228] TBDPSCl: tert-Butyldiphenylsilyl chloride

[0229] DMAP: 4-dimethylaminopyridine

[0230] TBAF: Tetra-N-butylammonium fluoride

[0231] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0232] HOBT: 1-hydroxybenzotriazole

[0233] EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0234] DIEA:N,N-diisopropylethylamine

[0235] DMF:N,N-dimethylformamide

[0236] DCM: Dichloromethane

[0237] DMSO: dimethyl sulfoxide

[0238] TFA: Trifluoroacetic acid

[0239] AcOH: acetic acid

[0240] EtOAc: ethyl acetate

[0241] NMP:N-methyl-2-pyrrolidone

[0242] HMPA: Hexamethylphosphoramide

[0243] CDI: 1,1'-carbonyldiimidazole

[0244] SEMCl: 2-(trimethylsilyl)ethoxymethyl chloride

[0245] TsOH:p-toluenesulfonic acid

[0246] Na2SO4: Sodium sulfate

[0247] CuI: Copper(I) iodide

[0248] Pd / C: Palladium / Carbon

[0249] (Pd(OH)2 / C: Palladium hydroxide / carbon

[0250] RuPhos-Pd-G3: (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate

[0251] NaH: Sodium hydride

[0252] TEA: Triethylamine

[0253] Pd(PPh3)4: Tetrakis(triphenylphosphine)palladium(0)

[0254] Ti(OEt)4: titanium(IV) ethoxide

[0255] Ti(i-PrO)4: titanium(IV) isopropoxide

[0256] Pd(PPh3)2Cl2: Bis(triphenylphosphine)palladium(II) dichloride

[0257] NaBH4: Sodium borohydride

[0258] NaOH: sodium hydroxide

[0259] (Pd(dppf)Cl2: [1,1'bis(diphenylphosphino)ferrocene]dichloropalladium(II)

[0260] Intermediate S1: Preparation of tert-butyl N-benzyl-N-(6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate

[0261]

[0262] Step 1: Preparation of tert-butyl (6-chloroimidazo[1,2-b]pyridazin-8-yl)glycinate

[0263] 8-Bromo-6-chloro-imidazo[1,2-b]pyridazine (24 g, 103.24 mmol) was dissolved in tert-butyl glycinate (240 mL) and stirred at 120 °C for 16 h. Water (2000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (800 mL). The organic layer was washed three times with brine (2000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give tert-butyl (6-chloroimidazo[1,2-b]pyridazin-8-yl)glycinate (26.6 g, 91.13% yield). MS: m / z = 283.1 (M+1, ESI+).

[0264] Step 2: Preparation of tert-butyl N-benzyl-N-(6-chloroimidazo[1,2-b]pyridazin-8-al)glycinate

[0265] The product of Step 1 (26.3 g, 93.02 mmol) and benzyl bromide (25.2 mL, 212 mmol) were dissolved in MeCN (200 mL), and then cesium carbonate (92.2 g, 283 mmol) was added and stirred at 60 °C for 16 h. Water (1200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (300 mL). The organic layer was washed three times with brine (1200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain tert-butyl N-benzyl- N-(6-chloroimidazo [1,2-b] pyridazin-8-yl) glycinate (24.22 g, 68.25% yield). MS: m / z = 373.1 (M+1, ESI+).

[0266] Step 3: Preparation of tert-butyl N-benzyl-N-(6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate

[0267] The product of Step 2 (14.2 g, 38.09 mmol) and morpholine (33.3 mL, 380.9 mmol) were dissolved in 1,4-dioxane (300 mL), then cesium carbonate (37.23 g, 114.3 mmol) and RuPhos-Pd-G3 (3.19 g, 3.81 mmol) were added and stirred at 110 °C for 16 h. Water (900 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (300 mL). The organic layer was washed three times with brine (150 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S1 (11.8 g, 73.16% yield). MS: m / z = 424.2 (M+1, ESI+).

[0268] Intermediate S2: Preparation of tert-butyl N-benzyl-N-(3-bromo-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate

[0269]

[0270] Intermediate S1 (5.31 g, 12.54 mmol) was dissolved in THF (80 mL), and then NBS (2.23 g, 12.54 mmol) was added several times at -60 °C and stirred at the same temperature under nitrogen gas for 6 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with sodium sulfite solution (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S2 (4.1 g, 65.09% yield). MS: m / z = 502.1 (M+1, ESI+).

[0271] Intermediate S3: Preparation of tert-butyl N-benzyl-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0272]

[0273] Step 1: Preparation of tert-butyl N-benzyl-N-(3-iodo-6-morpholinoimidazol-1,2-b]pyridazin-8-yl)glycinate

[0274] Intermediate S1 (5.1 g, 11.34 mmol) was dissolved in THF (80 mL), and NIS (3.72 g, 16.53 mmol) was added at 0 °C and stirred for 2 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give tert-butyl N-benzyl- N-(3-iodo-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (5.99 g, 72.13% yield). MS: m / z = 550.1 (M+1, ESI+).

[0275] Step 2: Preparation of tert-butyl N-benzyl-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0276] The product of Step 1 (5.95 g, 10.83 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (20.81 g, 108.30 mmol) were dissolved in DMF (150 mL), CuI (10.31 g, 54.15 mmol) was added, and the mixture was stirred at 100 °C for 16 h. Water (1000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (300 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S3 (1.4 g, 26.30% yield). MS: m / z = 492.2 (M+1, ESI+)

[0277] Intermediate S4: Preparation of tert-butyl N-(6-chloroimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0278]

[0279] Step 1: Preparation of tert-butyl (6-chloroimidazo[1,2-b]pyridazin-8-yl)glycinate

[0280] 8-Bromo-6-chloro-imidazo[1,2-b]pyridazine (100 g, 430.17 mmol) was dissolved in tert-butyl glycinate (1000 mL) and stirred at 120 °C for 16 h. Water (3000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (1000 mL). The organic layer was washed three times with brine (3000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give tert-butyl (6-chloroimidazo[1,2-b]pyridazin-8-yl)glycinate (102 g, 83.87% yield). MS: m / z = 283.1 (M+1, ESI+).

[0281] Step 2: Preparation of tert-butyl N-(6-chloroimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0282] The product of Step 1 (102 g, 361.7 mmol) and 4-methoxybenzyl chloride (62.1 g, 397.9 mmol) were dissolved in MeCN (1000 mL), cesium carbonate (178 g, 542.6 mmol) was added, and the mixture was stirred at 60 °C for 16 h. Water (5000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (1000 mL). The organic layer was washed three times with brine (5000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S4 (110.5 g, 76% yield). MS: m / z = 403.1 (M+1, ESI+).

[0283] Intermediate S5: Preparation of tert-butyl N-(3-bromo-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0284]

[0285] Step 1: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-morpholinoimidazol-1,2-b]pyridazin-8-yl)glycinate

[0286] Intermediate S4 (15 g, 37.23 mmol) and morpholine (33.1 mL, 372.33 mmol) were dissolved in 1,4-dioxane (300 mL), then cesium carbonate (37 g, 112 mmol) and Ruphos-Pd-G3 (3.12 g, 3.72 mmol) were added and stirred at 110 °C for 16 h. Water (800 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (300 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain tert-butyl N-(4-methoxybenzyl)-N-(6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (15.2 g, 90.01% yield). MS: m / z = 454.2 (M+1, ESI+).

[0287] Step 2: Preparation of tert-butyl N-(3-bromo-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0288] The product of Step 1 (15.2 g, 33.51 mmol) was dissolved in THF (200 mL), and then NBS (5.96 g, 33.51 mmol) was added at -60 °C and stirred at this temperature for 1 h under nitrogen. Water (800 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with sodium sulfite solution (800 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S5 (12 g, 67.25% yield). MS: m / z = 532.1 (M+1, ESI+).

[0289] Intermediate S6: Preparation of 6-chloro-N,N-bis(4-methoxybenzyl)imidazo[1,2-b]pyridazin-8-amine

[0290]

[0291] 8-Bromo-6-chloroimidazo[1,2-b]pyridazine (90 g, 390 mmol) and bis(4-methoxybenzyl)amine (120 g, 468 mmol) were dissolved in DMF (800 mL), DIEA (135.6 mL, 780 mmol) was added, and the mixture was stirred at 90 °C overnight. Water (4000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (800 mL). The organic layer was washed three times with brine (4000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give intermediate S6 (120 g, 75.95% yield). MS: m / z = 409.1 (M+1, ESI+).

[0292] Intermediate S7: Preparation of 6-chloro-N,N-bis(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0293]

[0294] Step 1: Preparation of 6-chloro-3-iodo-N,N-bis(4-methoxybenzyl)imidazo[1,2-b]pyridazin-8-amine

[0295] Intermediate S6 (120 g, 294 mmol) was dissolved in DMF (800 mL), NIS (66 g, 294 mmol) was added, and the mixture was stirred at 60 °C for 16 h. Sodium thiosulfate solution (8000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (1000 mL). The organic layer was washed three times with brine (8000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 6-chloro-3-iodo-N,N-bis(4-methoxybenzyl)imidazo[1,2-b]pyridazin-8-amine (139 g, 88.53% yield). MS: m / z = 535.1 (M+1, ESI+).

[0296] Step 2: Preparation of 6-chloro-N,N-bis(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0297] The product of Step 1 (139 g, 260.3 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (250 g, 1.3 mol) were dissolved in DMF (1000 mL), and then CuI (123.6 g, 650.8 mmol) and DIEA (226 mL, 1.3 mol) were added and stirred at 85 °C for 16 h. Water (8000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (2000 mL). The organic layer was washed three times with brine (8000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S7 (106 g, 85.48% yield). MS: m / z = 476.1 (M+1, ESI+).

[0298] Intermediate S8: Preparation of ethyl N-(6-chloro-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0299]

[0300] Step 1: Preparation of 6-chloro-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0301] Intermediate S7 (106 g, 222.7 mmol) was dissolved in DCM (600 mL), followed by the addition of TFA (200 mL) and stirring at 25 °C for 1 h. To the reaction mixture was added sodium bicarbonate solution (3000 mL), and the product was extracted three times with DCM (500 mL). The organic layer was washed three times with brine (3000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 6-chloro-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (72 g, 90.8% yield). MS: m / z = 357.2 (M+1, ESI+).

[0302] Step 2: Preparation of ethyl N-(6-chloro-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0303] The product of Step 1 (29 g, 81.5 mmol) and ethyl 2-bromoacetate (20.4 g, 122.2 mmol) were dissolved in THF (200 mL), cesium carbonate (53.14 g, 163 mmol) was added, and the mixture was stirred at 70 °C for 16 h. Water (800 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S8 (27 g, 75% yield). MS: m / z = 443.1 (M+1, ESI+).

[0304] Preparation of intermediate S9:N-(4-methoxybenzyl)-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0305]

[0306] Step 1: Preparation of N,N-bis(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0307] Morpholine (120 mL) was added to intermediate S7 (12 g, 25.2 mmol) and stirred at 120 °C for 16 h. The reaction mixture was cooled and concentrated, and then purified by silica gel chromatography to obtain N,N-bis(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (5 g, 37.23% yield). MS: m / z = 527.3 (M+1, ESI+).

[0308] Step 2: Preparation of N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0309] The product of Step 1 (36 g, 68.31 mmol) was dissolved in DCM (300 mL), then TFA (100 mL) was added, and the mixture was stirred at 25 °C for 1 h. Sodium bicarbonate solution (1000 mL) was added to the reaction mixture, and the product was extracted three times with DCM (100 mL). The organic layer was washed three times with brine (1000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (27 g, 97.12% yield). MS: m / z = 408.2 (M+1, ESI+).

[0310] Step 3: Preparation of ethyl N-(4-methoxybenzyl)-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0311] The product of Step 2 (8.4 g, 20.62 mmol) was dissolved in DMF (100 mL), and then NaH (1.24 g, 30.93 mmol, 60% purity) was added several times at 0 °C and stirred for 30 minutes. Ethyl 2-bromoacetate (5.17 g, 30.93 mmol) was added to the reaction mixture and stirred at 25 °C for 2 hours. Water (800 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain ethyl N-(4-methoxybenzyl)-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (8.2 g, 80.59% yield). MS: m / z = 494.2 (M+1, ESI+).

[0312] Step 4: Preparation of N-(4-methoxybenzyl)-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0313] The product of Step 3 (8.2 g, 16.62 mmol) was dissolved in 1,4-dioxane (80 mL) and water (20 mL), and then sodium hydroxide (1.6 g, 40 mmol) was added. The mixture was stirred at 100 °C for 1 h. 1 N hydrochloric acid (500 mL) was added to the reaction mixture, and the mixture was extracted three times with DCM (150 mL). The organic layer was washed three times with brine (500 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate S9 (7 g, 90.51% yield). MS: m / z = 466.1 (M+1, ESI+).

[0314] Preparation of intermediate S10: N-(4-methoxybenzyl)-N-(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinonyl chloride

[0315]

[0316] Intermediate S9 (600 mg, 1.29 mmol) was dissolved in DMF (10 uL) and DCM (6 mL), then oxalyl chloride ((COCl)2, 1.42 mmol, 0.12 mL) was added and stirred at 25 °C for 1 h. The reaction mixture was concentrated to obtain intermediate S10 (600 mg, crude) as a yellow solid.

[0317] Intermediate S11: Preparation of ethyl (6-(6-methyl-3,6-diazabicyclo[3.1.1]heptan-3-yl)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-8-yl)glycinate

[0318]

[0319] Step 1: tert-Butyl 3-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate

[0320] Intermediate S8 (8.5 g, 19.23 mmol) and intermediate A44 (11.42 g, 57.69 mmol) were dissolved in 1,4-dioxane (100 mL), and then Ruphos Pd-G3 (1.6 g, 1.92 mmol), Ruphos (1.8 g, 3.84 mmol), and cesium carbonate (18.75 g, 57.69 mmol) were added, and the mixture was stirred at 110 °C under nitrogen gas for 48 h. Water (500 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (500 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl 3-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (1.2 g, 10.34% yield) as a yellow solid. MS: m / z =605.3 (M+1, ESI+).

[0321] Step 2: Preparation of ethyl (6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0322] The product of Step 1 (1.2 g, 1.98 mmol) was dissolved in DCM (18 mL), TFA (6 mL) was added, and the mixture was stirred at 25 °C for 2 h. Aqueous sodium bicarbonate solution (200 mL) was added to the reaction mixture, and the product was extracted three times with DCM (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated, and the concentrate was purified by silica gel chromatography to give ethyl (6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)-3-(trifluoromethyl)imidazo[1,2-b] pyridazin-8-yl) glycinate (600 mg, 78.95% yield) as a yellow solid. MS: m / z = 385.0 (M+1, ESI+).

[0323] Step 3: Preparation of ethyl (6-(6-methyl-3,6-diazabicyclo[3.1.1]heptan-3-yl)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-8-yl)glycinate

[0324] The product of Step 2 (600 mg, 1.56 mmol) was dissolved in THF (12 mL), and formaldehyde (163.8 mg, 1.638 mmol) and AcOH (2 drops) were added and stirred at 25 °C for 0.5 h. After that, NaBH(OAc)3 (494 mg, 2.34 mmol) was added and stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated, and the concentrate was purified by silica gel chromatography to obtain intermediate S11 (500 mg, 80.26% yield) as a yellow solid. MS: m / z = 399.1 (M+1, ESI+).

[0325] Intermediate S12: Preparation of ethyl (6-(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0326]

[0327] Intermediate S12 (220 mg, 64.32% yield) was obtained as a yellow solid in the same manner as in steps 1 to 3 for the preparation of intermediate S11, except that intermediate A45 (1.88 g, 9.48 mmol) was used instead of intermediate A44 (11.42 g, 57.69 mmol) in step 1. MS: m / z = 399.1 (M+1, ESI+).

[0328] Intermediate A7: Preparation of 3-methyl-3,8-diazabicyclo[3.2.1]octane-8-ium trifluoroacetate salt

[0329]

[0330] Step 1: Preparation of tert-butyl 3-methyl-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

[0331] Tert-Butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (7 g, 33.13 mmol) and formaldehyde (38.15 g, 508.23 mmol, 35.00 mL, 40% purity) were dissolved in methanol (50 mL) and THF (50 mL), then AcOH (2.14 g, 16.56 mmol) was added and stirred at 25 °C for 1 h. To this reaction solution, NaBH(OAc)3 (10.53 g, 49.69 mmol) was added and stirred at 25 °C for 16 h. The reaction mixture was concentrated and the concentrate was purified by silica gel chromatography to obtain tert-butyl 3-methyl-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (6.6 g, 88.42% yield).

[0332] Step 2: Preparation of 3-methyl-3,8-diazabicyclo[3.2.1]octane-8-ium trifluoroacetate salt

[0333] The product of Step 1 (7.6 g, 33.58 mmol) was dissolved in DCM (30 mL), then TFA (12.9 mL, 167.91 mmol) was added and stirred at 25 °C for 16 h. The reaction mixture was concentrated to obtain intermediate A7 (9.4 g, crude).

[0334] Intermediate A16: Preparation of azetidine-3-carbonitrile trifluoroacetate salt

[0335]

[0336] Tert-Butyl 3-cyanoazetidine-1-carboxylate (3 g, 16.46 mmol) was dissolved in THF (30 mL), followed by the addition of TFA (10 mL) and stirring at 25 °C for 16 h. The reaction mixture was concentrated to obtain intermediate A16 (1.8 g, crude). MS: m / z = 83.2 (M+1, ESI+).

[0337] Intermediate A20: Preparation of 3-methyl-8-azabicyclo[3.2.1]octan-3-ol

[0338]

[0339] Step 1: Preparation of 8-benzyl-3-methyl-8-azabicyclo[3.2.1]octan-3-ol

[0340] 8-Benzyl-8-azabicyclo[3.2.1]octan-3-one (10.00 g, 46.45 mmol) was dissolved in THF (100 mL), and 3 M methylmagnesium bromide solution (39 mL) was slowly added at 0 °C over 30 min, and the mixture was stirred at 25 °C for 3 h. Ice water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 8-benzyl-3-methyl-8-azabicyclo[3.2.1]octan-3-ol (6 g, 55.84% yield). MS: m / z = 232.2 (M+1, ESI+).

[0341] Step 2: Preparation of 3-methyl-8-azabicyclo[3.2.1]octan-3-ol

[0342] The product of Step 1 (6 g, 25.94 mmol) was dissolved in methanol (50 mL), Pd / C (1 g) was added, and the mixture was stirred at 25°C for 16 h under a hydrogen gas atmosphere. The reaction mixture was filtered and concentrated to obtain intermediate A20 (3 g, 81.91% yield). MS: m / z = 142.1 (M+1, ESI+).

[0343] Intermediate A26: Preparation of 4-cyanopiperidin-1-ium trifluoroacetate salt

[0344]

[0345] Tert-Butyl 4-cyanopiperidine-1-carboxylate (3 g, 14.27 mmol) was dissolved in THF (20 mL), then TFA (5 mL) was added and stirred at 25 °C for 16 h. The reaction mixture was concentrated to obtain intermediate A26 (2.8 g, crude).

[0346] Intermediate A29: Preparation of 2-azaspiro[3.3]heptan-6-ol trifluoroacetate salt

[0347]

[0348] Tert-Butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (4 g, 18.76 mmol) was dissolved in DCM (30 mL), followed by the addition of TFA (10 mL) and stirring at 25 °C for 16 h. The reaction mixture was concentrated to obtain intermediate A29 (2.5 g, crude). MS: m / z = 113.9 (M+1, ESI+).

[0349] Intermediate A31: Preparation of pyrrolidine-3-carbonitrile trifluoroacetate salt

[0350]

[0351] Tert-Butyl 3-cyanopyrrolidine-1-carboxylate (1.8 g, 9.17 mmol) was dissolved in DCM (15 mL), followed by the addition of TFA (3 mL) and stirring at 25 °C for 1 h. The reaction mixture was concentrated to obtain intermediate A31 (1.12 g, crude). MS: m / z = 97.1 (M+1, ESI+).

[0352] Intermediate A32: Preparation of 1-(azetidin-3-yl)cyclopropan-1-ol

[0353]

[0354] Step 1: Preparation of methyl 1-benzylazetidine-3-carboxylate

[0355] Methyl azetidine-3-carboxylate hydrochloride (20 g, 132 mmol) and BnBr (17.22 mL, 145 mmol) were dissolved in DMF (300 mL), and DIEA (115 mL, 660 mmol) was added several times, followed by stirring at 25 °C for 16 h. Aqueous ammonium chloride solution (2000 mL) was added to the reaction mixture, and extracted three times with EtOAc (400 mL). The organic layer was washed three times with brine (1000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give methyl 1-benzylazetidine-3-carboxylate (22 g, 81.24% yield). MS: m / z = 205.9 (M+1, ESI+).

[0356] Step 2: Preparation of 1-(1-benzylazetidin-3-yl)cyclopropan-1-ol

[0357] The product of Step 1 (16 g, 77.95 mmol) was dissolved in THF (200 mL), and then Ti(i-PrO)4 (26.59 g, 93.54 mmol) was added. 1 M ethylmagnesium bromide solution (311.81 mL) was slowly added over 30 minutes at -60°C under argon gas. The reaction mixture was warmed to room temperature and stirred for 16 hours. Ammonium chloride aqueous solution (1000 mL) was added to the reaction mixture, and extracted three times with EtOAc (300 mL). The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 1-(1-benzylazetidin-3-yl)cyclopropan-1-ol (9.5 g, 59.95% yield). MS: m / z = 204.2 (M+1, ESI+).

[0358] Step 3: Preparation of 1-(azetidin-3-yl)cyclopropan-1-ol

[0359] The product of Step 2 (9.5 g, 46.73 mmol) was dissolved in methanol (120 mL), Pd / C (4.5 g, 10% purity) was added, and the mixture was stirred at 60°C for 16 hours under a hydrogen gas atmosphere. The reaction mixture was filtered and concentrated to obtain intermediate A32 (4.1 g, 77.53% yield). 1 H NMR (400 MHz, MeOD) δ 4.13 (dd, 4H), 2.76-2.62 (m, 1H), 0.79-0.76 (m, 2H), 0.54-0.47 (m, 2H); MS: m / z = 113.9 (M+1, ESI+).

[0360] Intermediate A36: Preparation of benzyl azetidin-3-yl(cyclopropyl)carbamate

[0361]

[0362] Step 1: Preparation of tert-butyl 3-((benzyloxy)carbonyl)(cyclopropyl)amino)azetidine-1-carboxylate

[0363] Tert-Butyl 3-(cyclopropylamino)azetidine-1-carboxylate (5 g, 23.55 mmol) and CbzOSu (8.80 g, 35.33 mmol) were dissolved in DCM (80 mL), TEA (9.85 mL, 70.66 mmol) was added, and the mixture was stirred at 25 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with DCM (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give tert-butyl 3-((benzyloxy)carbonyl)(cyclopropyl)amino)azetidine-1-carboxylate (7 g, 85.79% yield). MS: m / z = 347.1 (M+1, ESI+).

[0364] Step 2: Preparation of benzyl azetidin-3-yl(cyclopropyl)carbamate

[0365] The product of Step 1 (7 g, 20.21 mmol) was dissolved in DCM (60 mL), TFA (20 mL) was added, and the mixture was stirred at 25°C for 16 h. The reaction mixture was concentrated, adjusted to pH 8–9, and extracted three times with DCM (80 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate A36 (4 g, 80.37% yield). MS: m / z = 247.2 (M+1, ESI+).

[0366] Intermediate A37: Preparation of 3-((tert-butyldimethylsilyl)oxy)azetidine

[0367]

[0368] Azetidine-3-ol hydrochloride (5 g, 46 mmol) and TBDMSCl (13.8 g, 92 mmol) were dissolved in DCM (50 mL), and then TEA (19.10 mL, 137 mmol) was added and stirred at 25 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with DCM (80 mL). The organic layer was washed three times with brine (800 mL), and the organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give intermediate A37 (5.8 g, 67.83% yield). MS: m / z = 188.2 (M +1, ESI+).

[0369] Intermediate A38: Preparation of 3-(((tert-butyldiphenylsilyl)oxy)methyl)azetidine trifluoroacetate salt

[0370]

[0371] Step 1: Preparation of tert-butyl 3-(((tert-butyldiphenylsilyl)oxy)methyl)azetidine-1-carboxylate

[0372] 3-(Hydroxymethyl)azetidine-1-carboxylate (5 g, 26.70 mmol) and TBDPSCl (4.66 g, 40.06 mmol) were dissolved in DCM (80 mL), and then DMAP (326 mg, 2.67 mmol) and imidazole (2.73 g, 40.06 mmol) were added and stirred at 25 °C for 2 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with DCM (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give tert-butyl 3-(((tert-butyldiphenylsilyl)oxy)methyl)azetidine-1-carboxylate (8 g, 70.38% yield). MS: m / z = 426.0 (M+1, ESI+).

[0373] Step 2: Preparation of 3-(((tert-butyldiphenylsilyl)oxy)methyl)azetidine trifluoroacetate

[0374] The product of Step 1 (8 g, 18.80 mmol) was dissolved in DCM (60 mL), TFA (20 mL) was added, and the mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated to obtain intermediate A38 (6 g, crude). MS: m / z = 325.0 (M+1, ESI+).

[0375] Preparation of intermediate A40:(R)-3-((tert-butyldimethylsilyl)oxy)pyrrolidine

[0376]

[0377] Intermediate A40 (6.2 g, 76.10% yield) was obtained in the same manner as for Intermediate A37 using (R)-pyrrolidin-3-ol hydrochloride (5 g, 40.46 mmol) as a starting material. MS: m / z = 202.0 (M+1, ESI+).

[0378] Intermediate A41: Preparation of (S)-3-((tert-butyldimethylsilyl)oxy)pyrrolidine

[0379]

[0380] Intermediate A41 (15 g, 58.99% yield) was obtained in the same manner as for Intermediate A37 using (S)-pyrrolidin-3-ol hydrochloride (11 g, 126 mmol) as a starting material. MS: m / z = 202.2 (M+1, ESI+).

[0381] Intermediate A47: Preparation of 8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolane]

[0382]

[0383] Step 1: Preparation of benzyl 8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolane]-8-carboxylate

[0384] Benzyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (10 g, 38.61 mmol) was dissolved in toluene (60 mL), then TsOH (664 mg, 3.86 mmol) and ethylene glycol (11.97 g, 193.05 mmol) were added and stirred at 120 °C for 48 h. Water (500 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (500 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give benzyl 8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolane]-8-carboxylate (6.8 g, 58.27% yield) as a yellow solid. MS: m / z = 304.1 (M+1, ESI+).

[0385] Step 2: Preparation of 8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolane]

[0386] The product of Step 1 (6.8 g, 22.44 mmol) was dissolved in methanol (50 mL), Pd / C (1 g) was added, and the mixture was stirred at 25°C for 16 hours under a hydrogen gas atmosphere. The reaction mixture was concentrated and filtered, and the concentrate was purified by silica gel chromatography to obtain intermediate A47 (3.7 g, 97.63% yield) as a yellow solid. MS: m / z = 170.1 (M+1, ESI+).

[0387] Intermediate B6: Preparation of 4-chloro-5-methoxybenzene-1,2-diamine

[0388]

[0389] Step 1: Preparation of 4-chloro-5-methoxy-2-nitroaniline

[0390] 4,5-Dichloro-2-nitro-aniline (4 g, 19.32 mmol) was dissolved in methanol (25 mL), and then sodium methoxide (1.05 g, 19.32 mmol) was added and stirred at 70 °C for 4 h. Ice water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 4-chloro-5-methoxy-2-nitroaniline (3.4 g, 86.85% yield). MS: m / z = 203.1 (M+1, ESI+).

[0391] Step 2: Preparation of 4-chloro-5-methoxybenzene-1,2-diamine

[0392] The product of Step 1 (3.4 g, 16.78 mmol) was dissolved in ethanol (50 mL), and then ammonium chloride (13.5 g, 252 mmol), zinc (16.4 g, 252 mmol), and formic acid (1.98 mL, 50.4 mmol) were added at 0 °C and stirred at 25 °C for 2 h. The reaction mixture was filtered and concentrated, and ice water (200 mL) was added, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate B6 (2.3 g, 79.40% yield). MS: m / z = 173.1 (M+1, ESI+).

[0393] Intermediate B15: Preparation of 4-methyl-5-(trifluoromethyl)benzene-1,2-diamine

[0394]

[0395] Step 1: Preparation of 5-methyl-2-nitro-4-(trifluoromethyl)aniline

[0396] 5-Chloro-2-nitro-4-(trifluoromethyl)aniline (10 g, 41.57 mmol) was dissolved in dimethyl sulfoxide (30 mL), diethyl malonate (6.65 g, 41.57 mmol) and potassium tert-butoxide (11.66 g, 103.92 mmol) were added, and the mixture was stirred at 60 °C for 16 h. After the reaction solution was cooled to room temperature, potassium hydroxide (10.96 g, 195 mmol) and water (30 mL) were added, and the mixture was stirred at 60 °C for another 16 h. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain 5-methyl-2-nitro-4-(trifluoromethyl)aniline (5.97 g, 65.24% yield). MS: m / z = 220.2 (M+1, ESI+).

[0397] Step 2: Preparation of 4-methyl-5-(trifluoromethyl)benzene-1,2-diamine

[0398] The product of Step 1 (5.97 g, 27.12 mmol) was dissolved in methanol (20 mL), and then sodium hydrosulfite (23.61 g, 135.59 mmol) and sodium hydroxide (3.25 g, 81.35 mmol) were added and stirred at 25 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate B15 (2.13 g, 41.30% yield). MS: m / z = 191.1 (M+1, ESI+).

[0399] Intermediate B16: Preparation of 4,5-bis(trifluoromethyl)benzene-1,2-diamine

[0400]

[0401] Step 1: Preparation of 1-iodo-4-nitro-2-(trifluoromethyl)benzene

[0402] 4-Nitro-2-(trifluoromethyl)aniline (50 g, 242 mmol) was dissolved in water (600 mL) and sulfuric acid (400 mL) at 0 °C, and then a solution of sodium nitrite (33.48 g, 484 mmol) dissolved in water (160 mL) was slowly added. After stirring at the same temperature for 1 h, a solution of potassium iodide (KI, 181.8 g, 1212 mmol) dissolved in water (1000 mL) was added and stirred at 25 °C for 16 h. The reaction mixture was filtered, washed, and concentrated to obtain 1-iodo-4-nitro-2-(trifluoromethyl)benzene (74 g, 96.17% yield). 1 H NMR (400 MHz, CDCl3) δ 8.48 (d, 1H), 8.28 (d, 1H), 8.05 (dd, 1H).

[0403] Step 2: Preparation of 4-nitro-1,2-bis(trifluoromethyl)benzene

[0404] The product of Step 1 (74 g, 233.4 mmol), methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (Chen's reagent, 224.2 g, 1167.2 mmol) was dissolved in DMF (800 mL), CuI (44.4 g, 233.4 mmol) was added, and the mixture was stirred at 80°C under nitrogen gas for 16 h. Water (8000 mL) was added to the reaction mixture. The product was extracted three times with EtOAc (2000 mL). The organic layer was washed three times with brine (8000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 4-nitro-1,2-bis(trifluoromethyl)benzene (26 g, 42.97% yield).

[0405] Step 3: Preparation of 3,4-bis(trifluoromethyl)aniline

[0406] The product of Step 2 (26 g, 100.4 mmol) and tin(II) chloride (SnCl2, 114.2 g, 602.1 mmol) were dissolved in ethanol (2000 mL) and stirred at 70°C for 16 h under nitrogen gas. Water (800 mL) was added to the reaction mixture. The product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 3,4-bis(trifluoromethyl)aniline (21 g, 91.3% yield).

[0407] Step 4: Preparation of N-(3,4-bis(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide

[0408] The product of Step 3 (21.2 g, 92.54 mmol) was dissolved in 1,4-dioxane (150 mL), and then trifluoroacetic anhydride (TFAA, 38.88 g, 185.05 mmol, 25.73 mL) was added and stirred at 25 °C for 16 h. Water (1000 mL) was added to the reaction mixture. The product was extracted three times with EtOAc (300 mL). The organic layer was washed three times with brine (1000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(3,4-bis(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide (21.7 g, 72.14% yield). MS: m / z = 324.1 (M -1, ESI-).

[0409] Step 5: Preparation of 2,2,2-trifluoro-N-(2-nitro-4,5-bis(trifluoromethyl)phenyl)acetamide

[0410] The product of Step 4 (21.6 g, 66.31 mmol) was dissolved in sulfuric acid (200 mL), potassium nitrate (13.42 g, 132.62 mmol) was added at 0 °C, and the mixture was stirred at 25 °C for 16 h. Ice water (2000 mL) was added to the reaction mixture. The product was extracted three times with EtOAc (400 mL). The organic layer was washed three times with brine (2000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 2,2,2-trifluoro-N-(2-nitro-4,5-bis(trifluoromethyl)phenyl)acetamide (12.5 g, 50.83% yield). MS: m / z = 369.0 (M -1, ESI-).

[0411] Step 6: Preparation of 2-nitro-4,5-bis(trifluoromethyl)aniline

[0412] The product of Step 5 (12.5 g, 33.77 mmol) was dissolved in methanol (150 mL), potassium carbonate (23.3 g, 168.85 mmol) was added, and the mixture was stirred at 25°C for 16 hours. Water (300 mL) was added to the reaction mixture. The product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 2-nitro-4,5-bis(trifluoromethyl)aniline (10.4 g, crude). MS: m / z = 273.0 (M-1, ESI-).

[0413] Step 7: Preparation of 4,5-bis(trifluoromethyl)benzene-1,2-diamine

[0414] The product of Step 6 (10.4 g, 37.96 mmol) and tin(II) chloride (SnCl2, 43.18 g, 227.74 mmol) were dissolved in ethanol (100 mL) and stirred at 70°C for 16 h under nitrogen gas. Water (800 mL) was added to the reaction mixture. The product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate B16 (8.5 g, 94.55% yield). MS: m / z = 243.0 (M-1, ESI-).

[0415] Intermediate B20: Preparation of 5,6-dichloro-1-(phenylsulfonyl)-1H-indole-2-carboaldehyde

[0416]

[0417] Step 1: Preparation of 5,6-dichloro-1-(phenylsulfonyl)-1H-indole

[0418] 5,6-Dichloro-1H-indole (3 g, 16.13 mmol) was dissolved in DMF (30 mL), and NaH (774 mg, 19.35 mmol, 60% purity) was added portionwise at 0 °C and stirred at 0 °C for 1 h. Benzenesulfonyl chloride (4.27 g, 24.19 mmol) was then added and stirred at room temperature for 2 h. Water (300 mL) was added to the reaction mixture, and extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 5,6-dichloro-1-(phenylsulfonyl)-1H-indole (4.5 g, 85.56% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 8.09-8.03 (m, 2H), 7.97 (d, 1H), 7.93 (s, 1H), 7.74 (t, 1H), 7.64 (t, 2H), 6.87 (d, 1H).

[0419] Step 2: Preparation of 5,6-dichloro-1-(phenylsulfonyl)-1H-indole-2-carboaldehyde

[0420] The product of Step 1 (4.5 g, 13.80 mmol) was dissolved in THF (50 mL), and 2 M lithium diisopropylamide solution (2 M LDA, 13.8 mL, 27.6 mmol) was slowly added at -60 °C, and the mixture was stirred at -60 °C for 1 h. After that, DMF (2.02 g, 27.59 mmol, 1.42 mL) was added, and the mixture was stirred at -60 °C for 2 h. Water (200 mL) was added to the reaction mixture, and the mixture was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated, and the concentrate was purified by silica gel chromatography to obtain intermediate B20 (3 g, 61.39% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 8.05 (d, 2H), 7.98-7.93 (m, 2H), 7.74 (t, 1H), 7.63 (t, 2H), 6.86 (d, 1H). MS: m / z = 354.0 (M+1, ESI+).

[0421] Intermediate B22: Preparation of 5,6-dimethyl-1-(phenylsulfonyl)-1H-indole-2-carbaldehyde

[0422]

[0423] Intermediate B22 (1.3 g, 33.82% yield) was obtained as a yellow solid following the same method as in steps 1 and 2 for the preparation of intermediate B20, except that 5,6-dimethyl-1H-indole (2 g, 13.77 mmol) was used instead of 5,6-dichloro-1H-indole (3 g, 16.13 mmol) in step 1. 1 H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.94 (s, 1H), 7.92-7.87 (m, 2H), 7.70 (t, 1H), 7.61-7.55 (m, 3H), 7.52 (s, 1H), 2.41 (s, 3H), 2.28 (s, 3H). MS: m / z =314.1 (M+1, ESI+).

[0424] Intermediate B23: Preparation of 4-fluoro-1-(phenylsulfonyl)-1H-indole-2-carbaldehyde

[0425] Intermediate B23 (3.9 g, 64.36% yield) was obtained as a yellow oil following the same method as steps 1 and 2 for the preparation of intermediate B20, except that 4-fluoro-1H-indole (5.1 g, 37.74 mmol) was used instead of 5,6-dichloro-1H-indole (3 g, 16.13 mmol) in step 1.

[0426] Intermediate B24: Preparation of 4-fluoro-1-(phenylsulfonyl)-1H-indole-2-carbaldehyde

[0427]

[0428] Intermediate B24 (3 g, 78.24% yield) was obtained as a yellow oil following the same method as in steps 1 and 2 for the preparation of intermediate B20, except that 4,7-difluoro-1H-indole (2 g, 13.06 mmol) was used instead of 5,6-dichloro-1H-indole (3 g, 16.13 mmol) in step 1. MS: m / z = 322.1 (M+1, ESI+).

[0429] Intermediate B25: Preparation of 4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde

[0430]

[0431] 4,5,6,7-Tetrahydro-1H-indole (2 g, 16.50 mmol) was dissolved in DMF (20 mL), and phosphoryl chloride (POCl3, 2.46 mL, 26.41 mmol) was added several times at -20 °C, and the mixture was stirred at room temperature for 2 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain intermediate B25 (1.9 g, 77.16% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 9.27 (s, 1H), 6.68 (d, 1H), 2.56 (t, 2H), 2.45 (t, 2H), 1.75-1.65 (m, 4H); MS: m / z = 150.1 (M+1, ESI+).

[0432] Intermediate B26: Preparation of 7-hydroxy-1H-indole-2-carbaldehyde

[0433]

[0434] Intermediate B26 (3.1 g, 85.37% yield) was obtained as a red solid in the same manner as for Intermediate B25, except that 1H-indole-7-ol (3 g, 22.53 mmol) was used instead of 4,5,6,7-tetrahydro-1H-indole (2 g, 16.50 mmol). MS: m / z = 162.2 (M+1, ESI+).

[0435] Intermediate B27: Preparation of 6-hydroxy-1H-indole-2-carbaldehyde

[0436]

[0437] Intermediate B27 (3 g, 82.62% yield) was obtained as a yellow solid in the same manner as for Intermediate B25, except that 1H-indol-6-ol (3 g, 22.53 mmol) was used instead of 4,5,6,7-tetrahydro-1H-indole (2 g, 16.50 mmol). MS: m / z = 162.1 (M+1, ESI+).

[0438] Intermediate B28: Preparation of 7-nitro-1H-indole-2-carbaldehyde

[0439]

[0440] Intermediate B28 (5 g, 96.90% yield) was obtained as a yellow solid in the same manner as for Intermediate B25, except that 7-nitro-1H-indole (4.4 g, 27.14 mmol) was used instead of 4,5,6,7-tetrahydro-1H-indole (2 g, 16.50 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 10.07 (s, 1H), 8.57 (d, 1H), 8.48 (s, 1H), 8.24 (d, 1H), 7.47 (t, 1H). MS: m / z =191.1 (M+1, ESI+).

[0441] Intermediate B29: Preparation of 6-nitro-1H-indole-2-carbaldehyde

[0442]

[0443] Intermediate B29 (4 g, 50.76% yield) was obtained as a yellow solid in the same manner as for Intermediate B25, except that 6-nitro-1H-indole (4.2 g, 25.90 mmol) was used instead of 4,5,6,7-tetrahydro-1H-indole (2 g, 16.50 mmol). MS: m / z = 191.1 (M+1, ESI+).

[0444] Intermediate B30: Preparation of 4,5,6,7-tetrahydro-1H-benzo[d]imidazole-2-carbaldehyde

[0445]

[0446] Step 1: Preparation of 1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-benzo[d]imidazole

[0447] 4,5,6,7-Tetrahydro-1H-benzo[d]imidazole (2.2 g, 18.01 mmol) was dissolved in THF (20 mL), and NaH (792 mg, 19.81 mmol, 60% purity) was added several times at 0 °C, and the mixture was stirred at the same temperature for 1 h. After that, SEMCl (3.82 mL, 21.61 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give 1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-benzo[d]imidazole (1.5 g, 33.0% yield) as a yellow oil. MS: m / z = 253.3 (M+1, ESI+).

[0448] Step 2: Preparation of 1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-benzo[d]imidazole-2-carbaldehyde

[0449] The same procedure as in Step 2 for the preparation of intermediate B20 was followed, except that the product of Step 1 (1.5 g, 5.94 mmol) was used, to obtain 1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-benzo[d]imidazole-2-carbaldehyde (1.4 g, 84.01% yield) as a yellow oil. MS: m / z = 281.2 (M+1, ESI+).

[0450] Step 3: Preparation of 4,5,6,7-tetrahydro-1H-benzo[d]imidazole-2-carbaldehyde

[0451] The product of Step 2 (1.4 g, 4.99 mmol) was dissolved in DCM (12 mL), TFA (4 mL) was added, and the mixture was stirred at 25 °C for 16 h. Sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the mixture was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated to obtain intermediate B30 (700 mg, 93.37% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 13.11 (s, 1H), 9.46 (s, 1H), 2.56 (s, 4H), 1.76 (s, 4H); MS: m / z = 301.1 (M+1, ESI+).

[0452] Example 1: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-ethynyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0453]

[0454] Step 1: Preparation of tert-butyl (6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate

[0455] Intermediate S1 (4.9 g, 11.57 mmol) was dissolved in methanol (70 mL), and Pd / C (1 g) and Pd(OH)2 / C (1 g) were added. The mixture was stirred at 50 °C for 48 h under a hydrogen gas atmosphere. The reaction mixture was filtered, concentrated, and purified by silica gel chromatography to obtain tert-butyl (6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (3 g, 77.78% yield). MS: m / z = 334.1 (M+1, ESI+).

[0456] Step 2: Preparation of tert-butyl-N-(tert-butoxycarbonyl)-N-(6-morpholinoimidazol-1,2-b]pyridazin-8-yl)glycinate

[0457] The product of Step 1 (2.9 g, 10.26 mmol) and di-tert-butyl-dicarbonate (8.95 g, 41.03 mmol) were dissolved in MeCN (60 mL), then cesium carbonate (13.37 g, 41.03 mmol) was added and stirred at 70 °C for 16 h. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl-N-(tert-butoxycarbonyl)-N-(6-morpholinoimidazol-8-yl)glycinate (3.15 g, 80.22% yield). MS: m / z = 434.2 (M+1, ESI+).

[0458] Step 3: Preparation of tert-butyl N-(tert-butoxycarbonyl)-N-(3-iodo-6-morpholinoimidazol[1,2-b]pyridazin-8-yl)glycinate

[0459] The product of Step 2 (3.15 g, 8.23 ​​mmol) was dissolved in THF (50 mL), and then NIS (1.63 g, 7.27 mmol) was added at 0 °C and stirred at 25 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with sodium sulfite solution (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain tert-butyl N-(tert-butoxycarbonyl)-N-(3-iodo-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (3.83 g, 94.22% yield). MS: m / z = 560.1 (M+1, ESI+).

[0460] Step 4: Preparation of tert-butyl N-(tert-butoxycarbonyl)-N-(6-morpholino-3-((trimethylsilyl)ethynylimidazo[1,2-b]pyridazin-8-yl)glycinate

[0461] The product of Step 3 (3.83 g, 6.85 mmol) and ethynyl(trimethyl)silane (1.01 g, 10.27 mmol) were dissolved in 1,4-dioxane (50 mL), and then CuI (391 mg, 2.05 mmol), Pd(PPh3)2Cl2 (481 mg, 685 umol), and TEA (2.86 mL, 20.54 mmol) were added, and the mixture was stirred at 50 °C under nitrogen gas for 4 h. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(tert-butoxycarbonyl)-N-(6-morpholino-3-((trimethylsilyl)ethynylimidazo[1,2-b]pyridazin-8-yl)glycinate (2.86 g, 78.86% yield). MS: m / z = 530.2 (M+1, ESI+).

[0462] Step 5: Preparation of tert-butyl N-(tert-butoxycarbonyl)-N-(3-ethynyl-6-morpholinoimidazol[1,2-b]pyridazin-8-yl)glycinate

[0463] The product of Step 4 (2.86 g, 5.40 mmol) was dissolved in THF (30 mL), and then 1 M TBAF solution (21.6 mL, 21.60 mmol) was added and stirred at 25 °C for 16 h. Ammonium chloride aqueous solution (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain tert-butyl N-(tert-butoxycarbonyl)-N-(3-ethynyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (2 g, 80.96% yield). MS: m / z = 458.2 (M+1, ESI+).

[0464] Step 6: Preparation of (3-ethynyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycine

[0465] The product of Step 5 (2 g, 4.37 mmol) was dissolved in DCM (20 mL), TFA (3.34 mL, 43.71 mmol) was added, and the mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated, and the concentrate was purified by silica gel chromatography to obtain (3-ethynyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycine (900 mg, 68.33% yield). MS: m / z = 302.1 (M+1, ESI+).

[0466] Step 7: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((3-ethynyl-6-morpholinoimidazol[1,2-b]pyridazin-8-yl)amino)acetamide

[0467] The product of Step 6 (900 mg, 2.99 mmol) and intermediate B1 (517 mg, 3.58 mmol) were dissolved in DMF (20 mL), and HATU (1.70 g, 4.48 mmol) and DIEA (1.56 mL, 8.96 mmol) were added, and the mixture was stirred at 25 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(6-amino-2,3-difluorophenyl)-2-((3-ethynyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)amino)acetamide (1 g, 78.33% yield). MS: m / z = 428.1 (M+1, ESI+).

[0468] Step 8: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-ethynyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0469] The product of Step 7 (750 mg, 1.75 mmol) was dissolved in AcOH (10 mL) and stirred at 70°C for 16 hours. Sodium bicarbonate solution (80 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (80 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 1 (28 mg, 3.90% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 7.71-7.67 (m, 2H), 7.23-7.18 (m, 2H), 6.09 (s, 1H), 4.82 (d, 2H), 4.72 (s, 1H), 3.67 (s, 4H), 3.32 (s, 4H); MS: m / z = 410.0 (M+1, ESI+).

[0470] Example 2: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyradazin-8-amine

[0471]

[0472] Step 1: Preparation of 2-bromo-4,4,4-trifluorobutanal

[0473] 4,4,4-Trifluorobutanal (5 g, 39.66 mmol) was dissolved in 1,4-dioxane (50 mL), and bromine (2.62 mL, 47.59 mmol) was added at 0 °C and stirred under nitrogen at 25 °C for 1 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated to obtain 2-bromo-4,4,4-trifluorobutanal (4.5 g, crude).

[0474] Step 2: Preparation of 8-bromo-6-chloro-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazine

[0475] The product of Step 1 (4.5 g, 21.95 mmol) was dissolved in ethanol (50 mL), and 4-bromo-6-chloro-pyridazin-3-amine (915 mg, 4.39 mmol) was added, and the mixture was stirred at 120 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 8-bromo-6-chloro-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazine (890 mg, 64.45% yield). MS: m / z = 314.2 (M+1, ESI+).

[0476] Step 3: Preparation of tert-butyl (6-chloro-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0477] The product of Step 2 (890 mg, 2.83 mmol) was dissolved in tert-butyl glycinate (10 mL) and stirred at 120 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain tert-butyl (6-chloro-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (770 mg, 74.60% yield). MS: m / z = 365.1 (M+1, ESI+).

[0478] Step 4: Preparation of tert-butyl N-(6-chloro-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0479] The product of Step 3 (770 mg, 2.11 mmol) and 4-methoxybenzyl chloride (496 mg, 3.17 mmol) were dissolved in MeCN (20 mL), cesium carbonate (2.06 g, 6.33 mmol) was added, and the mixture was stirred at 60 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(6-chloro-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (470 mg, 45.91% yield). MS: m / z = 485.1 (M+1, ESI+).

[0480] Step 5: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0481] The product of Step 4 (470 mg, 969 μmol) and morpholine (0.85 mL, 9.69 mmol) were dissolved in 1,4-dioxane (20 mL), then cesium carbonate (948 mg, 2.91 mmol) and Ruphos-Pd-G3 (81 mg, 97 μmol) were added and stirred at 110 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(4-methoxybenzyl)-N-(6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (290 mg, 55.87% yield). MS: m / z = 536.2 (M+1, ESI+).

[0482] Step 6: Preparation of N-(4-methoxybenzyl)-N-(6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0483] The product of Step 5 (290 mg, 541.5 μmol) was dissolved in 1,4-dioxane (5 mL) and water (2 mL), and then sodium hydroxide (109 mg, 2.71 mmol) was added and stirred at 100 °C for 48 h. 1 N hydrochloric acid (50 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (15 mL). The organic layer was washed three times with brine (50 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(4-methoxybenzyl)-N-(6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)glycine (220 mg, 84.74% yield). MS: m / z = 480.1 (M+1, ESI+).

[0484] Step 7: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0485] The product of Step 6 (220 mg, 459 μmol) and intermediate B1 (2.20 g, 15.26 mmol) were dissolved in DMF (10 mL), and then HOBT (310 mg, 2.29 mmol), EDCI (440 mg, 2.29 mmol), and DIEA (0.4 mL, 2.29 mmol) were added and stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (230 mg, 82.77% yield). MS: m / z = 606.2 (M+1, ESI+).

[0486] Step 8: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-N-(4-methoxybenzyl)-6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-amine

[0487] The product of Step 7 (230 mg, 380 μmol) was dissolved in AcOH (10 mL) and stirred at 70 °C for 16 h. Sodium bicarbonate solution (60 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (20 mL). The organic layer was washed three times with brine (60 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-N-(4-methoxybenzyl)-6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-amine (300 mg, crude). MS: m / z = 588.2 (M+1, ESI+).

[0488] Step 9: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-6-morpholino-3-(2,2,2-trifluoroethyl)imidazo[1,2-b]pyridazin-8-amine

[0489] The product of Step 8 (300 mg, 509 μmol) was dissolved in DCM (10 mL), TFA (5 mL) was added, and the mixture was stirred at 25°C for 16 hours. Sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the product was extracted three times with DCM (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 2 (80 mg, 33.49% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 7.65 (t, 1H), 7.39 (s, 1H), 7.26-7.16 (m, 2H), 6.04 (s, 1H), 4.82 (d, 2H), 3.95 (dd, 2H), 3.67 (t, 4H), 3.32 (t, 4H); MS: m / z = 468.1 (M+1, ESI+).

[0490] Example 3: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-isopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0491]

[0492] Step 1: Preparation of 2-bromo-3-methylbutanal

[0493] 2-Bromo-3-methylbutanal (14.3 g crude) was obtained in the same manner as in Step 1 of Example 2, except that 3-methylbutanal (11 g, 127.71 mmol) was dissolved in DCM (300 mL), and then L-proline (1.47 g, 12.77 mmol) and NBS (29.55 g, 166.02 mmol) were substituted for bromine (7.61 g, 47.59 mmol) in Step 1 of Example 2, and the mixture was stirred for 16 hours.

[0494] Step 2: Preparation of 8-bromo-6-chloro-3-isopropylimidazo[1,2-b]pyridazine

[0495] 8-Bromo-6-chloro-3-isopropylimidazo[1,2-b]pyridazine (4.3 g, 90.37% yield) was obtained in the same manner as in Step 2 of Example 2, except that the reaction temperature was changed to 100°C and the mixture was stirred for 72 hours using the product of Step 1 (14.3 g, 86.65 mmol). MS: m / z = 274.2 (M+1, ESI+).

[0496] Step 3 to Step 9: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-isopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0497] Using the product of step 2 as a starting material, the compound of example 3 (210 mg, 67.26% yield) was obtained as a white solid in the same manner as steps 3 to 9 of example 2. However, the reaction times of steps 6, 7, and 9 of example 2 were changed to 16 hours, 2 hours, and 1 hour, respectively. 1 H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 7.50 (t, 1H), 7.22-7.13 (m, 3H), 5.97 (s, 1H), 4.79 (d, 2H), 3.68 (t, 4H), 3.29 (t, 4H), 3.25-3.19 (m, 1H), 1.31 (d, 6H); MS: m / z = 428.2 (M+1, ESI+).

[0498] Example 4: Preparation of N-(6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0499]

[0500] Step 1: Preparation of tert-butyl N-benzyl-N-(3-methyl-6-morpholinoimidazol-1,2-b]pyridazin-8-yl)glycinate

[0501] Intermediate S2 (2.1 g, 4.18 mmol) and 2,4,6-trimethyl-1,3,5,2,4,6-trioxatrivorinane (1.05 g, 8.36 mmol) were dissolved in 1,4-dioxane (20 mL) and water (5 mL), and then Pd(PPh3)4 (483 mg, 418 μmol) and potassium carbonate (1.73 g, 12.54 mmol) were added and stirred at 100 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (30 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain tert-butyl N-benzyl-N-(3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (4.1 g crude). MS: m / z = 438.2 (M+1, ESI+).

[0502] Step 2: Preparation of tert-butyl (3-methyl-6-morpholinoimidazol-1,2-b]pyridazin-8-yl)glycinate

[0503] The product of Step 1 (1.15 g, 2.63 mmol) was dissolved in methanol (20 mL), and then Pd / C (200 mg) and Pd(OH)2 / C (200 mg) were added. The mixture was stirred at 50°C for 48 h under a hydrogen gas atmosphere. The reaction mixture was filtered, concentrated, and purified by silica gel chromatography to obtain tert-butyl (3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycinate (550 mg, 60.23% yield). MS: m / z = 348.2 (M+1, ESI+).

[0504] Step 3: Preparation of (3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycine

[0505] The product of Step 2 (550 mg, 1.58 mmol) was dissolved in DCM (10 mL), then TFA (1.81 g, 15.83 mmol) was added and stirred at 25 °C for 16 h. The reaction mixture was concentrated to obtain (3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)glycine (830 mg, crude). MS: m / z = 292.1 (M+1, ESI+).

[0506] Step 4: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((3-methyl-6-morpholinoimidazol-1,2-b]pyridazin-8-yl)amino)acetamide

[0507] The product of Step 3 (830 mg, 2.85 mmol) and intermediate B1 (493 mg, 3.42 mmol) were dissolved in anhydrous DMF (10 mL), and HATU (1.63 g, 4.27 mmol) and DIEA (1.48 mL, 8.55 mmol) were added, and the mixture was stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(6-amino-2,3-difluorophenyl)-2-((3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)amino)acetamide (195 mg, 16.40% yield). MS: m / z = 418.1 (M+1, ESI+).

[0508] Step 5: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0509] The product of Step 4 (195 mg, 467 μmol) was dissolved in AcOH (5 mL) and stirred at 70 °C for 4 hours. Sodium bicarbonate solution (50 mL) was added to the reaction mixture, and the product was extracted three times with DCM (20 mL). The organic layer was washed three times with brine (50 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 4 (70 mg, 37.52% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H), 7.47 (s, 1H), 7.24-7.16 (m, 3H), 5.97 (s, 1H), 4.81 (d, 2H), 3.68 (t, 4H), 3.31 (t, 4H), 2.33 (s, 3H); MS: m / z = 400.1 (M+1, ESI+).

[0510] Example 5: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-ethyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0511]

[0512] The compound of Example 5 (35 mg, 25.72% yield) was obtained as a white solid in the same manner as Steps 1 to 5 of Example 4, except that vinylboronic acid (266 mg, 3.70 mmol) was used instead of 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (1.05 g, 8.36 mmol) in Step 1 of Example 4, using intermediate S2 (1.55 g, 3.09 mmol) as a starting material. 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 7.48 (t, 1H), 7.24-7.15 (m, 3H), 5.96 (s, 1H), 4.80 (d, 2H), 3.67 (t, 4H), 3.31 (t, 4H), 2.78 (dd, 2H), 1.26 (t, 3H); MS: m / z = 414.0 (M+1, ESI+).

[0513] Example 6: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0514]

[0515] Step 1: Preparation of tert-butyl (6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0516] Intermediate S3 (1.4 g, 2.85 mmol) was dissolved in methanol (20 mL), and Pd / C (200 mg) and Pd(OH)2 / C (200 mg) were added, followed by stirring at 50 °C for 48 h. The reaction mixture was filtered, concentrated, and purified by silica gel chromatography to obtain tert-butyl (6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (910 mg, 79.59% yield). MS: m / z = 402.1 (M+1, ESI+).

[0517] Step 2: Preparation of (6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0518] The product of Step 1 (910 mg, 2.27 mmol) was dissolved in DCM (10 ml), then TFA (1.74 mL, 22.67 mmol) was added and stirred at 25 °C for 16 h. The reaction mixture was concentrated, and the concentrate was purified by silica gel chromatography to obtain (6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine (970 mg, crude). MS: m / z = 346.1 (M+1, ESI+).

[0519] Step 3: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0520] The product of Step 2 (970 mg, 2.81 mmol) and intermediate B1 (486 mg, 3.37 mmol) were dissolved in anhydrous DMF (20 mL), and HATU (1.60 g, 4.21 mmol) and DIEA (1.47 mL, 8.43 mmol) were added, and the mixture was stirred at 25 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(6-amino-2,3-difluorophenyl)-2-((6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (890 mg, 67.21% yield). MS: m / z = 472.1 (M+1, ESI+).

[0521] Step 4: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0522] The product of Step 3 (890 mg, 1.89 mmol) was dissolved in AcOH (10 mL) and stirred at 70°C for 4 hours. Sodium bicarbonate solution (80 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 6 (570 mg, 66.59% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.88-7.84 (m, 2H), 7.28-7.15 (m, 2H), 6.21 (s, 1H), 4.84 (d, 2H), 3.67 (t, 4H), 3.35 (t, 4H); MS: m / z = 454.0 (M+1, ESI+).

[0523] Example 7: Preparation of 6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0524]

[0525] Step 1: Preparation of tert-butyl-N-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)imidazo[1,2-b]pyridazin-8-yl)-N-benzylglycinate

[0526] The product of step 2 of intermediate S1 (6 g, 16 mmol) and intermediate A1 (2.73 g, 24 mmol) were dissolved in 1,4-dioxane (60 mL), then Ruphos-Pd-G3 (674 mg, 805 μmol) and cesium carbonate (15.73 g, 48.28 mmol) were added and stirred at 110 °C under nitrogen gas for 16 h. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl-N-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)imidazo[1,2-b]pyridazin-8-yl)-N-benzylglycinate (5.1 g, 70.50% yield). MS: m / z = 450.2 (M+1, ESI+).

[0527] Step 2: Preparation of tert-butyl N-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-3-iodoimidazo[1,2-b]pyridazin-8-yl)-N-benzylglycinate

[0528] The product of Step 1 (5.1 g, 11.34 mmol) was dissolved in THF (30 mL), and then NIS (3.06 g, 13.61 mmol) was added at 0 °C and stirred at 25 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with DCM (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain tert-butyl N-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-3-iodoimidazo[1,2-b]pyridazin-8-yl)-N-benzylglycinate (5.2 g, 79.65% yield). MS: m / z = 576.1 (M+1, ESI+).

[0529] Step 3: Preparation of tert-butyl N-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-benzylglycinate

[0530] The product of Step 2 (5.2 g, 9.04 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (17.36 g, 90.4 mmol) were dissolved in DMF (30 mL), then CuI (8.61 g, 45.2 mmol) was added and stirred at 100 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-benzylglycinate (2.8 g, 59.87% yield). MS: m / z = 518.5 (M+1, ESI+).

[0531] Step 4 to Step 7: Preparation of 6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0532] Using the product of step 3 as a starting material, the compound of example 7 (58 mg, 14.33% yield) was obtained as a white solid in the same manner as steps 1 to 4 of example 6. However, the reaction time of step 7 of example 7 was changed to 2 hours. 1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.89-7.87 (m, 2H), 7.28-7.16 (m, 2H), 6.13 (s, 1H), 4.84 (d, 2H), 4.28 (s, 2H), 3.61 (d, 2H), 3.44 (d, 2H), 1.94-1.83 (m, 4H); MS: m / z = 480.1 (M+1, ESI+).

[0533]

[0534] Hereinafter, the compounds of Examples 8 to 22 were prepared using appropriate intermediates from the intermediate A series of [Table 1] according to the following reaction scheme I.

[0535] [Reaction Formula I]

[0536]

[0537] In the above reaction formula I, R 2 is the R of each example compound 2 have the same structure as. The intermediates referred to as Intermediate A1 to Intermediate A52 in Examples 8 to 22 as well as throughout the Examples have the structure shown in Table 1 below.

[0538] [Table 1]

[0539]

[0540]

[0541] Example 8: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0542]

[0543] Step 1: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0544] Intermediate S4 (6.1 g, 15.14 mmol) and intermediate A2 (4.54 g, 45.42 mmol) were dissolved in 1,4-dioxane (100 mL), and then cesium carbonate (9.87 g, 30.28 mmol) and Ruphos-Pd-G3 (1.27 g, 1.51 mmol) were added and stirred at 100 °C under nitrogen gas for 16 h. Water (500 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (500 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycinate (3.9 g, 55.21% yield). MS: m / z = 467.3 (M+1, ESI+).

[0545] Step 2: Preparation of tert-butyl N-(3-iodo-6-(4-methylpiperazin-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0546] The product of Step 1 (3.8 g, 8.14 mmol) was dissolved in DCM (80 mL), then NIS (1.03 g, 4.59 mmol) and TFA (1.25 mL, 16.28 mmol) were added at 0 °C, and the mixture was stirred for 2 h under nitrogen gas at 25 °C. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with DCM (60 mL). The organic layer was washed three times with sodium sulfite solution (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain tert-butyl N-(3-iodo-6-(4-methylpiperazin-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (2.3 g, 47.66% yield). MS: m / z = 593.2 (M+1, ESI+).

[0547] Step 3: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0548] The product of Step 2 (2.1 g, 3.85 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (7.40 g, 38.50 mmol) were dissolved in N-methyl-2-pyrrolidone (35 mL), then CuI (3.67 g, 19.25 mmol) and HMPA (3.45 g, 19.25 mmol) were added and stirred at 50 °C for 16 h. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (850 mg, 41.30% yield). MS: m / z = 535.3 (M+1, ESI+).

[0549] Step 4: Preparation of N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0550] The product of Step 3 (850 mg, 1.59 mmol) was dissolved in 1,4-dioxane (10 mL) and water (10 mL), sodium hydroxide (1.27 g, 31.80 mmol) was added, and the mixture was stirred at 100 °C for 16 h. 0.5 N hydrochloric acid (80 mL) was added to the reaction mixture, and the mixture was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine (500 mg, 65.72% yield). MS: m / z = 479.2 (M+1, ESI+).

[0551] Step 5: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0552] The product of Step 4 (500 mg, 1.05 mmol) and intermediate B1 (181 mg, 1.26 mmol) were dissolved in DMF (15 mL), and then HOBT (706 mg, 5.23 mmol), EDCI (1.00 g, 5.23 mmol), and DIEA (1.82 mL, 10.45 mmol) were added and stirred at 25 °C for 2 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (380 mg, 60.15% yield). MS: m / z = 605.2 (M+1, ESI+).

[0553] Step 6: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0554] The product of Step 5 (380 mg, 629 μmol) was dissolved in AcOH (10 mL) and stirred at 100 °C for 2 hours. Sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (330 mg, crude). MS: m / z = 587.2 (M+1, ESI+).

[0555] Step 7: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0556] The product of Step 6 (330 mg, 562.60 μmol) was dissolved in DCM (10 mL), TFA (5 mL) was added, and the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated and purified by Prep-HPLC to obtain the compound of Example 8 (55 mg, 20.96% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 7.86 (s, 1H), 7.82 (t, 1H), 7.27-7.15 (m, 2H), 6.19 (s, 1H), 4.83 (d, 2H), 3.37 (t, 4H), 2.35 (t, 4H), 2.18 (s, 3H); MS: m / z = 467.2 (M+1, ESI+).

[0557] Examples 9 to 18

[0558] Compounds of Examples 9 to 18 were prepared in the same manner as in Example 8 using the intermediates described in [Table 2].

[0559] [Table 2]

[0560]

[0561]

[0562]

[0563] Example 19: Preparation of 1-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-ol

[0564]

[0565] Step 1: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-(4-oxopiperidin-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0566] Tert-butyl N-(4-methoxybenzyl)-N-(6-(4-oxopiperidin-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycinate (5.4 g, 51.92% yield) was obtained by the same method as in step 1 of Example 8, except that intermediate S4 (9 g, 22.34 mmol) and intermediate A13 (3.32 g, 33.51 mmol) were used and reacted at 110°C. MS: m / z = 466.2 (M+1, ESI+).

[0567] Step 2: Preparation of tert-butyl N-(3-iodo-6-(4-oxopiperidin-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0568] Tert-butyl N-(3-iodo-6-(4-oxopiperidin-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (2.8 g, 40.81% yield) was obtained by the same method as in Step 2 of Example 8, except that THF (50 mL) was used instead of DCM (80 mL) and the mixture was stirred at 0 °C for 1 h without adding TFA. MS: m / z = 592.3 (M+3, ESI+).

[0569] Step 3: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-(4-oxopiperidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0570] The same procedure as step 3 of Example 8 was followed, except that DMF (80 mL) was used instead of NMP (35 mL) and DIEA (11.3 mL, 64.67 mmol) was used instead of HMPA (3.45 g, 19.25 mmol) and the mixture was stirred at 100 °C to obtain tert-butyl N-(4-methoxybenzyl)-N-(6-(4-oxopiperidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (1.7 g, 67.30% yield). MS: m / z = 534.3 (M+1, ESI+).

[0571] Step 4: Preparation of N-(4-methoxybenzyl)-N-(6-(4-oxopiperidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0572] N-(4-methoxybenzyl)-N-(6-(4-oxopiperidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine (1.2 g, 78.88% yield) was obtained in the same manner as in step 4 of Example 8, except that the reaction time was changed to 2 hours. MS: m / z = 492.2 (M+1, ESI+).

[0573] Step 5: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-(4-oxopiperidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0574] The same procedure as in Step 5 of Example 8 was followed, except that HATU (1.14 g, 3.01 mmol) was used instead of HOBT (706 mg, 5.23 mmol) and EDCI (1.00 g, 5.23 mmol), to obtain N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-(4-oxopiperidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (1.1 g, 72.85% yield). MS: m / z = 604.1 (M+1, ESI+).

[0575] Step 6: Preparation of 1-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-one

[0576] 1-(8-(((6,7-Difluoro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-one (700 mg, 65.60% yield) was obtained in the same manner as in step 6 of Example 8, except that the reaction temperature was changed to 70 ℃ and stirring was performed for 16 hours. MS: m / z = 586.2 (M+1, ESI+).

[0577] Step 7: Preparation of 1-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-ol

[0578] The product of Step 6 (700 mg, 1.2 mmol) was dissolved in methanol (10 mL), sodium borohydride (55 mg, 1.44 mmol) was added at 0 °C, and the mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated, and the concentrate was purified by Prep-HPLC to obtain 1-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-ol (550 mg, 78.57% yield). MS: m / z = 588.5 (M+1, ESI+).

[0579] Step 8: Preparation of 1-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-ol

[0580] The compound of Example 19 (200 mg, 45.76% yield) was obtained as a white solid in the same manner as step 7 of Example 8 except that the reaction time was changed to 2 hours. 1 H NMR (400 MHz, DMSO-d6) δ 12.73 (s, 1H), 7.85-7.80 (m, 2H), 7.25-7.16 (m, 2H), 6.16 (s, 1H), 4.83 (d, 2H), 4.67 (d, 1H), 3.80 (d, 2H), 3.66-3.63 (m, 1H), 3.02 (t, 2H), 1.71 (t, 2H), 1.34-1.27 (m, 2H); MS: m / z = 468.3 (M+1, ESI+).

[0581] Example 20: Preparation of 6-(2-aminopyrimidin-5-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine

[0582]

[0583] Step 1: Preparation of tert-butyl N-(6-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0584] Intermediate S4 (2.6 g, 6.45 mmol) and intermediate A14 (1.34 g, 9.68 mmol) were dissolved in 1,4-dioxane (50 mL) and water (50 mL), and then [1,1-bis(diphenylphosphino)ferrocine]dichloropalladium(II) (469 mg, 645 μmol) and cesium carbonate (4.21 g, 12.91 mmol) were added, and the mixture was stirred at 100 °C under nitrogen gas for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(6-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (2.5 g, 83.94% yield). MS: m / z = 462.2 (M+1, ESI+).

[0585] Step 2: Preparation of tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-bromoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0586] The product of Step 1 (2.5 g, 5.42 mmol) was dissolved in THF (50 mL), and then NBS (964 mg, 5.42 mmol) was added. The mixture was stirred at -60 °C for 3 h under nitrogen gas. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with sodium sulfite solution (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-bromoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (2.5 g, 85.40% yield). MS: m / z = 542.2 (M+3, ESI+).

[0587] Step 3: Preparation of tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0588] The product of Step 2 (2.5 g, 4.63 mmol) and prop-1-yne (3.71 g, 92.52 mmol) were dissolved in DMF (20 mL), and then CuI (176 mg, 925 μmol), Pd(PPh3)2Cl2 (325 mg, 463 μmol), and TEA (0.65 mL, 4.63 mmol) were added and stirred at 40 °C under nitrogen gas for 72 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (1.1 g, 47.60% yield). MS: m / z = 500.2 (M+1, ESI+).

[0589] Step 4: Preparation of N-(6-(2-aminopyrimidin-5-yl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0590] The product of step 3 (1.1 g, 2.20 mmol) was treated in the same manner as step 4 of Example 8 to obtain N-(6-(2-aminopyrimidin-5-yl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (300 mg, 30.72% yield). MS: m / z = 442.2 (M+1, ESI+).

[0591] Step 5: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((6-(2-aminopyrimidin-5-yl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide

[0592] N-(6-amino-2,3-difluorophenyl)-2-((6-(2-aminopyrimidin-5-yl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide (270 mg, 70.07% yield) was obtained by the same method as in step 5 of Example 8, except that DCM (20 mL) was replaced with DMF (15 mL) and the mixture was stirred for 16 h. MS: m / z = 570.3 (M+1, ESI+).

[0593] Step 6: Preparation of 6-(2-aminopyrimidin-5-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-N-(4-methoxybenzyl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine

[0594] 6-(2-Aminopyrimidin-5-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-N-(4-methoxybenzyl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine (250 mg, 95.62% yield) was obtained in the same manner as in step 6 of Example 8, except that the reaction temperature was changed to 70 ℃ and stirring was performed for 16 hours. MS: m / z = 552.2 (M+1, ESI+).

[0595] Step 7: Preparation of 6-(2-aminopyrimidin-5-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine

[0596] In the same manner as step 7 of Example 8, the compound of Example 20 (8 mg, 4.09% yield) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (s, 1H), 8.79 (s, 2H), 8.07 (s, 1H), 7.73 (s, 1H), 7.22-7.08 (m, 4H), 6.72 (s, 1H), 4.94 (d, 2H), 2.21 (s, 3H); MS: m / z = 432.1 (M+1, ESI+).

[0597] Example 21: Preparation of 6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-imidazo[1,2-b]pyridazin-8-amine

[0598]

[0599] Step 1: Preparation of tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0600] The product of step 2 of Example 20 (2.6 g, 4.81 mmol) and ethynylcyclopropane (318 mg, 4.81 mmol) were dissolved in MeCN (20 mL), and then tris(dibenzylideneacetone)dipalladium(0) (441 mg, 481 umol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos, 459 mg, 962 umol), potassium phosphate (K3PO4, 3.06 g, 14.43 mmol) were added and stirred at 70 °C under nitrogen gas for 48 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (900 mg, 35.59% yield). MS: m / z = 526.3 (M+1, ESI+).

[0601] Step 2: Preparation of N-(6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0602] N-(6-(2-Aminopyrimidin-5-yl)-3-(cyclopropylethynyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (780 mg, 67.17% yield) was obtained using the same method as in step 4 of Example 8. MS: m / z = 470.2 (M+1, ESI+).

[0603] Step 3: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)imidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide

[0604] N-(6-amino-2,3-difluorophenyl)-2-((6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)imidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide (660 mg, 66.70% yield) was obtained by the same method as in step 5 of Example 8, except that DCM (20 mL) was replaced with DMF (15 mL) and the mixture was stirred for 16 h. MS: m / z = 596.2 (M+1, ESI+).

[0605] Step 4: Preparation of 6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)imidazo[1,2-b]pyridazin-8-amine

[0606] 6-(2-Aminopyrimidin-5-yl)-3-(cyclopropylethynyl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)imidazo[1,2-b]pyridazin-8-amine (580 mg, 90.62% yield) was obtained in the same manner as in step 6 of Example 8, except that the reaction temperature was changed to 70 ℃ and stirring was performed for 16 hours. MS: m / z = 578.2 (M+1, ESI+).

[0607] Step 5: Preparation of 6-(2-aminopyrimidin-5-yl)-3-(cyclopropylethynyl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-imidazo[1,2-b]pyridazin-8-amine

[0608] In the same manner as in step 7 of Example 8, the compound of Example 21 (25 mg, 5.44% yield) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (s, 1H), 8.80 (s, 2H), 8.07-8.03 (m, 1H), 7.72 (s, 1H), 7.24-7.17 (m, 2H), 7.08 (s, 2H), 6.73 (s, 1H), 4.93 (s, 2H), 1.73-1.69 (m, 1H), 0.99-0.95 (m, 2H), 0.84-0.80 (m, 2H); MS: m / z = 458.2 (M+1, ESI+).

[0609] Example 22: Preparation of 6-(2-aminopyrimidin-5-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)-methyl)-3-ethynylimidazo[1,2-b]pyridazin-8-amine

[0610]

[0611] Step 1: Preparation of tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-iodoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0612] Tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-iodoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (3.7 g, 72.68% yield) was obtained in the same manner as in Step 2 of Example 8, except that the product of Step 1 of Example 20 (4 g, 8.67 mmol) was used as a starting material and the reaction time was changed to 1 hour. MS: m / z = 588.2 (M+1, ESI+).

[0613] Step 2: Preparation of tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-((trimethylsilyl)ethynyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0614] The product of Step 1 (3.7 g, 6.30 mmol) and ethynyl(trimethyl)silane (928 mg, 9.45 mmol) were dissolved in DMF (50 mL), and then CuI (240 mg, 1.26 mmol), Pd(PPh3)2Cl2 (577 mg, 630 umol), and TEA (2.63 mL, 18.90 mmol) were added, and the mixture was stirred at 50 °C under nitrogen gas for 2 h. Water (500 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (500 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(6-(2-aminopyrimidin-5-yl)-3-((trimethylsilyl)ethynyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (2.9 g, 82.55% yield). MS: m / z = 558.5 (M+1, ESI+).

[0615] Steps 3 to 6: Preparation of 6-(2-aminopyrimidin-5-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)-methyl)-3-ethynylimidazo[1,2-b]pyridazin-8-amine

[0616] The compound of Example 22 (30 mg, 9.66% yield) was obtained as a white solid in the same manner as steps 4 to 7 of Example 8. However, in step 5 of Example 8, the reaction time was changed to 16 hours, and in step 6 of Example 8, the reaction temperature was changed to 40°C and stirred for 16 hours. 1 H NMR (400 MHz, DMSO-d6) δ 12.74 (s, 1H), 8.80 (s, 2H), 8.15 (s, 1H), 7.88 (s, 1H), 7.22-7.11 (m, 4H), 6.78 (s, 1H), 4.95 (d, 2H), 4.86 (s, 1H); MS: m / z = 418.1 (M+1, ESI+).

[0617] Example 23: Preparation of 3-cyclopropyl-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0618]

[0619] Step 1: Preparation of tert-butyl N-(3-cyclopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0620] Intermediate S5 (2.6 g, 4.88 mmol) and cyclopropylboronic acid (1.26 g, 14.65 mmol) were dissolved in 1,4-dioxane / water (40 mL / 10 mL), then Pd(PPh3)4 (564 mg, 488 umol) and potassium carbonate (K2CO3, 2.02 g, 14.65 mmol) were added and stirred at 110 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(3-cyclopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (1.47 g, 60.99% yield). MS: m / z = 494.2 (M+1, ESI+).

[0621] Step 2: Preparation of N-(3-cyclopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0622] The product of Step 1 (1.47 g, 2.98 mmol) was dissolved in 1,4-dioxane / water (10 mL / 4 mL), and N-(3-cyclopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (620 mg, 47.59% yield) was obtained in the same manner as Step 4 of Example 8. MS: m / z = 438.2 (M+1, ESI+).

[0623] Step 3: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((3-cyclopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide

[0624] The product of Step 2 (620 mg, 1.42 mmol) and intermediate B1 (245 mg, 1.70 mmol) were dissolved in DMF (15 mL), HATU (802 mg, 2.13 mmol) and DIEA (550 mg, 4.25 mmol) were added, and the mixture was stirred at 70 °C for 16 hours. Water (150 mL) was added to the reaction mixture, and the product was extracted three times with DCM (40 mL). The organic layer was washed three times with brine (150 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(6-amino-2,3-difluorophenyl)-2-((3-cyclopropyl-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide (410 mg, 51.33% yield). MS: m / z = 564.2 (M+1, ESI+).

[0625] Step 4: Preparation of 3-cyclopropyl-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(morpholinoimidazo[1,2-b]pyridazin-8-amine

[0626] 3-Cyclopropyl-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(morpholinoimidazo[1,2-b]pyridazin-8-amine (350 mg, 87.88% yield) was obtained in the same manner as in step 6 of Example 8, except that the reaction temperature was changed to 70 ℃ and stirring was performed for 16 hours. MS: m / z = 546.2 (M+1, ESI+).

[0627] Step 5: Preparation of 3-cyclopropyl-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0628] The product of Step 4 (350 mg, 642 μmol) was dissolved in TFA (5 mL) and stirred at 25°C for 1 hour. The reaction mixture was concentrated and purified by Prep-HPLC to obtain the compound of Example 23 (120 mg, 43.97% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 7.47 (s, 1H), 7.26-7.16 (m, 2H), 7.09 (s, 1H), 6.00 (s, 1H), 4.80 (d, 2H), 3.68 (t, 4H), 3.34 (t, 4H), 2.09-2.04 (m, 1H), 0.96-0.92 (m, 2H), 0.80-0.76 (m, 2H); MS: m / z = 426.2 (M+1, ESI+).

[0629] Example 24: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine

[0630]

[0631] Step 1: Preparation of tert-butyl N-(4-methoxybenzyl)-N-(6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0632] Intermediate S5 (4 g, 7.51 mmol) and prop-1-yne (3 g, 75.1 mmol) were dissolved in DMF (30 mL), and then CuI (429 mg, 2.25 mmol), Pd(PPh3)2Cl2 (527 mg, 751 μmol), and TEA (3.14 mL, 22.54 mmol) were added, and the mixture was stirred at 40 °C under nitrogen gas for 48 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (80 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give tert-butyl N-(4-methoxybenzyl)-N-(6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycinate (1.45 g, 39.26% yield). MS: m / z = 492.2 (M+1, ESI+).

[0633] Step 2: Preparation of N-(4-methoxybenzyl)-N-(6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0634] The product of Step 1 (1.45 g, 2.95 mmol) was dissolved in 1,4-dioxane / water (15 mL / 15 mL), and N-(4-methoxybenzyl)-N-(6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)glycine (950 mg, 73.96% yield) was obtained in the same manner as Step 4 of Example 8. MS: m / z = 436.2 (M+1, ESI+).

[0635] Step 3: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0636] The product of Step 2 (900 mg, 2.07 mmol) was dissolved in DMF (15 mL), 1,1'-carbonyldiimidazole (312 mg, 2.17 mmol) was added, and the mixture was stirred at 25 °C for 2 h. Intermediate B1 (298 mg, 2.07 mmol) was added to the reaction mixture, and the mixture was stirred at 50 °C for 16 h. Water (150 mL) was added to the reaction mixture, and the product was extracted three times with DCM (40 mL). The organic layer was washed three times with brine (150 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give N-(6-amino-2,3-difluorophenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (470 mg, 40.50% yield). MS: m / z = 562.2 (M+1, ESI+).

[0637] Step 4: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine

[0638] N-((6,7-Difluoro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine (410 mg, 90.13% yield) was obtained in the same manner as in step 6 of Example 8, except that the reaction time was stirred for 1 hour. MS: m / z = 520.2 (M+1, ESI+).

[0639] Step 5: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(prop-1-yn-1-yl)imidazo[1,2-b]pyridazin-8-amine

[0640] The product of Step 4 (410 mg, 754 μmol) was dissolved in TFA (10 mL) and stirred at 25°C for 1 hour. The reaction mixture was concentrated and purified by Prep-HPLC to obtain the compound of Example 24 (50 mg, 15.67% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (t, 1H), 7.55 (s, 1H), 7.28-7.19 (m, 2H), 6.06 (s, 1H), 4.81 (d, 2H), 3.68 (t, 4H), 3.39 (t, 4H), 2.15 (s, 3H); MS: m / z = 424.2 (M+1, ESI+).

[0641] Example 25: Preparation of 3-(cyclopropylethynyl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0642]

[0643] Step 1: Preparation of tert-butyl N-(3-(cyclopropylethynyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0644] The same procedure as in Step 1 of Example 24 was followed except that ethynylcyclopropane (782 mg, 11.83 mmol) was used instead of prop-1-yne (3 g, 75.1 mmol) in Step 1 of Example 24 and stirring was performed at 90 °C, starting from intermediate S5 (4.2 g, 7.89 mmol) and tert-butyl N-(3-(cyclopropylethynyl)-6-morpholinoimidazol[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (2.66 g, 65.15% yield) was obtained. MS: m / z = 518.2 (M+1, ESI+).

[0645] Step 2: Preparation of N-(3-(cyclopropylethynyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0646] Using the product of Step 1 (2.6 g, 5.02 mmol) as a starting material, N-(3-(cyclopropylethynyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (1.5 g, 64.71% yield) was obtained in the same manner as in Step 4 of Example 8. MS: m / z = 462.2 (M+1, ESI+).

[0647] Step 3: Preparation of N-(6-amino-2,3-difluorophenyl)-2-((3-cyclopropylethynyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide

[0648] N-(6-amino-2,3-difluorophenyl)-2-((3-cyclopropylethynyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide (500 mg, 29.09% yield) was obtained in the same manner as in step 5 of Example 8, except that the reaction time was changed to 16 h. MS: m / z = 588.2 (M+1, ESI+).

[0649] Steps 4 and 5: Preparation of 3-(cyclopropylethynyl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0650] The compound of Example 25 (58 mg, 11.97% yield) was obtained as a white solid in the same manner as steps 6 and 7 of Example 8, except that the reaction times were changed to 4 hours and 2 hours, respectively. 1H NMR (400 MHz, DMSO-d6) δ 7.62 (t, 1H), 7.53 (s, 1H), 7.28-7.16 (m, 2H), 6.05 (s, 1H), 4.81 (d, 2H), 3.68 (s, 4H), 3.34 (s, 4H), 1.66-1.62 (m, 1H), 0.95-0.92 (m, 2H), 0.77-0.76 (m, 2H); MS: m / z = 450.4 (M+1, ESI+).

[0651] Example 26: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-iodo-6-morpholinoimidazo[1.2-b]pyridazin-8-amine

[0652]

[0653] Step 1: Preparation of 2-(chloromethyl)-6,7-difluoro-1H-benzo[d]imidazole

[0654] Intermediate B1 (10 g, 69.44 mmol) was dissolved in 6 M hydrochloric acid solution (100 mL), and 2-chloroacetic acid (9.84 g, 104.2 mmol) was added, followed by stirring at 100 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 2-(chloromethyl)-6,7-difluoro-1H-benzo[d]imidazole (10 g, 71.42% yield). MS: m / z = 203.2 (M+1, ESI+).

[0655] Step 2: Preparation of 2-(chloromethyl)-6,7-difluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazole

[0656] The product of Step 1 (10 g, 49.50 mmol) and DIEA (17.2 mL, 99 mmol) were dissolved in THF, and then TMSCl (11.85 g, 71.4 mmol) was added at 0 °C and stirred at 25 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 2-(chloromethyl)-6,7-difluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazole (16 g, 97.56% yield). MS: m / z = 333.2 (M+1, ESI+).

[0657] Step 3: Preparation of N,N-bis(4-methoxybenzyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0658] Using intermediate S6 (4 g, 9.78 mmol) as a starting material, N,N-bis(4-methoxybenzyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine (4 g, 88.98% yield) was obtained in the same manner as in step 5 of Example 2. MS: m / z = 460.2 (M+1, ESI+).

[0659] Step 4: Preparation of N-(4-methoxybenzyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0660] The product of Step 3 (4 g, 9.78 mmol) was dissolved in DCM (20 mL), TFA (10 mL) was added, and the mixture was stirred at 25 °C for 3 h. 2N sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the mixture was extracted three times with DCM (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give N-(4-methoxybenzyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine (2.8 g, 84.33% yield). MS: m / z = 340.1 (M+1, ESI+).

[0661] Step 5: Preparation of N-((6,7-difluoro-1-((2-trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-morpholinoimidazo[1,2-b]pyradazin-8-amine

[0662] The product of Step 4 (2.8 g, 8.25 mmol) was dissolved in DMF (30 mL), and NaH (990 mg, 24.75 mmol, 60% purity) was added several times at 0 °C and stirred for 30 min. The product of Step 2 (4.12 g, 12.38 mmol) was added to this reaction mixture and stirred at 25 °C for 16 h. Water (300 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give N-((6,7-difluoro-1-((2-trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-morpholinoimidazo[1,2-b]pyradazin-8-amine (2.6 g, 49.57% yield). MS: m / z = 636.5 (M+1, ESI+).

[0663] Step 6: Preparation of N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0664] The product of Step 5 (2.6 g, 4.09 mmol) was dissolved in DCM (20 mL), TFA (10 mL) was added, and the mixture was stirred at 25 °C for 1 h. 2N sodium bicarbonate solution (200 mL) was added to the reaction mixture, and the mixture was extracted three times with DCM (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine (1 g, 63.45% yield). MS: m / z = 386.2 (M+1, ESI+).

[0665] Step 7: Preparation of N-(6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-iodo-6-morpholinoimidazo[1.2-b]pyridazin-8-amine

[0666] The product of Step 6 (500 mg, 1.30 mmol) was dissolved in THF (10 mL), and NIS (263 mg, 1.17 mmol) was added at 0 °C and stirred for 1 hour. Water (80 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (20 mL). The organic layer was washed three times with sodium sulfite solution (80 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 26 (56 mg, 8.44% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d, 1H), 7.43 (d, 1H), 7.24-7.14 (m, 2H), 7.06 (t, 1H), 5.72 (d, 2H), 3.79 (t, 4H), 3.12 (t, 4H); MS: m / z = 512.0 (M+1, ESI+).

[0667] Example 27: Preparation of 3-chloro-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholinoimidazo[1,2-b]pyridazin-8-amine

[0668]

[0669] The product (300 mg, 778 μmol) of Step 6 of Example 26 was dissolved in THF (5 mL), and NCS (135 mg, 1.01 mmol) was added at 0 °C and stirred for 1 hour. Water (50 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (15 mL). The organic layer was washed three times with sodium sulfite solution (50 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain the compound of Example 27 (155 mg, 47.43% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.92 (d, 1H), 7.56 (t, 1H), 7.42 (s, 1H), 7.25-7.13 (m, 2H), 5.69 (d, 2H), 3.78 (t, 4H), 3.17 (t, 4H); MS: m / z = 421.7 (M+1, ESI+).

[0670] Example 28: Preparation of 6-(4-cyclopropylpiperazin-1-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0671]

[0672] Step 1: Preparation of 6-(4-cyclopropylpiperazin-1-yl)-N,N-bis(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0673] Intermediate S7 (2.1 g, 4.52 mmol) and intermediate A15 (1.17 g, 6.77 mmol) were dissolved in DMSO (15 mL), and then TsOH (722 mg, 4.19 mmol) was added and stirred at 140 ℃ for 16 h. Water (150 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (150 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain 6-(4-cyclopropylpiperazin-1-yl)-N,N-bis(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (1.33 g, 55.97% yield). MS: m / z = 567.5 (M+1, ESI+).

[0674] Step 2: Preparation of 6-(4-cyclopropyl)piperazin-1-yl)-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0675] The product of Step 1 (800 mg, 1.41 mmol) was dissolved in DCM (6 mL) and TFA (2 mL), and stirred at 25 °C for 3 h. Sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the product was extracted three times with DCM (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 6-(4-cyclopropyl)piperazin-1-yl)-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (500 mg, 79.32% yield). MS: m / z = 447.2 (M+1, ESI+).

[0676] Step 3: Preparation of 6-(4-cyclopropylpiperazin-1-yl)-N-((6,7-difluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)methyl-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0677] By the same method as in step 5 of Example 26, 6-(4-cyclopropylpiperazin-1-yl)-N-((6,7-difluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)methyl-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (650 mg, 78.13% yield) was obtained. MS: m / z = 743.5 (M+1, ESI+).

[0678] Step 4: Preparation of 6-(4-cyclopropylpiperazin-1-yl)-N-((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0679] The product of Step 3 (650 mg, 875 μmol) was dissolved in TFA (10 mL) and stirred at 25°C for 1 hour. The reaction mixture was concentrated and purified by Prep-HPLC to obtain the compound of Example 28 (140 mg, 32.49% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 7.87 (s, 2H), 7.26-7.17 (m, 2H), 6.20 (s, 1H), 4.85 (d, 2H), 3.33 (s, 4H), 2.57 (s, 4H), 1.60 (s, 1H), 0.42 (s, 2H), 0.33 (s, 2H); MS: m / z = 493.2 (M+1, ESI+).

[0680]

[0681] Hereinafter, the compounds of Examples 29 to 67 were prepared using appropriate intermediates from the intermediate A series of [Table 1] and the intermediate B series of [Table 3] according to the following reaction scheme II.

[0682] [Reaction Formula II]

[0683]

[0684] In the above reaction formula II, R 2 is the R of each example compound 2 and X1, X2 and X3 also represent the corresponding positional substituent structures of each example compound. Intermediates referred to as Intermediate B1 to Intermediate B30 in [Table 3] below, as well as in Examples 29 to 67, throughout the examples, have the structures shown in [Table 3] below.

[0685] [Table 3]

[0686]

[0687] Example 29: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0688]

[0689] Step 1: Preparation of N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine

[0690] Intermediate S8 (1 g, 2.26 mmol) and intermediate A2 (1.13 g, 11.29 mmol) were dissolved in 1,4-dioxane (20 mL), and then Ruphos-Pd-G3 (189 mg, 226 μmol), 2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl (RuPhos, 106 mg, 226 μmol), and cesium carbonate (3.68 g, 11.29 mmol) were added and stirred at 110 °C for 16 h. 1 N hydrochloric acid (150 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(4-methoxybenzyl)-N-(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycine (400 mg, 37.02% yield). MS: m / z = 479.2 (M+1, ESI+).

[0691] Step 2: Preparation of N-(2-amino-4,5-dichlorophenyl)-2-((4-methoxybenzyl)(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0692] The product of Step 1 (340 mg, 711 μmol) and intermediate B2 (629 mg, 3.55 mmol) were dissolved in DCM (10 mL), and then HOBT (480 mg, 3.55 mmol), EDCI (681 mg, 3.55 mmol), and DIEA (1.24 mL, 7.11 mmol) were added and stirred at 25 °C for 16 h. Water (80 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (80 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(2-amino-4,5-dichlorophenyl)-2-((4-methoxybenzyl)(6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (200 mg, 44.15% yield). MS: m / z = 637.1 (M+1, ESI+).

[0693] Step 3: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0694] The product of Step 2 (150 mg, 235 μmol) was dissolved in TFA (8 mL) and stirred at 70 °C for 16 h. Sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (130 mg, 89.19% yield). MS: m / z = 619.2 (M+1, ESI+).

[0695] Step 4: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0696] The product of Step 3 (139 mg, 204 μmol) was dissolved in DCM (4 mL), TFA (1 mL) was added, and the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated and purified by Prep-HPLC to obtain the compound of Example 29 (35 mg, 34.37% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.86 (s, 1H), 7.81 (t, 1H), 7.76 (s, 2H), 6.16 (s, 1H), 4.83 (d, 2H), 3.36 (t, 4H), 2.35 (t, 4H), 2.17 (s, 3H); MS: m / z = 499.1 (M+1, ESI+).

[0697] Example 30: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carbonitrile

[0698]

[0699] Step 1: Preparation of ethyl N-(6-(3-cyanoazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0700] Intermediate S8 (2 g, 4.52 mmol) and intermediate A16 (742 mg, 9.03 mmol) were dissolved in 1,4-dioxane (20 mL), and then Ruphos-Pd-G3 (189 mg, 226 μmol), 2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl (RuPhos, 106 mg, 226 μmol), and cesium carbonate (2.94 g, 9.03 mmol) were added and stirred at 110 °C for 48 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (20 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain ethyl N-(6-(3-cyanoazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (1.5 g, 67.99% yield). MS: m / z = 489.1 (M+1, ESI+).

[0701] Step 2: Preparation of N-(6-(3-cyanoazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0702] The product of Step 1 (1.5 g, 3.07 mmol) was dissolved in THF (10 mL) and water (5 mL), and then lithium hydroxide (368 mg, 15.35 mmol) was added and stirred at 25 °C for 2 h. 1 N hydrochloric acid (100 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(6-(3-cyanoazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (900 mg, 63.66% yield). MS: m / z = 461.1 (M+1, ESI+).

[0703] Step 3: Preparation of N-(2-amino-4,5-dichlorophenyl)-2-((6-(3-cyanoazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide

[0704] The product of Step 2 (900 mg, 1.95 mmol) and intermediate B2 (1.38 g, 7.8 mmol) were dissolved in DCM (20 mL), and then HOBT (1.32 g, 9.77 mmol), EDCI (1.87 g, 9.77 mmol), and DIEA (3.41 mL, 19.55 mmol) were added and stirred at 25 °C for 2 h. Water (80 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (80 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain N-(2-amino-4,5-dichlorophenyl)-2-((6-(3-cyanoazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)(4-methoxybenzyl)amino)acetamide (600 mg, 49.55% yield). MS: m / z = 619.2 (M+1, ESI+)

[0705] Step 4: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carbonitrile

[0706] The product of Step 3 (600 mg, 969 μmol) was dissolved in AcOH (10 mL) and stirred at 100 °C for 1 h. Sodium bicarbonate solution (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carbonitrile (400 mg, 68.66% yield). MS: m / z = 601.2 (M+1, ESI+)

[0707] Step 5: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carbonitrile

[0708] The product of Step 4 (400 mg, 665 μmol) was dissolved in DCM (6 mL), TFA (3 mL) was added, and the mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated and purified by Prep-HPLC to obtain the compound of Example 30 (55 mg, 17.18% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.98 (t, 1H), 7.89 (s, 1H), 7.77 (s, 2H), 5.71 (s, 1H), 4.81 (d, 2H), 4.18 (t, 2H), 4.04 (dd, 2H), 3.85-3.79 (m, 1H); MS: m / z = 481.1 (M+1, ESI+).

[0709] Examples 31 to 56

[0710] Intermediate A of [Table 1] and Intermediate B of [Table 3] corresponding to the structure of the target compound were used, and the compounds of Examples 31 to 56 were prepared in a similar manner to Example 30 by appropriately changing the amount of reagent, catalyst, and reaction conditions.

[0711] [Table 4]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719] Example 57: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-6-(piperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0720]

[0721] The compound of Example 51 (650 mg, 1.23 mmol) was dissolved in 1,4-dioxane (10 mL), 6 M hydrochloric acid solution (2 mL) was added, and the mixture was stirred at 100 °C for 48 hours. The reaction mixture was concentrated, and the concentrate was purified by Prep-HPLC to obtain the compound of Example 57 (120 mg, 20.06% yield) as a grayish white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.86 (s, 1H), 7.82-7.77 (m, 3H), 6.13 (s, 1H), 4.83 (d, 2H), 3.28 (s, 4H), 2.73 (s, 4H); MS: m / z = 485.0 (M+1, ESI+).

[0722] Example 58: Preparation of 6-(3-(cyclopropylamino)azetidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0723]

[0724] Steps 1 to 4: Preparation of benzyl cyclopropyl(1-(8-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-yl)carbamate

[0725] Benzyl cyclopropyl(1-(8-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-yl)carbamate (450 mg, 32.90% yield) was obtained in the same manner as in steps 1 to 4 of Example 30. However, in step 1 of Example 30, intermediate S8 (3 g, 6.77 mmol) and intermediate A36 (4.17 g, 16.94 mmol) were used and stirred for 16 hours, and in step 3 of Example 30, the reaction time was changed to 16 hours. MS: m / z = 765.3 (M+1, ESI+)

[0726] Step 5: Preparation of 6-(3-(cyclopropylamino)azetidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0727] The product of Step 4 (450 mg, 588 μmol) was dissolved in methanol (10 mL), Pd / C (45 mg) was added, and the mixture was stirred at 25°C under a hydrogen gas atmosphere for 3 hours. The reaction mixture was filtered and concentrated, and the concentrate was purified by silica gel chromatography to obtain 6-(3-(cyclopropylamino)azetidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (250 mg, 67.36% yield). MS: m / z = 631.2 (M+1, ESI+).

[0728] Step 6: Preparation of 6-(3-(cyclopropylamino)azetidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0729] The compound of Example 58 (65 mg, 32.11% yield) was obtained as a white solid in the same manner as step 5 of Example 30, except that the reaction time was changed to 1 hour.

[0730] 1 H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 7.86-7.83 (m, 2H), 7.77 (s, 2H), 5.57 (s, 1H), 4.78 (d, 2H), 4.01 (t, 2H), 3.69-3.66 (m, 1H), 3.57 (t, 2H), 2,91 (br s, 1H), 2.03-1.99 (m, 1H), 0.36-0.31 (m, 2H), 0.20-0.17 (m, 2H); MS: m / z = 511.1 (M+1, ESI+).

[0731] Example 59: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3-hydroxyazetidine-3-carbonitrile

[0732]

[0733] Steps 1 and 2: Preparation of N-(6-(3-((tert-butyldimethylsilyl)oxy)azetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0734] N-(6-(3-((tert-butyldimethylsilyl)oxy)azetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (2.5 g, crude) was obtained using the same method as in steps 1 and 2 of Example 30. However, in step 1, intermediate S8 (5 g, 11.29 mmol) and intermediate A37 (5.29 g, 28.23 mmol) were used and stirred for 16 hours, and in step 2, the reaction time was changed to 16 hours. MS: m / z = 566.2 (M +1, ESI+).

[0735] Step 3: Preparation of N-(6-hydroxyazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0736] The product of Step 2 (1.5 g, 2.65 mmol) was dissolved in THF (20 mL), and then 1 M TBAF solution (10.6 mmol, 10.6 mL) was added and stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(6-hydroxyazetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (700 mg, 58.48% yield). MS: m / z = 452.1 (M +1, ESI+).

[0737] Steps 4 and 5: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-ol

[0738] 1-(8-(((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-ol (780 mg, 90.31% yield) was obtained in the same manner as in steps 3 and 4 of Example 30. However, in step 3 of Example 30, the reaction time was changed to 16 hours, and in step 4 of Example 30, the reaction temperature was changed to 70°C and stirred for 3 hours. MS: m / z = 592.0 (M+1, ESI+).

[0739] Step 6: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-one

[0740] The product of Step 5 (730 mg, 1.23 mmol) was dissolved in DCM (20 mL), and Dess-Martin periodinane (1.57 g, 3.70 mmol) was added, followed by stirring at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with DCM (20 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine (550 mg, 75.60% yield). MS: m / z = 590.0 (M +1, ESI+).

[0741] Step 7. Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3-hydroxyazetidine-3-carbonitrile

[0742] The product of Step 6 (400 mg, 678 umol) and sodium cyanide (166 mg, 3.39 mmol) were dissolved in THF / water (10 mL / 10 mL), then sodium bicarbonate (285 mg, 3.39 mmol) was added and stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3-hydroxyazetidine-3-carbonitrile (250 mg, 59.76% yield). MS: m / z = 617.1 (M+1, ESI+).

[0743] Step 8: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3-hydroxyazetidine-3-carbonitrile

[0744] The compound of Example 59 (30 mg, 14.90% yield) was obtained as a white solid in the same manner as step 5 of Example 30, except that the reaction time was changed to 1 hour. 1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 8.00 (t, 1H), 7.90 (s, 1H), 7.77 (s, 2H), 7.53 (s, 1H), 5.78 (s, 1H), 4.82 (d, 2H), 4.38 (d, 2H), 3.96 (d, 2H); MS: m / z = 497.1 (M+1, ESI+).

[0745] Example 60: Preparation of (1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-yl)methanol

[0746]

[0747] Steps 1 to 5: Preparation of 6-(3-(((tert-butyldiphenylsilyl)oxy)methyl)azetidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0748] The same procedure as in Steps 1 to 5 of Example 30 was followed, except that intermediate S8 (2 g, 4.52 mmol) and intermediate A38 (7.35 g, 22.58 mmol) were used in Step 1 and the mixture was stirred for 16 h to obtain 6-(3-(((tert-butyldiphenylsilyl)oxy)methyl)azetidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (350 mg, 86.83% yield). MS: m / z = 724.2 (M+1, ESI+)

[0749] Step 6: Preparation of (1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidin-3-yl)methanol

[0750] The product of Step 5 (350 mg, 484 μmol) was dissolved in THF (10 mL), and 1 M TBAF solution (2.42 mL, 2.42 mmol) was added, followed by stirring at 25 °C for 1 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 60 (100 mg, 42.61% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 7.84-7.77 (m, 4H), 5.56 (s, 1H), 4.79-4.73 (m, 3H), 3.87 (t, 2H), 3.62 (dd, 2H), 3.52 (t, 2H), 2.77-2.70 (m, 1H); MS: m / z = 486.0 (M+1, ESI+).

[0751] Example 61: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3-hydroxypyrrolidine-3-carbonitrile

[0752]

[0753] Step 1: Preparation of ethyl N-(6-(1,4-dioxa-7-azaspiro[4.4]nonan-7-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0754] The same procedure as in Step 1 of Example 30 was followed, except that intermediate S8 (3.6 g, 8.13 mmol) and intermediate A39 (3.15 g, 24.29 mmol) were used and the mixture was stirred for 16 h to obtain ethyl N-(6-(1,4-dioxa-7-azaspiro[4.4]nonan-7-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (780 mg, 17.92% yield). MS: m / z = 535.2 (M+1, ESI+).

[0755] Step 2: Preparation of N-(6-(1,4-dioxa-7-azaspiro[4.4]nonan-7-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0756] The product of Step 1 (780 mg, 1.46 mmol) was dissolved in THF / water (10 mL / 5 mL), lithium hydroxide (185 mg, 4.38 mmol) was added, and the mixture was stirred at 50 °C for 2 h. 0.5 N hydrochloric acid solution (80 mL) was added to the reaction mixture, and the mixture was extracted three times with DCM (20 mL). The organic layer was washed three times with brine (80 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(6-(1,4-dioxa-7-azaspiro[4.4]nonan-7-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (410 mg, 55.25% yield). MS: m / z = 507.2 (M+1, ESI+).

[0757] Steps 3 to 5: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-one

[0758] 1-(8-(((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-one (200 mg, 62.30% yield) was obtained in the same manner as in steps 3 to 5 of Example 30, except that the reaction times in steps 3 and 5 of Example 30 were changed to 16 hours and 1 hour, respectively. MS: m / z = 484.0 (M+1, ESI+).

[0759] Step 6: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)-3-hydroxypyrrolidine-3-carbonitrile

[0760] The product of step 5 (200 mg, 413 umol) was treated in the same manner as step 7 of example 59 to obtain the compound of example 61 (95 mg, 42.03% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.89 (t, 1H), 7.85 (s, 1H), 7.79 (s, 2H). 6.96 (s, 1H), 5.83 (s, 1H), 4.87 (d, 2H), 3.74 (d, 1H), 3.64 (d, 1H), 3.54-3.49 (m, 1H), 3.44-3.38 (m, 1H), 2.46-2.39 (m, 1H), 2.33-2.28 (m, 1H); MS: m / z = 511.1 (M+1, ESI+).

[0761] Example 62: Preparation of (R)-1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-ol

[0762]

[0763] Step 1: Preparation of ethyl (R)-N-(6-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0764] The same procedure as in Step 1 of Example 30 was followed, except that intermediate S8 (2 g, 4.52 mmol) and intermediate A40 (2.73 g, 13.55 mmol) were used and the mixture was stirred for 16 h to obtain ethyl (R)-N-(6-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (560 mg, 20.40% yield). MS: m / z = 608.3 (M+1, ESI+).

[0765] Step 2: Preparation of ethyl (R)-N-(6-(3-hydroxypyrrolidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0766] The product of Step 1 (560 mg, 921 μmol) was dissolved in THF (5 mL), and then 1 M TBAF solution (1.84 mL, 1.84 mmol) was added and stirred at 25 °C for 3 h. Saturated aqueous ammonium chloride solution (50 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (10 mL). The organic layer was washed three times with brine (50 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain ethyl (R)-N-(6-(3-hydroxypyrrolidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (400 mg, 87.97% yield). MS: m / z = 494.0 (M+1, ESI+).

[0767] Steps 3 to 6: Preparation of (R)-1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-ol

[0768] The compound of Example 62 (56 mg, 25.93% yield) was obtained as a white solid in the same manner as steps 2 to 5 of Example 30. However, the reaction times of steps 2, 3, and 5 of Example 30 were changed to 16 hours, 16 hours, and 3 hours, respectively, and the reaction temperature in step 4 was changed to 65°C and stirred for 5 hours. 1 H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 7.86 (s, 1H), 7.80-7.76 (m, 2H), 7.68 (s, 1H), 5.72 (s, 1H), 4.93 (d, 1H), 4.81 (d, 2H), 4.33 (s, 1H), 3.39-3.35 (m, 2H), 3.19 (d, 1H), 1.98-1.92 (m, 1H), 1.85-1.81 (m, 1H); MS: m / z = 486.2 (M+1, ESI+).

[0769] Example 63: Preparation of (S)-1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-ol

[0770]

[0771] Steps 1 to 4: Preparation of (S)-6-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0772] (S)-6-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (850 mg, 87.12% yield) was obtained in the same manner as in steps 1 to 4 of Example 30. However, in step 1, intermediate S8 (3.1 g, 7.0 mmol) and intermediate A41 (2.82 g, 14.0 mmol) were used and the mixture was stirred for 16 hours, and in steps 2 and 3, the reaction time was changed to 16 hours. MS: m / z = 720.2 (M+1, ESI+).

[0773] Step 5: Preparation of (S)-1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-ol

[0774] (S)-1-(8-(((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-ol (600 mg, 83.89% yield) was obtained in the same manner as in step 2 of Example 62, except that the reaction time was changed to 16 hours. MS: m / z = 606.1 (M+1, ESI+).

[0775] Step 6: Preparation of (S)-1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-ol

[0776] The compound of Example 63 (290 mg, 59.86% yield) was obtained as a white solid in the same manner as step 5 of Example 30, except that the reaction time was changed to 16 hours.

[0777] 1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 7.80-7.76 (m, 4H), 5.72 (s, 1H), 4.93 (d, 1H), 4.81 (d, 2H), 4.33 (s, 1H), 3.39-3.33 (m, 2H), 3.20 (d, 1H), 1.98-1.94 (m, 1H), 1.85-1.82 (m, 1H); MS: m / z = 486.2 (M+1, ESI+).

[0778] Example 64: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxylic acid

[0779]

[0780] Step 1: Preparation of tert-butyl 1-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxylate

[0781] Tert-butyl 1-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxylate (1 g, 39.29% yield) was obtained by the same method as in Step 1 of Example 30, except that intermediate S8 (2 g, 4.52 mmol) and intermediate A42 (3.55 g, 22.58 mmol) were used and stirred for 16 h. MS: m / z = 564.2 (M+1, ESI+).

[0782] Step 2: Preparation of N-(6-(3-(tert-butoxycarbonyl)azetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0783] The product of Step 1 (1 g, 1.77 mmol) was dissolved in 1,2-dichloroethane (20 mL), and then trimethyltin hydroxide (1.60 g, 8.87 mmol) was added and stirred at 70 °C for 16 h. 0.5 N hydrochloric acid solution (100 mL) was added to the reaction mixture and extracted three times with DCM (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-(6-(3-(tert-butoxycarbonyl)azetidin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (750 mg, 78.93% yield). MS: m / z = 536.1 (M+1, ESI+).

[0784] Steps 3 to 5: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxylic acid

[0785] The compound of Example 64 (70 mg, 31.56% yield) was obtained as a white solid in the same manner as steps 3 to 5 of Example 30, except that the reaction time in step 5 of Example 30 was changed to 1 hour. 1 H NMR (400 MHz, DMSO-d6) δ 7.90 (t, 1H), 7.85 (s, 1H), 7.77 (s, 2H), 5.65 (s, 1H), 4.80 (d, 2H), 4.05 (t, 2H), 3.93 (t, 2H), 3.48-3.41 (m, 1H); MS: m / z = 500.0 (M+1, ESI+).

[0786] Example 65: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxamide

[0787]

[0788] Step 1: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxylic acid

[0789] The product of Step 4 of Example 64 (310 mg, 458 umol) was dissolved in 1,4-dioxane (8 mL) and water (4 mL), then sodium hydroxide (92 mg, 2.29 mmol) was added and stirred at 100 °C for 16 hours. 1 N hydrochloric acid solution (80 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxylic acid (250 mg, 87.94% yield). MS: m / z = 620.1 (M+1, ESI+).

[0790] Step 2: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxamide

[0791] 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxamide (150 mg, 60.10% yield) was obtained by the same method as in step 3 of Example 30, except that ammonium chloride (108 mg, 2.01 mmol) was used instead of intermediate B2 (1.38 g, 7.8 mmol). MS: m / z = 619.2 (M+1, ESI+)

[0792] Step 3: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)azetidine-3-carboxamide

[0793] The compound of Example 65 (50 mg, 41.35% yield) was obtained as a white solid in the same manner as step 5 of Example 30, except that the reaction time was changed to 1 hour. 1 H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.90-7.76 (m, 4H), 7.45 (s, 1H), 7.01 (s, 1H), 5.62 (s, 1H), 4.80 (d, 2H), 3.98 (t, 2H), 3.89 (t, 2H), 3.42-3.36 (m, 1H); MS: m / z = 499.0 (M+1, ESI+).

[0794] Example 66: Preparation of 4-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxamide

[0795]

[0796] Step 1: Preparation of benzyl 4-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate

[0797] Benzyl 4-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (700 mg, 24.73% yield) was obtained by the same method as in Step 1 of Example 30, except that intermediate S8 (2 g, 4.52 mmol) and intermediate A11 (4.97 g, 22.58 mmol) were used and stirred for 16 h. MS: m / z = 627.3 (M+1, ESI+).

[0798] Step 2: Preparation of ethyl N-(4-methoxybenzyl)-N-(6-(piperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0799] The product of Step 1 (700 mg, 1.12 mmol) was dissolved in methanol (15 mL), Pd / C (100 mg) was added, and the mixture was stirred at 25°C under a hydrogen gas atmosphere for 48 h. The reaction mixture was filtered and concentrated, and the concentrate was purified by silica gel chromatography to obtain ethyl N-(4-methoxybenzyl)-N-(6-(piperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (500 mg, 90.88% yield). MS: m / z = 493.2 (M+1, ESI+).

[0800] Step 3: Preparation of ethyl N-(6-(4-carbamoylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0801] The product of Step 2 (500 mg, 1.02 mmol) and isocyanatotrimethylsilane (351 mg, 3.05 mmol) were dissolved in THF (10 mL), DIEA (0.53 mL, 3.05 mmol) was added, and the mixture was stirred at 25 °C for 4 h. Water (80 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give ethyl N-(6-(4-carbamoylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (450 mg, 82.77% yield). MS: m / z = 536.3 (M+1, ESI+).

[0802] Step 4: Preparation of N-(6-(4-carbamoylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine

[0803] N-(6-(4-carbamoylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycine (280 mg, 65.66% yield) was obtained using the same method as in step 2 of Example 64. MS: m / z = 508.2 (M+1, ESI+).

[0804] Steps 5 to 7: Preparation of 4-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxamide

[0805] In step 4 of Example 30, the reaction temperature was changed to 70°C and stirring was performed for 3 hours, and in step 5, the reaction time was changed to 1 hour. In the same manner as in steps 3 to 5 of Example 30, the compound of Example 66 (39 mg, 28.16% yield) was obtained as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.58 (s, 1H), 7.88 (s, 1H), 7.84 (t, 1H), 7.77 (s, 2H), 6.22 (s, 1H), 6.05 (s, 2H), 4.84 (d, 2H), 3.35 (s, 8H); MS: m / z = 528.1 (M+1, ESI+).

[0806] Example 67: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidine-4-carboxamide

[0807]

[0808] Step 1: Preparation of tert-butyl 1-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidine-4-carboxylate

[0809] Intermediate S8 (1.1 g, 2.48 mmol) was dissolved in intermediate A43 (15 mL) and stirred at 120 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain tert-butyl 4-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (1.05 g, 71.42% yield). MS: m / z = 592.1 (M+1, ESI+).

[0810] Steps 2 to 5: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidine-4-carboxylic acid

[0811] 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidine-4-carboxylic acid (200 mg, 76.05% yield) was obtained in the same manner as in steps 2 to 5 of Example 30, except that the reaction times in steps 2, 3 and 5 of Example 30 were changed to 1 hour, 16 hours and 1 hour, respectively, and the reaction temperature in step 4 was changed to 70°C and stirred for 4 hours. MS: m / z = 528.2 (M+1, ESI+).

[0812] Step 6: Preparation of 1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]piperidine-4-carboxamide

[0813] The compound of Example 67 (10 mg, 5.00% yield) was obtained as a white solid in the same manner as step 3 of Example 30, except that ammonium chloride (102 mg, 1.90 mmol) was used instead of intermediate B2 (1.38 g, 7.8 mmol) and the mixture was stirred for 16 hours. 1 H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H), 7.85 (s, 1H), 7.79-7.72 (m, 3H), 7.24 (d, 1H), 6.76 (s, 1H), 6.16 (s, 1H), 4.83 (d, 2H), 4.06-4.01 (m, 2H), 2.82-2.67 (m, 2H), 2.44-2.28 (m, 1H), 1.71-1.66 (m, 2H), 1.53-1.44 (m, 2H); MS: m / z = 527.1 (M+1, ESI+).

[0814]

[0815] Hereinafter, the compounds of Examples 68 to 81 were prepared using appropriate intermediates from the intermediate B series of [Table 3] according to the following reaction scheme III.

[0816] [Reaction Formula III]

[0817]

[0818] (In the above reaction formula III, X 3a , X 4a , X 5a and X 6a At least one of them is N, the rest are CR 3d and R 3d are each independently H, halo, cyano, hydroxy, C1-C6 alkoxy or C1-C6 alkyl, and R 3dAny C1-C6 alkoxy group and C1-C6 alkyl group among them may be optionally substituted with halo, cyano or hydroxy.)

[0819] Example 68: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0820]

[0821] Step 1: Preparation of N-(2-amino-4,5-dichlorophenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0822] Except that the reaction time of step 3 of Example 30 was changed to 16 hours using intermediate S9 (1.9 g, 4.08 mmol) as the starting material, the same method was followed to obtain N-(2-amino-4,5-dichlorophenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (1.7 g, 66.69% yield). MS: m / z = 624.2 (M+1, ESI+).

[0823] Step 2: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0824] N-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (1.6 g, 96.91% yield) was obtained in the same manner as in step 4 of Example 30, except that the reaction temperature was changed to 70 ℃ and stirring was performed for 16 hours. MS: m / z = 606.2 (M+1, ESI+).

[0825] Step 3: Preparation of N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0826] In the same manner as in step 5 of Example 30, the compound of Example 68 (720 mg, 56.12% yield) was obtained as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.88-7.77 (m, 4H), 6.18 (s, 1H), 4.84 (d, 2H), 3.67 (t, 4H), 3.34 (t, 4H); MS: m / z = 486.1 (M+1, ESI+).

[0827] Examples 69 to 81

[0828] Among the intermediate B series, intermediates corresponding to the structure of the target compound were used, and the amounts of reagents, catalysts, and reaction conditions were appropriately changed to prepare compounds of Examples 69 to 81 in a similar manner to Example 68.

[0829] [Table 5]

[0830]

[0831]

[0832]

[0833] Example 82: Preparation of N-(imidazo[1,2-a]pyridin-2-ylmethyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0834]

[0835] Step 1: Preparation of 2-(chloromethyl)imidazo[1,2-a]pyridine

[0836] Pyridin-2-amine (2.2 g, 23.38 mmol) and 1,3-dichloropropan-2-one (2.97 g, 23.38 mmol) were dissolved in ethanol (30 mL) and stirred at 90 °C for 16 h. The reaction mixture was concentrated, and the concentrate was purified by silica gel chromatography to obtain 2-(chloromethyl)imidazo[1,2-a]pyridine (400 mg, 10.27% yield). MS: m / z = 167.0 (M+1, ESI+).

[0837] Step 2: Preparation of N-(imidazo[1,2-a]pyridin-2-ylmethyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0838] The product of Step 1 (245 mg, 1.47 mmol) and the product of Step 2 for the preparation of intermediate S9 (400 mg, 982 μmol) were dissolved in DMF (10 mL), then cesium carbonate (960 mg, 2.95 mmol) was added and stirred at 90 °C for 3 h. A saturated aqueous ammonium chloride solution (100 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give N-(imidazo[1,2-a]pyridin-2-ylmethyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (270 mg, 51.16% yield). MS: m / z = 538.3 (M+1, ESI+).

[0839] Step 3: Preparation of N-(imidazo[1,2-a]pyridin-2-ylmethyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0840] The product of Step 2 (270 mg, 502.3 μmol) was dissolved in DCM (6 mL), TFA (3 mL) was added, and the mixture was stirred at 25°C for 3 hours. The reaction mixture was concentrated, and the concentrate was purified by silica gel chromatography to obtain the compound of Example 82 (90 mg, 42.93% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (d, 1H), 7.84 (d, 2H), 7.74 (t, 1H), 7.50 (d, 1H), 7.23-7.19 (m, 1H), 6.87-6.83 (m, 1H), 6.20 (s, 1H), 4.68 (d, 2H), 3.68 (t, 4H), 3.36 (t, 4H); MS: m / z = 418.2 (M+1, ESI+).

[0841] Example 83: Preparation of N-(imidazo[1,2-a]pyrimidin-2-ylmethyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0842]

[0843] Step 1: Preparation of 2-(chloromethyl)imidazo[1,2-a]pyrimidine

[0844] Pyrimidin-2-amine (5 g, 52.57 mmol) was dissolved in diethylene glycol dimethyl ether (20 mL), 1,3-dichloropropan-2-one (20.03 g, 157.72 mmol) was added, and the mixture was stirred at 70 °C for 16 h. Water (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 2-(chloromethyl)imidazo[1,2-a]pyrimidine (2.5 g, 28.37% yield). MS: m / z = 167.8 (M+1, ESI+).

[0845] Step 2: Preparation of N-(imidazo[1,2-a]pyrimidin-2-ylmethyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0846] The product of Step 2 for the preparation of intermediate S9 (1 g, 2.45 mmol) was dissolved in DMF (5 mL), and then NaH (295 mg, 7.36 mmol, 60% purity) was added at 0 °C and stirred for 30 min. The product of Step 1 (411 mg, 2.45 mmol) was added to this reaction mixture and stirred at 25 °C for 3 h. Ice water (50 mL) was added to the reaction mixture, and the product was washed three times with EtOAc (10 mL). The organic layer was washed three times with brine (50 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give N-(imidazo[1,2-a]pyrimidin-2-ylmethyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (680 mg, 51.44% yield). MS: m / z = 539.3 (M+1, ESI+).

[0847] Step 3: Preparation of N-(imidazo[1,2-a]pyrimidin-2-ylmethyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0848] The product of Step 2 (680 mg, 1.26 mmol) was dissolved in DCM (10 mL), TFA (5 mL) was added, and the mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated, and the concentrate was purified by Prep-HPLC to obtain the compound of Example 83 (70 mg, 13.25% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.89 (dd, 1H), 8.49 (dd, 1H), 7.86 (s, 1H), 7.81 (t, 1H), 7.78 (s, 1H), 7.02 (dd, 1H), 6.18 (s, 1H), 4.72 (d, 2H), 3.68 (t, 4H), 3.35 (t, 4H); MS: m / z = 419.1 (M+1, ESI+).

[0849] Example 84: Preparation of N-((1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0850]

[0851] Step 1: Preparation of methyl 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate

[0852] Methyl 1H-pyrrolo[2,3-b]pyridine-2-carboxylate (3.2 g, 18.16 mmol) was dissolved in DMF (50 mL), and NaH (1.09 g, 27.25 mmol, 60% purity) was added several times at 0 °C and stirred at 25 °C for 1 h. 2-(Trimethylsilyl)ethoxymethyl chloride (3.86 mL, 21.80 mmol) was added to the reaction mixture and stirred at 25 °C for 16 h. Saturated aqueous ammonium chloride solution (500 mL) was added to the reaction mixture, and the product was washed three times with EtOAc (100 mL). The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain methyl 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (2.8 g, 50.31% yield). MS: m / z = 307.1 (M+1, ESI+).

[0853] Step 2: Preparation of (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methanol

[0854] The product of Step 1 (2.8 g, 9.14 mmol) was dissolved in THF (50 mL) and methanol (10 mL), and then sodium borohydride (491 mg, 13.71 mmol) was added and stirred at 50 °C for 5 h. Saturated aqueous ammonium chloride solution (200 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methanol (2.1 g, 82.55% yield). MS: m / z = 279.1 (M+1, ESI+).

[0855] Step 3: Preparation of 2-(chloromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine

[0856] The product of Step 2 (2.1 g, 7.54 mmol) was dissolved in DCM (15 mL), and then thionyl chloride (1.64 mL, 22.63 mmol) was slowly added at 0 °C and stirred at 25 °C for 3 h. The reaction mixture was concentrated, and the concentrate was purified by silica gel chromatography to obtain 2-(chloromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine (1.5 g, 66.99% yield). MS: m / z = 297.1 (M+1, ESI+).

[0857] Steps 4 and 5: Preparation of N-((1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0858] The compound of Example 84 (60 mg, 34.28% yield) was obtained as a white solid in the same manner as steps 2 and 3 of Example 82, except that the reaction time in step 2 of Example 82 was changed to 5 hours. 1 H NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H), 8.13 (dd, 1H), 7.86-7.83 (m, 2H), 7.70 (t, 1H), 7.00 (dd, 1H), 6.36 (s, 1H), 6.18 (s, 1H), 4.74 (d, 2H), 3.68 (t, 4H), 3.36 (t, 4H); MS: m / z = 418.3 (M+1, ESI+).

[0859] Example 85: Preparation of N-(imidazo[1,2-b]pyridazin-2-ylmethyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0860]

[0861] The compound of Example 85 (70 mg, 20.02% yield) was obtained as a white solid in the same manner as steps 1 to 3 of Example 82. However, in step 1 of Example 82, pyridazin-3-amine (5.2 g, 54.68 mmol) was used as a starting material and stirred at 85°C for 3 hours, and the reaction time of step 2 was changed to 5 hours. 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (dd, 1H), 8.21 (s, 1H), 8.07 (dd, 1H), 7.85 (s, 1H), 7.76 (t, 1H), 7.21 (dd, 1H), 6.21 (s, 1H), 4.72 (d, 2H), 3.69 (t, 4H), 3.36 (t, 4H); MS: m / z = 419.0 (M+1, ESI+).

[0862] Example 86: Preparation of 6-morpholino-N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0863]

[0864] Step 1: Preparation of methyl pyrazolo[1,5-a]pyridine-2-carboxylate

[0865] Pyrazolo[1,5-a]pyridine-2-carboxylic acid (1.1 g, 6.78 mmol) was dissolved in methanol (30 mL), and concentrated sulfuric acid (0.11 mL, 2.04 mmol) was added. The mixture was stirred at 75 °C for 5 h. The reaction mixture was concentrated, and sodium bicarbonate solution (100 mL) was added. The product was washed three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give methyl pyrazolo[1,5-a]pyridine-2-carboxylate (950 mg, 79.49% yield). MS: m / z = 177.1 (M+1, ESI+).

[0866] Steps 2 to 5: Preparation of 6-morpholino-N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0867] The compound of Example 86 (100 mg, 41.54% yield) was obtained as a white solid in the same manner as in Steps 2 to 5 of Example 84, except that the reaction times in Steps 3 and 4 of Example 84 were changed to 2 hours and 3 hours, respectively. 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (dd, 1H), 7.85-7.82 (m, 2H), 7.60 (d, 1H), 7.20-7.15 (m, 1H), 6.86-6.82 (m, 1H), 6.51 (s, 1H), 6.18 (s, 1H), 4.73 (d, 2H), 3.68 (t, 4H), 3.34 (t, 4H); MS: m / z = 418.1 (M+1, ESI+).

[0868] Example 87: Preparation of N-((1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0869]

[0870] In the same manner as steps 1 to 5 of Example 84, the compound of Example 87 (74 mg, 34.26% yield) was obtained as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.24 (d, 1H), 7.86 (s, 1H), 7.79 (s, 1H), 7.69 (d, 1H), 7.05-7.00 (m, 1H), 6.51 (s, 1H), 6.16 (d, 1H). 4.78 (s, 2H), 3.77-3.62 (m, 4H), 3.40-3.24 (m, 4H) MS: m / z =418.1 (M+1, ESI+).

[0871] Example 88: Preparation of N-((5,7-dimethylimidazo[1,2-a]pyrimidin-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0872]

[0873] The compound of Example 88 (28 mg, 10.14% yield) was obtained as a white solid in the same manner as steps 1 to 3 of Example 82. However, in step 1 of Example 82, 4,6-dimethyl-4,5-dihydropyrimidin-2-amine (6 g, 50 mmol) was dissolved in DME (80 mL) and stirred at 50°C, in step 2, NaH (145 mg, 6.03 mmol, 60% purity) was used and stirred at 25°C for 2 hours, and in step 3, the reaction time was changed to 1 hour. 1H NMR (400 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.73-7.67 (m, 1H), 7.66 (s, 1H), 6.84 (s, 1H), 6.19 (s, 1H), 4.68 (d, 2H), 3.72-3.64 (m, 4H), 3.38-3.33 (m, 4H), 2.56 (s, 3H), 2.48 (s, 3H); MS: m / z = 447.2 (M+1, ESI+).

[0874] Example 89: Preparation of 6-morpholino-N-(pyrazolo[1,5-a]pyrimidin-2-ylmethyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0875]

[0876] Step 1: Preparation of (5-nitro-1H-pyrazol-3-yl)methanol

[0877] Methyl 5-nitro-1H-pyrazole-3-carboxylate (9 g, 52.60 mmol) was dissolved in THF (200 mL), and lithium borohydride (LiBH4, 5.73 g, 263 mmol) was added several times within 1 h at 0 °C, and the mixture was heated to 40 °C and stirred for 48 h. Ice water (300 mL) was poured into the reaction mixture, slowly cooled to room temperature, and extracted three times with EtOAc (60 mL). The organic layer was washed three times with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give (5-nitro-1H-pyrazol-3-yl)methanol (4.5 g, 59.84% yield). MS: m / z = 144.1 (M+1, ESI+).

[0878] Step 2: Preparation of (5-amino-1H-pyrazol-3-yl)methanol

[0879] The product of Step 1 (4.50 g, 31.45 mmol) was dissolved in methanol (50 mL), Pd / C (1 g) was added, and the mixture was stirred at 25°C under a hydrogen gas atmosphere for 16 h. The reaction mixture was filtered and concentrated to obtain (5-amino-1H-pyrazol-3-yl)methanol (3.5 g, 98.39% yield). MS: m / z = 113.9 (M+1, ESI+).

[0880] Step 3: Preparation of pyrazolo[1,5-a]pyrimidin-2-ylmethanol

[0881] The product of Step 2 (3.5 g, 30.94 mmol) and 1,1,3,3-tetramethoxypropane (6.10 g, 37.13 mmol) were dissolved in ethanol (50 mL), TsOH (2.66 g, 15.47 mmol) was added, and the mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated, and the concentrate was purified by silica gel chromatography to obtain pyrazolo[1,5-a]pyrimidin-2-ylmethanol (360 mg, 7.80% yield). MS: m / z = 150.2 (M+1, ESI+).

[0882] Step 4: Preparation of 2-(chloromethyl)pyrazolo[1,5-a]pyrimidine

[0883] The product of Step 3 (360 mg, 2.41 mmol) was dissolved in toluene (5 mL), thionyl chloride (861 mg, 7.24 mmol, 0.53 mL) was added, and the mixture was stirred at 50 °C for 1 h. The reaction mixture was concentrated to obtain 2-(chloromethyl)pyrazolo[1,5-a]pyrimidine (360 mg, 2.15 mmol, 88.99% yield). MS: m / z = 168.0 (M+1, ESI+).

[0884] Steps 5 and 6: Preparation of 6-morpholino-N-(pyrazolo[1,5-a]pyrimidin-2-ylmethyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0885] In the same manner as steps 4 and 5 of Example 84, the compound of Example 89 (290 mg, 31.12% yield) was obtained as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.09-9.06 (m, 1H), 8.51 (dd, 1H), 7.89-7.85 (m, 2H), 7.00 (dd, 1H), 6.64 (s, 1H), 6.17 (s, 1H), 4.77 (d, 2H), 3.68 (t, 4H), 3.35 (t, 4H); MS: m / z = 419.2 (M+1, ESI+).

[0886] Example 90: Preparation of N-((5,6-dichloro-1H-indol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0887]

[0888] Step 1: Preparation of 6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0889] The product of Step 1 for the preparation of intermediate S9 (36 g, 68.31 mmol) was dissolved in DCM (300 mL), then TFA (300 mL) was added and stirred at 25 °C for 16 h. Sodium bicarbonate solution (2000 mL) was added to the reaction mixture, and the product was extracted three times with DCM (300 mL). The organic layer was washed three times with brine (2000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (18 g, 91.83% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.85 (s, 1H), 6.90 (br s, 2H), 6.09 (s, 1H), 3.71 (t, 4H), 3.33 (t, 4H). MS: m / z = 288.1 (M+1, ESI+).

[0890] Step 2: Preparation of N-((5,6-dichloro-1-(phenylsulfonyl)-1H-indol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyrazin-8-amine

[0891] The product of Step 1 (1.22 g, 4.23 mmol) and intermediate B20 (3 g, 8.47 mmol) were dissolved in THF (50 mL), and then Ti(OEt)4 (2.90 g, 12.70 mmol) was added and stirred at 90 °C for 16 h. After that, the reaction mixture was cooled to 0 °C, NaBH4 (641 mg, 16.94 mmol) was added, and stirred at 0 °C for 1 h. Water (400 mL) was added to the reaction mixture, and the mixture was extracted three times with EtOAc (80 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give N-((5,6-dichloro-1-(phenylsulfonyl)-1H-indol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyrazin-8-amine (250 mg, crude) as a yellow solid. MS: m / z = 625.2 (M+1, ESI+).

[0892] Step 3: Preparation of N-((5,6-dichloro-1H-indol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0893] The product of Step 2 (250 mg, crude) was dissolved in THF (10 mL), and then 4N NaOH (10 mL) aqueous solution was added and stirred at 90 °C for 16 h. After the reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted three times with DCM (5 mL). The organic layer was washed three times with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain N-((5,6-dichloro-1H-indol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (30 mg, 15.47% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H), 7.86 (s, 1H), 7.78 (t, 1H), 7.71 (s, 1H), 7.59 (s, 1H), 6.40 (s, 1H), 6.15 (s, 1H), 4.73 (d, 2H), 3.68 (t, 4H), 3.60-3.34 (m, 4H); MS: m / z = 485.1 (M+1, ESI+).

[0894] Example 91: Preparation of N-((5,6-dichlorobenzo[d]oxazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0895]

[0896] Step 1: Preparation of N-(4,5-dichloro-2-hydroxyphenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide

[0897] The same procedure as in Step 3 of Example 30 was followed, except that intermediate S9 (2 g, 4.34 mmol) and 2-amino-4,5-dichlorophenyl (1.91 g, 10.74 mmol) were used and stirred for 16 h to obtain N-(4,5-dichloro-2-hydroxyphenyl)-2-((4-methoxybenzyl)(6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)acetamide (1.3 g, 48.37% yield) as a yellow solid. MS: m / z = 625.1 (M+1, ESI+).

[0898] Step 2: Preparation of N-((5,6-dichlorobenzo[d]oxazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0899] The product of Step 1 (1.3 g, 2.08 mmol) was dissolved in toluene (15 mL), and then TsOH (1.79 g, 10.39 mmol) was added and stirred at 120°C for 2 hours. The reaction mixture was cooled to room temperature, ice water (200 mL) was added, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain the compound of Example 91 (30 mg, 2.68% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H), 8.09 (s, 1H), 7.94 (t, 1H), 7.88 (s, 1H), 6.31 (s, 1H), 4.97 (d, 2H), 3.68 (t, 4H), 3.37 (t, 4H); MS: m / z = 487.1 (M+1, ESI+).

[0900] Example 92: Preparation of 4-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxamide

[0901]

[0902] Step 1: Preparation of benzyl 4-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate

[0903] Except that intermediate A11 (4.48 g, 20.37 mmol) was used and stirred for 16 h, the same procedure as in step 1 of Example 30 was followed to obtain benzyl 4-(8-((2-ethoxy-2-oxoethyl)(4-methoxybenzyl)amino)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (1.5 g, 35.29% yield) as a yellow solid. MS: m / z = 627.1 (M+1, ESI+).

[0904] Step 2: Preparation of ethyl N-(4-methoxybenzyl)-N-(6-(piperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate

[0905] The product of Step 1 (1.5 g, 2.40 mmol) was dissolved in methanol (20 mL), Pd / C (200 mg) was added, and the mixture was stirred at 25°C under a hydrogen gas atmosphere for 48 h. The reaction mixture was filtered and concentrated, and the concentrate was purified by silica gel chromatography to obtain ethyl N-(4-methoxybenzyl)-N-(6-(piperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)glycinate (970 mg, 82.20% yield) as a yellow solid. MS: m / z = 493.1 (M+1, ESI+).

[0906] Step 3: Preparation of ethyl N-(6-(4-carbamoylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0907] The product of Step 2 (970 mg, 1.97 mmol) and trimethylsilyl isocyanate (295 mg, 2.56 mmol) were dissolved in THF (30 mL), DIEA (763 mg, 5.91 mmol, 1.04 mL) was added, and the mixture was stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with EtOAc (40 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give ethyl N-(6-(4-carbamoylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b] pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (450 mg, 42.73% yield) as a yellow solid. MS: m / z = 536.1 (M+1, ESI+).

[0908] Steps 4 to 7: Preparation of 4-(8-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxamide

[0909] The compound of Example 92 (60 mg, 31.09% yield) was obtained as a white solid in the same manner as steps 2 to 5 of Example 30. However, in step 5, the product of step 3 (450 mg, 840 μmol) and intermediate B1 (583 mg, 4.05 mmol) were dissolved in DMF (15 mL), stirred for 16 hours, and in step 7, the reaction temperature was changed to 70 °C and stirred for 16 hours. 1 H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 7.88-7.85 (m, 2H), 7.25-7.16 (m, 2H), 6.23 (s, 1H), 6.05 (s, 2H), 4.85 (d, 2H), 3.36 (s, 8H); MS: m / z = 496.0 (M+1, ESI+).

[0910] Example 93: Preparation of N-((5,6-dichlorobenzo[d]thiazol-2-yl)methyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0911]

[0912] Step 1: Preparation of 6,6'-disulfanedialbis(3,4-dichloroaniline)

[0913] 5,6-Dichlorobenzo[d]thiazol-2-amine (3.6 g, 16.43 mmol) was dissolved in water (40 mL), and NaOH (19.7 g, 493 mmol) was added at 0 °C and stirred at 130 °C for 16 h. The reaction mixture was concentrated, methanol was added, filtered, and concentrated to obtain 6,6'-disulfanedialbis(3,4-dichloroaniline) (1.9 g, 29.94% yield) as a yellow solid. MS: m / z = 386.8 (M+1, ESI+).

[0914] Step 2: Preparation of N-((5,6-dichlorobenzo[d]thiazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0915] The product of Step 1 (480 mg, 1.24 mmol) was dissolved in DMF (10 mL), 1,4-dithiothreitol (DTT, 959 mg, 6.22 mmol) was added, and the mixture was stirred at 25 °C for 30 min. After this, intermediate S10 (600 mg, crude) was added and stirred at 25 °C for 16 h. Water (100 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to give N-((5,6-dichlorobenzo[d]thiazol-2-yl)methyl)-N-(4-methoxybenzyl)-6-morpholino-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (330 mg, 42.58% yield) as a yellow solid. MS: m / z = 623.1 (M+1, ESI+).

[0916] Step 3: Preparation of N-((5,6-dichlorobenzo[d]thiazol-2-yl)methyl)-6-morpholino87-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0917] The compound of Example 93 (85 mg, 31.91% yield) was obtained as a white solid in the same manner as step 5 of Example 30, except that the reaction time was changed to 2 hours. 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 8.29 (s, 1H), 8.25 (t, 1H), 7.90 (s, 1H), 6.29 (s, 1H), 5.07 (d, 2H), 3.67 (t, 4H), 3.35 (t, 4H); MS: m / z = 502.9 (M+1, ESI+).

[0918] Examples 94 to 108

[0919] Intermediate S series and intermediate A series of [Table 1] were used, respectively, to prepare compounds of Examples 94 to 108, using intermediates and intermediate B2 corresponding to the structure of the target compound, and appropriately changing the amount of reagent, catalyst, and reaction conditions in a manner similar to Example 30.

[0920] [Table 6]

[0921]

[0922]

[0923]

[0924]

[0925] Examples 109 to 113

[0926] Along with intermediate B5, an appropriate intermediate corresponding to the structure of the target compound was selected and used among the products obtained during the preparation process of the above example compounds, and the amounts of reagents, catalysts, and reaction conditions were appropriately changed to prepare compounds of Examples 109 to 113 in a manner similar to Example 30.

[0927] [Table 7]

[0928]

[0929]

[0930] Example 114: Preparation of 1-(1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-6-yl)piperidin-4-yl)-2-hydroxyethan-1-one

[0931]

[0932] Step 1: Preparation of 1-(piperidin-4-yl)ethan-1-one hydrochloride

[0933] 4 M hydrochloric acid solution (219.97 mmol, 200 mL) was added to tert-butyl 4-acetylpiperidine-1-carboxylate (50 g, 219.97 mmol) and stirred at 25 °C for 16 h. The reaction mixture was concentrated to obtain 1-(piperidin-4-yl)ethan-1-one hydrochloride (70 g, crude) as a light yellow solid. MS: m / z = 128.1 (M+1, ESI+).

[0934] Step 2: Preparation of benzyl 4-acetylpiperidine-1-carbolsylate

[0935] The product of Step 1 (70 g, 428 mmol) was dissolved in a Na2CO3 aqueous solution (500 mL), and CbzOSu (170.57 g, 684 mmol) was added and stirred at 25 °C for 16 h. The reaction mixture was filtered, and the resulting solid was dried to obtain benzyl 4-acetylpiperidine-1-carbolsylate (55 g, 49.20% yield) as a light yellow solid. MS: m / z = 262.0 (M+1, ESI+).

[0936] Step 3: Preparation of benzyl 4-(2-bromoacetyl)piperidine-1-carboxylate

[0937] The product of Step 2 (55 g, 210.47 mmol) was dissolved in methanol (500 mL), and then bromine (Br2, 10.8 mL, 210.47 mmol) was slowly added at 0 °C and stirred at 25 °C for 16 h. Water (2000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (500 mL). The organic layer was washed three times with brine (2000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain benzyl 4-(2-bromoacetyl)piperidine-1-carboxylate (30 g, 41.90% yield) as a light yellow solid. MS: m / z = 340.0 (M+1, ESI+).

[0938] Step 4: Preparation of benzyl 4-(2-hydroxyacetyl)piperidine-1-carboxylate

[0939] The product of Step 3 (30 g, 88.18 mmol) was dissolved in THF (80 mL), potassium hydroxide (KOH, 9.90 g, 176.36 mmol) was added, and the mixture was stirred at 25 °C for 1 h. Water (600 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with brine (600 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain benzyl 4-(2-hydroxyacetyl)piperidine-1-carboxylate (12 g, 49.07% yield) as a light yellow solid. MS: m / z = 278.0 (M+1, ESI+).

[0940] Step 5: Preparation of benzyl 4-(2-(hydroxymethyl)-1,3-dioxolan-2-yl)piperidine-1-carbolsylate

[0941] The product of Step 4 (12 g, 43.32 mmol) was dissolved in toluene (150 mL), and then TsOH (225 mg, 4.33 mmol) and ethane-1,2-diol (13.42 g, 216.6 mmol) were added and stirred at 120 °C for 48 h. After cooling the reaction mixture, water (800 mL) was added, and the product was extracted three times with EtOAc (200 mL). The organic layer was washed three times with brine (800 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain benzyl 4-(2-(hydroxymethyl)-1,3-dioxolan-2-yl)piperidine-1-carbolsylate (8.8 g, 63.31% yield) as a light yellow solid. MS: m / z = 321.9 (M+1, ESI+).

[0942] Step 6: Preparation of benzyl 4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-dioxolan-2-yl)piperidine-1-carbolsylate

[0943] The product of Step 5 (8.8 g, 33.72 mmol) was dissolved in DMF (100 mL), and then imidazole (4.6 g, 67.44 mmol) and TBDMSCl (8.8 g, 67.44 mmol) were added and stirred at 25 °C for 16 h. Water (1000 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (300 mL). The organic extract was washed three times with brine (1000 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to obtain benzyl 4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-dioxolan-2-yl)piperidine-1-carbolsylate (8.4 g, 57.53% yield) as a light yellow solid. MS: m / z = 436.1 (M+1, ESI+).

[0944] Step 7: Preparation of 4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-dioxolan-2-yl)piperidine

[0945] The product of Step 6 (8.4 g, 19.31 mmol) was dissolved in methanol (100 mL), Pd / C (1.16 g, 9.66 mmol) was added, and the mixture was stirred at 25 °C under a hydrogen gas atmosphere for 2 h. The reaction mixture was filtered and concentrated to obtain 4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-dioxolan-2-yl)piperidine (5.26 g, 90.53% yield) as a light yellow solid. MS: m / z = 302.1 (M+1, ESI+).

[0946] Steps 8 to 12: 1-(1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-6-yl)piperidin-4-yl)-2-hydroxyethan-1-one

[0947] The compound of Example 114 (70 mg, 30.3% yield) was obtained as a yellow solid in the same manner as steps 1 to 5 of Example 30. However, in step 8, the product of step 7 (5.26 g, 17.48 mmol) was used and stirred for 96 hours. 1 H NMR (400 MHz, DMSO-d6) δ 12.58 (s, 1H), 7.86 (s, 1H), 7.82-7.76 (m, 3H), 6.15 (s, 1H), 5.01 (t, 1H), 4.83 (d, 2H), 4.16 (d, 2H), 4.04 (d, 2H), 2.85 (t, 2H), 2.78-2.72 (m, 1H), 1.74 (d, 2H), 1.45-1.35 (m, 2H); MS: m / z = 542.2 (M+1, ESI+).

[0948] Example 115: Preparation of N-(1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl) imidazo[1,2-b]pyridazin-6-yl)piperidin-4-yl)-2-hydroxyacetamide

[0949]

[0950] Steps 1 to 5: Preparation of 6-(4-aminopiperidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0951] 6-(4-Aminopiperidin-1-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (150 mg, 71.43% yield) was obtained as a yellow solid in the same manner as in steps 1 to 5 of Example 30. However, in step 1 of Example 30, intermediate A12 (4.61 g, 23.07 mmol) was used and stirred for 16 hours, and in step 2, the reaction temperature was changed to 50 °C and stirred for 2 hours. In addition, in step 4, the reaction conditions were changed to stirring at 70 °C for 4 hours. MS: m / z = 499.0 (M+1, ESI+).

[0952] Step 6: Preparation of N-(1-(8-(((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-4-yl)-2-hydroxyacetamide

[0953] The product of Step 5 (300 mg, 595 μmol) and 2-hydroxyacetamide (46 mg, 0.26 mmol) were dissolved in DCM (10 mL), and then HOBT (176 mg, 1.3 mmol), EDCI (248 mg, 1.3 mmol), and DIEA (336 mg, 2.6 mmol, 0.45 mL) were added and stirred at 25 °C for 16 h. Water (80 mL) was added to the reaction mixture, and the product was extracted three times with DCM (30 mL). The organic layer was washed three times with brine (80 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain the compound of Example 115 (32 mg, 22.22% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 7.85 (d, 1H), 7.82-7.71 (m, 3H), 7.59 (d, 1H), 6.18 (s, 1H), 5.36 (t, 1H), 4.83 (d, 2H), 4.02 (d, 2H), 3.88-3.80 (m, 1H), 3.77 (d, 2H), 2.88 (t, 2H), 1.70 (d, 2H), 1.52-1.42 (m, 2H); MS: m / z = 557.0 (M+1, ESI+).

[0954] Example 116: Preparation of N-((1H-indol-2-yl)methyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0955]

[0956] Step 1: Preparation of N,N-bis(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0957] Intermediate S7 (6 g, 12.58 mmol) was dissolved in intermediate A2 (15 mL) and stirred at 120 ℃ for 16 h. Water (250 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give N,N-bis(4-methoxybenzyl-1)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (6 g, 88.22% yield). MS: m / z = 541.2 (M+1, ESI+).

[0958] Step 2: Preparation of 6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0959] The product of Step 1 (2 g, 3.7 mmol) was dissolved in DCM (15 mL), followed by the addition of TFA (5 mL) and stirring at 25 °C for 16 h. The reaction mixture was concentrated, adjusted to pH 8-9 with sodium bicarbonate solution, and extracted three times with DCM (50 mL). The organic layer was washed three times with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by silica gel chromatography to give 6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (750 mg, 67.57% yield) as a yellow solid. MS: m / z = 301.1 (M+1, ESI+).

[0960] Step 3: Preparation of N-((1H-indol-2-yl)methyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0961] The product of Step 2 (600 mg, 2 mmol) and intermediate B21 (580.64 mg, 4 mmol) were dissolved in THF (10 mL), and then Ti(OEt)4 (1.7 g, 6 mmol) was added and stirred at 90 °C for 16 hours. After that, the reaction mixture was cooled to 0 °C, and NaBH4 (302.64 mg, 8 mmol) was added and stirred at 0 °C for 1 hour. Water (150 mL) was added to the reaction mixture, and the mixture was extracted three times with EtOAc (50 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 116 (60 mg, 5.55% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 7.84 (s, 1H), 7.64 (t, 1H), 7.44 (d, 1H), 7.33 (d, 1H), 7.04-7.00 (m, 1H), 6.96-6.92 (m, 1H), 6.36 (s, 1H), 6.15 (s, 1H), 4.71 (d, 2H), 3.37 (t, 4H), 2.36 (t, 4H), 2.18 (s, 3H); MS: m / z = 430.2 (M+1, ESI+).

[0962] Examples 117 to 125

[0963] Among the intermediate B series, intermediates corresponding to the structure of the target compound were used, and the amounts of reagents, catalysts, and reaction conditions were appropriately changed to prepare compounds of Examples 117 to 125 in a similar manner to Example 116.

[0964] [Table 8]

[0965]

[0966]

[0967] Example 126: Preparation of 2-(((6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)methyl)-1H-benzo[d]imidazol-7-ol

[0968]

[0969] Steps 1 and 2: Preparation of N-((7-methoxy-1H-benzo[d]imidazo-2-yl)methyl)-N-(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0970] Using intermediate B8 (718.5 mg, 5.2 mmol), the same method as steps 5 and 6 of Example 8 was used to obtain N-((7-methoxy-1H-benzo[d]imidazo-2-yl)methyl)-N-(4-methoxybenzyl)-6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine (410 mg, 82.89% yield) as a yellow solid. However, in step 1, the reaction time was changed to 16 h, and in step 2, the reaction temperature was changed to 70 °C and stirred for 16 h. MS: m / z = 581.3 (M+1, ESI+).

[0971] Step 3: Preparation of 2-(((6-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)amino)methyl)-1H-benzo[d]imidazol-7-ol

[0972] The product of Step 2 (570 mg, 0.97 mmol) was dissolved in a 30% aqueous hydrobromonic acid solution (HBr, 10 mL) and stirred at 100°C for 16 hours. After cooling the reaction mixture to room temperature, an aqueous sodium bicarbonate solution (100 mL) was added, and the product was extracted three times with EtOAc (30 mL). The organic layer was washed three times with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The concentrate was purified by Prep-HPLC to obtain the compound of Example 126 (65 mg, 20.62% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.72 (s, 1H), 6.92 (t, 2H), 6.51 (t, 1H), 6.18 (s, 1H), 4.77 (s, 2H), 3.36 (t, 4H), 2.35 (t, 4H), 2.17 (s, 3H); MS: m / z = 447.3 (M+1, ESI+)

[0973] Examples 127 to 129

[0974] Intermediate B of [Table 3] corresponding to the structure of the target compound was used, and compounds of Examples 127 to 129 were prepared in a similar manner to Example 121 by appropriately changing the amount of reagent, catalyst, and reaction conditions.

[0975] [Table 9]

[0976]

[0977] Example 130: Preparation of 6-(5-aminopyridin-2-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0978]

[0979] Step 1: Preparation of 6-(tributylstannyl)pyridin-3-amine

[0980] 6-Bromopyridin-3-amine (2.2 g, 12.72 mmol) and 1,1,1,2,2,2-hexabutyldistannane (25.83 g, 44.52 mmol) were dissolved in 1,4-dioxane (40 mL), P(Cy)3Pd G3 (827 mg, 1.27 mmol) was added, and the mixture was stirred at 120 °C for 48 h under nitrogen gas. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain 6-(tributylstannyl)pyridin-3-amine (3.2 g, 65.68% yield) as a yellow solid. MS: m / z = 384.9 (M+1, ESI+)

[0981] Step 2: Preparation of ethyl N-(6-(5-aminopyridin-2-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate

[0982] The product of Step 1 (3.2 g, 8.36 mmol) and intermediate S8 (1.48 g, 3.34 mmol) were dissolved in 1,4-dioxane (20 mL), Pd(PPh3)4 (386 mg, 0.33 mmol) was added, and the mixture was stirred at 120 °C for 72 h under nitrogen gas. Water (400 mL) was added to the reaction mixture, and the product was extracted three times with EtOAc (100 mL). The organic layer was washed three times with brine (400 mL), dried over Na2SO4, filtered, concentrated, and the concentrate was purified by silica gel chromatography to obtain ethyl N-(6-(5-aminopyridin-2-yl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-yl)-N-(4-methoxybenzyl)glycinate (800 mg, 70.78% yield) as a yellow solid. MS: m / z = 501.3 (M+1, ESI+)

[0983] Steps 3 to 6: Preparation of 6-(5-aminopyridin-2-yl)-N-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)imidazo[1,2-b]pyridazin-8-amine

[0984] Using the product of Step 2 (300 mg, 0.6 mmol) of the above, the compound of Example 130 (33 mg, 18.65% yield) was obtained as a white solid in the same manner as Steps 2 to 5 of Example 30. However, in Step 3, the reaction time was changed to 16 hours, and in Step 4, the reaction solvent was changed to DCM and stirred for 16 hours. In addition, in Step 5, the reaction temperature was changed to 70°C and stirred for 16 hours. 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 8.20 (t, 1H), 8.10 (s, 1H), 7.95 (d, 1H), 7.90 (d, 1H), 7.76 (s, 2H), 7.11 (s, 1H), 7.03 (dd, 1H), 5.82 (s, 2H), 4.92 (d, 2H); MS: m / z = 493.1 (M+1, ESI+).

[0985] Examples 131 to 133

[0986] Compounds of Examples 131 to 133 were prepared in a similar manner to Example 130 by using a starting material corresponding to the structure of the target compound instead of the starting material 6-bromopyridin-3-amine and appropriately changing the amount of reagent, catalyst, and reaction conditions.

[0987] [Table 10]

[0988]

[0989] Experimental Example 1: CDK12 and CDK13 kinase inhibitory activity assay

[0990] Before starting the CDK12, CDK13 kinase reaction, the diluted solution of each example compound in a range of nine concentrations in DMSO as a solvent was incubated with the mixture of kinase and substrate (and cofactor, if necessary) for 20 minutes at room temperature. Then, radioisotope-labeled ATP ( 33 P-γ-ATP) was added and the kinase reaction was carried out at room temperature for 120 minutes. After the reaction was completed, the reaction mixture was treated with a radioisotope-labeled catalyst product ( 33 Transfer to a filter paper that binds the P-substrate and the unreacted 33P-ATP was removed by washing with 0.75% phosphoric acid, and the radioactive phosphorylated substrate remaining on the filter paper was measured. Kinase activity data were expressed as the percentage of kinase activity remaining in the test sample compared to the DMSO control, and the half maximal inhibitory concentration (IC) for each kinase was calculated using Prism (Graphpad) software. 50 was produced.

[0991] The CDK12 and CDK13 kinase inhibitory activities of the example compounds measured by the above method were evaluated based on the following criteria, and the results are shown in Table 11.

[0992] A: IC 50 ≤ 0.1 μM

[0993] B: 0.1 μM < IC 50 ≤ 0.5 μM

[0994] C: IC 50 > 0.5 μM

[0995] [Table 11]

[0996]

[0997]

[0998]

[0999] In Table 11 above, the example compounds of the present invention exhibited CDK12 and CDK13 kinase inhibitory activity superior to SR-4835.

[1000] Experimental Example 2: Intracellular CDK12 Inhibitory Activity Assay

[1001] Using MDA-MB-231 breast cancer cells, the phosphorylation level of Ser2 in the carboxy-terminal domain of RNA polymerase II subunit B1 was analyzed to determine whether the example compounds inhibited intracellular CDK12. 100 μL of the MDA-MB-231 cell suspension was dispensed into 96-well plates using L-15 (Invitrogen) medium containing 10% FBS and incubated overnight in a cell incubator at 0% CO2 and 37°C. The following day, each example compound and DMSO control dissolved in DMSO at a range of nine concentrations were treated with MDA-MB-231 cells for 6 hours at 0% CO2 and 37°C. After compound treatment, the medium in the 96-well plates was removed, and 60 μL of fixative solution (4.0% paraformaldehyde in PBS) was added and incubated for 20 minutes. After removing the fixative solution, the wells were washed with PBS, and 60 μL of PBS containing 0.1% Triton X-100 was added, followed by shaking at room temperature for 15 minutes. After washing with PBS, 100 μL of blocking buffer was added, and the wells were shaken at room temperature for 1 hour. After removing the blocking buffer, 60 μL of the primary antibodies, carboxy-terminal domain Ser2 phosphorylation of RNA polymerase II subunit B1 (1:500, Cell signaling technology) and carboxy-terminal domain of RNA polymerase II subunit B1 (1:200, Cell signaling technology), were added to the wells, and the reaction was carried out overnight at 4°C. 150 μL of PBS containing 0.1% Triton X-100 was added at each time, and the sections were washed at least twice with shaking for 5 minutes at room temperature. 60 μL of secondary antibody was added, and the sections were shaken for 1 hour at room temperature, blocking light. 150 μL of PBS containing 0.1% Triton X-100 was added at each time, and the sections were washed at least twice with shaking for 5 minutes at room temperature. 150 μL of PBS was added at each time, and the sections were washed at least twice with shaking for 5 minutes at room temperature.The plate was scanned on a Li-COR Odyssey machine with the wells filled with PBS. IC. 50 Values ​​were calculated by fitting dose-response curves using a four-parameter model in Prism (Graphpad) software.

[1002] The intracellular CDK12 inhibitory activity of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 12.

[1003] IC 50 Value range

[1004] A: IC 50 ≤ 0.3 μM

[1005] B: 0.3 μM < IC 50 ≤ 3 μM

[1006] C: IC 50 > 3 μM

[1007] [Table 12]

[1008]

[1009]

[1010]

[1011] In Table 12 above, the example compound of the present invention exhibited superior intracellular CDK12 inhibitory activity than SR-4835.

[1012] Experimental Example 3: Proliferation inhibition activity assay for MDA-MB-231 breast cancer cells

[1013] To measure the cell proliferation inhibitory activity of each example compound, an experiment was performed using the MDA-MB-231 breast cancer cell line. MDA-MB-231 cells were cultured in L-15 (Invitrogen) medium containing 10% FBS. 100 μL of the MDA-MB-231 cell suspension was dispensed into 96 wells and incubated in a cell incubator at 0% CO2 and 37°C. The following day, each example compound and the DMSO control, diluted in DMSO at nine concentrations, were treated with the cells for 72 hours. After compound treatment, 100 μL of CellTiter Glo (Promega) reagent was added to each 96 well, shaken for 10 minutes, and left to stand at room temperature for 10 minutes. The 96-well plate was inserted into an Envision plate reader (PerkinElmer) and the luminescence signal was measured. Relative IC 50 Values ​​were calculated by fitting dose-response curves using XLfit (IDBS Ltd) software.

[1014] The breast cancer cell proliferation inhibitory activity of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 13.

[1015] IC 50 Value range

[1016] A: IC 50 ≤ 0.05 μM

[1017] B: 0.05 μM < IC 50 ≤ 0.5 μM

[1018] C: IC 50 > 0.5 μM

[1019] [Table 13]

[1020]

[1021]

[1022]

[1023] In Table 13 above, the example compound of the present invention showed a superior inhibitory effect on MDA-MB-231 breast cancer cells than SR-4835.

[1024] Experimental Example 4: CDK1, CDK2 kinase inhibitory activity assay and selectivity analysis for CDK12 kinase.

[1025] Before starting the CDK1, CDK2 kinase reaction, the diluted solution of each example compound in a range of nine concentrations in DMSO as a solvent was incubated with the mixture of kinase and substrate (and cofactor, if necessary) for 20 minutes at room temperature. Then, radioisotope-labeled ATP ( 33 P-γ-ATP) was added and the kinase reaction was carried out at room temperature for 120 minutes. After the reaction was completed, the reaction mixture was treated with a radioisotope-labeled catalyst product ( 33 Transfer to a filter paper that binds the P-substrate and the unreacted 33 P-ATP was removed by washing with 0.75% phosphoric acid, and the radioactive phosphorylated substrate remaining on the filter paper was measured. Kinase activity data were expressed as the percentage of kinase activity remaining in the test sample compared to the DMSO control, and the half maximal inhibitory concentration (IC) for each kinase was calculated using Prism software. 50 was produced.

[1026] The CDK1 and CDK2 kinase inhibitory activities of the example compounds measured by the above method were evaluated based on the following criteria, and the results are shown in Table 14.

[1027] IC 50 Value range

[1028] A: IC 50 ≤ 0.1 μM

[1029] B: 0.1 μM < IC 50 ≤ 1 μM

[1030] C: IC 50 > 1 μM

[1031] The selectivity assay criteria for CDK12 were CDK1 IC 50 and CDK12 IC 50 of the CDK2 IC or 50 and CDK12 IC 50 It was calculated as a ratio and evaluated based on the following criteria, and the results are summarized in Table 14 below.

[1032] A: CDK1 / CDK12 or CDK2 / CDK12 > 500x

[1033] B: 100x < CDK1 / CDK12 or CDK2 / CDK12 ≤ 500x

[1034] C: CDK1 / CDK12 or CDK2 / CDK12 ≤ 100-fold

[1035] [Table 14]

[1036]

[1037]

[1038] In Table 14 above, the example compounds of the present invention showed excellent selectivity for CDK12.

[1039] Experimental Example 5: Test for inhibition of breast cancer cell proliferation

[1040] To measure the cell proliferation inhibitory activity of each example compound, experiments were performed using three breast cancer cell lines. HCC70 cells were seeded in 96-well plates using RPMI1640 (Invitrogen) medium containing 10% FBS, and 100 μL of HCC70 cell suspension was dispensed, and then incubated in a cell incubator at 5% CO2 and 37°C. BT-20 cells were seeded in 96-well plates using EMEM (Invitrogen) medium containing 10% FBS, and then 100 μL of BT-20 cell suspension was dispensed, and then incubated in a cell incubator at 5% CO2 and 37°C. MDA-MB-436 cells were cultured in 96-well plates using L-15 (Invitrogen) medium containing 10% FBS. 100 μL of the MDA-MB-436 cell suspension was dispensed and incubated in a cell incubator at 0% CO2 and 37°C. The following day, the cells were treated with each example compound and the DMSO control, which were diluted in DMSO at nine concentrations, for 72 hours. After compound treatment, 100 μL of CellTiter Glo (Promega) reagent was added to each 96-well plate, shaken for 10 minutes, and left at room temperature for 10 minutes. The 96-well plate was inserted into an Envision plate reader (PerkinElmer), and the luminescence signal was measured. Relative IC 50 Values ​​were calculated by fitting dose-response curves using XLfit (IDBS Ltd) software.

[1041] The inhibition of breast cancer cell proliferation of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 15.

[1042] IC 50 Value range

[1043] A: IC 50 ≤ 0.05 μM

[1044] B: 0.05 μM < IC 50 ≤ 1 μM

[1045] C: IC 50 > 1 μM

[1046] [Table 15]

[1047]

[1048] In Table 15 above, Examples 8, 29, and 30 of the present invention showed a superior breast cancer cell proliferation inhibition effect than the comparative compounds palbociclib, olaparib, and SR-4835.

[1049] Experimental Example 6: Test for inhibition of gastric cancer cell proliferation

[1050] To measure the cell proliferation inhibitory activity of each example compound, experiments were performed using four gastric cancer cell lines (AGS, SUN-1, KATO-Ⅲ, MKN45). AGS cells were cultured in F-12K (Gibco) medium containing 10% FBS, and 90 μL of each cell suspension was dispensed into a 96-well plate, followed by incubation in a cell incubator at 5% CO2 and 37°C. SNU-1 and MKN45 cells were cultured in RPMI1640 (Gibco) medium containing 10% FBS, and 90 μL of each cell suspension was dispensed into a 96-well plate, followed by incubation in a cell incubator at 5% CO2 and 37°C. KATO-Ⅲ cells were cultured in IMDM (Gibco) medium containing 10% FBS. Each cell suspension was dispensed into a 96-well plate (90 μL each) and incubated in a cell incubator at 37°C and 5% CO2. The following day, 10 μL of each example compound and DMSO control, diluted in a range of nine concentrations in DMSO, were added to each 96-well plate and treated for 72 hours. After compound treatment, 100 μL of CellTiter Glo (Promega) reagent was added to each 96-well plate, shaken for 10 minutes, and left at room temperature for 10 minutes. The 96-well plate was inserted into an Envision plate reader (PerkinElmer) and the luminescence signal was measured. Relative IC 50 Values ​​were calculated by fitting dose-response curves using XLfit (IDBS Ltd) software.

[1051] The inhibition of gastric cancer cell proliferation of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 16.

[1052] IC 50 Value range

[1053] A: IC 50 ≤ 0.2 μM

[1054] B: 0.2 μM < IC 50 ≤ 1 μM

[1055] C: IC 50 > 1 μM

[1056] [Table 16]

[1057]

[1058] In Table 16 above, Examples 8, 29, and 30 of the present invention showed a superior effect on suppressing gastric cancer cell proliferation compared to the comparative compound Cisplatin.

[1059] Experimental Example 7: Cyclin K Resolution Analysis

[1060] To confirm the cyclin K degradation ability of the example compound, HCC70 breast cancer cells and MKN45 gastric cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS, and then the cells were harvested to prepare a cell suspension. The HCC70 breast cancer cell suspension was seeded at 2.0 X 10 per 6 wells. 6 After dividing into the cell number, the cells were cultured in a 5% CO2 cell incubator at 37°C for 24 hours, and the MKN45 gastric cancer cell suspension was seeded at 5.0 X 10 per each 6 well. 6After dividing into cell numbers, they were cultured in a 5% CO2 cell incubator at 37℃ for 24 hours. The next day, the example compounds and the negative control group (DMSO treatment group) diluted in DMSO at a range of 4 to 5 concentrations were added to 6 wells, and HCC70 breast cancer cells were treated for 8 hours, and MKN45 gastric cancer cells were treated with the compounds for 8 hours or 24 hours. After compound treatment, the medium was removed, each cell was washed with PBS, and 100 μL of RIPA buffer for cell lysis was added to each 6 wells to collect the cell lysate. The protein concentration of the cell lysate was quantified by the BSA method, and a loading buffer was added to adjust the protein concentration to 30 μg, and the reaction was performed at 85℃ for 10 minutes, centrifuged, and left to stand at room temperature. After electrophoresis on a 4-12% Bis-Tris gel, proteins were transferred to a nitrocellulose membrane. The membrane was labeled with primary antibodies, cyclin K (1:1000, Bethyl Lab) and vinculin (1:1000, Sigma), respectively, and then labeled with secondary antibodies (1:10000, LI-COR). Signals were detected using an Odyssey imager (LI-COR Biosciences). The results are shown in Figures 1 to 5.

[1061] In FIGS. 1 to 3, Examples 8, 30, and 40 were shown to strongly decompose cyclin K in HCC70 cells compared to the negative control group, and in FIGS. 4 and 5, Examples 8 and 40 were shown to strongly decompose cyclin K in MKN45 cells compared to the negative control group.

[1062] Experimental Example 8: Zebrafish Xenograft Breast and Stomach Cancer Models

[1063] Transgenic Tg(fli1:EGFP)y1 zebrafish embryos were cultured in E3 embryo medium with 0.2 mM 1-phenyl-2-thiourea, also known as PTU, at 28°C for 48 hours. HCC70 breast cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS, and AGS gastric cancer cells were cultured in F-12K medium supplemented with 10% FBS. Each cell was harvested, labeled with Dil red fluorescent dye, and then transplanted subcutaneously into 2-day-old zebrafish embryos. Embryos with tumors were selected and divided into experimental groups (20 embryos / group). The compounds of the examples, DMSO control, or comparative compounds were added, dissolved and diluted in DMSO at 2-3 concentrations, and incubated at 35.5°C for 48 or 72 hours. In the breast cancer model, primary tumors were collected immediately after transplantation (day 0) and 48 hours (day 2), and in the gastric cancer model, primary tumors were obtained immediately after transplantation (day 0) and 72 hours (day 3). The collected tumor photographs were analyzed for tumor size reduction compared to the DMSO control group using image software, and the number of seeded cancer cells was manually counted through the captured images after 3 days, and the results are shown in Figs. 6, 7, and 8. In the breast cancer model, one-way ANOVA test and Dunnett's multiple comparison method were performed for statistical analysis of the Example compound treatment group compared to the vehicle group (***p<0.001). In the gastric cancer model, the Kruskal-Wallis test and Dunn's multiple comparison method were performed for statistical analysis of the Example compound treatment group compared to the vehicle group (*p<0.05). In the gastric cancer model, statistical analysis of the treatment groups of the example compound compared to the control compound 5-FU group was performed using the Mann-Whitney test (*p< 0.05).

[1064] In Fig. 6, the compound of Example 30 showed a dose-dependent tumor size reduction effect compared to the vehicle in the HCC70 breast cancer model. In Fig. 7, the compound of Example 8 showed a dose-dependent tumor size reduction effect compared to the vehicle in the AGS gastric cancer model. In Fig. 8, the compound of Example 8 showed a significant effect of reducing the number of metastatic gastric cancer cells compared to the comparative compound 5-FU 1 mM when Example 8 was treated at 0.1 μM in the AGS gastric cancer model.

[1065] Experimental Example 9: Test for inhibition of lung cancer cell proliferation

[1066] To measure the cell proliferation inhibitory activity of the compounds of the present invention, experiments were performed using three lung cancer cell lines. NCI-H23 and NCI-H358 cells were cultured in RPMI1640 medium containing 10% FBS and 1% PS. 100 μL of each cell suspension was dispensed into a 96-well plate and incubated in a cell incubator at 5% CO2 and 37°C. A427 cells were cultured in DMEM medium containing 10% FBS and 1% PS. 100 μL of each cell suspension was dispensed into a 96-well plate and incubated in a cell incubator at 5% CO2 and 37°C. The following day, the compounds of Example 8 and the DMSO control, which were dissolved and diluted in DMSO at a range of 10 concentrations, were treated to the cells for 72 hours. After compound treatment, 10 μL of Cell Counting Kit-8 (Dojindo) reagent was added to each 96-well plate and incubated for 2 hours. The 96-well plate was inserted into an Envision plate reader (Thermo Fisher) and the luminescence signal was measured. Relative IC 50 Values ​​were calculated by fitting dose-response curves using Graphpad PRISM software.

[1067] The inhibition of lung cancer cell proliferation of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 17.

[1068] IC 50 Value range

[1069] A: IC 50 ≤ 0.2 μM

[1070] B: 0.2 μM < IC 50 ≤ 1 μM

[1071] C: IC 50 > 1 μM

[1072] [Table 17]

[1073]

[1074] In Table 17 above, the compound of Example 8 showed an excellent lung cancer cell proliferation inhibitory effect.

[1075] Experimental Example 10: Test for inhibition of pancreatic cancer cell proliferation

[1076] To measure the cell proliferation inhibitory activity of the compounds of the present invention, experiments were conducted using three pancreatic cancer cell lines. AsPC-1, MIA-PaCA2-Control, and Gemcitabine-resistant-MIA-PaCa2 cells were cultured in DMEM medium containing 10% FBS and 1% PS. 100 μL of each cell suspension was dispensed into a 96-well plate and incubated in a cell incubator at 37°C and 5% CO2. The following day, the cells were treated with the compound of Example 8 and the DMSO control, which were diluted in DMSO at a concentration range of 10, for 72 hours. After compound treatment, 10 μL of the Cell Counting Kit-8 (Dojindo) reagent was added to each 96-well plate and incubated for 2 hours. The 96-well plate was inserted into an Envision plate reader (Thermo Fisher) and the luminescence signal was measured. Relative IC 50 Values ​​were calculated by fitting dose-response curves using Graphpad PRISM software.

[1077] The inhibition of pancreatic cancer cell proliferation of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 18.

[1078] IC 50 Value range

[1079] A: IC 50 ≤ 0.2 μM

[1080] B: 0.2 μM < IC 50 ≤ 1 μM

[1081] C: IC 50 > 1 μM

[1082] [Table 18]

[1083]

[1084] In Table 18 above, the compound of Example 8 showed an excellent effect of inhibiting the proliferation of pancreatic cancer cells.

[1085] Experimental Example 11: Test for inhibition of colon cancer cell proliferation

[1086] To measure the cell proliferation inhibitory activity of each example compound, experiments were conducted using seven colon cancer cell lines. DLD-1, LoVo, SW620, SW480, HCT116, HCT15, and HT29 cells were cultured in RPMI1640 medium containing 10% FBS and 1% PS. 100 μL of each cell suspension was dispensed into a 96-well plate and incubated in a cell incubator at 37°C and 5% CO2. The following day, the cells were treated with each example compound and DMSO control, which were diluted in a 10-concentration range, for 72 hours. After compound treatment, 10 μL of Cell Counting Kit-8 (Dojindo) reagent was added to each 96-well plate and incubated for 2 hours. The 96-well plate was inserted into an Envision plate reader (Thermo Fisher) and the luminescence signal was measured. Relative IC 50Values ​​were calculated by fitting dose-response curves using Graphpad PRISM software.

[1087] The inhibition of colon cancer cell proliferation of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 19.

[1088] IC 50 Value range

[1089] A: IC 50 ≤ 0.2 μM

[1090] B: 0.2 μM < IC 50 ≤ 1 μM

[1091] C: IC 50 > 1 μM

[1092] [Table 19]

[1093]

[1094] In Table 19 above, the compounds of Examples 8 and 40 showed excellent colon cancer cell proliferation inhibition effects.

[1095] Experimental Example 12: Test for inhibition of gastric cancer cell proliferation

[1096] To measure the cell proliferation inhibitory activity of each example compound, experiments were performed using six gastric cancer cell lines. NCI-N87, SNU601, KATO-Ⅲ, MKN1, and AGS cells were cultured in RPMI1640 medium containing 10% FBS and 1% PS. 100 μL of each cell suspension was dispensed into a 96-well plate and incubated in a cell incubator at 5% CO2 and 37°C. HS746T cells were cultured in DMEM medium containing 10% FBS and 1% PS. 100 μL of each cell suspension was dispensed into a 96-well plate and incubated in a cell incubator at 5% CO2 and 37°C. The following day, the cells were treated with each compound and DMSO control, which were dissolved and diluted in DMSO at a range of 10 concentrations, for 72 hours. After compound treatment, 10 μL of Cell Counting Kit-8 (Dojindo) reagent was added to each 96-well plate and incubated for 2 hours. The 96-well plate was inserted into an Envision plate reader (Thermo Fisher) and the luminescence signal was measured. Relative IC 50 Values ​​were calculated by fitting dose-response curves using Graphpad PRISM software.

[1097] The inhibition of gastric cancer cell proliferation of the example compound measured by the above method was evaluated based on the following criteria, and the results are shown in Table 20.

[1098] IC 50 Value range

[1099] A: IC 50 ≤ 0.2 μM

[1100] B: 0.2 μM < IC 50 ≤ 1 μM

[1101] C: IC 50 > 1 μM

[1102] [Table 20]

[1103]

[1104] In Table 20 above, the compounds of Examples 8 and 40 showed excellent gastric cancer cell proliferation inhibition effects.

Claims

1. A compound of the following formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above chemical formula I, R 1 Silver halo, hydroxy, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkyne, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkyl-C 1 -C 6 Alkyl-, C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkenyl-, or C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkynyl- and the above R 1 Any C among 1 -C 6 Alkoxy group, C 1 -C 6 Alkyl group, C 2 -C 6 Alkenyl group, C 2 -C 6 Alkyne and C 3 -C 6 The cycloalkyl group may be optionally substituted with halo, hydroxy or cyano; R 2 is C 6 -C 10 Aryl; 5- or 6-membered heteroaryl comprising 1 to 2 nitrogen atoms; or a monocyclic or bridged or spiro 4- to 12-membered heterocyclyl comprising 1 nitrogen atom and connected to an imidazopyridazine ring of formula I through said nitrogen atom, wherein said heterocyclyl may optionally comprise 1 additional nitrogen atom or 1 oxygen atom, Above R 2 may be optionally substituted with one or more substituents selected from the following: (i) H, halo, hydroxy, cyano, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, (ii) C substituted with H, halo, hydroxy or cyano; 1 -C 6 Alkyl, (iii) C substituted with halo, hydroxy or cyano; 3 -C 6 Cycloalkyl, (iv) Amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, (C 3 -C 6 Cycloalkyl)amino, (C 1 -C 6 Hydroxyalkyl)amino, (C 1 -C 6 alkyl)carbonylamino, (C 1 -C 6 Hydroxyalkyl)carbonylamino or (C 1 -C 6 Alkoxy)(C 1 -C 6 alkyl)carbonylamino, and (v) (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 Alkyl)aminocarbonyl, di(C 1 -C 6 alkyl)aminocarbonyl, carboxy or (C 1 -C 6 alkoxy)carbonyl; R 3 silver , or and; X 1a Silver NR 3a , O or S, and X 2a is N or CR 3a But, X 2a Go CR 3a Back side X 1a Silver NR 3a and; X 1b and X 2b One of them is N and the other is CR 3b and; X 2c is N or CR 3c and; X 3a , X 4a , X 5a and X 6a At least one of them is N, the rest are CR 3d and; X 3b , X 4b , X 5b and X 6b At least one of them is N, the rest are CR 3e and; X 3c , X 4c , X 5c and X 6c 1 or less of these are NR 3f and the rest are CR 3f R 3g and; R 3a , R 3b and R 3c are each independently H or C 1 -C 6 It is alkyl, R 3d , R 3e , R 3f and R 3g are each independently H, halo, cyano, hydroxy, amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, C 1 -C 6 Alkoxy or C 1 -C 6 Alkyl, R 3d , R 3e , R 3f and R 3g Any C among 1 -C 6 Alkoxy group and C 1 -C 6 The alkyl group may be optionally substituted with halo, cyano or hydroxy.

2. In paragraph 1, R 1 C randomly substituted with halo, halo 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkyne, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkyl-C 1 -C 6 Alkyl-, C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkenyl-, or C 3 -C 6 Cycloalkyl-C 2 -C 6 A compound of formula I, which is an alkynyl-, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof.

3. In paragraph 1, R 1 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein said compound is ethynyl, methylethynyl, cyclopropylethynyl, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, trifluoroethyl, iodo or chloro.

4. In paragraph 1, R 2 is phenyl; pyrimidinyl, pyridinyl, pyrrolyl or imidazolyl; azetidinyl, pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, wherein any two non-adjacent carbon atoms of said morpholinyl, piperidinyl or piperazinyl are optionally C 1 -C 3 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is a 7 to 11 membered azaspiro ring which may be linked to each other by alkylene groups to form a bridged ring; or which comprises one nitrogen atom and optionally comprises one additional nitrogen atom or one oxygen atom.

5. In paragraph 4, The above 7 to 11 membered azaspiro ring is selected from the group consisting of 2-azaspiro[3.3]heptan-2-yl, 2,6-diazaspiro[3.3]heptan-2-yl, 6-oxa-2-azaspiro[3.3]heptan-2-yl, 2-azaspiro[3.4]octane-2-yl, 2,6-diazaspiro[3.4]octane-2-yl, 6-oxa-2-azaspiro[3.4]octane-2-yl, 6-azaspiro[3.4]octane-6-yl, 2,6-diazaspiro[3.4]octane-6-yl, 2-oxa-6-azaspiro[3.4]octane-6-yl, 2-azaspiro[4.4]nonan-2-yl, 2,7-diazaspiro[4.4]nonan-2-yl or 2-oxa-7-azaspiro[4.4]nonan-7-yl, The above-mentioned bridged ring is selected from the group consisting of 3-oxa-8-azabicyclo[3.2.1]octane-8-yl, 8-oxa-3-azabicyclo[3.2.1]octane-3-yl, 3,8-diazabicyclo[3.2.1]octane-3-yl, 3,8-diazabicyclo[3.2.1]octane-8-yl, 2-oxa-5-azabicyclo[2.2.2]octane-5-yl, 8-azabicyclo[3.2.1]octane-8-yl, 3-azabicyclo[3.1.1]heptan-3-yl, 6-oxa-3-azabicyclo[3.1.1]heptan-3-yl, 3,6-diazabicyclo[3.1.1]heptan-3-yl or A compound of formula I, which is 3,6-diazabicyclo[3.1.1]heptan-6-yl, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof.

6. In paragraph 1, R 2 is C 6 -C 10 An aryl or a 5- or 6-membered heteroaryl containing 1 to 2 nitrogen atoms, wherein said aryl and heteroaryl are H, hydroxy, amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino and (C 3 -C 6 R selected from the group consisting of cycloalkyl)amino 2d is arbitrarily substituted with; or R 2 is selected from the following chemical formula A or chemical formula B: [Chemical Formula A] In the above chemical formula A, n1 and n2 are each independently 1 or 2, and the carbon atoms of the ring are optionally C 1 -C 6 may be substituted with alkyl, If both n1 and n2 are 2, Y 1 Silver CR 2a R 2b , NR 2c , or O, and any two non-adjacent carbon atoms of said ring are optionally C 1- C 3 Can be linked to each other by alkylene to form a bridged ring, If one or both of n1 and n2 are 1, Y 1 Silver CR 2a R 2b and; [Chemical Formula B] In the above chemical formula B, n3, n4, n5 and n6 are each independently 1 or 2, and the carbon atoms of the ring are optionally C 1 -C 6 may be substituted with alkyl, Y 2 is CR 2a R 2b , NR 2c or O, In the above chemical formulas A and B, R 2a and R 2b are each independently H, halo, hydroxy, cyano, C 1 -C 6 Alkyl, or C 3 -C 6 Cycloalkyl; C substituted with halo, hydroxy or cyano 1 -C 6 C substituted with alkyl, halo, hydroxy or cyano 3 -C 6 Cycloalkyl; amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, (C 3 -C 6 Cycloalkyl)amino, (C 1 -C 6 alkyl)carbonylamino, (C 1 -C 6 hydroxyalkyl)carbonylamino; and (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 Alkyl)aminocarbonyl, di(C 1 -C 6 alkyl) is selected from the group consisting of aminocarbonyl and carboxy; R 2c is H, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 alkyl)aminocarbonyl or di(C 1 -C 6 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein said compound is (alkyl)aminocarbonyl.

7. In paragraph 6, R 2a and R 2b One of them is H, halo, hydroxy, cyano, C 1 -C 6 Alkyl, or C 3 -C 6 Cycloalkyl; C substituted with halo, hydroxy or cyano 1 -C 6 C substituted with alkyl, halo, hydroxy or cyano 3 -C 6 Cycloalkyl; amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, (C 3 -C 6 Cycloalkyl)amino, (C 1 -C 6 alkyl)carbonylamino, (C 1 -C 6 hydroxyalkyl)carbonylamino; and (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 Alkyl)aminocarbonyl, di(C 1 -C 6 alkyl) selected from the group consisting of aminocarbonyl and carboxy, R 2a and R 2b The remaining one is H, halo, hydroxy, cyano or C 1 -C 6 A compound of formula I, which is an alkyl group, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof.

8. In paragraph 6, R 2a and R 2b is selected from the group consisting of H, hydroxy, amino, cyclopropylamino, hydroxyacetamido, methyl, hydroxymethyl, 2-hydroxy-isopropyl, hydroxycyclopropyl, carboxy, carbamoyl, hydroxyacetyl and cyano, R 2c is selected from the group consisting of H, methyl, cyclopropyl, acetyl, hydroxyacetyl and carbamoyl, R 2d A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of hydroxy and amino.

9. In paragraph 6, R 2 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: (R in the above structure 2a , R 2b , R 2c and R 2d ) is as described in Article 6.

10. In paragraph 1, R 2 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

11. In paragraph 1, R 3 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following structures: (In the above structure, X 3a , X 4a , X 5a and X 6a At least one of them is N, the rest are CR 3d And, X 3b , X 4b , X 5b and X 6b At least one of them is N, the rest are CR 3e And, X 3c , X 4c , X 5c and X 6c 1 or less of these are NR 3f and the rest are CR 3f R 3g And, R 3a , R 3b and R 3c are each independently H or C 1 -C 6 is alkyl, and R 3d , R 3e , R 3f and R 3g are each independently H, halo, cyano, hydroxy, amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, C 1 -C 6 Alkoxy or C 1 -C 6 Alkyl, R 3d , R 3e , R 3f and R 3g Any C among 1 -C 6 Alkoxy group and C 1 -C 6 The alkyl group may be optionally substituted with halo, cyano or hydroxy.) 12. In paragraph 11, R 3 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following structures: (In the above structure, R 3a , R 3b and R 3c are each independently H or C 1 -C 6 It is alkyl, R 3d , R 3e , R 3f and R 3g are each independently H, halo, hydroxy, amino, C 1 -C 6 Alkoxy, C 1 -C 6 Alkyl or C 1 -C 6 (It is haloalkyl.) 13. In paragraph 12, R 3d , R 3e , R 3f and R 3g A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein each independently is selected from the group consisting of H, F, Cl, hydroxy, amino, methoxy, methyl and trifluoromethyl.

14. In paragraph 1, R 3 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following structures:

15. In paragraph 1, A compound, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following chemical formula IA: [Chemical Formula IA] In the above chemical formula IA, R 1 Silver halo, hydroxy, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkyne, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkyl-C 1 -C 6 Alkyl-, C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkenyl-, or C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkynyl- and the above R 1 Any C among 1 -C 6 Alkoxy group, C 1 -C 6 Alkyl group, C 2 -C 6 Alkenyl group, C 2 -C 6 Alkyne and C 3 -C 6 The cycloalkyl group may be optionally substituted with halo, hydroxy or cyano; Ring A is a monocyclic or bridged or spiro 4- to 12-membered heterocyclyl which may optionally contain one additional nitrogen atom or one additional oxygen atom; Ring A may be optionally substituted with one or more substituents selected from the following: (i) H, halo, hydroxy, cyano, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, (ii) C substituted with halo, hydroxy or cyano; 1 -C 6 Alkyl, (iii) C substituted with halo, hydroxy or cyano; 3 -C 6 Cycloalkyl, (iv) Amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, (C 3 -C 6 Cycloalkyl)amino, (C 1 -C 6 Hydroxyalkyl)amino, (C 1 -C 6 alkyl)carbonylamino, (C 1 -C 6 Hydroxyalkyl)carbonylamino or (C 1 -C 6 Alkoxy)(C 1 -C 6 alkyl)carbonylamino, and (v) (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 Alkyl)aminocarbonyl, di(C 1 -C 6 alkyl)aminocarbonyl, carboxy or (C 1 -C 6 alkoxy)carbonyl; R 3d is H, halo, cyano, hydroxy, amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, C 1 -C 6 Alkoxy or C 1 -C 6 Alkyl, R 3d Any C among 1 -C 6 Alkoxy group and C 1 -C 6 The alkyl group may be optionally substituted with halo, cyano or hydroxy; p is an integer from 0 to 3.

16. In paragraph 1, A compound, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following chemical formula IB: [Chemical Formula IB] In the above chemical formula, R 1 Silver halo, hydroxy, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkyne, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkyl-C 1 -C 6 Alkyl-, C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkenyl-, or C 3 -C 6 Cycloalkyl-C 2 -C 6 Alkyne- and R 1 Any C among 1 -C 6 Alkoxy group, C 1 -C 6 Alkyl group, C 2 -C 6 Alkenyl group, C 2 -C 6 Alkyne and C 3 -C 6 The cycloalkyl group may be optionally substituted with halo, hydroxy or cyano; Ring B is is selected from; R 2d is H, hydroxy, amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino or (C 3 -C 6 cycloalkyl)amino; R 3d is H, halo, cyano, hydroxy, C 1 -C 6 Alkoxy or C 1 -C 6 Alkyl, R 3d Any C among 1 -C 6 Alkoxy group and C 1 -C 6 The alkyl group may be optionally substituted with halo, cyano or hydroxy; p is an integer from 0 to 3.

17. In paragraph 16, this And, R 2d Ga amino, (C 1 -C 6 Alkyl)amino or di(C 1 -C 6 A compound, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein said compound is an alkyl)amino group.

18. In paragraph 1, A compound, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following chemical formula IC-1 or chemical formula IC-2: In the above chemical formula IC-1, n1 and n2 are each independently 1 or 2, and Y 1 The carbon atoms of the ring containing N are optionally C 1 -C 6 may be substituted with alkyl, If both n1 and n2 are 2, Y 1 Silver CR 2a R 2b , NR 2c , or O, If one or both of n1 and n2 are 1, Y 1 Silver CR 2a R 2b and; In the above chemical formula IC-2, Y 2 is CR 2a R 2b , NR 2c or O; In the above chemical formulas IC-1 and IC-2, R 1 Silver halo, hydroxy, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkyl or C 1 -C 6 It is haloalkyl, R 3d are each independently H, halo, hydroxy, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkyl, or C 1 -C 6 It is haloalkyl, p is an integer from 0 to 2; Above R 2a and R 2b are each independently H, halo, hydroxy, cyano, C 1 -C 6 Alkyl, or C 3 -C 6 Cycloalkyl; C substituted with halo, hydroxy or cyano 1 -C 6 C substituted with alkyl, halo, hydroxy or cyano 3 -C 6 Cycloalkyl; amino, (C 1 -C 6 Alkyl)amino, di(C 1 -C 6 Alkyl)amino, (C 3 -C 6 Cycloalkyl)amino, (C 1 -C 6 alkyl)carbonylamino, (C 1 -C 6 hydroxyalkyl)carbonylamino; and (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 Alkyl)aminocarbonyl, di(C 1 -C 6 alkyl) selected from the group consisting of aminocarbonyl and carboxy; Above R 2c is H, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, (C 1 -C 6 alkyl)carbonyl, (C 1 -C 6 hydroxyalkyl)carbonyl, aminocarbonyl, (C 1 -C 6 alkyl)aminocarbonyl or di(C 1 -C 6 A compound of formula I, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein said compound is (alkyl)aminocarbonyl.

19. In paragraph 1, A compound of formula I selected from the following, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof:

20. A pharmaceutical composition comprising a compound of any one of claims 1 to 19, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof as an active ingredient.

21. In paragraph 20, A pharmaceutical composition for the treatment of a disease caused by overexpression, excessive activity, mutation, and / or activation of signaling pathways associated with cyclin K of cyclin-dependent protein kinase (CDK)12 and / or CDK13.

22. In paragraph 21, A pharmaceutical composition, wherein the disease is cancer, a proliferative disease, a neurodegenerative disease, an autoimmune disease or a disease caused by an abnormality in protein translation function within a cell.

23. In paragraph 22, A pharmaceutical composition wherein the disease is cancer.

24. In paragraph 23, A pharmaceutical composition wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, head and neck cancer, thyroid cancer, skin cancer, biliary tract cancer, esophageal cancer, hematopoietic tumors of the lymphoid and myeloid systems, tumors of the central and peripheral nervous systems, and sarcoma.

25. In paragraph 23, A pharmaceutical composition for the treatment of advanced cancer or cancer that has become resistant to treatment with previously administered therapeutic agents.

26. In paragraph 23, The pharmaceutical composition is administered in combination with at least one therapeutic agent selected from the group consisting of radiotherapy, taxane derivatives, platinum compounds, antimetabolites, anti-CTLA4 therapy, anti-PD1 therapy, anti-PD-L1 therapy, anti-VEGF therapy, anti-EGFR therapy, topoisomerase inhibitors, anti-HER2 therapy, antihormone therapy, estrogen receptor inhibitors, ERK inhibitors, PARP inhibitors, mTOR inhibitors, CDK4 / 6 inhibitors, EGFR inhibitors, HER2 inhibitors, ALK inhibitors, tyrosine kinase inhibitors, MEK inhibitors, BCR-ABL inhibitors, PI3K inhibitors, FGFR inhibitors, ROS1 inhibitors, androgen biosynthesis inhibitors, androgen receptor inhibitors, Hedgehog inhibitors, MET inhibitors, AXL inhibitors, NTRK1 inhibitors, RET inhibitors, KRAS inhibitors, and RAF inhibitors.

27. In paragraph 22, The above neurodegenerative disease is Alzheimer's disease, Parkinson's disease or Lou Gehrig's disease; A pharmaceutical composition, wherein the above autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, psoriasis or Sjogren's syndrome.

28. A method for treating a disease caused by overexpression, excessive activity, mutation and / or activation of a signaling pathway associated with CDK12 and / or CDK13, comprising administering to a subject a compound of any one of claims 1 to 19, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof.

29. A method for inhibiting CDK12 and / or CDK13 or degrading cyclin K, comprising the step of contacting an effective amount of a compound of any one of claims 1 to 19, a stereoisomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, with a cyclin-dependent kinase.

Images